{{Short description|Modular approach to chemical synthesis}} {{TopicTOC-Chemistry}} '''Click chemistry''' is an approach to chemical synthesis that is used to join two molecules. The application of the term, click, as a qualifier to the type of chemistry 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.&nbsp;Barry Sharpless, Hartmuth C.&nbsp;Kolb, and M.&nbsp;G.&nbsp;Finn of The Scripps Research Institute in 2001.<ref name="Sharpless">{{cite journal |author1=H. C. Kolb |author2=M. G. Finn |author3=K. B. Sharpless | title = Click Chemistry: Diverse Chemical Function from a Few Good Reactions | year = 2001 | journal = Angewandte Chemie International Edition | volume = 40 | issue = 11 | pages = 2004–2021 | doi = 10.1002/1521-3773(20010601)40:11<2004::AID-ANIE2004>3.0.CO;2-5 | pmid=11433435| doi-access = }}</ref><ref>{{cite journal | author = R. A. Evans | title = The Rise of Azide–Alkyne 1,3-Dipolar 'Click' Cycloaddition and its Application to Polymer Science and Surface Modification | year = 2007 | journal = Australian Journal of Chemistry | volume = 60 | issue = 6 | pages = 384–395 | doi = 10.1071/CH06457}}</ref> 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.<ref>The term "click chemistry" was coined in 1998 by Sharpless' wife, Jan Dueser,{{Citation |title=Nobel Prize lecture: Barry Sharpless, Nobel Prize in Chemistry 2022 | date=3 February 2023 |url=https://www.youtube.com/watch?v=55trnYorBhQ |access-date=2024-01-04 |language=en}}</ref>

Many perspectives have been offered on the concepts or principles of Click Chemistry.<ref name="Devaraj">{{cite journal|last1=Devaraj |first1=Neal K. |last2=Finn |first2=M. G. |title=Introduction: Click Chemistry |journal=Chemical Reviews |date=2021 |volume=121 |issue=12 |pages=6697–6698 |doi=10.1021/acs.chemrev.1c00469 |pmid=34157843 }}</ref><ref name=pdcs>{{Cite journal|title=Additive manufacturing of ceramics from preceramic polymers: A versatile stereolithographic approach assisted by thiol-ene click chemistry|first1=Xifan|last1=Wang|first2=Franziska|last2=Schmidt|first3=Dorian|last3=Hanaor|first4=Paul H.|last4=Kamm|first5=Shuang|last5=Li|first6=Aleksander|last6=Gurlo|date=May 6, 2019|journal=Additive Manufacturing|volume=27|pages=80–90|doi=10.1016/j.addma.2019.02.012|arxiv=1905.02060|s2cid=104470679 }}</ref> 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.

Click Chemistry was founded on the recognition that most compounds in biology are "stitched together" by formation of C-heteroatom bonds (heteroatom = N, O, S). Few major classes of structurally complex organic compounds have more than six contiguous C-C bonds (except aryl derivatives). C-heteroatom bond forming reactions are often highly favorable and thus these bonds are strong. Of these C-heteroatom bond forming reactions, a few are particularly easy to implement. Sharpless et al. recommended a focus on certain cycloadditions, nucleophilic substitutions involving strained substrates, formation of ureas and ethers, and additions of heteroatoms to 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 Sharpless's review, Tornøe, Christensen, and Meldal at the Carlsberg Laboratory, Denmark described the copper-catalyzed addition of azides to alkynes, with no mention of Sharpless et al. nor of Click Chemistry.<ref name=Tornoe/> Meldal's innovation became the paradigmatic reaction of Click Chemistry. One set of particularly significant reports regarding azide couplings came from the Bertozzi lab who conducted the reaction within living cells.

In 2022, the Nobel Prize in Chemistry was jointly awarded to Carolyn R.&nbsp;Bertozzi, Morten P.&nbsp;Meldal, and Karl Barry Sharpless, "for the development of click chemistry and bioorthogonal chemistry".<ref>{{Cite web |title=The Nobel Prize in Chemistry 2022 |url=https://www.nobelprize.org/prizes/chemistry/2022/press-release/ |access-date=2022-10-05 |website=NobelPrize.org |language=en-US}}</ref>

