Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Bioorthogonal chemistry

Abstract

Bioorthogonal chemistry represents a class of high-yielding chemical reactions that proceed rapidly and selectively in biological environments without side reactions towards endogenous functional groups. Rooted in the principles of physical organic chemistry, bioorthogonal reactions are intrinsically selective transformations not commonly found in biology. Key reactions include native chemical ligation and the Staudinger ligation, copper-catalysed azide–alkyne cycloaddition, strain-promoted [3 + 2] reactions, tetrazine ligation, metal-catalysed coupling reactions, oxime and hydrazone ligations as well as photoinducible bioorthogonal reactions. Bioorthogonal chemistry has significant overlap with the broader field of ‘click chemistry’ — high-yielding reactions that are wide in scope and simple to perform, as recently exemplified by sulfuryl fluoride exchange chemistry. The underlying mechanisms of these transformations and their optimal conditions are described in this Primer, followed by discussion of how bioorthogonal chemistry has become essential to the fields of biomedical imaging, medicinal chemistry, protein synthesis, polymer science, materials science and surface science. The applications of bioorthogonal chemistry are diverse and include genetic code expansion and metabolic engineering, drug target identification, antibody–drug conjugation and drug delivery. This Primer describes standards for reproducibility and data deposition, outlines how current limitations are driving new research directions and discusses new opportunities for applying bioorthogonal chemistry to emerging problems in biology and biomedicine.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: Different classes of bioorthogonal reactions.
Fig. 2: The native chemical ligation and oxime and hydrazine ligations.
Fig. 3: The Staudinger ligation types and the copper-catalysed azide–alkyne reaction.
Fig. 4: Cycloaddition-based bioorthogonal chemical reaction types.
Fig. 5: Light-activated click chemistry and metal-catalysed coupling reactions.
Fig. 6: Applications for labelling different molecule types in cells.
Fig. 7: Examples of biorthogonal chemistry applications in vitro and in vivo.

References

  1. 1.

    Nienhaus, G. U. The green fluorescent protein: a key tool to study chemical processes in living cells. Angew. Chem. Int. Ed. 47, 8992–8994 (2008).

    Google Scholar 

  2. 2.

    Winter, G. & Milstein, C. Man-made antibodies. Nature 349, 293–299 (1991).

    ADS  Google Scholar 

  3. 3.

    Lang, K. & Chin, J. W. Bioorthogonal reactions for labeling proteins. ACS Chem. Biol. 9, 16–20 (2014).

    Google Scholar 

  4. 4.

    Kolb, H. C., Finn, M. G. & Sharpless, K. B. Click chemistry: diverse chemical function from a few good reactions. Angew. Chem. Int. Ed. 40, 2004–2021 (2001). This seminal review article outlines the concept and requirements of click chemistry.

    Google Scholar 

  5. 5.

    Dong, J., Krasnova, L., Finn, M. G. & Sharpless, K. B. Sulfur(VI) fluoride exchange (SuFEx): another good reaction for click chemistry. Angew. Chem. Int. Ed. 53, 9430–9448 (2014). This article presents an initial description of SuFEx chemistry as an efficient biocompatible click chemistry tool.

    Google Scholar 

  6. 6.

    Hoyle, C. E. & Bowman, C. N. Thiol–ene click chemistry. Angew. Chem. Int. Ed. 49, 1540–1573 (2010).

    Google Scholar 

  7. 7.

    Griffin, B. A., Adams, S. R. & Tsien, R. Y. Specific covalent labeling of recombinant protein molecules inside live cells. Science 281, 269–272 (1998).

    ADS  Google Scholar 

  8. 8.

    Zhang, Y., Park, K.-Y., Suazo, K. F. & Distefano, M. D. Recent progress in enzymatic protein labelling techniques and their applications. Chem. Soc. Rev. 47, 9106–9136 (2018).

    Google Scholar 

  9. 9.

    Lopez Aguilar, A. et al. Tools for studying glycans: recent advances in chemoenzymatic glycan labeling. ACS Chem. Biol. 12, 611–621 (2017).

    Google Scholar 

  10. 10.

    Liss, V., Barlag, B., Nietschke, M. & Hensel, M. Self-labelling enzymes as universal tags for fluorescence microscopy, super-resolution microscopy and electron microscopy. Sci. Rep. 5, 17740 (2015).

    ADS  Google Scholar 

  11. 11.

    Dawson, P., Muir, T., Clark-Lewis, I. & Kent, S. Synthesis of proteins by native chemical ligation. Science 266, 776–779 (1994). This article presents an initial description of native chemical ligation, which enabled the use of fully deprotected peptides through bioorthogonal reaction of a thioester motif with an N-terminal cysteine residue.

    ADS  Google Scholar 

  12. 12.

    Agouridas, V. et al. Native chemical ligation and extended methods: mechanisms, catalysis, scope, and limitations. Chem. Rev. 119, 7328–7443 (2009).

    Google Scholar 

  13. 13.

    Kent, S. B. H. Total chemical synthesis of proteins. Chem. Soc. Rev. 38, 338–351 (2009).

    Google Scholar 

  14. 14.

    Wieland, T., Bokelmann, E., Bauer, L., Lang, H. U. & Lau, H. Über Peptidsynthesen. 8. Mitteilung Bildung von S-haltigen Peptiden durch intramolekulare Wanderung von Aminoacylresten. Justus Liebigs Ann. Chem. 583, 129–149 (1953).

    Google Scholar 

  15. 15.

    Dawson, P. E., Churchill, M. J., Ghadiri, M. R. & Kent, S. B. H. Modulation of reactivity in native chemical ligation through the use of thiol additives. J. Am. Chem. Soc. 119, 4325–4329 (1997).

    Google Scholar 

  16. 16.

    Yan, L. Z. & Dawson, P. E. Synthesis of peptides and proteins without cysteine residues by native chemical ligation combined with desulfurization. J. Am. Chem. Soc. 123, 526–533 (2001).

    Google Scholar 

  17. 17.

    Wan, Q. & Danishefsky, S. J. Free-radical-based, specific desulfurization of cysteine: a powerful advance in the synthesis of polypeptides and glycopolypeptides. Angew. Chem. Int. Ed. 46, 9248–9252 (2007).

    Google Scholar 

  18. 18.

    Metanis, N., Keinan, E. & Dawson, P. E. Traceless ligation of cysteine peptides using selective deselenization. Angew. Chem. Int. Ed. 49, 7049–7053 (2010).

    Google Scholar 

  19. 19.

    Kulkarni, S. S., Sayers, J., Premdjee, B. & Payne, R. J. Rapid and efficient protein synthesis through expansion of the native chemical ligation concept. Nat. Rev. Chem. 2, 0122 (2018).

    Google Scholar 

  20. 20.

    Muir, T. W., Sondhi, D. & Cole, P. A. Expressed protein ligation: a general method for protein engineering. Proc. Natl Acad. Sci. USA 95, 6705–6710 (1998). This article presents the first description of expressed protein ligation — the application of native chemical ligation to ligate small synthetic sequences to much larger recombinant protein fragments.

    ADS  Google Scholar 

  21. 21.

    Tam, J. P. & Miao, Z. Stereospecific pseudoproline ligation of N-terminal serine, threonine, or cysteine-containing unprotected peptides. J. Am. Chem. Soc. 121, 9013–9022 (1999).

    Google Scholar 

  22. 22.

    Zhang, Y., Xu, C., Lam, H. Y., Lee, C. L. & Li, X. Protein chemical synthesis by serine and threonine ligation. Proc. Natl Acad. Sci. USA 110, 6657–6662 (2013).

    ADS  Google Scholar 

  23. 23.

    Bode, J. W., Fox, R. M. & Baucom, K. D. Chemoselective amide ligations by decarboxylative condensations of N-alkylhydroxylamines and α-ketoacids. Angew. Chem. Int. Ed. 45, 1248–1252 (2006).

    Google Scholar 

  24. 24.

    Noda, H., Erős, G. & Bode, J. W. Rapid ligations with equimolar reactants in water with the potassium acyltrifluoroborate (KAT) amide formation. J. Am. Chem. Soc. 136, 5611–5614 (2014).

    Google Scholar 

  25. 25.

    Geoghegan, K. F. & Stroh, J. G. Site-directed conjugation of nonpeptide groups to peptides and proteins via periodate oxidation of a 2-amino alcohol. Application to modification at N-terminal serine. Bioconjugate Chem. 3, 138–146 (1992).

    Google Scholar 

  26. 26.

    Zhang, L. & Tam, J. P. Thiazolidine formation as a general and site-specific conjugation method for synthetic peptides and proteins. Anal. Biochem. 233, 87–93 (1996).

    Google Scholar 

  27. 27.

    Agarwal, P., van der Weijden, J., Sletten, E. M., Rabuka, D. & Bertozzi, C. R. A Pictet–Spengler ligation for protein chemical modification. Proc. Natl Acad. Sci. USA 110, 46–51 (2013).

    ADS  Google Scholar 

  28. 28.

    Ren, H. et al. A biocompatible condensation reaction for the labeling of terminal cysteine residues on proteins. Angew. Chem. Int. Ed. 48, 9658–9662 (2009).

    Google Scholar 

  29. 29.

    Gilmore, J. M., Scheck, R. A., Esser-Kahn, A. P., Joshi, N. S. & Francis, M. B. N-terminal protein modification through a biomimetic transamination reaction. Angew. Chem. Int. Ed. 45, 5307–5311 (2006).

    Google Scholar 

  30. 30.

    Witus, L. S. et al. Site-specific protein transamination using N-methylpyridinium-4-carboxaldehyde. J. Am. Chem. Soc. 135, 17223–17229 (2013).

    Google Scholar 

  31. 31.

    MacDonald, J. I., Munch, H. K., Moore, T. & Francis, M. B. One-step site-specific modification of native proteins with 2-pyridinecarboxyaldehydes. Nat. Chem. Biol. 11, 326–331 (2015).

    Google Scholar 

  32. 32.

    Rideout, D. Self-assembling cytotoxins. Science 233, 561–563 (1986).

    ADS  Google Scholar 

  33. 33.

    Kalia, J. & Raines, R. T. Hydrolytic stability of hydrazones and oximes. Angew. Chem. Int. Ed. 47, 7523–7526 (2008).

    Google Scholar 

  34. 34.

    Brudno, Y. et al. Refilling drug delivery depots through the blood. Proc. Natl Acad. Sci. USA 111, 12722–12727 (2014).

    ADS  Google Scholar 

  35. 35.

