A Brief Introduction to Click Chemistry

At 11:45 on October 5, 2022, local time in Sweden (17:45 on October 5, Beijing time), the Nobel Prize Committee announced that the 2022 Nobel Prize in Chemistry will be awarded to American chemist Carolyn R. Bertozzi, Danish chemist Morten Meldal, an American chemist, and Karl Barry Sharpless, an American chemist, for their outstanding contributions to the development of click chemistry and bioorthogonal chemistry. The concept of click chemistry was first proposed by Sharpless and has been used to link organic and bioorganic molecules. Click chemistry refers to a set of efficient, robust, and stereoselective reactions that can exploit simple reaction conditions and readily available starting materials to develop promising building blocks.

Looking back at the development of organic synthesis before the concept of click chemistry was put forward, the United States dominated the frontier of this field after World War II. The research work focused on the synthesis of complex molecular structures (especially natural products) through the construction of carbon-carbon bonds (CC). and EJ Corey and other full-synthetic masters represented. Their work embodies the courage of people to challenge nature, and some novel synthetic methods reported also make the content of organic chemistry more abundant and systematic, but these reactions are often difficult to study in other fields because of high operational difficulty or low yield. are widely used. Nucleic acids and proteins are common biomacromolecules in nature. Complex chemical structures and rich biological functions are realized by small molecular units linked by carbon-heteroatom bonds (phosphate bonds and peptide bonds). Inspired by this, Sharpless proposed the concept of click chemistry in 2001, emphasizing the rapid and reliable chemical synthesis of various molecules based on the synthesis of carbon-heteroatom bonds (CXC) and even inorganic linkages.

Immediately following the concept of click chemistry, the monovalent copper-catalyzed azide-alkyne cycloaddition reaction was independently reported by the Sharpless and Medal groups in 2002. This reaction can be regarded as the first classic work in click chemistry. Azides and terminal alkynes are stable under most chemical conditions but can be efficiently and specifically converted to 1,3-substituted triazoles (Formula 1) under monovalent copper-catalyzed conditions. The linking group that is completely consistent with its structure has not been found in nature, but the characteristics of mild conditions, high yield, high chemoselectivity, and no interference from water and oxygen have become the outstanding advantages of this reaction.

2. Strain-promoted azide-alkyne click chemistry (SPAAC)

Strain-promoted azide-alkyne cycloaddition (SPAAC) was developed by Bertozzi et al. in 2004, which does not require the use of metal catalysts, reducing agents, or stable ligands. Instead, this reaction utilizes the enthalpy released by ring strain to cyclooctynes (e.g., OCT, BCN, DBCO, DIBO, and DIFO) to form stable triazoles (Eq. 2). Although the reaction kinetics of SPAAC is slower than that of CuAAC, its biocompatibility in living cells is beyond doubt. So far, this reaction has been widely used in the fields of hybrid and block polymer formation, metabolic engineering, nanoparticle functionalization, oligonucleotide labeling, etc.

3. Linkage between tetrazine and alkenes (trans-cyclooctene)

Trans-cyclooctene TCO reacts in the inverse electron demand Diels Alder (IEDDA), and the reaction under physiological conditions has the characteristics of no catalyst, fast reaction rate, and good biocompatibility. Trans-cyclooctene is widely used in the research of biology and materials science, especially the pre-targeting method and related kits for targeted medical imaging or therapy. Tetrazine is a class of click chemistry labeling reagents containing reactive tetrazine groups, a six-membered heterocyclic compound containing four nitrogen atoms, and has three isomers: 1,2,3,4-tetrazine, 1, 2,4,5-tetrazine, 1,2,3,5-tetrazine. Tetrazine reagents are highly reactive with TCO (trans-cyclooctene) in the inverse electron demand Diels Alder reaction and the reverse Diels Alder reaction to eliminate nitrogen. This is a very fast response for bioconjugation at low concentrations in labeling live cells, molecular imaging, and other bioconjugation applications.

(1) Reaction modularization, such as azide and alkynyl can generate triazole compounds;

(2) Raw materials are easy to obtain and have a wide range of applications;

(3) High reaction yield, good regio and stereoselectivity;

(4) Simple operation, mild reaction conditions, not afraid of water and oxygen;

(5) The product is easy to separate and purify, and can be separated by recrystallization or distillation without chromatographic column separation;

(6) Most reactions involve the formation of carbon-heteroatom (mainly nitrogen, oxygen, sulfur) bonds;

(7) The reaction requires a high thermodynamic driving force (>84kJ/mol);

(8) Click reactions are generally chemical compounds (no by-products) or condensation reactions (products are small molecules such as water).

1. Drugs

Studies by Buckle et al. have shown that triazole derivatives are powerful anti-skin allergic drugs, and they have shown good drug activity with mice as receptors. 1,2,3-triazole-substituted benzenesulfonamide compounds are strong selective constrictors of human β-adrenergic hormone receptors. Pharmacological screening experiments have proved that 4-trifluoromethylbenzyl homologs have extraordinary effects. The selective small-molecule anticoagulant drug ticagrelor is a 1,2,3-triazole derivative, which can reversibly act on the purine subtype P2Ym on vascular smooth muscle cells and affects ADP-induced platelet aggregation. It has an obvious inhibitory effect and takes effect quickly after oral administration, so it can effectively improve the symptoms of patients with acute coronary heart disease.