== Click reactions == === Copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) === {{main|Azide alkyne Huisgen cycloaddition}} The classic click reaction<ref>{{Cite journal |last=Gregory C. |first=Patton |date=November 8, 2004 |title=Development and Applications of Click Chemistry |url=https://chemistry.illinois.edu/research/organic/seminar_extracts/2004_2005/08_Patton_Abstract.pdf |department=Department of Chemistry |journal= |publisher=College of Liberal Arts & Sciences, University of Illinois at Urbana-Champaign |archive-url=https://web.archive.org/web/20100709120839/http://chemistry.illinois.edu/research/organic/seminar_extracts/2004_2005/08_Patton_Abstract.pdf |archive-date=2010-07-09}}</ref><ref>{{cite journal|last=Kolb|first=H.C.|author2=Sharpless, B.K.|title=The growing impact of click chemistry on drug discovery|year=2003|volume=8|issue=24|pages=1128–1137|doi=10.1016/S1359-6446(03)02933-7|pmid=14678739|journal=Drug Discov Today|doi-access=free}}</ref> is the copper-catalyzed reaction of an azide with an alkyne to form a 5-membered C<sub>2</sub>N<sub>3</sub> ring. This reaction is the Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC). The first triazole synthesis, from diethyl acetylenedicarboxylate and phenyl azide, was reported by Arthur Michael in 1893.<ref name="D. Astruc 2011">L. Liang and D. Astruc: "The copper(I)-catalysed alkyne-azide cycloaddition (CuAAC) "click" reaction and its applications. An overview", 2011; 255, 23–24, 2933–2045, p. 2934</ref> Later, in the middle of the twentieth century, this family of 1,3-dipolar cycloadditions took on Rolf Huisgen's name after his studies of their reaction kinetics and conditions.

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

The copper-catalysis of the Huisgen 1,3-dipolar cycloaddition was discovered concurrently and independently by the groups of Fokin and Sharpless<ref>{{cite journal|last=Rostovtsev|first=Vsevolod V.|author2=Green, Luke G |author3=Fokin, Valery V. |author4= Sharpless, K. Barry |title=A Stepwise Huisgen Cycloaddition Process: Copper(I)-Catalyzed Regioselective "Ligation" of Azides and Terminal Alkynes|journal=Angewandte Chemie International Edition|year=2002|volume=41|issue=14|pages=2596–2599|doi=10.1002/1521-3773(20020715)41:14<2596::aid-anie2596>3.0.co;2-4 |pmid=12203546}}</ref> and Meldal.<ref name=Tornoe> {{cite journal |author1=Tornoe, C. W. |author2=Christensen, C. |author3=Meldal, M. | title = Peptidotriazoles on Solid Phase: [1,2,3]-Triazoles by Regiospecific Copper(I)-Catalyzed 1,3-Dipolar Cycloadditions of Terminal Alkynes to Azides | year = 2002 | journal = Journal of Organic Chemistry | volume = 67 | issue = 9 | pages = 3057–3064 | doi = 10.1021/jo011148j | pmid=11975567|s2cid=11957672 |url=https://figshare.com/articles/Peptidotriazoles_on_Solid_Phase_1_2_3_-Triazoles_by_Regiospecific_Copper_I_-Catalyzed_1_3-Dipolar_Cycloadditions_of_Terminal_Alkynes_to_Azides/3691059 | url-access =subscription }} </ref> 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 and requires a temperature of 100 °C.<ref name="D. Astruc 2011"/>

thumb|The two-copper mechanism of the CuAAC catalytic cycle

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 minimize the dosage needed,<ref name="Brotherton, W. S. 2009">Brotherton, W. S.; Michaels, H. A.; Simmons, J. T.; Clark, R.J.; Dalal, N. S.; Zhu, L. Org. Lett. 2009, 11, 4954.</ref><ref>Kuang, G.-C.; Michaels, H. A.; Simmons, J. T.; Clark, R. J.; Zhu, L" ''J. Org. Chem.'' 2010; 75, 6540.</ref> An analogous "RuAAC reaction" (catalyzed by ruthenium, instead of copper) allows for the selective production of 1,5-isomers.<ref>{{cite journal |last1=Zhang |first1=Li |last2=Chen |first2=Xinguo |last3=Xue |first3=Peng |last4=Sun |first4=Herman H. Y. |last5=Williams |first5=Ian D. |last6=Sharpless |first6=K. Barry |last7=Fokin |first7=Valery V. |last8=Jia |first8=Guochen |title=Ruthenium-Catalyzed Cycloaddition of Alkynes and Organic Azides |journal=Journal of the American Chemical Society |date=November 2005 |volume=127 |issue=46 |pages=15998–15999 |doi=10.1021/ja054114s|pmid=16287266 |bibcode=2005JAChS.12715998Z |url=https://figshare.com/articles/Ruthenium_Catalyzed_Cycloaddition_of_Alkynes_and_Organic_Azides/3255712 |url-access=subscription }}</ref>

