The 2022 Nobel Prize in chemistry was awarded to scientists Carolyn R. Bertozzi, Morten Meldal and K. Barry Sharpless for their development of click chemistry and bioorthogonal chemistry. 
Figure 1. 2022 Nobel Prize in chemistry, soucce: reference 
Barry Sharpless and Morten Meldal brought chemistry into the age of functionalism and laid the foundation for click chemistry. While Carolyn Bertozzi took click chemistry into a new dimension and began using it to map cells. Among many other applications, her bioorthogonal chemistry contributes to more targeted cancer therapies, such as antibody-drug conjugates (ADCs), fluorescent imaging, and targeted drug delivery, etc.
What Is Click Chemistry & Bioorthogonal Chemistry?
Click chemistry: A chemical synthesis method for the rapid and efficient synthesis of useful new molecules based on the heteroatom link (C-X-C). 
Bioorthogonal Chemistry: Chemical reactions that use the principles of click chemistry to occur inside of living systems without interfering with native biochemical processes.
In 2001, an examination of nature's favorite molecules reveals a striking preference for making carbon-heteroatom bonds over carbon-carbon bonds. The concept of "Click Chemistry" was inspired by the fact that nucleic acids, proteins and polysaccharides are condensed polymers held together by carbon-heteroatom bonds. Click chemistry is a chemical synthesis method that allows for the rapid and efficient synthesis of useful new molecules based on carbon-heteroatom link (C-X-C).
Prior to this, chemical synthesis was complicated and difficult but with low yields. Until the first generation of click chemistry, the monovalent copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction was proposed, the complex reactions started to be simplified by building functional molecules in a patterned reaction manner. However, the cytotoxicity of copper catalysts limited the application of CuAAC reactions in vitro and in vivo.
Chemists have since discovered a strain-promoted alkyne-azide cycloaddition (SPAAC) reaction that allows the azide-alkyne reaction to occur without the need for a cytotoxic copper catalyst. This reaction has been used to label glycoproteins on the cell surface in vitro and in vivo with no apparent cytotoxicity.
However, some chemists were not satisfied with the second-order reaction rate constants of SPAAC. Therefore, Blackman et al. developed the inverse electron-demand Diels-Alder (iEDDA) reaction between the cycloaddition reactions of s-tetrazine and trans-cyclooctene (TCO) derivatives to produce faster copper-free click chemistry than the SPAAC reaction.
Figure 2. Characteristics of currently used click chemistry reactions, source: reference 
Click Chemistry in Biomedical Applications
Click chemistry has made important advances in the field of biomedical research, particularly copper-free click chemistry, including SPAAC and iEDDA reactions. In in vitro studies, click chemistry allows for the specific labeling of cellular target proteins and the study of the interaction of drug targets with drug surrogates in living cells. In addition, cell membrane lipids and proteins can be selectively labeled in vitro and cells can be linked together by click chemistry. In vivo studies, click chemistry makes molecular imaging and drug delivery for diagnosis and therapy efficient and effective. 
Following, we present several specific applications of click chemistry in biomedical research.
Click Chemistry for Fluorescence Imaging
One of the most interesting applications of copper-free click chemistry might be the uorescence imaging of target of interest (TOI) proteins inside cells. Especially with iEDDA reactions, innate TOI proteins in live cells could be successfullyvisualized with a TCO–ligand conjugate and subsequent treatment of Tz containing uorophores (FLTz).
For example, the clinical drug AZD2281 was combined with TCO to develop a bioprobe for the study of PARP1 protein (known to be an important cellular protein for DNA repair). TCO was coupled to the anticancer agent Taxol and tubulin proteins inside cells were successfully visualized with the Taxel-TCO/Tz-BODIPY FL combination. Later multiligand-TCO conjugates such as BI2536, Foretinib, Dasatinib and Ibrutinib were also used to develop targeting various TOI proteins such as polo-like kinase 1 (PLK1), MET, and BTK proteins. 
