Coined by K. Barry Sharpless, Hartmuth C. Kolb, and M.G. Finn in 2001, click chemistry is a term used to describe reactions that rapidly and selectively react or “click” with each other in a predictable way to form physiologically stable products with heteroatom links (C-X-C). Widely used in the modification of biomolecules, surfaces, particles, and organic compounds, click chemistry offers many advantages:

  • Broad in scope  
  • Modular nature   
  • Mild reaction conditions  
  • Reliable in both small- and large-scale applications 
  • High yielding (proceeding rapidly to completion)  
  • Simple product isolation (requiring little to no purification) 
  • Inoffensive byproduct generation (adhering to the 12 principles of green chemistry 
  • Compatibility in living systems (allowing the chemoselective modification of biomolecules with little perturbation), serving to probe biology better.2  


Out of about 10 different types of click reactions, 3 are the most heavily used in various life science applications, ranging from “simple” biomolecule labeling & detection to advanced CRISPER applications. For more information refer to The 3 most common click reactions and their reagents. Here, we highlight the top 9 (most current) applications that you need to know to get the full scope of what this chemistry has to offer.  

1) Biomolecule labeling & detection  

2) (Solid & solution phase) biomolecule modification/ligation 

3) Construction of analog compound libraries for structure activity relationship profiling  

4) In situ click chemistry for drug lead discovery 

5) Drug delivery 

6) Material optimization (polymer modification)  

7) Probe for viral studies 

8) CRISPER sgRNA synthesis and target gene labelling 

9) Novel applications including ‘click to release’  


1) Biomolecule labeling & detection  

One of the most useful features of click chemistry is its ability to label and visualize biomolecules such as lipids,3 peptides4, glycans,4,5 glycoproteins,6 nucleic acids,7,8 and synthetic molecules (e.g. Taxol9) with minimal physiological perturbation (in vitro and in vivo).10 In a two-step process, target biomolecules are first tagged/labelled (either genetically, enzymatically, metabolically,11,12 or synthetically9,13) with a biorthogonal click handle such as an alkyne or azide. Detection/visualization then occurs when a complementary click handle present on a molecule with a fluorescent or affinity group engages the target molecule in a click reaction.   


For example, surface glycans have been visualized in live developing zebrafish at subcellular resolution; not normally seen with conventional molecular imaging approaches reliant upon genetically encoded reporters.14 In this study, Bertozzi et al. used metabolic glycoengineering in tandem with a multicolor detection strategy to reveal differences in cell-surface expression, intracellular trafficking, and tissue distribution of glycans throughout zebrafish embryogenesis.14 Similar studies have also been conducted with mice to track transplanted cells, 11 and the uptake of peptides into cells has also been measured, helpful for structure-activity-permeability relationship optimization studies. 15 Labelling biomolecules at two sites (called dual-site labelling) is now also facilitating the study of complicated biological systems.16,17,18,19,20 


2) (Solid- & solution- phase) biomolecule modification/ligation 

Peptides, nucleotides, small molecules, supramolecular structures, etc. have all been readily modified using solid-phase or solution-phase click chemistry with little to no product purification or protecting group manipulation.3,21,22 In general, solid phase synthesis is faster and requires little to no purification, but there are pros and cons associated with each method. 2324   


3) Construction of analog compound libraries   

Analog compound libraries can be rapidly and reliably constructed via click chemistry with little synthetic effort, followed by in-situ high-throughput screening (HTS) to facilitate molecule structure activity relationship (SAR) profiling necessary for the optimization and discovery of bioactive molecules. Already many (focused combinatorial) fragment libraries based on the click (triazole) scaffold have been assembled25; for instance, a Janus Kinase inhibitor ruxolitinib-derived triazole library was constructed to evaluate JAK3 inhibitors.24 


4) In situ click chemistry for drug lead discovery 

In situ click chemistry is a (kinetic) target-guided synthesis approach coined and applied for the first time by Sharpless and co-workers in 2002 for the discovery of a potent inhibitor of acetylcholinesterase. 26 This approach uses the target biomolecule itself as a scaffold upon which binding ligands engage in a click reaction if brought in close enough proximity and proper orientation to react. In this way, the best ligands leading to a stable complex with the target can be screened from a pool of fragments bearing complementary reactive functional groups. 27  Prior synthesis, purification, and biochemical evaluation of the library members is not necessary enabling large numbers of compounds to be rapidly and cost efficiently screened.28,29   


Inhibitors of carbonic anhydrase,30 HIV protease,31 chitinase,32 ligands of nucleotides,33 protein-protein interactions (via sulfo-click chemistry),34 antibody-like protein-capture agents,35,36 transcriptional factors,37 channels,38 etc. have also been characterized.  