=== 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.<ref>{{Cite journal |last1=Ramírez-López |first1=Pedro |last2=Suárez |first2=José Ramón |last3=Flores |first3=Aida |last4=Hernáiz |first4=María J. |date=2025-08-20 |title=Exploring Metal-Free Click Reactions: New Frontiers in Glycochemistry and Bioconjugation |journal=Bioconjugate Chemistry |volume=36 |issue=8 |pages=1553–1581 |doi=10.1021/acs.bioconjchem.5c00049 |pmid=40673383 |issn=1043-1802|pmc=12371699 }}</ref>

Versions of Huisgen's copper-free click reactions minimize the cytotoxicity.<ref>Huisgen, R. ''Angew. Chem. Int. Ed.'' Engl. 1963, 2, 565<p> {{cite journal | last1 = Agard | first1 = N. J. | last2 = Baskin | first2 = J. M. | last3 = Prescher | first3 = J. A. | last4 = Lo | first4 = A. | last5 = Bertozzi | first5 = C. R. | year = 2006 | title = A Comparative Study of Bioorthogonal Reactions with Azides| journal = ACS Chem. Biol. | volume = 1 | issue = 10| pages = 644–648 | doi=10.1021/cb6003228| pmid = 17175580 }}</p></ref><ref>{{cite journal | last1 = Gordon | first1 = C. G. | last2 = Mackey | first2 = J. L. | last3 = Jewett | first3 = J. C. | last4 = Sletten | first4 = E. M. | last5 = Houk | first5 = K. N. | last6 = Bertozzi | first6 = C. R. | year = 2012 | title = Reactivity of Biarylazacyclooctynones in Copper-Free Click Chemistry| journal = J. Am. Chem. Soc. | volume = 134 | issue = 22| pages = 9199–9208 | doi=10.1021/ja3000936| pmid = 22553995 | pmc = 3368396 | bibcode = 2012JAChS.134.9199G }}</ref>

thumb|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.<ref name="ReferenceB">{{cite journal | last1 = MacKenzie | first1 = DA | last2 = Sherratt | first2 = AR | last3 = Chigrinova | first3 = M | last4 = Cheung | first4 = LL | last5 = Pezacki | first5 = JP | date = Aug 2014 | title = Strain-promoted cycloadditions involving nitrones and alkynes—rapid tunable reactions for bioorthogonal labeling| doi = 10.1016/j.cbpa.2014.05.023 | pmid = 25022431 | journal = Curr Opin Chem Biol | volume = 21 | pages = 81–8 }}</ref>

thumb|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.<ref>(64) (a) Ning, X.; Temming, R. P.; Dommerholt, J.; Guo, J.; Ania, D.B.; Debets, M. F.; Wolfert, M. A.; Boons, G.-J.; van Delft, F. L" ''Angew. Chem. Int. Ed.'' 2010; 49, 3065. (b) McKay, C. S.; Moran, J.; Pezacki, J. P. Chem. Commun. (Cambridge, U. K.) 2010, 46, 931. (c) Debets, M. F.; van Berkel, S. S.; Dommerholt, J.; Dirks, A. T. J.; Rutjes, F. P. J. T.; van Delft, F. L. Acc. Chem. Res. 2011, 44, 805. (d) McKay, C. S.; Chigrinova, M.; Blake, J. A.; Pezacki, J. P. Org. Biomol. Chem. 2012, 10, 3066.</ref>

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.<ref name="ReferenceB"/>

The applications of this reaction include labeling proteins containing serine as the first residue: the serine is oxidized to aldehyde with NaIO<sub>4</sub> and then converted to nitrone with p-methoxybenzenethiol, N-methylhydroxylamine and p-ansidine, and finally is incubated with cyclooctyne to give a click product. The SPANC also allows for multiplex labeling.<ref name="ReferenceA">{{cite journal | last1 = Lang | first1 = K. | last2 = Chin | first2 = J. | year = 2014 | title = Bioorthogonal Reactions for Labeling Proteins | journal = ACS Chem. Biol. | volume = 9 | issue = 1| pages = 16–20 | doi=10.1021/cb4009292| pmid = 24432752 }}</ref><ref>{{cite journal | last1 = MacKenzie | first1 = DA | last2 = Pezacki | first2 = JP | year = 2014 | title = Kinetics studies of rapid strain- promoted [3+2] cycloadditions of nitrones with bicyclo[6.1.0]nonyne | journal = Can J Chem | volume = 92 | issue = 4| pages = 337–340 | doi=10.1139/cjc-2013-0577 | bibcode = 2014CaJCh..92..337M }}</ref>