Figure 3. Copper-free click reaction between AZD2281-TCO and Texas Red-Tz in MDA-MB436 cells. Source: reference 
Click Chemistry in Targeted Drug Delivery
Click chemistry has emerged as a powerful chemical tool for the targeted delivery of drugs in the study of living organisms. The fast second-order reaction rate constants, simplicity and orthogonality of click chemistry can be used for polymer synthesis or for site-specific modification of bioligands during drug carrier development. For example, in 2012, Koo and Lee et al. provides evidence, for the first time, that click chemistry in vivo could be used for nanoparticle delivery. In the study, azide group labeling of tumor cells by Ac4ManNAz-loaded nanoparticles and second tumor targeting by DBCO-modified nanoparticles containing photosensitizers, were injected into mice sequentially, and tumor-targeting was enhanced by SPAAC between azide groups and DBCO. 
Figure 4. Application of click chemistry for tumor-targeted drug delivery. Source: reference 
Click Chemistry-based ADC Synthesis
Cu(I)-catalyzed alkyne-azide cycloaddition (CuAAC) has great potential in the synthesis of antibody-drug conjugates (ADCs) . Researchers have now devised efficient and cost-effective CuAAC-based ADC conjugation methods and demonstrated that ADCs can be synthesized rapidly, which led to the development of the GlycoConnect coupling technology. GlycoConnect enables targeted conjugation using native glycosylation sites and can convert monoclonal antibodies into stable conjugated ADCs in just a few days. The technology is based on two processes: first, enzymatic remodeling (modification and tagging with azide), followed by ligation of the payload based on copper-free click chemistry. Synaffix has partnered with several companies with its next-generation ADC technology platform, including GlycoConnect.
Figure 5. Next-generation ADCs being developed under a license agreement
ADC Therapeutics was the earlier company to license Synaffix ADC platform technology and is currently the representative with the largest number of products developed using this technology, of which ADCT-601 is already in the clinical study phase.  Currently, ADC Therapeutics' publicly available products in the solid tumor space (3/5) is using Synaffix's ADC technology.
Figure 6. ADC Therapeutics Pipelines
Figure 7. Structure of ADCT-601
Click Chemistry-based PROTAC Synthesis
Click chemistry is often used in the linker of PROTAC molecules to connect the two ends of the molecule due to milder reaction conditions and higher efficiency. Ryan P Wurz et al. demonstrates the utility of this approach with the bromodomain and extraterminal domain-4 (BRD4) ligand JQ-1 (3) and ligase binders targeting cereblon (CRBN) and Von Hippel–Lindau (VHL) proteins .
Figure 8. Click Chemistry-based PROTAC Synthesis, source: reference 
Click Chemistry-based Diagnosis
Click chemistry can also be used to develop molecular tools for understanding tissue development, disease diagnosis and treatment monitoring. Many cancers release membrane-bound microvesicles (MVs) into the peripheral circulation, and the analysis of MVs such as glioblastomas (GBMs) is a promising method for disease diagnosis. For example, Lee et al. reported a microfluidic system combining iEDDA-type click chemistry and small micro-nuclear magnetic resonance (μNMR) for the analysis of MVs in the blood of GBM patients .
Figure 9. Click Chemistry-based Diagnosis. source: reference 
Click chemistry and non-copper bioorthogonal reactions have made important advances in the field of biomedical research. Click chemistry allows for the specific labeling of cellular target proteins and can be used to adhere cells together and also enables efficient and effective molecular imaging and drug delivery for diagnostic and therapeutic purposes. Click chemistry can also be used to develop molecular tools such as DNA nanocatalysts, chemical synthesis of genomic DNA, assisted CRISPR-Cas gene editing, ADC and PROTAC synthesis, etc. Overall, click chemistry has become a valuable tool in the biomedical field and in organic chemistry.
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. Zammarchi F, Havenith KE, Chivers S, Hogg P, Bertelli F, et al. Preclinical Development of ADCT-601, a Novel Pyrrolobenzodiazepine Dimer-based Antibody-drug Conjugate Targeting AXL-expressing Cancers. Mol Cancer Ther. 2022 Apr 1;21(4):582-593.
 Wurz RP, Dellamaggiore K, Dou H, Javier N, Lo MC, McCarter JD, Mohl D, Sastri C, Lipford JR, Cee VJ. A "Click Chemistry Platform" for the Rapid Synthesis of Bispecific Molecules for Inducing Protein Degradation. J Med Chem. 2018 Jan 25;61(2):453-461.