5) Drug delivery  

The controlled administration of drugs into the body is an important aspect of effective drug design. Click chemistry has been used to construct polymeric nano- and microparticulate drug delivery systems (DDS) such as polymeric micelles, liposomes, capsules, carbon nanotubes, etc.6,39 


6) Material optimization (modification of polymers) 

From linear- and graft-polymers to the synthesis of more complex architectures (e.g. star-polymers, block-copolymers, and dendrimers) to the functionalization of surfaces and interfaces,40  click chemistry has had an enormous impact in the material making field. 


For instance, since small molecule byproducts are not produced, click chemistry minimizes the formation of bubbles, cavities, and irregularities that deteriorate the appearance and properties of newly synthesized thermosetting materials, as other polycondensation reactions would.41   


CuAAC click chemistry is also being used as an efficient, environmentally friendly cross-linking strategy to improve the performance of waterborne polymers, suitable for coatings and adhesives (figure below).42  Broadly applicable for polyurethanes (WPU), polyester dispersions (PED), and polyacrylate emulsions (PAE), this strategy is advantageous over other available cross-linking strategies (including self-cross-linking systems based upon N-methylolacrylamide (NMA), pendent acetoacetate groups, and reversible keto-hydrazide reactions) that cannot be broadly applied to all three WPU, PED, and PAE systems. Click cross-linked polymer films exhibited significantly improved mechanical strength, hardness, and water/solvent resistance, providing an alternative to the use of hardeners with the potential to reduce costs when used in industrial coating applications.  


Formation of click cross-linked waterborne polymers 

 Yang, et al (2016). Click Cross-Linking-Improved Waterborne Polymers for Environment-Friendly Coatings and Adhesives. ACS Appl. Mater. Interfaces 8(27): 17499-17510

Image adapted from Yang, et al (2016). Click Cross-Linking-Improved Waterborne Polymers for Environment-Friendly Coatings and Adhesives. ACS Appl. Mater. Interfaces 8(27): 17499-17510 


Moreover, the synthesis of various (1D, 2D, 3D) dimensional biomaterials (e.g. hydrogels) is gaining traction in the fields of tissue engineering43,44,45,46, regenerative medicine47, drug delivery48, and gene therapy.49   


7) Probe for viral studies 

Virus-related research over the last few decades,50 including viral (protein, nucleic acid, or virion) tracking,5152 antiviral agent design,53,54 diagnosis,55,56,57and virus-based delivery systems58,59 have all used click chemistry. A copper-free click reaction for instance was used to label enveloped viruses [vaccinia virus (VACV) and avian influenza A virus (H9N2)] by linking virions modified with azide to quantum dots (QDs) derived with dibenzocyclooctynes (DBCO).  A labeling efficiency of more than 80% was achieved that did not interfere with the virus’ ability to infect, and the fluorescence was strong enough to realize single-virion tracking.60 


8) CRISPER sgRNA synthesis and target gene labelling 

Click chemistry has now even found its place in the CRISPR toolbox in the synthesis of individual or pools of single-guide RNA (sgRNA), circumventing existing synthetic limitations related to (longer) oligo length and promoting a short turnaround time between sgRNA design and application. 

Instead of fabricating the entire sgRNA in one go, this approach (coined ‘split-and-click’) simply ligates two smaller (more readily synthesizable) chunks: a ~20-mer (crRNA) targeting sequence prepared on demand and in high purity and a generic 79-mer CRISPR endonuclease protein (Cas9) recruiting sequence (tracrRNA) made cost-effectively in a large scale. The resultant ~99-mer with a triazole linkage was found to enable effective Cas9-mediated DNA cleavage in vitro and in cells, with a comparable off-target profile to in vitro transcribed sgRNA.61  

Click chemistry has also been used to label target genes with a functional tag (called sgRNA-Click (sgR-CLK)). 62  This technique involves the installation of a click handle to the 3′ end of an in vitro transcribed CRISPR sgRNA to form an azide-labeled-ternary complex (consisting of dCas9, sgRNA, and the target gene). Functionalization of this ternary complex is then realized upon a click reaction with an alkyne counterpart. 

Moreover, click chemistry was used to engineer a flexible dendritic polymer for delivery of zinc fingers, TALEs and CRISPR/dCas9 platforms. High transfection efficiencies and packaging capacity were observed using this method.63 


9) Novel applications including ‘click to release’   

Beyond ligation, click chemistry is now being explored for uncaging or ‘click-to-release’ applications, which have enabled new strategies for probe activation and therapeutic delivery.64,65,66 An inverse electron demand Diels Alder pyridazine elimination reaction for instance was used to provoke rapid release of doxorubicin from an antibody-drug conjugate (ADC) in vitro and in tumor-bearing mice. 67 

Click chemistry has also been used to develop state-of-the-art microchip and capillary-based systems68 such as microfluidic “click chip”69 and graphene-based “Click-A+Chip”. 70 Moreover, the “electro-click” conjugation method has been used to immobilize enzymes (for use in biosensors), fabricate electrochemical immunosensors, and control protein conjugation spatially and temporally. 7172,73 


By QiChuck


Just added to your wishlist:
My Wishlist
You've just added this product to the cart:
Go to cart page