New Reactions with Click Chemistry

Wed, 10/10/2012 - 10:51am
Jyothi Thundimadathil, PhD, American Peptide Co. Inc.

Simple and elegant chemical reactions have far-ranging applications in R&D.

Click Chemistry Article Image 1

Figure 1: (A). Synthesis of block copolymers utilizing click chemistry. Alkyne and azide modified polymer chains react quickly in the presence of a copper catalyst to form triazole linked material. (B): SAMs assembled on gold surface are functionalized with an azide moiety which can “click” with a sugar molecule modified with an alkyne group. All figures: American Peptide Co. Inc.

The term click chemistry was introduced in 2001 by Valery V. Fokin and K.B. Sharpless to describe chemistry tailored to generate substances quickly and reliably by joining small units together, similar to the modular strategy adopted by nature. The click reactions are highly efficient, wide in scope, stereospecific, simple to perform using inexpensive reagents; and can be conducted in benign solvents such as water. In addition, product isolation is easy.

The copper-catalyzed variant of Huisgen azide-alkyne cycloaddition (CuAAC) fits the concept well and is one of the most popular prototype click reactions to date. The CuAAC click reaction between an azide and alkyne takes place in presence of a Cu (I) catalyst under mild conditions resulting in the formation of a triazole link connecting two molecules. Click chemistry is finding a number of applications in the broad areas of materials science, drug discovery, and bioconjugation.

Materials science
The concept of click chemistry is becoming a common and efficient tool for material scientists in the design of a variety of materials with interesting properties and applications. Synthesis of macromolecular structures such as dendrimers, calixarenes, and functionalized rotaxanes have been achieved with great success using this chemistry. Mechanically interlocked compounds such as rotaxanes and catenanes are functional molecular media for a host of applications including information storage, mechanical actuation, and drug release. More complex, higher-order, and functional architectures—once only a dream—are now comfortably within reach.

Click chemistry is also revolutionizing the area of surface patterning and has been developed into a standard way to functionalize single-walled nanotubes (SWNTs), gold nanoparticles, and various surfaces modified with self-assembled monolayers (SAMs). The controlled decoration of surfaces and design of bio-interfaces are exemplified by the preparation of SAMs containing azido groups on well-defined electrode surfaces for reaction with alkynyl substrates, and immobilization of surface arrays of acetylene-containing oligonucleotides on to azide functionalized SAMs on gold.

Carbohydrate-based sensors made by clicking azide functionalized sugars onto alkyne terminated SAMs on gold could lead to potential applications in high-throughput characterization of carbohydrate-protein interactions. Peptide-based SAMs—excellent model systems for biomimetic surfaces—that resist the adsorption of proteins have been created by click chemistry.

In a universal surface modification approach, alkyne-containing vapor deposited polymer coatings by chemical vapor deposition ( CVD) were shown to possess remarkable reactivity towards azide functionalized moieties. Recently, DNA microarrays were made by clicking DNA modified with pentynyl groups at its 5'-end with azide-loaded slides and successfully employed in biological model experiments. DNA microarrays (DNA chip or biochip) are used to measure the expression levels of large numbers of genes simultaneously or to genotype multiple regions of a genome. Given its compatibility with the conditions required for biological molecules, click chemistry has been proven to be a reliable means of attaching biomolecules to surfaces, including sugars, proteins, and peptides.

Polymer science has also benefited from the use of click chemistry due to the functional group tolerance of the click reaction, rendering it ideal for the introduction of functionality into polymers pre- or post-polymerization. Click reaction has been employed in combination with living polymerization techniques such as ring-opening polymerization (ROP), ring-opening metathesis polymerization (ROMP), cationic polymerization, nitric-oxide mediated radical polymerization (NMP), atom transfer radical polymerization (ATRP), and reversible addition fragmentation chain transfer polymerization (RAFT).

It is now possible to apply this concept in the synthesis of block copolymers, cyclic polymers, hyper-branched macromolecules, star-shaped polymers, cross-linked polymeric networks, and in the end-functionalization of well-defined polymers and side-chain functionalization. Cross-linked polymeric adhesives were made from polyvalent azide and alkyne building blocks, owing their adhesiveness to the strong affinity of triazoles for metal ions and surfaces. Poly (vinyl alcohol)-based hydrogels were synthesized by mixing azido modified PVA and acetylene appended PVA in the presence of a Cu (1) catalyst. Click chemistry provides a controlled atmosphere to form cross-links (which enhance the gel’s thermo stability) at low temperature and under mild conditions in the synthesis of organogels and hydrogels.

Peptide-based polymers are interesting materials, since they can be applied for a variety of purposes such as drug delivery devices, scaffolds for tissue engineering and repair, and as novel biomaterials. Polypeptide-based polymers are difficult to synthesize by conventional methods. Click chemistry techniques are making an impact in this area as exemplified by the synthesis of polypeptide polymers with repetitive peptide units having several amino acids. Peptide-based block copolymers, biocompatible amphiphilic graft polyesters, polymers that can self-assemble into hierarchical nanostructures, lower critical solution temperature polymers, and supra-molecular polymers containing cyclic oligomers are designed with the help of click chemistry.