    Deygen, I. M. et al. Novel prodrug of doxorubicin modified by stearoylspermine encapsulated into PEG-chitosan-stabilized liposomes. Langmuir 32, 10861–10869 (2016).

    Google Scholar 

  36. 36.

    Matson, J. B. & Stupp, S. I. Drug release from hydrazone-containing peptide amphiphiles. ChemComm 47, 7962–7964 (2011).

    Google Scholar 

  37. 37.

    Kölmel, D. K. & Kool, E. T. Oximes and hydrazones in bioconjugation: mechanism and catalysis. Chem. Rev. 117, 10358–10376 (2017).

    Google Scholar 

  38. 38.

    Saito, F., Noda, H. & Bode, J. W. Critical evaluation and rate constants of chemoselective ligation reactions for stoichiometric conjugations in water. ACS Chem. Biol. 10, 1026–1033 (2015).

    Google Scholar 

  39. 39.

    Gaertner, H. F. et al. Construction of protein analogs by site-specific condensation of unprotected fragments. Bioconjugate Chem. 3, 262–268 (1992).

    Google Scholar 

  40. 40.

    Jencks, W. P. Studies on the mechanism of oxime and semicarbazone formation1. J. Am. Chem. Soc. 81, 475–481 (1959).

    Google Scholar 

  41. 41.

    Sander, E. G. & Jencks, W. P. Equilibria for additions to the carbonyl group. J. Am. Chem. Soc. 90, 6154–6162 (1968).

    Google Scholar 

  42. 42.

    Dirksen, A., Hackeng, T. M. & Dawson, P. E. Nucleophilic catalysis of oxime ligation. Angew. Chem. Int. Ed. 45, 7581–7584 (2006).

    Google Scholar 

  43. 43.

    Dirksen, A., Dirksen, S., Hackeng, T. M. & Dawson, P. E. Nucleophilic catalysis of hydrazone formation and transimination:  implications for dynamic covalent chemistry. J. Am. Chem. Soc. 128, 15602–15603 (2006).

    Google Scholar 

  44. 44.

    Crisalli, P. & Kool, E. T. Importance of ortho proton donors in catalysis of hydrazone formation. Org. Lett. 15, 1646–1649 (2013).

    Google Scholar 

  45. 45.

    Crisalli, P. & Kool, E. T. Water-soluble organocatalysts for hydrazone and oxime formation. J. Org. Chem. 78, 1184–1189 (2013).

    Google Scholar 

  46. 46.

    Larsen, D. et al. Exceptionally rapid oxime and hydrazone formation promoted by catalytic amine buffers with low toxicity. Chem. Sci. 9, 5252–5259 (2018).

    Google Scholar 

  47. 47.

    Agarwal, P. et al. Hydrazino-Pictet–Spengler ligation as a biocompatible method for the generation of stable protein conjugates. Bioconjugate Chem. 24, 846–851 (2013).

    MathSciNet  Google Scholar 

  48. 48.

    Sudalai, A., Khenkin, A. & Neumann, R. Sodium periodate mediated oxidative transformations in organic synthesis. Org. Biomol. Chem. 13, 4374–4394 (2015).

    Google Scholar 

  49. 49.

    Zeng, Y., Ramya, T. N. C., Dirksen, A., Dawson, P. E. & Paulson, J. C. High-efficiency labeling of sialylated glycoproteins on living cells. Nat. Methods 6, 207–209 (2009).

    Google Scholar 

  50. 50.

    Chelius, D. & Shaler, T. A. Capture of peptides with N-terminal serine and threonine:  a sequence-specific chemical method for peptide mixture simplification. Bioconjugate Chem. 14, 205–211 (2003).

    Google Scholar 

  51. 51.

    Hansske, F., Sprinzl, M. & Cramer, F. Reaction of the ribose moiety of adenosine and AMP with periodate and carboxylic acid hydrazides. Bioorg. Chem. 3, 367–376 (1974).

    Google Scholar 

  52. 52.

    Haney, C. M. & Horne, W. S. Oxime side-chain cross-links in an α-helical coiled-coil protein: structure, thermodynamics, and folding-templated synthesis of bicyclic species. Chem. Eur. J. 19, 11342–11351 (2013).

    Google Scholar 

  53. 53.

    Haney, C. M., Loch, M. T. & Horne, W. S. Promoting peptide α-helix formation with dynamic covalent oxime side-chain cross-links. ChemComm 47, 10915–10917 (2011).

    Google Scholar 

  54. 54.

    Hardisty, R. E., Kawasaki, F., Sahakyan, A. B. & Balasubramanian, S. Selective chemical labeling of natural T modifications in DNA. J. Am. Chem. Soc. 137, 9270–9272 (2015).

    Google Scholar 

  55. 55.

    Köhn, M. & Breinbauer, R. The Staudinger ligation — a gift to chemical biology. Angew. Chem. Int. Ed. 43, 3106–3116 (2004).

    Google Scholar 

  56. 56.

    Bednarek, C., Wehl, I., Jung, N., Schepers, U. & Bräse, S. The Staudinger ligation. Chem. Rev. 120, 4301–4354 (2020).

    Google Scholar 

  57. 57.

    Staudinger, H. & Meyer, J. Über neue organische phosphorverbindungen III. phosphinmethylenderivate und phosphinimine. Helv. Chim. Acta 2, 635–646 (1919).

    Google Scholar 

  58. 58.

    Saxon, E. & Bertozzi, C. R. Cell surface engineering by a modified Staudinger reaction. Science 287, 2007–2010 (2000). This article presents the first description of cell surface engineering using azido reporters and bioorthogonal chemistry.

    ADS  Google Scholar 

  59. 59.

    Shah, L., Laughlin, S. T. & Carrico, I. S. Light-activated Staudinger–Bertozzi ligation within living animals. J. Am. Chem. Soc. 138, 5186–5189 (2016).

    Google Scholar 

  60. 60.

    Schilling, C. I., Jung, N., Biskup, M., Schepers, U. & Bräse, S. Bioconjugation via azide–Staudinger ligation: an overview. Chem. Soc. Rev. 40, 4840–4871 (2011).

    Google Scholar 

  61. 61.

    Kiick, K. L., Saxon, E., Tirrell, D. A. & Bertozzi, C. R. Incorporation of azides into recombinant proteins for chemoselective modification by the Staudinger ligation. Proc. Natl Acad. Sci. USA 99, 19–24 (2002).

    ADS  Google Scholar 

  62. 62.

    Ovaa, H. et al. Chemistry in living cells: detection of active proteasomes by a two-step labeling strategy. Angew. Chem. Int. Ed. 42, 3626–3629 (2003).

    Google Scholar 

  63. 63.

    Tsao, M.-L., Tian, F. & Schultz, P. G. Selective Staudinger modification of proteins containing p-azidophenylalanine. ChemBioChem 6, 2147–2149 (2005).

    Google Scholar 

  64. 64.

    Wang, C. C. Y., Seo, T. S., Li, Z., Ruparel, H. & Ju, J. Site-specific fluorescent labeling of DNA using Staudinger ligation. Bioconjugate Chem. 14, 697–701 (2003).

    Google Scholar 

  65. 65.

    Vocadlo, D. J., Hang, H. C., Kim, E.-J., Hanover, J. A. & Bertozzi, C. R. A chemical approach for identifying O-GlcNAc-modified proteins in cells. Proc. Natl Acad. Sci. USA 100, 9116–9121 (2003).

    ADS  Google Scholar 

  66. 66.

    Martin, D. D. et al. Rapid detection, discovery, and identification of post-translationally myristoylated proteins during apoptosis using a bio-orthogonal azidomyristate analog. FASEB J. 22, 797–806 (2008).

    Google Scholar 

  67. 67.

    Hang, H. C., Wilson, J. P. & Charron, G. Bioorthogonal chemical reporters for analyzing protein lipidation and lipid trafficking. Acc. Chem. Res. 44, 699–708 (2011).

    Google Scholar 

  68. 68.

    Lemieux, G. A., de Graffenried, C. L. & Bertozzi, C. R. A fluorogenic dye activated by the Staudinger ligation. J. Am. Chem. Soc. 125, 4708–4709 (2003).

    Google Scholar 

  69. 69.

    Hangauer, M. J. & Bertozzi, C. R. A FRET-based fluorogenic phosphine for live-cell imaging with the Staudinger ligation. Angew. Chem. Int. Ed. 47, 2394–2397 (2008).

    Google Scholar 

  70. 70.

    Lukasak, B., Morihiro, K. & Deiters, A. Aryl azides as phosphine-activated switches for small molecule function. Sci. Rep. 9, 1–6 (2019).

    Google Scholar 

  71. 71.

    Luo, J., Liu, Q., Morihiro, K. & Deiters, A. Small-molecule control of protein function through Staudinger reduction. Nat. Chem. 8, 1027 (2016).

    Google Scholar 

  72. 72.

    Prescher, J. A., Dube, D. H. & Bertozzi, C. R. Chemical remodelling of cell surfaces in living animals. Nature 430, 873–877 (2004). This article presents the first description of bioorthogonal chemistry in live mice.

    ADS  Google Scholar 

  73. 73.

    Dube, D. H., Prescher, J. A., Quang, C. N. & Bertozzi, C. R. Probing mucin-type O-linked glycosylation in living animals. Proc. Natl Acad. Sci. USA 103, 4819–4824 (2006).

    ADS  Google Scholar 

  74. 74.

    Lin, F. L., Hoyt, H. M., van Halbeek, H., Bergman, R. G. & Bertozzi, C. R. Mechanistic investigation of the Staudinger ligation. J. Am. Chem. Soc. 127, 2686–2695 (2005).

    Google Scholar 

  75. 75.

    Ren, G., Zheng, Q. & Wang, H. Aryl fluorosulfate trapped Staudinger reduction. Org. Lett. 19, 1582–1585 (2017).

    Google Scholar 

  76. 76.

    Tam, A. & Raines, R. T. Protein engineering with the traceless Staudinger ligation. Methods Enzymol. 462, 25–44 (2009).

    Google Scholar 

  77. 77.

    Saxon, E., Armstrong, J. I. & Bertozzi, C. R. A “traceless” Staudinger ligation for the chemoselective synthesis of amide bonds. Org. Lett. 2, 2141–2143 (2000).

    Google Scholar 

  78. 78.

    Nilsson, B. L., Kiessling, L. L. & Raines, R. T. Staudinger ligation: a peptide from a thioester and azide. Org. Lett. 2, 1939–1941 (2000).

    Google Scholar 

  79. 79.

