Click chemistry

Click chemistry is an approach to chemical synthesis that is used to join two molecules. Clicking refers to an emphasis on efficiency and simplicity. To link two molecular components, each is first fitted with appropriate functional groups, such as azide and alkyne groups. These components are then "clicked" together in a process that is highly favorable and which tolerates many functional groups that might complicate other coupling processes.

History

Click chemistry was first fully described by K. Barry Sharpless, Hartmuth C. Kolb, and M. G. Finn of The Scripps Research Institute in 2001.^[1][2] The paper argued that synthetic chemistry could emulate the way nature constructs complex molecules, using efficient reactions to join together simple, non-toxic building blocks. The term "click chemistry" was coined in 1998 by Sharpless' wife, Jan Dueser,^[3] who found the simplicity of this approach to chemical synthesis akin to clicking together Lego blocks.

Many perspectives have been offered on the concepts or principles of Click Chemistry.^[4][5] Some of these attributes include the quest for selectivity and ease of implementation, which of course is a goal for many or most chemical reactions.

A foundational motivation for the Click Chemistry was the recognition that most compounds in biology are "stitched together" by formation of C-heteroatom bonds. Few major classes of structurally complex organic compounds have more than six C-C bonds (except aryl derivatives) C-heteroatom bond forming reactions are often highly favorable and thus virtually irreversible. Of these C-heteroatom bond forming reactions, a few are particularly easy to implement. Sharpless et al. originally recommended a focus on certain cycloadditions, nucleophilic substitutions involving strained substrates, formation of ureas and ethers, additions of heteroatoms to C=C and C≡C bonds. Not mentioned in this landmark review was the Huisgen 1,3-dipolar cycloaddition, possibly because it was slow. The year after publication of the review by Sharpless et al., Tornøe, Christensen, Meldal described the copper-catalyzed addition of azides to alkynes, with no mention of Sharpless et al. nor of Click Chemistry.^[6] Meldal's innovation became the paradigmatic reaction of Click Chemistry, for which he shared the Nobel Prize.

In 2022, the Nobel Prize in Chemistry was jointly awarded to Carolyn R. Bertozzi, Morten P. Meldal and Karl Barry Sharpless, "for the development of click chemistry and bioorthogonal chemistry".^[7]

Click reactions

Copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC)

Main article: Azide alkyne Huisgen cycloaddition

The classic^[8][9] click reaction is the copper-catalyzed reaction of an azide with an alkyne to form a 5-membered heteroatom ring: a Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC). The first triazole synthesis, from diethyl acetylenedicarboxylate and phenyl azide, was reported by Arthur Michael in 1893.^[10] Later, in the middle of the 20th century, this family of 1,3-dipolar cycloadditions took on Rolf Huisgen's name after his studies of their reaction kinetics and conditions.

A comparison of the Huisgen and the copper-catalyzed Azide-Alkyne cycloadditions

The copper(I)-catalysis of the Huisgen 1,3-dipolar cycloaddition was discovered concurrently and independently by the groups of Valery V. Fokin and K. Barry Sharpless at the Scripps Research Institute in California^[11] and Morten Meldal in the Carlsberg Laboratory, Denmark.^[6] The copper-catalyzed version of this reaction gives only the 1,4-isomer, whereas Huisgen's non-catalyzed 1,3-dipolar cycloaddition gives both the 1,4- and 1,5-isomers, is slow, and requires a temperature of 100 degrees Celsius.^[10]

The two-copper mechanism of the CuAAC catalytic cycle

Moreover, this copper-catalyzed "click" does not require special ligands on the metal, although accelerating ligands such as tris(triazolyl)methyl amine ligands with various substituents have been reported and used with success in aqueous solution.^[10] Other ligands such as PPh3 and TBIA can also be used, even though PPh₃ is liable to Staudinger ligation with the azide substituent. Cu₂O in water at room temperature was found also to catalyze the same reaction in 15 minutes with 91% yield.^[12]

Various mechanisms have been proposed.^{[13][14][15][16][17]} Even though this reaction proceeds effectively at biological conditions, copper in this range of dosage is cytotoxic. Solutions to this problem have been presented, such as using water-soluble ligands on the copper to enhance cell penetration of the catalyst and thereby reduce the dosage needed,^[18][19][20] or to use chelating ligands to further increase the effective concentration of Cu(I) and thereby decreasing the actual dosage.^[21][22][23]

Although the Cu(I)-catalyzed variant was first reported by Meldal and co-workers for the synthesis of peptidotriazoles on solid support, their conditions were far from the true spirit of click chemistry and were overtaken by the publicly more recognized Sharpless. Meldal and co-workers also chose not to label this reaction type "click chemistry" which allegedly caused their discovery to be largely overlooked by the mainstream chemical society. Fokin and Sharpless independently described it as a reliable catalytic process offering "an unprecedented level of selectivity, reliability, and scope for those organic synthesis endeavors which depend on the creation of covalent links between diverse building blocks".