=== Reactions of strained alkenes === Strained alkenes also use 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 may interact specifically with the strained alkene, staying bioorthogonal to endogenous alkenes found in lipids, fatty acids, cofactors, and other natural products.<ref name="ReferenceA"/>

==== Alkene and tetrazine inverse-demand Diels-Alder ====

thumb|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).<ref name="Houk et al, 2013, 135, 15642-15649">{{cite journal|first1= Fang|last1= Liu|first2= Robert S. |last2= Paton|first3 = Seonah|last3 = Kim|first4= Yong|last4 = Liang|first5 =K. N. |last5 = Houk|date= 2013|volume= 135|pages= 15642–15649 |title = Diels–Alder Reactivities of Strained and Unstrained Cycloalkenes with Normal and Inverse-Electron-Demand Dienes: Activation Barriers and Distortion/Interaction Analysis|journal = J. Am. Chem. Soc.|issue = 41|doi= 10.1021/ja408437u|pmid= 24044412|bibcode= 2013JAChS.13515642L}}</ref> Three-membered and four-membered cycloalkenes, due to their high ring strain, also make ideal alkene substrates.<ref name="Houk et al, 2013, 135, 15642-15649"/>

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, may often be attached to electron-donating alkyl groups on target molecules, thus making the dienophile more suitable for the reaction.<ref>{{cite journal|last1= Rieder|first1= Ulrike|last2= Luedtke|first2= Nathan W. |journal = Angew Chem Int Ed Engl|date= 25 August 2014|volume = 53|issue=35|pages = 9168–9172|title = Alkene-tetrazine ligation for imaging cellular DNA|doi= 10.1002/anie.201403580|pmid= 24981416|bibcode= 2014ACIE...53.9168R}}</ref>

==== 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 may be activated by UV light at 365&nbsp;nm (365 does not damage cells) react quickly (so that the UV light does not have to be on for a long time, usually approximately 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&nbsp;nm damages cells minimally. Moreover, the reaction proceeds quickly, so that the UV light may be administered for short durations. Quantum yields for short wavelength UV light may 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.<ref>{{cite journal |last1=Menzel |first1=Jan P. |last2=Feist |first2=Florian |last3=Tuten |first3=Bryan |last4=Weil |first4=Tanja |author-link4=Tanja Weil|last5=Blinco |first5=James P. |last6=Barner-Kowollik |first6=Christopher |title=Light-Controlled Orthogonal Covalent Bond Formation at Two Different Wavelengths |journal=Angewandte Chemie International Edition |date=2019 |volume=58 |issue=22 |pages=7470–7474 |doi=10.1002/anie.201901275 |pmid= 30916368|doi-access=free |bibcode=2019ACIE...58.7470M |hdl=21.11116/0000-0003-99B4-5 |hdl-access=free }}</ref> 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, although usually cis (as in fatty acids) they can still react with the activated tetrazole.<ref>{{cite journal|last1=Ramil|first1= Carlo P|last2= Lin|first2= Qing|journal = Current Opinion in Chemical Biology|date=August 2014 |volume = 21|pages = 89–95|title = Photoclick chemistry: a fluorogenic light-triggered ''in vivo'' ligation reaction|doi = 10.1016/j.cbpa.2014.05.024|pmid= 25022432|pmc= 4149939}}</ref>

The PQ-ERA reaction has been optimized through thiophene substitution to enhance its reactivity, enabling more efficient photocycloaddition processes.<ref>{{Cite journal |last1=Fu |first1=Youxin |last2=Alachouzos |first2=Georgios |last3=Simeth |first3=Nadja A. |last4=Donato |first4=Mariangela Di |last5=Hilbers |first5=Michiel F. |last6=Buma |first6=Wybren Jan |last7=Szymanski |first7=Wiktor |last8=Feringa |first8=Ben L. |date=2023-07-12 |title=Establishing PQ-ERA photoclick reactions with unprecedented efficiency by engineering of the nature of the phenanthraquinone triplet state |journal=Chemical Science |language=en |volume=14 |issue=27 |pages=7465–7474 |doi=10.1039/D3SC01760E |issn=2041-6539 |pmc=10337724 |pmid=37449069}}</ref>