    Kleineweischede, R. & Hackenberger, C. P. Chemoselective peptide cyclization by traceless Staudinger ligation. Angew. Chem. Int. Ed. 47, 5984–5988 (2008).

    Google Scholar 

  80. 80.

    Merkx, R., Rijkers, D. T., Kemmink, J. & Liskamp, R. M. Chemoselective coupling of peptide fragments using the Staudinger ligation. Tetrahedron Lett. 44, 4515–4518 (2003).

    Google Scholar 

  81. 81.

    Böhrsch, V., Serwa, R., Majkut, P., Krause, E. & Hackenberger, C. P. Site-specific functionalisation of proteins by a Staudinger-type reaction using unsymmetrical phosphites. ChemComm 46, 3176–3178 (2010).

    Google Scholar 

  82. 82.

    Vallée, M. R. J. et al. Staudinger-phosphonite reactions for the chemoselective transformation of azido-containing peptides and proteins. Org. Lett. 13, 5440–5443 (2011).

    Google Scholar 

  83. 83.

    Lee, Y. J., Kurra, Y. & Liu, W. R. Phospha-Michael addition as a new click reaction for protein functionalization. ChemBioChem 17, 456–461 (2016).

    Google Scholar 

  84. 84.

    Bos, J. & Muir, T. W. A chemical probe for protein crotonylation. J. Am. Chem. Soc. 140, 4757–4760 (2018).

    Google Scholar 

  85. 85.

    Shih, H.-W. & Prescher, J. A. A bioorthogonal ligation of cyclopropenones mediated by triarylphosphines. J. Am. Chem. Soc. 137, 10036–10039 (2015).

    Google Scholar 

  86. 86.

    Row, R. D., Shih, H.-W., Alexander, A. T., Mehl, R. A. & Prescher, J. A. Cyclopropenones for metabolic targeting and sequential bioorthogonal labeling. J. Am. Chem. Soc. 139, 7370–7375 (2017).

    Google Scholar 

  87. 87.

    Row, R. D. & Prescher, J. A. A cyclopropenethione–phosphine ligation for rapid biomolecule labeling. Org. Lett. 20, 5614–5617 (2018).

    Google Scholar 

  88. 88.

    Heiss, T. K. & Prescher, J. A. Cyclopropeniminium ions exhibit unique reactivity profiles with bioorthogonal phosphines. J. Org. Chem. 84, 7443–7448 (2019).

    Google Scholar 

  89. 89.

    Tornøe, C. W., Christensen, C. & Meldal, M. Peptidotriazoles on solid phase:  [1,2,3]-triazoles by regiospecific copper(I)-catalyzed 1,3-dipolar cycloadditions of terminal alkynes to azides. J. Org. Chem. 67, 3057–3064 (2002).

    Google Scholar 

  90. 90.

    Rostovtsev, V. V., Green, L. G., Fokin, V. V. & Sharpless, K. B. A stepwise Huisgen cycloaddition process: copper(I)-catalyzed regioselective “ligation” of azides and terminal alkynes. Angew. Chem. Int. Ed. 41, 2596–2599 (2002). Together with Tornøe et al. (2002), these articles present the initial description of copper-catalysed azide–alkyne cycloaddition chemistry.

    Google Scholar 

  91. 91.

    Himo, F. et al. Copper(I)-catalyzed synthesis of azoles. DFT study predicts unprecedented reactivity and intermediates. J. Am. Chem. Soc. 127, 210–216 (2005).

    Google Scholar 

  92. 92.

    Presolski, S. I., Hong, V., Cho, S.-H. & Finn, M. G. Tailored ligand acceleration of the Cu-catalyzed azide–alkyne cycloaddition reaction: practical and mechanistic implications. J. Am. Chem. Soc. 132, 14570–14576 (2010).

    Google Scholar 

  93. 93.

    Chan, T. R., Hilgraf, R., Sharpless, K. B. & Fokin, V. V. Polytriazoles as copper(I)-stabilizing ligands in catalysis. Org. Lett. 6, 2853–2855 (2004).

    Google Scholar 

  94. 94.

    Lipshutz, B. H. & Taft, B. R. Heterogeneous copper-in-charcoal-catalyzed click chemistry. Angew. Chem. Int. Ed. 45, 8235–8238 (2006).

    Google Scholar 

  95. 95.

    Chen, J. et al. Enzyme-like click catalysis by a copper-containing single-chain nanoparticle. J. Am. Chem. Soc. 140, 13695–13702 (2018).

    Google Scholar 

  96. 96.

    Wang, F. et al. A biocompatible heterogeneous MOF–Cu catalyst for in vivo drug synthesis in targeted subcellular organelles. Angew. Chem. Int. Ed. 58, 6987–6992 (2019).

    Google Scholar 

  97. 97.

    Zhu, X., Liu, J. & Zhang, W. De novo biosynthesis of terminal alkyne-labeled natural products. Nat. Chem. Biol. 11, 115–120 (2015).

    Google Scholar 

  98. 98.

    Marchand, J. A. et al. Discovery of a pathway for terminal-alkyne amino acid biosynthesis. Nature 567, 420–424 (2019).

    ADS  Google Scholar 

  99. 99.

    Hong, V., Presolski, S. I., Ma, C. & Finn, M. G. Analysis and optimization of copper-catalyzed azide–alkyne cycloaddition for bioconjugation. Angew. Chem. Int. Ed. 48, 9879–9883 (2009).

    Google Scholar 

  100. 100.

    Hong, V., Steinmetz, N. F., Manchester, M. & Finn, M. G. Labeling live cells by copper-catalyzed alkyne–azide click chemistry. Bioconjugate Chem. 21, 1912–1916 (2010).

    Google Scholar 

  101. 101.

    Soriano del Amo, D. et al. Biocompatible copper(I) catalysts for in vivo imaging of glycans. J. Am. Chem. Soc. 132, 16893–16899 (2010).

    Google Scholar 

  102. 102.

    Kuang, G.-C., Michaels, H. A., Simmons, J. T., Clark, R. J. & Zhu, L. Chelation-assisted, copper(II)-acetate-accelerated azide–alkyne cycloaddition. J. Org. Chem. 75, 6540–6548 (2010).

    Google Scholar 

  103. 103.

    Besanceney-Webler, C. et al. Increasing the efficacy of bioorthogonal click reactions for bioconjugation: a comparative study. Angew. Chem. Int. Ed. 50, 8051–8056 (2011).

    Google Scholar 

  104. 104.

    Uttamapinant, C. et al. Fast, cell-compatible click chemistry with copper-chelating azides for biomolecular labeling. Angew. Chem. Int. Ed. 51, 5852–5856 (2012).

    Google Scholar 

  105. 105.

    Bevilacqua, V. et al. Copper-chelating azides for efficient click conjugation reactions in complex media. Angew. Chem. Int. Ed. 53, 5872–5876 (2014).

    Google Scholar 

  106. 106.

    Su, Y., Li, L., Wang, H., Wang, X. & Zhang, Z. All-in-one azides: empowered click reaction for in vivo labeling and imaging of biomolecules. ChemComm 52, 2185–2188 (2016).

    Google Scholar 

  107. 107.

    Inoue, N., Onoda, A. & Hayashi, T. Site-specific modification of proteins through N-terminal azide labeling and a chelation-assisted CuAAC reaction. Bioconjugate Chem. 30, 2427–2434 (2019).

    Google Scholar 

  108. 108.

    Li, S. et al. Copper-catalyzed click reaction on/in live cells. Chem. Sci. 8, 2107–2114 (2017).

    Google Scholar 

  109. 109.

    Speers, A. E., Adam, G. C. & Cravatt, B. F. Activity-based protein profiling in vivo using a copper(I)-catalyzed azide–alkyne [3 + 2] cycloaddition. J. Am. Chem. Soc. 125, 4686–4687 (2003).

    Google Scholar 

  110. 110.

    Yang, M. et al. Biocompatible click chemistry enabled compartment-specific pH measurement inside E. coli. Nat. Commun. 5, 4981 (2014).

    ADS  Google Scholar 

  111. 111.

    Clavadetscher, J. et al. Copper catalysis in living systems and in situ drug synthesis. Angew. Chem. Int. Ed. 55, 15662–15666 (2016).

    Google Scholar 

  112. 112.

    Sun, D. E. et al. Click-ExM enables expansion microscopy for all biomolecules. Nat. Methods 18, 107–113 (2021).

    Google Scholar 

  113. 113.

    Morgan, M. T. et al. Ratiometric two-photon microscopy reveals attomolar copper buffering in normal and Menkes mutant cells. Proc. Natl Acad. Sci. USA 116, 12167 (2019).

    Google Scholar 

  114. 114.

    Kennedy, D. C. et al. Cellular consequences of copper complexes used to catalyze bioorthogonal click reactions. J. Am. Chem. Soc. 133, 17993–18001 (2011).

    Google Scholar 

  115. 115.

    Agard, N. J., Prescher, J. A. & Bertozzi, C. R. A strain-promoted [3 + 2] azide–alkyne cycloaddition for covalent modification of biomolecules in living systems. J. Am. Chem. Soc. 126, 15046–15047 (2004). This article presents the first use of strained molecules to facilitate rapid, catalyst-free bioorthogonal labelling.

    Google Scholar 

  116. 116.

    Baskin, J. M. et al. Copper-free click chemistry for dynamic in vivo imaging. Proc. Natl Acad. Sci. USA 104, 16793–16797 (2007).

    ADS  Google Scholar 

  117. 117.

    Dommerholt, J., Rutjes, F. P. J. T. & van Delft, F. L. Strain-promoted 1,3-dipolar cycloaddition of cycloalkynes and organic azides. Top. Curr. Chem. 374, 16 (2016).

    Google Scholar 

  118. 118.

    Chupakhin, E. G. & Krasavin, M. Y. Achievements in the synthesis of cyclooctynes for ring strain-promoted [3 + 2] azide–alkyne cycloaddition. Chem. Heterocycl. Compd. 54, 483–501 (2018).

    Google Scholar 

  119. 119.

    Codelli, J. A., Baskin, J. M., Agard, N. J. & Bertozzi, C. R. Second-generation difluorinated cyclooctynes for copper-free click chemistry. J. Am. Chem. Soc. 130, 11486–11493 (2008).

    Google Scholar 

  120. 120.

    Dommerholt, J. et al. Readily accessible bicyclononynes for bioorthogonal labeling and three-dimensional imaging of living cells. Angew. Chem. Int. Ed. 49, 9422–9425 (2010).

    Google Scholar 

  121. 121.