An analogous "RuAAC reaction" (catalyzed by ruthenium, instead of copper) allows for the selective production of 1,5-isomers.^[24]

Strain-promoted azide-alkyne cycloaddition (SPAAC)

Metal-free click reactions have gained prominence due to their enhanced biocompatibility and reduced cytotoxicity. Notably, the strain-promoted azide–alkyne cycloaddition (SPAAC) and inverse electron-demand Diels–Alder (IEDDA) reactions have been widely adopted for bioorthogonal labeling in living systems. These reactions offer high specificity without the need for metal catalysts, making them ideal for applications in living organisms and complex environments.^[25]

Versions of Huisgen's copper-free click reactions minimize the cytotoxicity.^[26][27]

Scheme of the Strain-promoted Azide-Alkyne Cycloaddition

Strain-promoted alkyne-nitrone cycloaddition (SPANC)

Diaryl-strained-cyclooctynes including dibenzylcyclooctyne (DIBO) have also been used to react with 1,3-nitrones in strain-promoted alkyne-nitrone cycloadditions (SPANC) to yield N-alkylated isoxazolines.^[28]

The SPAAC vs SpANC reaction

Because this reaction is metal-free and proceeds with fast kinetics (k2 as fast as 60 1/Ms, faster than both the CuAAC or the SPAAC) SPANC can be used for live cell labeling. Moreover, substitution on both the carbon and nitrogen atoms of the nitrone dipole, and acyclic and endocyclic nitrones are all tolerated. This large allowance provides a lot of flexibility for nitrone handle or probe incorporation.^[29]

However, the isoxazoline product is not as stable as the triazole product of the CuAAC and the SpAAC, and can undergo rearrangements at biological conditions. Regardless, this reaction is still very useful as it has notably fast reaction kinetics.^[28]

The applications of this reaction include labeling proteins containing serine as the first residue: the serine is oxidized to aldehyde with NaIO₄ and then converted to nitrone with p-methoxybenzenethiol, N-methylhydroxylamine and p-ansidine, and finally incubated with cyclooctyne to give a click product. The SPANC also allows for multiplex labeling.^[30][31]

Reactions of strained alkenes

Strained alkenes also utilize strain-relief as a driving force that allows for their participation in click reactions. Trans-cycloalkenes (usually cyclooctenes) and other strained alkenes such as oxanorbornadiene react in click reactions with a number of partners including azides, tetrazines and tetrazoles. These reaction partners can interact specifically with the strained alkene, staying bioorthogonal to endogenous alkenes found in lipids, fatty acids, cofactors and other natural products.^[30]

Alkene and azide [3+2] cycloaddition

Oxanorbornadiene (or another activated alkene) reacts with azides, giving triazoles as a product. However, these product triazoles are not aromatic as they are in the CuAAC or SPAAC reactions, and as a result are not as stable. The activated double bond in oxanobornadiene makes a triazoline intermediate that subsequently spontaneously undergoes a retro Diels-alder reaction to release furan and give 1,2,3- or 1,4,5-triazoles. Even though this reaction is slow, it is useful because oxabornodiene is relatively simple to synthesize. The reaction is not, however, entirely chemoselective.^[32]

Alkene and tetrazine inverse-demand Diels-Alder

A Tetrazine-Alkene reaction between a generalized tetrazine and a strained, trans-cyclooctene

Strained cyclooctenes and other activated alkenes react with tetrazines in an inverse electron-demand Diels-Alder reaction followed by a retro [4+2] cycloaddition (see figure).^[33] Three-membered and four-membered cycloalkenes, due to their high ring strain, also make ideal alkene substrates.^[33]

Similar to other [4+2] cycloadditions, electron-donating substituents on the dienophile and electron-withdrawing substituents on the diene accelerate the inverse-demand Diels-Alder reaction. The diene, the tetrazine, by virtue of having the additional nitrogens, is a good diene for this reaction. The dienophile, the activated alkene, can often be attached to electron-donating alkyl groups on target molecules, thus making the dienophile more suitable for the reaction.^[34]

Alkene and tetrazole photoclick reaction

The tetrazole-alkene "photoclick" reaction is another dipolar addition that Huisgen first introduced in the late 1960s ChemBioChem 2007, 8, 1504. (68) Clovis, J. S.; Eckell, A.; Huisgen, R.; Sustmann, R. Chem. Ber. 1967, 100, 60.) Tetrazoles with amino or styryl groups that can be activated by UV light at 365 nm (365 does not damage cells) react quickly (so that the UV light does not have to be on for a long time, usually around 1–4 minutes) to make fluorogenic pyrazoline products. This reaction scheme is well suited for the purpose of labeling in live cells, because UV light at 365 nm damages cells minimally. Moreover, the reaction proceeds quickly, so that the UV light can be administered for short durations. Quantum yields for short wavelength UV light can be higher than 0.5. This allows tetrazoles to be used wavelength selectively in combination with another photoligation reaction, where at the short wavelength the tetrazole ligation reaction proceeds nearly exclusively and at longer wavelength another reaction (ligation via o-quinodimethanes) proceeds exclusively.^[35] Finally, the non-fluorogenic reactants give rise to a fluorogenic product, equipping the reaction with a built-in spectrometry handle.

