What is Click Chemistry?

On October 4, 2022, 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 and American chemist Karl Barry Sharpless in recognition of their outstanding contributions to click chemistry and bioorthogonal chemistry. As a set of efficient, robust, and stereoselective reactions, click chemistry can exploit simple reaction conditions and readily available starting materials to develop promising building blocks. Click chemistry has opened up a new method of combinatorial chemistry based on the synthesis of carbon-heteroatom bonds (C-X-C), and with the help of these reactions (click reactions) to obtain molecular diversity simply and efficiently.

What is Meant by Click Chemistry?

Click chemistry is a synthesis concept introduced by American chemist Professor Sharpless in 2001. Its main purpose is to quickly and reliably complete the chemical synthesis of various molecules through the splicing of small units. It is an imitation of organic synthesis in nature. The core of click chemistry is to make the reaction as simple and efficient as possible. Anyone can use click reactions to realize the diversity of molecules and thereby realize their functions. In the development process of traditional organic synthesis, the formation and breakage of C-C bonds are more involved. Click chemistry emphasizes new combinatorial chemistry methods based on carbon-heteroatom bond (C-X) synthesis, and uses these reactions (click reactions) to obtain molecular diversity simply and efficiently. Click chemistry is currently most widely used not in the field of chemistry but in biology, materials and other fields, such as bioconjugation technology, biomedicine, DNA self-assembly, supramolecular chemistry, dendrimers, functional polymers, proteomics, etc. It shows broad application prospects. Generally, click responses have the following common characteristics:

(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 (>84 kJ/mol).

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

Click Chemistry Tools

Click chemistry tools are widely used in drug discovery, materials science, and bioconjugation, where rapid and efficient synthesis of diverse chemical libraries is critical for identifying new lead compounds. The ability to quickly and easily generate different molecular structures using click chemistry has significantly accelerated the drug discovery process, allowing the identification of new drug candidates for a variety of diseases. BOC Sciences offers a variety of lipid click chemistry reagents to support lipid labeling, lipid functionalization, and lipid conjugation applications. Our click reagents are of extremely high purity and quality, with each reagent carefully synthesized and purified to ensure they are free of impurities that could interfere with experimental results.

Click Chemistry Reaction

Cu(I)-Catalyzed Azide-Alkyne Click Chemistry (CuAAC)

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 (Eq. 1) under the catalysis of monovalent copper. The advantages of this reaction are mild reaction conditions, high yield, high chemoselectivity and no interference from water and oxygen.

Strain-promoted Azide-Alkyne Click Chemistry (SPAAC)

The SPAAC reaction does not require metal catalysts, reducing agents, or stable ligands. This reaction utilizes the enthalpy released by ring strain to cyclooctynes (such as 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.

Inverse Electron Demand Diels-Alder (IEDDA)

The reaction of trans-cyclooctene (TCO) through IEDDA under physiological conditions has the characteristics of no catalyst, fast reaction rate and good biocompatibility. Tetrazine is a class of click chemistry labeling reagents containing reactive tetrazine groups, six-membered heterocyclic compounds containing four nitrogen atoms. Tetrazine 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 in IEDDA reactions to eliminate nitrogen. Currently, the reaction of trans-cyclooctene with Tetrazine is widely used in the research of biology and material science, especially targeted medical imaging or other bioconjugation applications.

What Can Click Chemistry be Used For?

Click Chemistry-based Drug Discovery

Click chemistry can be combined with high-throughput enzymatic assay technologies such as microarrays, which changes lead discovery and lead optimization in drug discovery. Due to the efficiency and water compatibility of the "click reaction", the assembled products can be directly screened for inhibition without any purification. Small molecule libraries synthesized by click chemistry have been successfully used to generate unique inhibitors and activity-guided fingerprints of important enzymes, which may lead to the identification and characterization of new enzyme subclasses. In addition, click chemistry is also one of the most practical approaches to develop fragment-based inhibitors due to its highly modular and efficient reactive nature. With the help of click chemistry, pharmaceutical compositions of closely related but fragmented prodrugs can be designed to specifically interact with molecular targets (e.g., proteins or genes), thereby enabling personalized therapy. The highly modular and efficient reaction properties of click chemistry enable synthetic chemists to construct libraries of diverse classes of compounds through fragment-based enzyme inhibitors.

Click Chemistry-based Nucleic Acid Functionalization

The modularity provided by azide-alkyne cycloaddition may serve as a nucleic acid assembly method for biological and nanotechnology applications. Strategies for the chemical synthesis of genomic DNA fragments, particularly those containing modified and epigenetic bases, have been developed in vitro and in cells. Genes assembled using this method function in both prokaryotic and eukaryotic systems. Furthermore, click ligation of oligonucleotides is an efficient tool for generating antisense oligonucleotides. In this case, the presence of triazole increases the stability of the oligonucleotide against nuclease degradation and reduces the anionic charge of the oligonucleotide, which may facilitate cellular uptake. Chemical synthesis and click ligation of antisense oligonucleotides have been used to introduce base modifications and nucleoside derivatives (i.e., locked nucleic acids, G-clamps) that enhance target binding and mismatch sensitivity.

