Mimicking Nature with the Click of a Reaction

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Click chemistry has become a powerful tool in drug discovery, chemical biology, and proteomic applications.

In biological systems, complex molecules are constructed by forming multiple carbon-heteroatom bonds with the aid of enzymes and using modular building blocks (amino acids). First introduced by K. Barry Sharpless of The Scripps Research Institute in 2001, click chemistry encompasses reactions that mimic this modular approach. The most famous example is the Cu(I)-catalyzed version of the Huisgen 1,3-dipolar cycloaddition reaction between azides and terminal alkynes to form 1,2,3-triazoles.

These reactions have several features in common that qualify them as click chemistry reactions. First, they are modular, wide in scope, give very high yields, and generate minimal byproducts that are nonhazardous and readily removed without the need for complex separations (i.e., chromatography), according to Dr. Prakasam Thirumurugan, who previously worked in the Laboratory of Medicinal Chemistry and Neuroengineering at the Medical University of Lublin in Poland and who currently is a postdoctoral associate at the New York University Center of Science and Engineering in the United Arab Emirates.

In addition, click reactions are stereoselective (but not always enantioselective), can be run using simple reaction conditions (ideally in air at room temperature) and readily available and inexpensive starting materials, require no or a benign solvent such as water, and generate products that are easy to isolate using only simple purification processes if necessary, such as crystallization and distillation. Furthermore, the products of click chemistry reactions are stable under physiological conditions.

Significant impact on synthetic efficiency
In recent years, drug development has become more focused on biology-oriented synthesis with the goal of producing molecules and systems that exhibit well-understood biological activity. In addition, the sequencing of the human genome and the genomes of numerous types of harmful microbes has uncovered large numbers of potentially novel biological targets with no obvious and efficient synthetic routes. 


“Conventional organic synthetic approaches are often slow, costly, and labor-intensive, and thus very inefficient for the practical preparation of these complex compounds,” Thirumurugan notes. “Click chemistry, because it mimics the synthetic approach used in biological systems, is a particularly promising strategy for the synthesis of carbon−heteroatom−carbon bonds in an aqueous environment, especially when used in conjunction with combinatorial and high throughput techniques,” he explains. He adds that because click reactions are facile, selective, and reliable, they enable faster discovery and optimization of drug candidates, and should be readily scalable for commercial manufacture.

The key copper-catalyzed cycloaddition
Probably the most widely recognized click reaction is the Cu(I)-catalyzed [2+3] cycloaddition of azides with terminal acetylenes to form 1,2,3-triazoles. “This reaction is very attractive because it proceeds at room temperature, is highly dependable, provides complete specificity to the 1,2,3-triazole scaffold, and uses reactants that are biocompatible,” explains Thirumurugan. As importantly, the triazole structure is found in many different natural products that exhibit biological activity.

In situ reactions for optimum results
An interesting development in click chemistry has been its used in target-guided synthesis (TGS). In this approach, different building blocks are brought together in the presence of the biological target (an enzyme, DNA, a protein) so that it can act as a template and lead to formation, ideally, of the best ligand. The target is incubated with a mixture of reagents. In thermodynamic TGS (also known as dynamic combinatorial chemistry), different pairs of building block reversibly join, and those that bind most tightly to the target are retained, thus driving the reaction. With kinetic TGS, the building blocks combine irreversibly at different reaction rates that are influenced by the target, and the combinations that react the fastest are produced in higher concentrations.

It has been suggested that, in the future, it might be possible to use such in situ click reactions to have a drug assembled inside the patient in response to the specific conditions of the disease within that particular patient. This approach has been investigated particularly for the development of enzyme inhibitors for the treatment of a number of medical problems, such as AIDS, cancer, anthrax poisoning, and Huntington’s disease, according to Thirumurugan.

Practical use
More conventional (non in situ) click chemistry has also been used for the fragment-based assembly of a number of compounds with specific enzyme inhibition activity, including ultra-potent inhibitors of serine hydrolase. Other compounds, including agonists, antagonists, and selective ligands for receptor−ligand binding studies, have also been readily prepared using click reactions. “What is attractive about these bioorthogonal click chemistry reactions is that the reagents can form covalent bonds within functionalized biological systems. As a result, they have even been used for the site-specific bioconjugation of proteins, glycans, lipids, and cell surfaces inside living animals under physiological conditions,” observes Thirumurugan.

Of course, the click reactions used in biological systems do not involve copper, which is a limitation of the [3+2] cycloaddition reaction described above. For in-vitro and in-vivo applications in particular, the development of copper-free, and preferably metal-free-ligation chemistries, is important. Copper-free reactions involving azides and strained alkynes, and strain-promoted cycloadditions of cyclic alkenes and alkynes have been shown to be powerful tools in bioconjugations, according to Thirumurugan.

Chemical labeling of biomolecules
Because click reactions are highly selective and modular and involve the use of reactants that do not interfere with biological systems, they are also ideal for the labeling of biomolecules, both inside and outside of living systems. Azides are useful functional groups that can be incorporated into biological molecules and then tracked based on their click chemistry. The reaction of the azides with strained alkynes, such as cyclooctynes, leads to the formation of triazoles without the use of a toxic metal and enables the determination of their abundance, location, and dynamics. More recently, according to Thirumurugan, other metal-free click reactions have been introduced that are useful for this application, including thiol−ene reactions, alkene−azamethine cycloadditions, and the cycloaddition of tetrazine and trans-cyclooctene.

Additional information on click chemistry in pharmaceutical synthesis may be found in this recent review: “Click Chemistry for Drug Development and Diverse Chemical−Biology Applications”. P. Thirumurugan, D. Matosiuk, and K. Jozwiak, Chem. Rev., Article ASAP,  DOI: 10.1021/cr200409f, Publication Date (Web): March 27, 2013.