    Debets, M. F., van der Doelen, C. W. J., Rutjes, F. P. J. T. & van Delft, F. L. Azide: a unique dipole for metal-free bioorthogonal ligations. ChemBioChem 11, 1168–1184 (2010).

    Google Scholar 

  122. 122.

    Jewett, J. C., Sletten, E. M. & Bertozzi, C. R. Rapid Cu-free click chemistry with readily synthesized biarylazacyclooctynones. J. Am. Chem. Soc. 132, 3688–3690 (2010).

    Google Scholar 

  123. 123.

    Ning, X., Guo, J., Wolfert, M. A. & Boons, G.-J. Visualizing metabolically labeled glycoconjugates of living cells by copper-free and fast Huisgen cycloadditions. Angew. Chem. Int. Ed. 47, 2253–2255 (2008).

    Google Scholar 

  124. 124.

    Nainar, S. et al. Temporal labeling of nascent RNA using photoclick chemistry in live cells. J. Am. Chem. Soc. 139, 8090–8093 (2017).

    Google Scholar 

  125. 125.

    Friscourt, F. et al. Polar dibenzocyclooctynes for selective labeling of extracellular glycoconjugates of living cells. J. Am. Chem. Soc. 134, 5381–5389 (2012).

    Google Scholar 

  126. 126.

    Sletten, E. M. & Bertozzi, C. R. A hydrophilic azacyclooctyne for Cu-free click chemistry. Org. Lett. 10, 3097–3099 (2008).

    Google Scholar 

  127. 127.

    Stöckmann, H. et al. Development and evaluation of new cyclooctynes for cell surface glycan imaging in cancer cells. Chem. Sci. 2, 932–936 (2011).

    Google Scholar 

  128. 128.

    Almeida, G. D., Townsend, L. C. & Bertozzi, C. R. Synthesis and reactivity of dibenzoselenacycloheptynes. Org. Lett. 15, 3038–3041 (2013).

    Google Scholar 

  129. 129.

    Almeida, G. D., Sletten, E. M., Nakamura, H., Palaniappan, K. K. & Bertozzi, C. R. Thiacycloalkynes for copper-free click chemistry. Angew. Chem. Int. Ed. 51, 2443–2447 (2012).

    Google Scholar 

  130. 130.

    Weterings, J. et al. TMTHSI, a superior 7-membered ring alkyne containing reagent for strain-promoted azide–alkyne cycloaddition reactions. Chem. Sci. 11, 9011–9016 (2020).

    Google Scholar 

  131. 131.

    Friscourt, F., Fahrni, C. J. & Boons, G.-J. A fluorogenic probe for the catalyst-free detection of azide-tagged molecules. J. Am. Chem. Soc. 134, 18809–18815 (2012).

    Google Scholar 

  132. 132.

    Jewett, J. C. & Bertozzi, C. R. Synthesis of a fluorogenic cyclooctyne activated by Cu-free click chemistry. Org. Lett. 13, 5937–5939 (2011).

    Google Scholar 

  133. 133.

    Ess, D. H. & Houk, K. N. Distortion/interaction energy control of 1,3-dipolar cycloaddition reactivity. J. Am. Chem. Soc. 129, 10646–10647 (2007).

    Google Scholar 

  134. 134.

    Liang, Y., Mackey, J. L., Lopez, S. A., Liu, F. & Houk, K. N. Control and design of mutual orthogonality in bioorthogonal cycloadditions. J. Am. Chem. Soc. 134, 17904–17907 (2012).

    Google Scholar 

  135. 135.

    Liu, F., Liang, Y. & Houk, K. N. Bioorthogonal cycloadditions: computational analysis with the distortion/interaction model and predictions of reactivities. Acc. Chem. Res. 50, 2297–2308 (2017).

    Google Scholar 

  136. 136.

    McKay, C. S., Blake, J. A., Cheng, J., Danielson, D. C. & Pezacki, J. P. Strain-promoted cycloadditions of cyclic nitrones with cyclooctynes for labeling human cancer cells. ChemComm 47, 10040–10042 (2011).

    Google Scholar 

  137. 137.

    McKay, C. S., Chigrinova, M., Blake, J. A. & Pezacki, J. P. Kinetics studies of rapid strain-promoted [3 + 2]-cycloadditions of nitrones with biaryl-aza-cyclooctynone. Org. Biomol. Chem. 10, 3066–3070 (2012).

    Google Scholar 

  138. 138.

    McKay, C. S., Moran, J. & Pezacki, J. P. Nitrones as dipoles for rapid strain-promoted 1,3-dipolar cycloadditions with cyclooctynes. ChemComm 46, 931–933 (2010). This article presents the first description of strain-promoted bioorthogonal reactions of nitrones with strained cycloalkynes.

    Google Scholar 

  139. 139.

    Sherratt, A. R. et al. Dual strain-promoted alkyne–nitrone cycloadditions for simultaneous labeling of bacterial peptidoglycans. Bioconjugate Chem. 27, 1222–1226 (2016).

    Google Scholar 

  140. 140.

    Gutsmiedl, K., Wirges, C. T., Ehmke, V. & Carell, T. Copper-free “click” modification of DNA via nitrile oxide–norbornene 1,3-dipolar cycloaddition. Org. Lett. 11, 2405–2408 (2009).

    Google Scholar 

  141. 141.

    McGrath, N. A. & Raines, R. T. Diazo compounds as highly tunable reactants in 1,3-dipolar cycloaddition reactions with cycloalkynes. Chem. Sci. 3, 3237–3240 (2012).

    Google Scholar 

  142. 142.

    Moran, J., McKay, C. S. & Pezacki, J. P. Strain-promoted 1,3-dipolar cycloadditions of diazo compounds with cyclooctynes. Can. J. Chem. 89, 148–151 (2011).

    Google Scholar 

  143. 143.

    Sanders, B. C. et al. Metal-free sequential [3 + 2]-dipolar cycloadditions using cyclooctynes and 1,3-dipoles of different reactivity. J. Am. Chem. Soc. 133, 949–957 (2011).

    Google Scholar 

  144. 144.

    Bernard, S. et al. Bioorthogonal click and release reaction of iminosydnones with cycloalkynes. Angew. Chem. Int. Ed. 56, 15612–15616 (2017).

    Google Scholar 

  145. 145.

    Richard, M. et al. New fluorine-18 pretargeting PET imaging by bioorthogonal chlorosydnone–cycloalkyne click reaction. ChemComm 55, 10400–10403 (2019).

    Google Scholar 

  146. 146.

    Wallace, S. & Chin, J. W. Strain-promoted sydnone bicyclo-[6.1.0]-nonyne cycloaddition. Chem. Sci. 5, 1742–1744 (2014).

    Google Scholar 

  147. 147.

    Sletten, E. M. & Bertozzi, C. R. A bioorthogonal quadricyclane ligation. J. Am. Chem. Soc. 133, 17570–17573 (2011).

    Google Scholar 

  148. 148.

    Carboni, R. A. & Lindsey, R. V. Reactions of tetrazines with unsaturated compounds. a new synthesis of pyridazines. J. Am. Chem. Soc. 81, 4342–4346 (1959).

    Google Scholar 

  149. 149.

    Zhang, J., Shukla, V. & Boger, D. L. Inverse electron demand Diels–Alder reactions of heterocyclic azadienes, 1-aza-1,3-butadienes, cyclopropenone ketals, and related systems. A retrospective. J. Org. Chem. 84, 9397–9445 (2019).

    Google Scholar 

  150. 150.

    Thalhammer, F., Wallfahrer, U. & Sauer, J. Reaktivität einfacher offenkettiger und cyclischer dienophile bei Diels–Alder-reaktionen mit inversem elektronenbedarf. Tetrahedron Lett. 31, 6851–6854 (1990).

    Google Scholar 

  151. 151.

    Blackman, M. L., Royzen, M. & Fox, J. M. Tetrazine ligation: fast bioconjugation based on inverse-electron-demand Diels–Alder reactivity. J. Am. Chem. Soc. 130, 13518–13518 (2008). This article presents an initial report of tetrazine ligation with TCO and the first example of very rapid kinetics in bioorthogonal chemistry (k2 > 103 M–1 s–1).

    Google Scholar 

  152. 152.

    Devaraj, N. K., Weissleder, R. & Hilderbrand, S. A. Tetrazine-based cycloadditions: application to pretargeted live cell imaging. Bioconjugate Chem. 19, 2297–2299 (2008). This article presents an initial report of tetrazine ligation with norbornene and the first example of tetrazine ligation in a live-cell application.

    Google Scholar 

  153. 153.

    Pipkorn, R. et al. Inverse-electron-demand Diels–Alder reaction as a highly efficient chemoselective ligation procedure: synthesis and function of a BioShuttle for temozolomide transport into prostate cancer cells. J. Pept. Sci. 15, 235–241 (2009).

    Google Scholar 

  154. 154.

    Mao, W. et al. Organocatalytic and scalable syntheses of unsymmetrical 1,2,4,5-tetrazines by thiol-containing promotors. Angew. Chem. Int. Ed. 58, 1106–1109 (2019).

    Google Scholar 

  155. 155.

    Qu, Y., Sauvage, F.-X., Clavier, G., Miomandre, F. & Audebert, P. Metal-free synthetic approach to 3-monosubstituted unsymmetrical 1,2,4,5-tetrazines useful for bioorthogonal reactions. Angew. Chem. Int. Ed. 57, 12057–12061 (2018).

    Google Scholar 

  156. 156.

    Yang, J., Karver, M. R., Li, W., Sahu, S. & Devaraj, N. K. Metal-catalyzed one-pot synthesis of tetrazines directly from aliphatic nitriles and hydrazine. Angew. Chem. Int. Ed. 51, 5222–5225 (2012).

    Google Scholar 

  157. 157.

    Lambert, W. D. et al. Installation of minimal tetrazines through silver-mediated Liebeskind–Srogl coupling with arylboronic acids. J. Am. Chem. Soc. 141, 17068–17074 (2019).

    Google Scholar 

  158. 158.

    Wu, H., Yang, J., Šečkutė, J. & Devaraj, N. K. In Situ synthesis of alkenyl tetrazines for highly fluorogenic bioorthogonal live-cell imaging probes. Angew. Chem. 126, 5915–5919 (2014).

    Google Scholar 

  159. 159.

    Xie, Y. et al. Divergent synthesis of monosubstituted and unsymmetrical 3,6-disubstituted tetrazines from carboxylic ester precursors. Angew. Chem. Int. Ed. 59, 16967–16973 (2020).

    Google Scholar 

  160. 160.