Click Chemistry-based Cell Labeling

Since biological components such as sugars, amino acids, or lipids are used and metabolized in living cells, the use of chemically tagged biomolecules allows the introduction of chemical tags into proteins, glycans, and lipids in living cells. Metabolic glycoengineering using sugar analogs is particularly useful for introducing SPAAC and iEDDA chemical substrates into living cells. For example, chemically tagged monosaccharides and N-azidoacetylmannosamine (Ac4ManNAz) in metabolic sugar engineering are widely used to engineer cell surfaces. The combination of metabolic glycoengineering and copper-free click chemistry allows cells to be stably labeled with a variety of molecules without affecting the cell's properties.

Click Chemistry-based Drug Delivery

Currently, bioorthogonal chemistry receptors and SPAAC reactions have been used for successful tumor-targeted drug delivery: DBCO-modified drugs can target azide-expressing tumor cells through the SPAAC reaction. Cancer cell-specific azide labeling methods using cancer overexpressed enzyme-cleavable Ac3ManNAz analogues and SPAAC reactivity can be used for drug delivery for cancer cell-specific imaging. Additionally, modifying nanoparticles with click chemistry chemicals such as BCN and DBCO increases the affinity between the nanoparticles and azide-labeled cancer cells. The iEDDA response has also been studied for cancer diagnosis, achieving higher tumor-to-background contrast at early time points. The pretargeting approach based on iEDDA response reduces radiation dose to non-target tissues.

Click Chemistry-based ADC Synthesis

Cu(I)-catalyzed alkyne-azide cycloaddition (CuAAC) has great potential in the synthesis of antibody-drug conjugates (ADCs). At present, researchers have designed an efficient and cost-effective ADC conjugation method based on CuAAC, and demonstrated that ADC can be rapidly synthesized, and thus developed GlycoConnect coupling technology. It enables site-specific conjugation using natural glycosylation sites, converting monoclonal antibodies into stable conjugated ADCs in just a few days. The technology is based on two processes: first enzymatic remodeling (modification and labeling with azide), followed by ligation of the payload based on copper-free click chemistry.

Click Chemistry-based PROTAC Synthesis

Preparation of PROTAC libraries using a click chemistry platform using cereblon and VHL ligase ligands with bromodomain and terminal ectodomain 4 (BRD4) ligand JQ-1 and targeting cereblon (CRBN) and Von Hippel-Lindau (VHL) proteins ligase binding agents, the ability of these molecules to form a ternary complex between ligase/PROTAC/BRD4 protein and their activity in targeting BRD4 protein degradation in two lung cancer cell lines.

Click Chemistry-based Fluorescence Imaging

One of the most interesting capabilities of click chemistry is the fluorescence imaging of intracellular target proteins (TOIs). In iEDDA reactions, TOI proteins intrinsic to living cells can be visualized by treatment with TCO ligand conjugates and Tz-containing fluorophores (FLTz). For example, the clinical drug AZD2281 was combined with TCO to develop a biological probe for studying PARP1 protein (a known important cellular protein for DNA repair). TCO was coupled to the anticancer agent paclitaxel, and intracellular tubulin was successfully visualized using the paclitaxel-TCO/Tz-BODIPY FL combination. Later, multi-ligand-TCO conjugates such as BI2536, Foretinib, Dasatinib and Ibrutinib were also used to develop targeting various TOI proteins.

Click Chemistry-based Diagnostic Analysis

Click chemistry can also be used to develop molecular tools to understand tissue development, disease diagnosis, and treatment monitoring. Many cancers release small membrane-bound microvesicles (MVs) into the peripheral circulation, and analysis of MVs, such as glioblastomas (GBMs), is a promising approach to disease diagnosis. For example, Lee et al. reported a microfluidic system combining iEDDA-type click chemistry and small micro-nuclear magnetic resonance (μNMR) for analyzing MVs in the blood of GBM patients.

Click Chemistry and Bioorthogonal Chemistry

Bioorthogonal chemistry refers to the set of chemical reactions that occur in living systems without interfering with natural biochemical processes. These reactions are often highly selective and can be performed under physiological conditions, allowing researchers to introduce specific chemical modifications in biological systems without causing major damage or disruption. Many click chemistry reactions are bioorthogonal and have been widely used in various fields such as bioimaging, drug delivery, proteomics, and chemical biology. For example, bioorthogonal reactions can be used to attach fluorescent dyes or other imaging probes to specific biomolecules, allowing researchers to visualize and track their localization and dynamics in living cells or organisms. Likewise, bioorthogonal chemistry can be used to selectively deliver drugs or therapeutics to specific targets in vivo, thereby minimizing off-target effects. Currently, the types of click reactions that can be used in bioorthogonal chemistry include strain-promoted azido-alkyne cyclodition (SPAAC), Cu-catalyzed azido-alkyne cyclodition (CuAAC), Staudinger ligation, cyclodition reactions between a tetrazine and a trans-alkene or a trained alkyne, reactions between an isocyanopropyl group and tetrazine, and Diels-Alder reactions between a cyclopentadienone and a trained alkyne. Each type of reaction has its own characteristics and is suitable for certain types of applications, such as drug delivery or bioimaging.

PEG for Click Chemistry

BOC Sciences is a leading customer-focused biotechnology company based in New York, USA. We provide a wide range of high-quality PEG linkers to customers worldwide. With a stock of over 3,000 low dispersion and high purity PEG reagents (such as DBCO PEG and Azide PEG), we provide customer with convenient and efficient solutions ready for immediate shipment. The diversity and availability of our products can greatly benefit researchers and professionals in various fields who require PEG connectors for their work. In addition, we also provide one-stop PEG & click chemistry solutions to facilitate customers' applications in drug delivery, medical diagnostics, and material chemistry.