Applications of Click Chemistry

Click chemistry has made important progress in scientific research fields such as biomedicine, especially copper-free click chemistry.In in vitro studies, click chemistry enables specific labeling of cellular target proteins and studies the interaction of drug targets with drug substitutes in living cells. In addition, cell membrane lipids and proteins can be selectively labeled in vitro, and cells can be chemically linked together by clicks. In in vivo research, click chemistry makes molecular imaging and drug delivery for diagnosis and treatment efficient and effective. This article describes several specific applications of click chemistry in biomedical research.

Drug development

Molecular backbone

The structure-activity relationship of drug molecules is an important research content of medicinal chemistry. The triazole structure obtained by the classical CuAAC click chemistry is actually a common backbone group in drugs. Current studies have shown that the triazole structure may play a variety of roles in drug molecules, and there are many approved or promising drug molecules that contain the triazole structure. For example, the Buckle team's research showed that triazole derivatives are powerful anti-skin allergenic drugs, and the study subjects in mice showed good drug activity. The selective small-molecule anticoagulant ticagrelor is a 1,2,3-triazole derivative, which can reversibly act on vascular smooth muscle cells and effectively improve the symptoms of patients with acute coronary heart disease.

Fig.1 Approved drugs or drug candidates containing 1,2,3-triazole structure. (Jiang Xiangyi, et al., 2019)

Antibody-drug conjugates (ADCs)

Antibody-drug conjugates (ADCs) are forms of drugs in which potent small-molecule drugs are conjugated to monoclonal antibodies via chemical linkers to enable targeted therapies. Click chemistry has become an important tool for ADC design and synthesis due to its high efficiency and selectivity. For example, MedImmune, an ADC drug development arm of AstraZeneca, has developed a series of ADC drug candidates through CuAAC click chemical linkage. In the series of ADC candidates, the azide group was modified on the monoclonal antibody targeting HER2/neu (a member of the EGFR family), and the tenoalkyne was modified on the toxin of Auristatin F, a cytotoxic tubulin inhibitor belonging to the nudibranch. In addition, click chemistry is also used to modify drugs at different locations to optimize their pharmacokinetic and pharmacodynamic properties.

PROTAC

Because click chemistry is good at molecular linking, it is also commonly used in the development of popular proteolytic targeted chimera (PROTAC) drugs. Amgen has reported a general strategy for the development of PROTACs using click chemistry and using this strategy to construct a library of PROTAC molecules. Astex Pharmaceuticals reported intracellular conjugation of two drug precursors to heterobifunctional molecules via iEDDA click chemistry, which was successfully used to degrade two cancer targets, BRD4 and ERK1/2.

Drug discovery

Click chemistry excels at efficient ligation of molecular fragments and is a practical approach in the field of high-throughput screening of HTS) as well as fragment-based drug design (FBDD). The straightforward use of the click-and-click component library is ideal for rapid combinatorial synthesis of novel molecular drugs, which can greatly reduce the time required for lead discovery and structure optimization. After proposing the concept of click chemistry and the CuAAC method, Prof. Sharpless's group has also practiced click chemistry in the field of fragment-based drug design. They chose acetylcholinesterase (AchE) as the research target, and in one of the pockets, the two types of molecular fragments were linked in situ through click chemistry, and incidentally discovered a highly active inhibitor of AchE.

Fig.2 AchE inhibitors screened using Click Chemistry. (Krasinski Antoni, et al., 2005)

Targeted drug delivery

Click chemistry has emerged as a powerful chemical tool for targeted drug delivery in biological research. The rapid second-order reaction rate constant, simplicity, and orthogonality of click chemistry can be used in polymer synthesis or for positional modification of bioligands during drug carrier development. For example, in Lee et al.'s study, a second intravenous injection of a nanoparticle containing a photosensitizer and BCN resulted in the effective delivery of a SPAAC to the tumor tissue in vivo compared to a bare nanoparticle or a drug-loaded nanoparticle that was not injected for the first time with Ac4ManNAz.

Biomarkers

Biological probes

Functionalized planes play an important role in today's biotechnology, for example in sugar, DNA or protein microarrays, biosensors, or microfluidic devices. With the development of technology, people are no longer limited to static observations, but also gradually turn to real-time dynamic observations, in which how to accurately and efficiently label biological targets is the core link of all observations. Prof. Bertozzi's team used a mouse model to first inject mice with tetraacylated N-azide acetylmannosamine (Ac4ManNAz), and then injected the mouse model with an alkyne-modified FLAG peptide, so that SiaNAz can be labeled in vivo through SPACC click chemistry. Subsequently, the fluorescent molecule-labeled FLAG monoclonal antibody can be used to study the distribution and metabolism of SicNAz molecules in different tissues and organs in mouse models through fluorescent detection signals.

Fig.3 Click chemistry to label sugars on the surface of cell membranes. (Chang Pamela V., et al., 2010)

Immunofluorescence detection

Sun's team reported a novel immunofluorescence labeling method and its application in cell fluorescence detection. Firstly, the sensitivity of the labeling method was measured by copper ionization of the alkyne group in 4-ethynyl-N-ethyl-1,8-naphthalimide with the azide group of the labeled antibody, and the sensitivity of the labeling method was measured with the NHS-FITC and NHS-Rhodamine labeled antibodies as the positive control, and the results were comparable to the positive control. The detection limit of this assay was up to 0.1 μg when stained at the cellular level, indicating that azide-labeled antibodies can be effectively used for immunofluorescence staining analysis.

Summary

Click chemistry and bioorthogonal chemistry provide powerful chemical tools for life science research, as well as reliable and efficient modular synthesis methods for drug discovery, bridges to link pharmacophores or functional molecular fragments, and in-situ exploration of drug-target binding. In the future, with the continuous progress and application of click chemistry technology, click chemistry will play a more important role in various fields.