== Potential applications == === Selective labeling and tracking of biomolecules === Click chemistry is often employed to attach visualizing tags. In many potential applications, click reactions join a biomolecule and a reporter molecule or other molecular probe, a process called bioconjugation.<ref>{{cite journal | author=B. Stump | title = Click Bioconjugation: Modifying Proteins Using Click-Like Chemistry | year = 2022 | journal = ChemBioChem | volume = 23 | issue = 16 | article-number = e202200016 | doi = 10.1002/cbic.202200016 | pmid=35491526| s2cid = 248494718 }}</ref> The possibility of attaching fluorophores and other reporter molecules has made click chemistry a tool for identifying, locating, and characterizing biomolecules. 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.

Examples of bioconjugation for labelling include the use of azidocoumarin to label alkyne groups in modified proteins and biomolecules.<ref>{{Cite journal |last1=Sletten |first1=Ellen M. |last2=Bertozzi |first2=Carolyn R. |date=2009-09-07 |title=Bioorthogonal Chemistry: Fishing for Selectivity in a Sea of Functionality |journal=Angewandte Chemie International Edition |language=en |volume=48 |issue=38 |pages=6974–6998 |doi=10.1002/anie.200900942 |issn=1433-7851 |pmc=2864149 |pmid=19714693 |bibcode=2009ACIE...48.6974S }}</ref> The fluorophore rhodamine has been coupled onto norbornene, and reacted with tetrazine in living systems.<ref>{{cite journal |last1= Devaraj|first1= Neal K.|last2= Weissleder|first2= Ralph|last3= Hilderbrand|first3= Scott A.|journal = Bioconjugate Chem.|date=December 2008|volume = 19|pages= 2297–2299|doi = 10.1021/bc8004446|pmid= 19053305|title = Tetrazine-based cycloadditions: application to pretargeted live cell imaging|pmc=2677645|issue = 12}}</ref> In other cases, SPAAC between a cyclooctyne-modified fluorophore and azide-tagged proteins allowed the selection of these proteins in cell lysates.<ref>{{cite journal | last1 = Ding | first1 = H. | last2 = Demple | first2 = B | year = 2000 | title = Direct nitric oxide signal transduction via nitrosylation of iron-sulfur centers in the SoxR transcription activator| doi = 10.1073/pnas.97.10.5146 | pmid = 10805777 | journal = Proc. Natl. Acad. Sci. U.S.A. | volume = 97 | issue = 10| pages = 5146–5150 | bibcode = 2000PNAS...97.5146D | pmc = 25796 | doi-access = free }}</ref>

thumb|Unnatural Amino Acids

Methods for the incorporation of click reaction partners into systems ''in'' and ''ex vivo'' contribute to the scope of possible bioconjugation 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, azidohomoalanine (AHA) is a methionine analog with an azide side group. This azide side group allows cycloalkynes to react to proteins that incorporate this "AHA" unnatural amino acid.<ref>{{cite journal | last1 = Dieterich |display-authors=et al | year = 2007 | title = Labeling, detection and identification of newly synthesized proteomes with bioorthogonal non-canonical amino-acid tagging| journal = Nature Protocols | volume = 2 | issue = 3| pages = 532–540 | doi=10.1038/nprot.2007.52| pmid = 17406607|s2cid=2833184 }}</ref> In another example, "CpK" is a lysine analog. 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.<ref>{{cite journal | last1 = Yu |display-authors=et al | year = 2012 | title = Genetically Encoded Cyclopropene Directs Rapid, Photoclick-Chemistry-Mediated Protein Labeling in Mammalian Cells| journal = Angew Chem Int Ed Engl | volume = 51 | issue = 42| pages = 10600–10604 | doi=10.1002/anie.201205352| pmid = 22997015| pmc = 3517012}}</ref>

[[File:Luciferin Reaction Figure.png|thumb|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 may then be 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 may be cleaved to yield a fragment with a N' Cys that is vulnerable to the 2-CBT.<ref>(a) Liang, G.; Ren, H.; Rao, J. Nat. Chem. 2010, 2, 54. (b) Ren, H.; Xiao, F.; Zhan, K.; Kim, Y.-P.; Xie, H.; Xia, Z.; Rao, J. Angew.Chem., Int. Ed. 2009, 48, 9658.</ref>

=== 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.<ref>{{Cite journal |last1=Pasieka |first1=Anna |last2=Diamanti |first2=Eleonora |last3=Uliassi |first3=Elisa |last4=Laura Bolognesi |first4=Maria |date=2023 |title=Click Chemistry and Targeted Degradation: A Winning Combination for Medicinal Chemists? |url=https://onlinelibrary.wiley.com/doi/abs/10.1002/cmdc.202300422 |journal=ChemMedChem |language=en |volume=18 |issue=20 |article-number=e202300422 |doi=10.1002/cmdc.202300422 |pmid=37706617 |issn=1860-7187|hdl=11585/952551 |hdl-access=free }}</ref> The simplicity of click chemistry has had significant impact in many themes, especially pharmaceutical development.