Both tetrazoles and the alkene groups have been incorporated as protein handles as unnatural amino acids, but this benefit is not unique. Instead, the photoinducibility of the reaction makes it a prime candidate for spatiotemporal specificity in living systems. Challenges include the presence of endogenous alkenes, though usually cis (as in fatty acids) they can still react with the activated tetrazole.^[36]

The PQ-ERA reaction has been optimized through thiophene substitution to enhance its reactivity, enabling more efficient photocycloaddition processes.^[37]

Potential applications

The simplicity of click chemistry has had significant impact in many themes, especially pharmaceutical development.

In many potential applications, click reactions join a biomolecule and a reporter molecule or other molecular probe, a process called bioconjugation.^[38] The possibility of attaching fluorophores and other reporter molecules has made click chemistry a tool for identifying, locating, and characterizing biomolecules. This approach has been used in numerous studies, and discoveries include that salinomycin localizes to lysosomes to initiate ferroptosis in cancer stem cells^[39] and that metformin derivatives accumulate in mitochondria to chelate copper(II), affecting metabolism and epigenetic changes downstream in inflammatory macrophages.^[40]

Click chemistry is often employed to attach visualizing tags. One of the earliest and most important methods in bioconjugation was to express a reporter gene, such as the gene green fluorescent protein (GFP), on the same genetic sequence as a protein of interest.

Click chemistry is not limited to biological conditions: the concept of a "click" reaction has been used in chemoproteomic, pharmacological, biomimetic and molecular machinery applications.^[41]

The fluorophore rhodamine has been coupled onto norbornene, and reacted with tetrazine in living systems.^[42] In other cases, SPAAC between a cyclooctyne-modified fluorophore and azide-tagged proteins allowed the selection of these proteins in cell lysates.^[43]

Unnatural Amino Acids

Methods for the incorporation of click reaction partners into systems in and ex vivo contribute to the scope of possible reactions. The development of unnatural amino acid incorporation by ribosomes has allowed for the incorporation of click reaction partners as unnatural side groups on these unnatural amino acids. For example, an UAA with an azide side group provides convenient access for cycloalkynes to proteins tagged with this "AHA" unnatural amino acid.^[44] In another example, "CpK" has a side group including a cyclopropane alpha to an amide bond that serves as a reaction partner to tetrazine in an inverse diels-alder reaction.^[45]

Scheme of the synthesis of firefly luciferin

The synthesis of luciferin exemplifies another strategy of isolating reaction partners, which is to take advantage of rarely-occurring, natural groups such as the 1,2-aminothiol, which appears only when a cysteine is the final N' amino acid in a protein. Their natural selectivity and relative bioorthogonality is thus valuable in developing probes specific for these tags. The above reaction occurs between a 1,2-aminothiol and a 2-cyanobenzothiazole to make luciferin, which is fluorescent. This luciferin fluorescence can be then quantified by spectrometry following a wash, and used to determine the relative presence of the molecule bearing the 1,2-aminothiol. If the quantification of non-1,2-aminothiol-bearing protein is desired, the protein of interest can be cleaved to yield a fragment with a N' Cys that is vulnerable to the 2-CBT.^[46]

Additional applications include:

• two-dimensional gel electrophoresis separation^[47]
• preparative organic synthesis of 1,4-substituted triazoles
• modification of peptide function with triazoles
• modification of natural products and pharmaceuticals
• natural product discovery ^[48]
• drug discovery
• macrocyclizations using Cu(I) catalyzed triazole couplings
• modification of DNA and nucleotides by triazole ligation
• supramolecular chemistry: calixarenes, rotaxanes, and catenanes
• dendrimer design
• carbohydrate clusters and carbohydrate conjugation by Cu(1) catalyzed triazole ligation reactions
• polymers and biopolymers^[49]
• surfaces^[50]
• material science
• nanotechnology,^[51]
• bioconjugation, for example, azidocoumarin, and
• biomaterials^[52]

In combination with combinatorial chemistry, high-throughput screening, and building chemical libraries, click chemistry has hastened new drug discoveries by making each reaction in a multistep synthesis fast, efficient, and predictable.

Drug discovery and bio-conjugation

Click chemistry has become a cornerstone in drug discovery and bioconjugation. Its ability to rapidly and selectively form stable covalent bonds has facilitated the development of targeted therapeutics and diagnostic agents. Recent studies have explored the use of click chemistry in creating multifunctional drug delivery systems, enhancing the specificity and efficacy of treatments.^[53]

Technology license

The Scripps Research Institute has a portfolio of click-chemistry patents.^[54] Licensees include Invitrogen,^[55] Allozyne,^[56] Aileron,^[57] Integrated Diagnostics,^[58] and the biotech company baseclick,^[59] a BASF spin-off created to sell products made using click chemistry.^[60] Moreover, baseclick holds a worldwide exclusive license for the research and diagnostic market for the nucleic acid field. Fluorescent azides and alkynes are also produced by companies such as Cyandye.^[61]