    Pigga, J. E. & Fox, J. M. Flow photochemical syntheses of trans-cyclooctenes and trans-cycloheptenes driven by metal complexation. Isr. J. Chem. 60, 207–218 (2020).

    Google Scholar 

  161. 161.

    Royzen, M., Yap, G. P. A. & Fox, J. M. A photochemical synthesis of functionalized trans-cyclooctenes driven by metal complexation. J. Am. Chem. Soc. 130, 3760–3761 (2008).

    Google Scholar 

  162. 162.

    Darko, A. et al. Conformationally strained trans-cyclooctene with improved stability and excellent reactivity in tetrazine ligation. Chem. Sci. 5, 3770–3776 (2014). This report describes ultrarapid bioorthogonal reactions with rates as fast as k2 = 3.3 × 106 M–1 s–1.

    Google Scholar 

  163. 163.

    Taylor, M. T., Blackman, M. L., Dmitrenko, O. & Fox, J. M. Design and synthesis of highly reactive dienophiles for the tetrazine–trans-cyclooctene ligation. J. Am. Chem. Soc. 133, 9646–9649 (2011).

    Google Scholar 

  164. 164.

    Lang, K. et al. Genetic encoding of bicyclononynes and trans-cyclooctenes for site-specific protein labeling in vitro and in live mammalian cells via rapid fluorogenic Diels–Alder reactions. J. Am. Chem. Soc. 134, 10317–10320 (2012). This article presents the first example of site-specific protein labelling via IEDDA in living cells with rapidly reacting cyclooctyne and TCO dienophiles.

    Google Scholar 

  165. 165.

    Patterson, D. M., Nazarova, L. A., Xie, B., Kamber, D. N. & Prescher, J. A. Functionalized cyclopropenes as bioorthogonal chemical reporters. J. Am. Chem. Soc. 134, 18638–18643 (2012).

    Google Scholar 

  166. 166.

    Ramil, C. P. et al. Spirohexene–tetrazine ligation enables bioorthogonal labeling of class B G protein-coupled receptors in live cells. J. Am. Chem. Soc. 139, 13376–13386 (2017).

    Google Scholar 

  167. 167.

    Niederwieser, A. et al. Two-color glycan labeling of live cells by a combination of Diels–Alder and click chemistry. Angew. Chem. Int. Ed. 52, 4265–4268 (2013).

    Google Scholar 

  168. 168.

    Rieder, U. & Luedtke, N. W. Alkene–tetrazine ligation for imaging cellular. DNA. Angew. Chem. Int. Ed. 53, 9168–9172 (2014).

    Google Scholar 

  169. 169.

    Stöckmann, H., Neves, A. A., Stairs, S., Brindle, K. M. & Leeper, F. J. Exploring isonitrile-based click chemistry for ligation with biomolecules. Org. Biomol. Chem. 9, 7303–7305 (2011).

    Google Scholar 

  170. 170.

    Engelsma, S. B. et al. Acylazetine as a dienophile in bioorthogonal inverse electron-demand Diels–Alder ligation. Org. Lett. 16, 2744–2747 (2014).

    Google Scholar 

  171. 171.

    Liu, K. et al. A genetically encoded cyclobutene probe for labelling of live cells. ChemComm 53, 10604–10607 (2017).

    Google Scholar 

  172. 172.

    Kamber, D. N. et al. 1,2,4-Triazines are versatile bioorthogonal reagents. J. Am. Chem. Soc. 137, 8388–8391 (2015).

    Google Scholar 

  173. 173.

    Carlson, J. C. T., Meimetis, L. G., Hilderbrand, S. A. & Weissleder, R. BODIPY–tetrazine derivatives as superbright bioorthogonal turn-on probes. Angew. Chem. Int. Ed. 52, 6917–6920 (2013).

    Google Scholar 

  174. 174.

    Devaraj, N. K., Hilderbrand, S., Upadhyay, R., Mazitschek, R. & Weissleder, R. Bioorthogonal turn-on probes for imaging small molecules inside living cells. Angew. Chem. Int. Ed. 49, 2869–2872 (2010). This article presents an initial description of fluorogenic tetrazine–dye conjugates for live-cell imaging applications.

    Google Scholar 

  175. 175.

    Meimetis, L. G., Carlson, J. C., Giedt, R. J., Kohler, R. H. & Weissleder, R. Ultrafluorogenic coumarin-tetrazine probes for real-time biological imaging. Angew. Chem. Int. Ed. 53, 7531–7534 (2014).

    Google Scholar 

  176. 176.

    Beliu, G. et al. Bioorthogonal labeling with tetrazine-dyes for super-resolution microscopy. Commun. Biol. 2, 261 (2019).

    ADS  Google Scholar 

  177. 177.

    Werther, P. et al. Live-cell localization microscopy with a fluorogenic and self-blinking tetrazine probe. Angew. Chem. Int. Ed. 59, 804–810 (2020).

    Google Scholar 

  178. 178.

    Ehret, F., Wu, H., Alexander, S. C. & Devaraj, N. K. Electrochemical control of rapid bioorthogonal tetrazine ligations for selective functionalization of microelectrodes. J. Am. Chem. Soc. 137, 8876–8879 (2015).

    Google Scholar 

  179. 179.

    Mayer, S. V., Murnauer, A., von Wrisberg, M.-K., Jokisch, M.-L. & Lang, K. Photo-induced and rapid labeling of tetrazine-bearing proteins via cyclopropenone-caged bicyclononynes. Angew. Chem. Int. Ed. 58, 15876–15882 (2019).

    Google Scholar 

  180. 180.

    Zhang, H. et al. Rapid bioorthogonal chemistry turn-on through enzymatic or long wavelength photocatalytic activation of tetrazine ligation. J. Am. Chem. Soc. 138, 5978–5983 (2016).

    Google Scholar 

  181. 181.

    Lim, R. K. & Lin, Q. Photoinducible bioorthogonal chemistry: a spatiotemporally controllable tool to visualize and perturb proteins in live cells. Acc. Chem. Res. 44, 828–839 (2011).

    Google Scholar 

  182. 182.

    Clovis, J. S., Eckell, A., Huisgen, R. & Sustmann, R. 1.3-Dipolare cycloadditionen, XXV. der nachweis des freien diphenylnitrilimins als zwischenstufe bei cycloadditionen. Chem. Ber. 100, 60–70 (1967).

    Google Scholar 

  183. 183.

    Wang, Y., Rivera Vera, C. I. & Lin, Q. Convenient synthesis of highly functionalized pyrazolines via mild, photoactivated 1,3-dipolar cycloaddition. Org. Lett. 9, 4155–4158 (2007).

    Google Scholar 

  184. 184.

    Song, W., Wang, Y., Qu, J., Madden, M. M. & Lin, Q. A photoinducible 1,3-dipolar cycloaddition reaction for rapid, selective modification of tetrazole-containing proteins. Angew. Chem. Int. Ed. 47, 2832–2835 (2008). This article presents the first report of tetrazole-based photoclick chemistry as a new bioorthogonal reaction for biological applications.

    Google Scholar 

  185. 185.

    Song, W., Wang, Y., Qu, J. & Lin, Q. Selective functionalization of a genetically encoded alkene-containing protein via “photoclick chemistry” in bacterial cells. J. Am. Chem. Soc. 130, 9654–9655 (2008).

    Google Scholar 

  186. 186.

    Kumar, G. S. & Lin, Q. Light-triggered click chemistry. Chem. Rev. https://doi.org/10.1021/acs.chemrev.0c00799 (2020).

    Article  Google Scholar 

  187. 187.

    Wang, Y., Song, W., Hu, W. J. & Lin, Q. Fast alkene functionalization in vivo by photoclick chemistry: HOMO lifting of nitrile imine dipoles. Angew. Chem. Int. Ed. 48, 5330–5333 (2009).

    Google Scholar 

  188. 188.

    Lee, Y. J. et al. A genetically encoded acrylamide functionality. ACS Chem. Biol. 8, 1664–1670 (2013).

    Google Scholar 

  189. 189.

    Wang, J. et al. A biosynthetic route to photoclick chemistry on proteins. J. Am. Chem. Soc. 132, 14812–14818 (2010).

    Google Scholar 

  190. 190.

    Kamber, D. N. et al. Isomeric cyclopropenes exhibit unique bioorthogonal reactivities. J. Am. Chem. Soc. 135, 13680–13683 (2013).

    Google Scholar 

  191. 191.

    An, P., Lewandowski, T. M., Erbay, T. G., Liu, P. & Lin, Q. Sterically shielded, stabilized nitrile imine for rapid bioorthogonal protein labeling in live cells. J. Am. Chem. Soc. 140, 4860–4868 (2018). This report describes the exploitation of the steric shielding effect to improve bioorthogonality of the tetrazole photoclick chemistry in cellular systems.

    Google Scholar 

  192. 192.

    An, P., Yu, Z. & Lin, Q. Design and synthesis of laser-activatable tetrazoles for a fast and fluorogenic red-emitting 1,3-dipolar cycloaddition reaction. Org. Lett. 15, 5496–5499 (2013).

    Google Scholar 

  193. 193.

    Yu, Z., Ohulchanskyy, T. Y., An, P., Prasad, P. N. & Lin, Q. Fluorogenic, two-photon-triggered photoclick chemistry in live mammalian cells. J. Am. Chem. Soc. 135, 16766–16769 (2013).

    Google Scholar 

  194. 194.

    Lederhose, P. et al. Near-infrared photoinduced coupling reactions assisted by upconversion nanoparticles. Angew. Chem. Int. Ed. 55, 12195–12199 (2016).

    Google Scholar 

  195. 195.

    Yu, Z., Pan, Y., Wang, Z., Wang, J. & Lin, Q. Genetically encoded cyclopropene directs rapid, photoclick-chemistry-mediated protein labeling in mammalian cells. Angew. Chem. Int. Ed. 51, 10600–10604 (2012).

    Google Scholar 

  196. 196.

    Yu, Z. & Lin, Q. Design of spiro[2.3]hex-1-ene, a genetically encodable double-strained alkene for superfast photoclick chemistry. J. Am. Chem. Soc. 136, 4153–4156 (2014). This report describes an unprecedented double-strained alkene for accelerated tetrazole photoclick chemistry and the effect of chloride ion on reaction kinetics.

    Google Scholar 

  197. 197.

    An, P., Wu, H. Y., Lewandowski, T. M. & Lin, Q. Hydrophilic azaspiroalkenes as robust bioorthogonal reporters. ChemComm 54, 14005–14008 (2018).

    Google Scholar 

  198. 198.