2. Synthesis of lead compound library

The construction process of directly using the click component module library is ideal for the rapid combinatorial synthesis of new molecular drugs, which can greatly shorten the time required for lead compound discovery and structure optimization. Using some short reaction sequences, click chemistry can prepare a large number of complex, novel, and diverse compound libraries in the laboratory, such as 1,2-disubstituted ethane derivative library, five-membered aromatic heterocyclic compound library, 1, 2.3-triazole derivative library and non-aromatic heterocyclic compound library, etc. For example, Khanetskyy et al. have created a compound library containing 27 pyrimidinones as basic skeletons. The reaction process includes methyl bromination, azidation, and dipolar cycloaddition reactions to form a ring. The microwave-promoted technology was used in multiple steps in the reaction, and cu(D was used to catalyze the final ring-forming reaction, and good results were obtained.

3. Target-directed active small molecule synthesis

Target-guided click chemistry can be used to find high-affinity inhibitors. Azido compounds and alkynyl compounds with appropriate structures can quickly generate stereospecific triazole compounds under the catalysis of the active center of the enzyme. Inhibitor of synthetase itself. Sharpless et al. used inert reactants under physiological conditions to perform irreversible target-directed synthesis to obtain high-affinity AChE inhibitors.

4. Glycoproteins

Glycoproteins play a very important role in the field of biopharmaceuticals. Usually, glycoproteins contain oligosaccharides linked to proteins in the form of N or O, but glycopeptide bonds are very sensitive to enzymatic hydrolysis, which limits their metabolic stability. In addition, O-glycoprotein synthesis assembly is easily inhibited due to the elimination of the glycosyl moiety. Click chemistry can overcome the instability of synthesis and metabolism, so the use of click chemistry in the synthesis of glycoproteins is very suitable for Rutjes et al. to synthesize triazole sugar amino acids under mild conditions by using azido amino acids and alkyne glycosides through click reactions. And the yield is higher. Chemoselective alkyne group substitution of cysteine thiols by Macmillan and Blanc demonstrates the compatibility of click chemistry and native chemical strategies. Westermann et al. combined click chemistry with ring-closing metathesis to prepare a macrocyclic glycolipid mimic library.

5. Biological probes and microarrays

Functionalized planes play an important role in today's biotechnology, such as applications in sugar, DNA or protein microarrays, biosensors, or microfluidic devices; while efficient triazole linkages are very suitable for modifying organic or inorganic surfaces, Therefore, a lot of click chemistry methods for modifying substrate planes have been reported in recent years. Glycoarrays are a high-performance means for screening hemagglutinin proteins. Wong's group first used click chemistry to construct glycosyl microarrays, to develop an achievable, high-performance screening method for the identification of hemagglutinin-like proteins. Among them, azide-modified galactose undergoes a cycloaddition reaction with hydrophobic acrylamide under the catalysis of CuI/DIPEA at room temperature, and then acrylamide is attached to the polystyrene carrier in a non-covalent manner. Experiments showed that D-galactoside-bound ricin B hemagglutinin protein could be successfully screened by glycan array. Wong's group also reported a non-covalent sugar array using click chemistry as the main immobilization approach, which can be used for high-efficiency screening of Fuc-T inhibitors. To develop a more stable high-efficiency screening program.

6. Immunofluorescence Detection

Sun Dan et al. reported a new immunofluorescence labeling method and its application in autofluorescence detection. First, two key compounds, 6-azido-hexanoic acid succinimide active ester and 4-ethynyl-IV-ethyl-1,8-naphthalimide, were synthesized, and the synthesized 6-azide -Coupling of hexanoic acid succinimide active ester with the free amino group of anti-her2 antibody Anti—HPl5 to obtain azide IgG; subsequent ionization of 4-ethynyl-N-ethyl-1,8-naphthalimide by copper The alkynyl group in and the azide group of the labeled antibody were click chemically reacted. At the same time, the NHS-FITC and NHS-Rhodamine labeled antibodies were used as positive controls to measure the sensitivity of the labeling method, and the results were comparable to the positive controls. Then, the detection limit of staining at the cellular level can reach 0.1 μg, and the results show that the azide-labeled antibody can be effectively applied to immunofluorescence staining analysis. Finally, the laser confocal three-channel composite fluorescence analysis method was used to study different labeling methods and their corresponding immunofluorescence chromogenic methods and confirmed that the antibodies labeled by this method can be used simultaneously with other immunofluorescence techniques without interfering with each other. In this study, by developing a new antibody labeling technique, a new immunofluorescence antibody analysis method was established, and the application verification was carried out at the cellular level, which enriched the immunofluorescence antibody detection methods. This method has development potential and broad application prospects in future immune research.