Click chemistry been used in numerous drug studies to study their mechanism of action. Discoveries include that salinomycin localizes to lysosomes to initiate ferroptosis in cancer stem cells<ref>{{cite journal |last1=Mai |first1=Trang Thi |last2=Hamaï |first2=Ahmed |last3=Hienzsch |first3=Antje |last4=Cañeque |first4=Tatiana |last5=Müller |first5=Sebastian |last6=Wicinski |first6=Julien |last7=Cabaud |first7=Olivier |last8=Leroy |first8=Christine |last9=David |first9=Amandine |last10=Acevedo |first10=Verónica |last11=Ryo |first11=Akihide |last12=Ginestier |first12=Christophe |last13=Birnbaum |first13=Daniel |last14=Charafe-Jauffret |first14=Emmanuelle |last15=Codogno |first15=Patrice |last16=Mehrpour |first16=Maryam |last17=xRodriguez |first17=Raphaël Rodriguez |title=Salinomycin kills cancer stem cells by sequestering iron in lysosomes |journal=Nature Chemistry |date=Oct 2017 |volume=9 |issue=10 |pages=1025–1033 |doi=10.1038/nchem.2778 |pmid=28937680 |pmc=5890907 |bibcode=2017NatCh...9.1025M }}</ref> and that metformin derivatives accumulate in mitochondria to chelate copper(II), affecting metabolism and epigenetic changes downstream in inflammatory macrophages.<ref>{{cite journal |last1=Solier |first1=Stéphanie |last2=Müller |first2=Sebastian |last3=Tatiana |first3=Cañeque |last4=Antoine |first4=Versini |last5=Arnaud |first5=Mansart |last6=Fabien |first6=Sindikubwabo |last7=Leeroy |first7=Baron |last8=Laila |first8=Emam |last9=Pierre |first9=Gestraud |last10=G. Dan |first10=Pantoș |last11=Vincent |first11=Gandon |last12=Christine |first12=Gaillet |last13=Ting-Di |first13=Wu |last14=Florent |first14=Dingli |last15=Damarys |first15=Loew |last16=Sylvain |first16=Baulande |last17=Sylvère |first17=Durand |last18=Valentin |first18=Sencio |last19=Cyril |first19=Robil |last20=François |first20=Trottein |last21=David |first21=Péricat |last22=Emmanuelle |first22=Näser |last23=Céline |first23=Cougoule |last24=Etienne |first24=Meunier |last25=Anne-Laure |first25=Bègue |last26=Hélène |first26=Salmon |last27=Nicolas |first27=Manel |last28=Alain |first28=Puisieux |last29=Sarah |first29=Watson |last30=Mark A. |first30=Dawson |last31=Nicolas |first31=Servant |last32=Guido |first32=Kroemer |last33=Djillali |first33=Annane |last34=Raphaël |first34=Rodriguez |title=A druggable copper-signalling pathway that drives inflammation |journal=Nature |date=2023 |volume=617 |issue=7960 |pages=386–394 |doi=10.1038/s41586-023-06017-4 |pmid=37100912 |pmc=10131557 |bibcode=2023Natur.617..386S }}</ref>

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.