    Arndt, S. & Wagenknecht, H. A. “Photoclick” postsynthetic modification of DNA. Angew. Chem. Int. Ed. 53, 14580–14582 (2014).

    Google Scholar 

  199. 199.

    Holstein, J. M., Stummer, D. & Rentmeister, A. Enzymatic modification of 5′-capped RNA with a 4-vinylbenzyl group provides a platform for photo-click and inverse electron-demand Diels–Alder reaction. Chem. Sci. 6, 1362–1369 (2015).

    Google Scholar 

  200. 200.

    Schart, V. F. et al. Triple orthogonal labeling of glycans by applying photoclick chemistry. ChemBioChem 20, 166–171 (2019).

    Google Scholar 

  201. 201.

    Yu, Z., Ho, L. Y. & Lin, Q. Rapid, photoactivatable turn-on fluorescent probes based on an intramolecular photoclick reaction. J. Am. Chem. Soc. 133, 11912–11915 (2011).

    Google Scholar 

  202. 202.

    Kulkarni, R. A. et al. Photoinducible oncometabolite detection. ChemBioChem 20, 360–365 (2019).

    Google Scholar 

  203. 203.

    Poloukhtine, A. A., Mbua, N. E., Wolfert, M. A., Boons, G.-J. & Popik, V. V. Selective labeling of living cells by a photo-triggered click reaction. J. Am. Chem. Soc. 131, 15769–15776 (2009). This article describes the first use of dibenzocyclopropenones as the photo-masked precursor of dibenzycyclooctyne and the subsequent click reactions with azides.

    Google Scholar 

  204. 204.

    Orski, S. V. et al. High density orthogonal surface immobilization via photoactivated copper-free click chemistry. J. Am. Chem. Soc. 132, 11024–11026 (2010).

    Google Scholar 

  205. 205.

    Jiang, T. et al. Modular enzyme- and light-based activation of cyclopropene–tetrazine ligation. ChemBioChem 20, 2222–2226 (2019).

    Google Scholar 

  206. 206.

    Arumugam, S. & Popik, V. V. Light-induced hetero-Diels–Alder cycloaddition: a facile and selective photoclick reaction. J. Am. Chem. Soc. 133, 5573–5579 (2011).

    Google Scholar 

  207. 207.

    Feist, F., Menzel, J. P., Weil, T., Blinco, J. P. & Barner-Kowollik, C. Visible light-induced ligation via o-quinodimethane thioethers. J. Am. Chem. Soc. 140, 11848–11854 (2018).

    Google Scholar 

  208. 208.

    Li, J. et al. Visible light-initiated bioorthogonal photoclick cycloaddition. J. Am. Chem. Soc. 140, 14542–14546 (2018).

    Google Scholar 

  209. 209.

    Zhang, L. et al. Discovery of fluorogenic diarylsydnone–alkene photoligation: conversion of ortho-dual-twisted diarylsydnones into planar pyrazolines. J. Am. Chem. Soc. 140, 7390–7394 (2018).

    Google Scholar 

  210. 210.

    Ojida, A., Tsutsumi, H., Kasagi, N. & Hamachi, I. Suzuki coupling for protein modification. Tetrahedron Lett. 46, 3301–3305 (2005).

    Google Scholar 

  211. 211.

    Kodama, K. et al. Regioselective carbon–carbon bond formation in proteins with palladium catalysis; new protein chemistry by organometallic chemistry. ChemBioChem 7, 134–139 (2006).

    Google Scholar 

  212. 212.

    Kodama, K. et al. Site-specific functionalization of proteins by organopalladium reactions. ChemBioChem 8, 232–238 (2007).

    Google Scholar 

  213. 213.

    Brustad, E. et al. A genetically encoded boronate-containing amino acid. Angew. Chem. Int. Ed. 47, 8220–8223 (2008).

    Google Scholar 

  214. 214.

    Jbara, M., Maity, S. K. & Brik, A. Palladium in the chemical synthesis and modification of proteins. Angew. Chem. Int. Ed. 56, 10644–10655 (2017).

    Google Scholar 

  215. 215.

    Isenegger, P. G. & Davis, B. G. Concepts of catalysis in site-selective protein modifications. J. Am. Chem. Soc. 141, 8005–8013 (2019).

    Google Scholar 

  216. 216.

    Chalker, J. M., Wood, C. S. & Davis, B. G. A convenient catalyst for aqueous and protein Suzuki–Miyaura cross-coupling. J. Am. Chem. Soc. 131, 16346–16347 (2009). This article describes the first example of palladium-catalysed cross-coupling reactions for selective protein modification.

    Google Scholar 

  217. 217.

    Li, N., Lim, R. K., Edwardraja, S. & Lin, Q. Copper-free Sonogashira cross-coupling for functionalization of alkyne-encoded proteins in aqueous medium and in bacterial cells. J. Am. Chem. Soc. 133, 15316–15319 (2011).

    Google Scholar 

  218. 218.

    Simmons, R. L., Yu, R. T. & Myers, A. G. Storable arylpalladium(II) reagents for alkene labelling in aqueous media. J. Am. Chem. Soc. 133, 15870–15873 (2011).

    Google Scholar 

  219. 219.

    Yusop, R. M., Unciti-Broceta, A., Johansson, E. M. V., Sánchez-Martín, R. M. & Bradley, M. Palladium-mediated intracellular chemistry. Nat. Chem. 3, 239–243 (2011). This article presents the first report of the effective use of palladium-based uncaging inside cells.

    Google Scholar 

  220. 220.

    Ma, X., Wang, H. & Chen, W. N-Heterocyclic carbene-stabilized palladium complexes as organometallic catalysts for bioorthogonal cross-coupling reactions. J. Org. Chem. 79, 8652–8658 (2014).

    Google Scholar 

  221. 221.

    Li, N., Ramil, C. P., Lim, R. K. V. & Lin, Q. A genetically encoded alkyne directs palladium-mediated protein labelling on live mammalian cell surface. ACS Chem. Biol. 10, 379–384 (2015).

    Google Scholar 

  222. 222.

    Lim, R. K. V., Li, N., Ramil, C. P. & Lin, Q. Fast and sequence-specific palladium-mediated cross-coupling reaction identified from phage display. ACS Chem. Biol. 9, 2139–2148 (2014).

    Google Scholar 

  223. 223.

    Cheng, G., Lim, R. K. V., Li, N. & Lin, Q. Storable palladacycles for selective functionalization of alkyne-containing proteins. Chem. Commun. 49, 6809–6811 (2013).

    Google Scholar 

  224. 224.

    Dumas, A. et al. Self-liganded Suzuki–Miyaura coupling for site-selective protein PEGylation. Angew. Chem. Int. Ed. 52, 3916–3921 (2013).

    Google Scholar 

  225. 225.

    Spicer, C. D., Triemer, T. & Davis, B. G. Palladium-mediated cell-surface labelling. J. Am. Chem. Soc. 134, 800–803 (2012).

    Google Scholar 

  226. 226.

    Lercher, L., McGouran, J. F., Kessler, B. M., Schofield, C. J. & Davis, B. G. DNA modification under mild conditions by Suzuki–Miyaura cross-coupling for the generation of functional probes. Angew. Chem. Int. Ed. 52, 10553–10558 (2013).

    Google Scholar 

  227. 227.

    Spicer, C. D. & Davis, B. G. Palladium-mediated site-selective Suzuki–Miyaura protein modification at genetically encoded aryl halides. Chem. Commun. 47, 1698–1700 (2011).

    Google Scholar 

  228. 228.

    Lin, Y. A., Chalker, J. M. & Davis, B. G. Olefin cross-metathesis on proteins: investigation of allylic chalcogen effects and guiding principles in metathesis partner selection. J. Am. Chem. Soc. 132, 16805–16811 (2010).

    Google Scholar 

  229. 229.

    Lin, Y. A., Chalker, J. M., Floyd, N., Bernardes, G. J. L. & Davis, B. G. Allyl sulfides are privileged substrates in aqueous cross-metathesis: application to site-selective protein modification. J. Am. Chem. Soc. 130, 9642–9643 (2008). This article describes the first example of selective ruthenium-mediated cross-metathesis of olefins with protein substrates.

    Google Scholar 

  230. 230.

    Chalker, J. M., Lin, Y. A., Boutureira, O. & Davis, B. G. Enabling olefin metathesis on proteins: chemical methods for installation of S-allyl cysteine. Chem. Commun. 25, 3714–3716 (2009).

    Google Scholar 

  231. 231.

    Lin, Y. A. et al. Rapid cross-metathesis for reversible protein modifications via chemical access to Se-allyl-selenocysteine in proteins. J. Am. Chem. Soc. 135, 12156–12159 (2013).

    Google Scholar 

  232. 232.

    Bhushan, B. et al. Genetic incorporation of olefin cross-metathesis reaction tags for protein modification. J. Am. Chem. Soc. 140, 14599–14603 (2018).

    Google Scholar 

  233. 233.

    Hruby, V. J., Boteju, L. & Li, G. in Chemical & Engineering News Vol. 71, 2 (American Chemical Society, Safety Letters, 1993).

  234. 234.

    Niemeier, J. K. & Kjell, D. P. Hydrazine and aqueous hydrazine solutions: evaluating safety in chemical processes. Org. Process. Res. Dev. 17, 1580–1590 (2013).

    Google Scholar 

  235. 235.

    Richardson, M. B. et al. Synthesis and explosion hazards of 4-azido-l-phenylalanine. J. Org. Chem. 83, 4525–4536 (2018).

    Google Scholar 

  236. 236.

    Sperry, J. B. et al. Thermal stability assessment of peptide coupling reagents commonly used in pharmaceutical manufacturing. Org. Process. Res. Dev. 22, 1262–1275 (2018). This study provides an excellent overview of the different techniques for analysing the energetics of materials.

    Google Scholar 

  237. 237.

    Green, S. P. et al. On the use of differential scanning calorimetry for thermal hazard assessment of new chemistry: avoiding explosive mistakes. Angew. Chem. Int. Ed. 59, 15798–15802 (2020).

    Google Scholar 

  238. 238.

    Gordon, A. J. & Ford, R. A. The Chemist’s Companion: A Handbook of Practical Data, Techniques, and References (Wiley, 1972).

  239. 239.

    Grammel, M. & Hang, H. C. Chemical reporters for biological discovery. Nat. Chem. Biol. 9, 475–484 (2013).

    Google Scholar 

  240. 240.

    Parker, C. G. & Pratt, M. R. Click chemistry in proteomic investigations. Cell 180, 605–632 (2020).

    Google Scholar 

  241. 241.