It is expected that future research of click chemistry in polymer science will be directed towards library preparation and screening to optimize selected polymer properties. Excellent reviews are available highlighting the application of click chemistry in materials science. See the Additional Resources box for references.

Click Chemistry Article Image 2

Figure 2: Examples of substrates that can be attached via “click” reaction is growing and is embracing every scientific field.

Drug discovery and development
Click chemistry has been proven to be a powerful tool in drug discovery and development for the screening of large libraries of simple compounds in the search for specific properties. Large libraries of novel diverse compounds can be made owing to the simplicity of click reaction. Compounds can be screened directly from the crude solution of the reaction mixture. High-throughput methodology using click chemistry has led to the discovery of a novel inhibitor of human r-1, 3-fucosyltransferase, telomerase inhibitors, and potent HIV protease inhibitors. Click chemistry also provides new routes to many complex compounds, particularly in the field of carbohydrate synthesis. Multivalent triazole linked neo-glycoconjugates such as heptavalent manno-beta-cyclodextrins were made using click reaction.

The most innovative application of click reaction in drug discovery is kinetically controlled target-guided synthesis (TGS), also known as in situ click chemistry. In this method, a target (for example: enzyme, protein) is incubated with several building blocks (selected inhibitors modified with azide and alkyne groups) and the target facilitates the formation of an irreversible bonding between two selected building blocks (via click reaction), resulting in the formation of a more potent inhibitor that deactivates the target which recruited them.

Bioconjugation is the process by which synthetic molecules are attached to biological targets, or by which biomolecules are linked together. The CuAAC is an invaluable tool for labeling DNA and for making DNA-protein conjugates. Arginine-rich TAT peptides (capable of penetrating plasma membrane directly) modified with clickable azido group can be conjugated to oligonucleotides, cytotoxic drugs, or kinase inhibitors to facilitate cell penetration for therapeutic applications. The click reaction is very useful for conjugating fluorescent molecules to peptides and proteins under mild conditions and has applications in the emerging field of cell biology and functional proteomics.

The cytotoxicity of copper remains a concern and a limiting factor for widespread in vivo application of CuAAC-click reaction. The presence of copper and/or reducing agents can cause degradation or aggregation of the targeted biomolecules. Fortunately, these challenges can be overcome by the use of copper-free ‘click’ chemistry. This technique is based on the reaction of cyclooctynes (such as OCT, MOFO, DIBO, DIFO) with azides in the absence of Cu catalyst at low temperature, giving better efficiency than CuAAC reaction.

Application of click chemistry in several areas is rapidly increasing with possibilities of exciting discoveries in the horizon. Apart from the general areas of materials science, nanoscience, medicinal chemistry, drug discovery, and bio-conjugation, the concept has already made huge impact in synthetic chemistry. A constant drive towards simpler chemical reactions such as CuAAC with a wide scope is in progress. Synthesis of large molecules such as peptides, proteins, and supramolecular structures, as well as their modifications, will be positively influenced by the progress in click chemistry. Due to the simplicity of click reactions, biochemists and molecular biologists are able to make complex molecular systems without much bothering about the synthetic chemistry. Click chemistry is approaching the level of ready-made chemistry as many kits are already available in the market for an easy mix and shake approach.

Additional Resources on Click Chemistry
Clicking polymers: a straightforward approach to novel macromolecular architectures. David Fournier, Richard Hoogenboom, and Ulrich S. Schubert; Chem. Soc. Rev., 2007;36:1369–1380.
Click Chemistry: Versatility and Control in the Hands of Materials Scientists. Himabindu Nandivada, Xuwei Jiang, and Joerg Lahann; Adv. Mater. 2007;19: 2197–2208.
‘Click’ Chemistry in Polymer and Materials Science. Wolfgang H. Binder, Robert Sachsenhofer; Macromol. Rapid Commun. 2007; 28:5–54.
Applications of Orthogonal “Click” Chemistries in the Synthesis of Functional Soft Materials. Rhiannon K. Iha, et al. Chem. Rev. 2009;109:5620–5686.
Click Chemistry: 1,2,3-Triazoles as Pharmacophores.; Sandip G. Agalave, Suleman R. Maujan, and Vandana S. Pore; Chem. Asian J. 2011;6:2696-2718.
The growing impact of click chemistry on drug discovery. Hartmuth C. Kolb and K. Barry Sharpless; DDT. 2003;8 (24).
Click Chemistry, A Powerful Tool for Pharmaceutical Sciences. Christopher D. Hein, Xin-Ming Liu, and Dong Wang. Pharmaceutical Research., 2008; 25 (10)2008.
In situ click chemistry: probing the binding landscapes of biological molecules. Sreeman K. Mamidyala and M. G. Finn. Chem. Soc. Rev. 2010; 39:1252–1261.
Growing Applications of ‘‘Click Chemistry’’ for Bioconjugation in Contemporary Biomedical Research. Kido Nwe and Martin W. Brechbiel; Cancer Biotherapy and Radiopharmaceuticals.2009;24 (3).
Recent Applications of Click Chemistry for the Synthesis of Radiotracers for Molecular Imaging. Constantin Mamat, Theres Ramenda and Frank R. Wuest. Mini-Reviews in Organic Chemistry. 2009;6:21-34.


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