=== Other possible applications === Additional applications and potential applications include: *ClickSeq, a method for generating next generation sequencing libraries<ref>{{Cite journal |last1=Routh |first1=Andrew |last2=Head |first2=Steven R. |last3=Ordoukhanian |first3=Phillip |last4=Johnson |first4=John E. |date=2015-08-14 |title=ClickSeq: Fragmentation-Free Next-Generation Sequencing via Click Ligation of Adaptors to Stochastically Terminated 3'-Azido cDNAs |journal=Journal of Molecular Biology |volume=427 |issue=16 |pages=2610–2616 |doi=10.1016/j.jmb.2015.06.011 |issn=1089-8638 |pmc=4523409 |pmid=26116762}}</ref> *two-dimensional gel electrophoresis separation<ref>{{cite journal |author1=Ilya A. Osterman |author2=Alexey V. Ustinov |author3=Denis V. Evdokimov |author4=Vladimir A. Korshun |author5=Petr V. Sergiev |author6=Marina V. Serebryakova |author7=Irina A. Demina |author8=Maria A. Galyamina |author9=Vadim M. Govorun |author10=Olga A. Dontsova |title=A nascent proteome study combining click chemistry with 2DE |journal=Proteomics |volume=13 |issue=1 |pages=17–21 |date=January 2013 |pmid=23161590 |doi=10.1002/pmic.201200393 |s2cid=9002232 |url=https://www.cyandye.com/A_nascent_proteome_study_combining_click_chemistry_with_2DE.pdf |access-date=2015-02-11 |archive-url=https://web.archive.org/web/20150630075256/https://www.cyandye.com/A_nascent_proteome_study_combining_click_chemistry_with_2DE.pdf |archive-date=2015-06-30 }}</ref> *preparative organic synthesis of 1,4-substituted triazoles *modification of peptide function with triazoles *modification of natural products and pharmaceuticals *natural product discovery <ref>{{cite journal|last1=Cox|first1=Courtney L.|last2=Tietz|first2=Jonathan I.|last3=Sokolowski|first3=Karol|last4=Melby|first4=Joel O.|last5=Doroghazi|first5=James R.|last6=Mitchell|first6=Douglas A.|title=Nucleophilic 1,4-Additions for Natural Product Discovery|journal=ACS Chemical Biology|date=17 June 2014|pages=2014–2022|doi=10.1021/cb500324n|pmid=24937678|volume=9|issue=9|pmc=4168802}}</ref> *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 *synthesis of polymers and biopolymers<ref>{{cite journal |author1=Michael Floros |author2=Alcides Leão |author3=Suresh Narine | title = Vegetable Oil Derived Solvent, and Catalyst Free "Click Chemistry" Thermoplastic Polytriazoles | year = 2014 | journal = BioMed Research International | doi = 10.1155/2014/792901 |pmid=25032224 | volume = 2014 | pages=1–14| pmc=4085725 |doi-access=free }}</ref> *attachment of molecular machinery to solid surfaces<ref>{{Cite journal |doi = 10.1002/chem.201300500|pmid = 23784916|title = Towards Dynamic Control of Wettability by Using Functionalized Altitudinal Molecular Motors on Solid Surfaces|journal = Chemistry: A European Journal|volume = 19|issue = 32|pages = 10690–10697|year = 2013|last1 = London|first1 = Gábor|last2 = Chen|first2 = Kuang-Yen|last3 = Carroll|first3 = Gregory T.|last4 = Feringa|first4 = Ben L.| bibcode=2013ChEuJ..1910690L | s2cid=5759186 |url = https://www.rug.nl/research/portal/en/publications/towards-dynamic-control-of-wettability-by-using-functionalized-altitudinal-molecular-motors-on-solid-surfaces(d37ada79-57a1-4d72-a96b-4862185a3f8f).html|hdl = 11370/d37ada79-57a1-4d72-a96b-4862185a3f8f|hdl-access = free}}</ref><ref>{{Cite journal |last1=Carroll |first1=Gregory T. |last2=London |first2=Gábor |last3=Landaluce |first3=Tatiana Fernández |last4=Rudolf |first4=Petra |last5=Feringa |first5=Ben L. |date=2011-01-25 |title=Adhesion of photon-driven molecular motors to surfaces via 1,3-dipolar cycloadditions: effect of interfacial interactions on molecular motion |journal=ACS Nano |volume=5 |issue=1 |pages=622–630 |doi=10.1021/nn102876j |issn=1936-086X |pmid=21207983 |bibcode=2011ACSNa...5..622C |url=https://research.rug.nl/en/publications/028d268e-cd1b-4d68-a715-80d8325f9557 }}</ref> *material science *nanotechnology<ref>{{cite journal |author1=John E. Moses |author2=Adam D. Moorhouse | title = The growing applications of click chemistry | year = 2007 | journal = Chem. Soc. Rev. | issue = 8 | pages = 1249–1262 | doi = 10.1039/b613014n | pmid = 17619685 | volume = 36 |bibcode=2007CSRev..36.1249M }}</ref> *biomaterials<ref>{{cite journal |author1=Jean-François Lutz |author2=Zoya Zarafshani | title = Efficient construction of therapeutics, bioconjugates, biomaterials and bioactive surfaces using azide–alkyne "click" chemistry | year = 2008 | journal = Advanced Drug Delivery Reviews | issue = 9 | pages = 958–970 | doi = 10.1016/j.addr.2008.02.004 |pmid=18406491 |volume = 60}}</ref> <!-- most of this content does not describe a application (commercial use), but summarize publication with aspirations of commercial success--> == Technology license == The Scripps Research Institute has a portfolio of click-chemistry patents.<ref>{{cite web |url=http://www.scripps.edu/research/technology/clickchem.html |title=Click Chemistry |access-date=2012-06-05 |archive-url=https://web.archive.org/web/20120515074634/http://www.scripps.edu/research/technology/clickchem.html |archive-date=2012-05-15 }}</ref> Licensees include Invitrogen,<ref>{{cite web |url=https://ir.lifetechnologies.com/releasedetail.cfm?ReleaseID=538901 |title=Invitrogen Exclusively Licenses Novel Click Chemistry-Based Cell Proliferation Assays from Harvard University (NASDAQ:LIFE) |access-date=2012-06-05 |archive-url=https://archive.today/20121217163245/https://ir.lifetechnologies.com/releasedetail.cfm?ReleaseID=538901 |archive-date=2012-12-17 }}</ref> Allozyne,<ref>{{Cite web | url=http://www.xconomy.com/seattle/2010/07/14/allozyne-licenses-scripps-chemistry/ | title=Xconomy: Allozyne Licenses Scripps Chemistry| work=Xconomy| date=2010-07-15}}</ref> Aileron,<ref>{{Cite web | url=http://www.xconomy.com/boston/2010/11/30/aileron-and-scripps-ink-deal/ |title = Xconomy: Aileron and Scripps Ink Deal| work=Xconomy |date = 2010-11-30}}</ref> Integrated Diagnostics,<ref>{{cite web |url=http://www.integrated-diagnostics.com/press-releases/barry-sharpless-click-chemistry/ |title=Integrated Diagnostics Licenses "Click Chemistry" from the Scripps Research Institute, Strengthening Partner Network |access-date=2012-06-05 |archive-url=https://web.archive.org/web/20120430070833/http://www.integrated-diagnostics.com/press-releases/barry-sharpless-click-chemistry/ |archive-date=2012-04-30 }}</ref> and the biotech company {{proper name|baseclick}},<ref>{{Cite web |title=baseclick GmbH :: We enable nucleic acid labeling bioconjugation |url=https://www.baseclick.eu/ |access-date=2022-03-21 |website=baseclick GmbH |language=en-US}}</ref> a BASF spin-off created to sell products made using click chemistry.<ref>http://www.basf.com/group/pressrelease/P-10-427 {{Dead link|date=July 2019 |bot=InternetArchiveBot |fix-attempted=yes }}</ref> Moreover, {{proper name|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.<ref>{{Cite web |date=2018-10-03 |title=CYANDYE |url=http://www.cyandye.us/ |access-date=2022-03-21 |website= |archive-url=https://web.archive.org/web/20181003073347/http://www.cyandye.us/ |archive-date=3 October 2018 }}</ref>