    Madl, C. M. & Heilshorn, S. C. Bioorthogonal strategies for engineering extracellular matrices. Adv. Funct. Mater. 28, 1706046 (2018).

    Google Scholar 

  242. 242.

    Tu, J., Xu, M. & Franzini, R. M. Dissociative bioorthogonal reactions. ChemBioChem 20, 1615–1627 (2019).

    Google Scholar 

  243. 243.

    Lim, R. K. V. & Lin, Q. Bioorthogonal chemistry: recent progress and future directions. ChemComm 46, 1589–1600 (2010).

    Google Scholar 

  244. 244.

    Sletten, E. M. & Bertozzi, C. R. Bioorthogonal chemistry: fishing for selectivity in a sea of functionality. Angew. Chem. Int. Ed. 48, 6974–6998 (2009).

    Google Scholar 

  245. 245.

    McKay, C. S. & Finn, M. G. Click chemistry in complex mixtures: bioorthogonal bioconjugation. Chem. Biol. 21, 1075–1101 (2014).

    Google Scholar 

  246. 246.

    Laughlin, S. T., Baskin, J. M., Amacher, S. L. & Bertozzi, C. R. In vivo imaging of membrane-associated glycans in developing zebrafish. Science 320, 664–667 (2008).

    ADS  Google Scholar 

  247. 247.

    Agarwal, P., Beahm, B. J., Shieh, P. & Bertozzi, C. R. Systemic fluorescence imaging of zebrafish glycans with bioorthogonal chemistry. Angew. Chem. Int. Ed. 54, 11504–11510 (2015).

    Google Scholar 

  248. 248.

    Salic, A. & Mitchison, T. J. A chemical method for fast and sensitive detection of DNA synthesis in vivo. Proc. Natl Acad. Sci. USA 105, 2415–2420 (2008).

    ADS  Google Scholar 

  249. 249.

    Nainar, S. et al. Metabolic incorporation of azide functionality into cellular RNA. ChemBioChem 17, 2149–2152 (2016).

    Google Scholar 

  250. 250.

    Alvarez-Castelao, B. et al. Cell-type-specific metabolic labelling of nascent proteomes in vivo. Nat. Biotechnol. 35, 1196–1201 (2017).

    Google Scholar 

  251. 251.

    Yuet, K. P. et al. Cell-specific proteomic analysis in Caenorhabditis elegans. Proc. Natl Acad. Sci. USA 112, 2705–2710 (2015).

    ADS  Google Scholar 

  252. 252.

    Chang, P. V., Prescher, J. A., Hangauer, M. J. & Bertozzi, C. R. Imaging cell surface glycans with bioorthogonal chemical reporters. J. Am. Chem. Soc. 129, 8400–8401 (2007).

    Google Scholar 

  253. 253.

    Liang, D. et al. A real-time, click chemistry imaging approach reveals stimulus-specific subcellular locations of phospholipase D activity. Proc. Natl Acad. Sci. USA 116, 15453–15462 (2019).

    Google Scholar 

  254. 254.

    Jiang, H., Kim, J. H., Frizzell, K. M., Kraus, W. L. & Lin, H. Clickable NAD analogues for labeling substrate proteins of poly(ADP-ribose) polymerases. J. Am. Chem. Soc. 132, 9363–9372 (2010).

    Google Scholar 

  255. 255.

    Cañeque, T., Müller, S. & Rodriguez, R. Visualizing biologically active small molecules in cells using click chemistry. Nat. Rev. Chem. 2, 202–215 (2018).

    Google Scholar 

  256. 256.

    Tian, Y. & Lin, Q. Fitness factors for bioorthogonal chemical probes. ACS Chem. Biol. 14, 2489–2496 (2019).

    Google Scholar 

  257. 257.

    Patterson, D. M., Nazarova, L. A. & Prescher, J. A. Finding the right (bioorthogonal) chemistry. ACS Chem. Biol. 9, 592–605 (2014).

    Google Scholar 

  258. 258.

    Kim, E. J. Chemical reporters and their bioorthogonal reactions for labeling protein O-GlcNAcylation. Molecules 23, 2411 (2018).

    Google Scholar 

  259. 259.

    Thirumurugan, P., Matosiuk, D. & Jozwiak, K. Click chemistry for drug development and diverse chemical–biology applications. Chem. Rev. 113, 4905–4979 (2013).

    Google Scholar 

  260. 260.

    Zhang, X. & Zhang, Y. Applications of azide-based bioorthogonal click chemistry in glycobiology. Molecules 18, 7145–7159 (2013).

    Google Scholar 

  261. 261.

    Mahal, L. K., Yarema, K. J. & Bertozzi, C. R. Engineering chemical reactivity on cell surfaces through oligosaccharide biosynthesis. Science 276, 1125–1128 (1997).

    Google Scholar 

  262. 262.

    Cole, C. M., Yang, J., Šečkutė, J. & Devaraj, N. K. Fluorescent live-cell imaging of metabolically incorporated unnatural cyclopropene-mannosamine derivatives. ChemBioChem 14, 205–208 (2013).

    Google Scholar 

  263. 263.

    Nguyen, K. et al. Cell-selective bioorthogonal metabolic labeling of RNA. J. Am. Chem. Soc. 139, 2148–2151 (2017).

    Google Scholar 

  264. 264.

    Chang, P. V., Dube, D. H., Sletten, E. M. & Bertozzi, C. R. A strategy for the selective imaging of glycans using caged metabolic precursors. J. Am. Chem. Soc. 132, 9516–9518 (2010).

    Google Scholar 

  265. 265.

    Xie, R., Hong, S. & Chen, X. Cell-selective metabolic labeling of biomolecules with bioorthogonal functionalities. Curr. Opin. Chem. Biol. 17, 747–752 (2013).

    Google Scholar 

  266. 266.

    Wang, H. et al. Selective in vivo metabolic cell-labeling-mediated cancer targeting. Nat. Chem. Biol. 13, 415–424 (2017).

    Google Scholar 

  267. 267.

    Ngo, J. T. et al. Cell-selective metabolic labeling of proteins. Nat. Chem. Biol. 5, 715–717 (2009).

    Google Scholar 

  268. 268.

    Debets, M. F. et al. Metabolic precision labeling enables selective probing of O-linked N-acetylgalactosamine glycosylation. Proc. Natl Acad. Sci. USA 117, 25293–25301 (2020).

    Google Scholar 

  269. 269.

    Islam, K. et al. Defining efficient enzyme–cofactor pairs for bioorthogonal profiling of protein methylation. Proc. Natl Acad. Sci. USA 110, 16778–16783 (2013).

    ADS  Google Scholar 

  270. 270.

    Stone, S. E., Glenn, W. S., Hamblin, G. D. & Tirrell, D. A. Cell-selective proteomics for biological discovery. Curr. Opin. Chem. Biol. 36, 50–57 (2017).

    Google Scholar 

  271. 271.

    Lang, K. & Chin, J. W. Cellular incorporation of unnatural amino acids and bioorthogonal labeling of proteins. Chem. Rev. 114, 4764–4806 (2014).

    Google Scholar 

  272. 272.

    Young, D. D. & Schultz, P. G. Playing with the molecules of life. ACS Chem. Biol. 13, 854–870 (2018).

    Google Scholar 

  273. 273.

    Lee, K. J., Kang, D. & Park, H. S. Site-specific labeling of proteins using unnatural amino acids. Mol. Cell 42, 386–396 (2019).

    Google Scholar 

  274. 274.

    Mayer, S. & Lang, K. Tetrazines in inverse-electron-demand Diels–Alder cycloadditions and their use in biology. Synthesis 49, 830–848 (2017).

    Google Scholar 

  275. 275.

    Lang, K. et al. Genetically encoded norbornene directs site-specific cellular protein labelling via a rapid bioorthogonal reaction. Nat. Chem. 4, 298 (2012). This article present the first example of site-specific protein labelling via IEDDA in living cells.

    Google Scholar 

  276. 276.

    Plass, T. et al. Amino acids for Diels–Alder reactions in living cells. Angew. Chem. Int. Ed. 51, 4166–4170 (2012).

    Google Scholar 

  277. 277.

    Jang, H. S., Jana, S., Blizzard, R. J., Meeuwsen, J. C. & Mehl, R. A. Access to faster eukaryotic cell labeling with encoded tetrazine amino acids. J. Am. Chem. Soc. 142, 7245–7249 (2020).

    Google Scholar 

  278. 278.

    Blizzard, R. J. et al. Ideal bioorthogonal reactions using a site-specifically encoded tetrazine amino acid. J. Am. Chem. Soc. 137, 10044–10047 (2015).

    Google Scholar 

  279. 279.

    Nikić, I. et al. Minimal tags for rapid dual-color live-cell labeling and super-resolution microscopy. Angew. Chem. Int. Ed. 53, 2245–2249 (2014).

    Google Scholar 

  280. 280.

    Peng, T. & Hang, H. C. Site-specific bioorthogonal labeling for fluorescence imaging of intracellular proteins in living cells. J. Am. Chem. Soc. 138, 14423–14433 (2016).

    Google Scholar 

  281. 281.

    Lahann, J. (ed.) Click Chemistry for Biotechnology and Materials Science 411 (Wiley, 2009).

  282. 282.

    Mideksa, Y. G. et al. Site-specific protein labeling with fluorophores as a tool to monitor protein turnover. ChemBioChem 21, 1861–1867 (2020).

    Google Scholar 

  283. 283.

    Sachdeva, A., Wang, K., Elliott, T. & Chin, J. W. Concerted, rapid, quantitative, and site-specific dual labeling of proteins. J. Am. Chem. Soc. 136, 7785–7788 (2014).

    Google Scholar 

  284. 284.

    Wang, K. et al. Optimized orthogonal translation of unnatural amino acids enables spontaneous protein double-labelling and FRET. Nat. Chem. 6, 393–403 (2014).

    Google Scholar 

  285. 285.

    Tsai, Y.-H., Essig, S., James, J. R., Lang, K. & Chin, J. W. Selective, rapid and optically switchable regulation of protein function in live mammalian cells. Nat. Chem. 7, 554–561 (2015).

    Google Scholar 

  286. 286.

    Liang, H. et al. Metabolic labelling of the carbohydrate core in bacterial peptidoglycan and its applications. Nat. Commun. 8, 15015 (2017).

    ADS  Google Scholar 

  287. 287.

    Laughlin, S. T. & Bertozzi, C. R. In vivo imaging of Caenorhabditis elegans glycans. ACS Chem. Biol. 4, 1068–1072 (2009).

    Google Scholar 

  288. 288.