== References == {{reflist}}

== External links == * [https://www.organic-chemistry.org/namedreactions/click-chemistry.shtm Click Chemistry: Short Review and Recent Literature] * [https://www.nsf.gov/news/news_summ.jsp?cntn_id=110681&org=NSF&from=newsField National Science Foundation: Feature "Going Live with Click Chemistry"] * [http://pubs.acs.org/cen/coverstory/8006/8006clickchemistry.html Chemical and Engineering News: Feature "In-Situ Click Chemistry"] * [http://pubs.acs.org/cen/news/85/i43/8543notw8.html Chemical and Engineering News: Feature "Copper-free Click Chemistry"] * [https://archive.today/20130105054019/http://www3.interscience.wiley.com/journal/122408457/abstract Metal-free click chemistry review] * [http://pubs.rsc.org/en/journals/journalissues/cs#!issueid=cs039004&type=current&issnprint=0306-0012 Click Chemistry]{{snd}} a [http://www.rsc.org/Publishing/Journals/CS/Index.asp ''Chem Soc Rev''] themed issue highlighting the latest applications of click chemistry, guest edited by M. G. Finn and Valery Fokin. Published by the Royal Society of Chemistry {{BranchesofChemistry}}

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{{DEFAULTSORT:Click Chemistry}} Category:Organic chemistry