    Nikić, I. et al. Debugging eukaryotic genetic code expansion for site-specific click-PAINT super-resolution microscopy. Angew. Chem. Int. Ed. 55, 16172–16176 (2016).

    Google Scholar 

  289. 289.

    Uttamapinant, C. et al. Genetic code expansion enables live-cell and super-resolution imaging of site-specifically labeled cellular proteins. J. Am. Chem. Soc. 137, 4602–4605 (2015).

    Google Scholar 

  290. 290.

    Elliott, T. S., Bianco, A., Townsley, F. M., Fried, S. D. & Chin, J. W. Tagging and enriching proteins enables cell-specific proteomics. Cell Chem. Biol. 23, 805–815 (2016).

    Google Scholar 

  291. 291.

    Elliott, T. S. et al. Proteome labeling and protein identification in specific tissues and at specific developmental stages in an animal. Nat. Biotechnol. 32, 465–472 (2014).

    Google Scholar 

  292. 292.

    Krogager, T. P. et al. Labeling and identifying cell-specific proteomes in the mouse brain. Nat. Biotechnol. 36, 156–159 (2018).

    Google Scholar 

  293. 293.

    Shieh, P., Hangauer, M. J. & Bertozzi, C. R. Fluorogenic azidofluoresceins for biological imaging. J. Am. Chem. Soc. 134, 17428–17431 (2012).

    Google Scholar 

  294. 294.

    Mao, W. et al. A general strategy to design highly fluorogenic far-red and near-infrared tetrazine bioorthogonal probes. Angew. Chem. Int. Ed. 60, 2393–2397 (2021).

    Google Scholar 

  295. 295.

    Shieh, P. et al. CalFluors: a universal motif for fluorogenic azide probes across the visible spectrum. J. Am. Chem. Soc. 137, 7145–7151 (2015).

    Google Scholar 

  296. 296.

    Ngo, J. T. et al. Click-EM for imaging metabolically tagged nonprotein biomolecules. Nat. Chem. Biol. 12, 459–465 (2016).

    Google Scholar 

  297. 297.

    Hong, S., Lin, L., Xiao, M. & Chen, X. Live-cell bioorthogonal Raman imaging. Curr. Opin. Chem. Biol. 24, 91–96 (2015).

    Google Scholar 

  298. 298.

    Bunnage, M. E., Chekler, E. L. P. & Jones, L. H. Target validation using chemical probes. Nat. Chem. Biol. 9, 195–199 (2013).

    Google Scholar 

  299. 299.

    Cravatt, B. F., Wright, A. T. & Kozarich, J. W. Activity-based protein profiling: from enzyme chemistry to proteomic chemistry. Annu. Rev. Biochem. 77, 383–414 (2008).

    Google Scholar 

  300. 300.

    Cravatt, B. F., Hsu, K.-L. & Weerapana, E. Activity-Based Protein Profiling Vol. 420 (Springer, 2019).

  301. 301.

    Geurink, P. P., Prely, L. M., van der Marel, G. A., Bischoff, R. & Overkleeft, H. S. in Activity-Based Protein Profiling 85–113 (Springer, 2011). This book provides a summary of ABPP and photoaffinity labelling with relevant applications in natural product target discovery and microbial pathogenesis.

  302. 302.

    Jessani, N., Liu, Y., Humphrey, M. & Cravatt, B. F. Enzyme activity profiles of the secreted and membrane proteome that depict cancer cell invasiveness. Proc. Natl Acad. Sci. USA 99, 10335–10340 (2002).

    ADS  Google Scholar 

  303. 303.

    Nomura, D. K. et al. Monoacylglycerol lipase regulates a fatty acid network that promotes cancer pathogenesis. Cell 140, 49–61 (2010).

    Google Scholar 

  304. 304.

    Barglow, K. T. & Cravatt, B. F. Activity-based protein profiling for the functional annotation of enzymes. Nat. Methods 4, 822–827 (2007).

    Google Scholar 

  305. 305.

    Nomura, D. K., Dix, M. M. & Cravatt, B. F. Activity-based protein profiling for biochemical pathway discovery in cancer. Nat. Rev. Cancer 10, 630–638 (2010).

    Google Scholar 

  306. 306.

    Smith, E. & Collins, I. Photoaffinity labeling in target-and binding-site identification. Future Med. Chem. 7, 159–183 (2015).

    Google Scholar 

  307. 307.

    Preston, G. W. & Wilson, A. J. Photo-induced covalent cross-linking for the analysis of biomolecular interactions. Chem. Soc. Rev. 42, 3289–3301 (2013).

    Google Scholar 

  308. 308.

    Zuhl, A. M. et al. Chemoproteomic profiling reveals that cathepsin D off-target activity drives ocular toxicity of β-secretase inhibitors. Nat. Commun. 7, 1–14 (2016).

    Google Scholar 

  309. 309.

    Hur, J.-Y. et al. The innate immunity protein IFITM3 modulates γ-secretase in Alzheimer’s disease. Nature 586, 735–740 (2020).

    ADS  Google Scholar 

  310. 310.

    Speers, A. E., Adam, G. C. & Cravatt, B. F. Activity-based protein profiling in vivo using a copper (I)-catalyzed azide–alkyne [3 + 2] cycloaddition. J. Am. Chem. Soc. 125, 4686–4687 (2003).

    Google Scholar 

  311. 311.

    Speers, A. E. & Cravatt, B. F. Profiling enzyme activities in vivo using click chemistry methods. Chem. Biol. 11, 535–546 (2004).

    Google Scholar 

  312. 312.

    van der Linden, W. A. et al. Two-step bioorthogonal activity-based proteasome profiling using copper-free click reagents: a comparative study. Bioorg. Med. Chem. 20, 662–666 (2012).

    Google Scholar 

  313. 313.

    Jessani, N. & Cravatt, B. F. The development and application of methods for activity-based protein profiling. Curr. Opin. Chem. Biol. 8, 54–59 (2004).

    Google Scholar 

  314. 314.

    Van Esbroeck, A. C. et al. Activity-based protein profiling reveals off-target proteins of the FAAH inhibitor BIA 10-2474. Science 356, 1084–1087 (2017).

    ADS  Google Scholar 

  315. 315.

    Medina-Cleghorn, D., Heslin, A., Morris, P. J., Mulvihill, M. M. & Nomura, D. K. Multidimensional profiling platforms reveal metabolic dysregulation caused by organophosphorus pesticides. ACS Chem. Biol. 9, 423–432 (2014).

    Google Scholar 

  316. 316.

    Huang, Z. et al. Global portrait of protein targets of metabolites of the neurotoxic compound BIA 10–2474. ACS Chem. Biol. 14, 192–197 (2019).

    Google Scholar 

  317. 317.

    Weerapana, E., Speers, A. E. & Cravatt, B. F. Tandem orthogonal proteolysis-activity-based protein profiling (TOP-ABPP) — a general method for mapping sites of probe modification in proteomes. Nat. Protoc. 2, 1414 (2007).

    Google Scholar 

  318. 318.

    Weerapana, E. et al. Quantitative reactivity profiling predicts functional cysteines in proteomes. Nature 468, 790–795 (2010).

    ADS  Google Scholar 

  319. 319.

    Roberts, A. M., Ward, C. C. & Nomura, D. K. Activity-based protein profiling for mapping and pharmacologically interrogating proteome-wide ligandable hotspots. Curr. Opin. Biotechnol. 43, 25–33 (2017).

    Google Scholar 

  320. 320.

    Vinogradova, E. et al. An activity-guided map of electrophile–cysteine interactions in primary human immune cells. Cell https://doi.org/10.2139/ssrn.3476689 (2019).

    Article  Google Scholar 

  321. 321.

    Zhang, X., Crowley, V. M., Wucherpfennig, T. G., Dix, M. M. & Cravatt, B. F. Electrophilic PROTACs that degrade nuclear proteins by engaging DCAF16. Nat. Chem. Biol. 15, 737–746 (2019).

    Google Scholar 

  322. 322.

    Lambert, J. M. & Morris, C. Q. Antibody–drug conjugates (ADCs) for personalized treatment of solid tumors: a review. Adv. Ther. 34, 1015–1035 (2017).

    Google Scholar 

  323. 323.

    Agarwal, P. & Bertozzi, C. R. Site-specific antibody–drug conjugates: the nexus of bioorthogonal chemistry, protein engineering, and drug development. Bioconjugate Chem. 26, 176–192 (2015). This article describes key advancements in the ADC field with a focus on bioorthogonal chemistry and protein engineering.

    Google Scholar 

  324. 324.

    Azoulay, M., Tuffin, G., Sallem, W. & Florent, J.-C. A new drug-release method using the Staudinger ligation. Bioorg. Med. Chem. Lett. 16, 3147–3149 (2006).

    Google Scholar 

  325. 325.

    Brakel, R. V., Vulders, R. C., Bokdam, R. J., Grüll, H. & Robillard, M. S. A doxorubicin prodrug activated by the Staudinger reaction. Bioconjugate Chem. 19, 714–718 (2008).

    Google Scholar 

  326. 326.

    Matikonda, S. S. et al. Bioorthogonal prodrug activation driven by a strain-promoted 1,3-dipolar cycloaddition. Chem. Sci. 6, 1212–1218 (2015).

    Google Scholar 

  327. 327.

    Zheng, Y. et al. Enrichment-triggered prodrug activation demonstrated through mitochondria-targeted delivery of doxorubicin and carbon monoxide. Nat. Chem. 10, 787–794 (2018).

    Google Scholar 

  328. 328.

    Versteegen, R. M., Rossin, R., ten Hoeve, W., Janssen, H. M. & Robillard, M. S. Click to release: instantaneous doxorubicin elimination upon tetrazine ligation. Angew. Chem. Int. Ed. 52, 14112–14116 (2013). This article presents an initial description of bioorthogonal uncaging of small molecules.

    Google Scholar 

  329. 329.

    Wu, H., Alexander, S. C., Jin, S. & Devaraj, N. K. A bioorthogonal near-infrared fluorogenic probe for mRNA detection. J. Am. Chem. Soc. 138, 11429–11432 (2016).

    Google Scholar 

  330. 330.

    Neumann, K. et al. Tetrazine-responsive self-immolative linkers. ChemBioChem 18, 91–95 (2017).

    Google Scholar 

  331. 331.

    Jiménez-Moreno, E. et al. Vinyl ether/tetrazine pair for the traceless release of alcohols in cells. Angew. Chem. Int. Ed. 56, 243–247 (2017).

    Google Scholar 

  332. 332.

    Tu, J., Xu, M., Parvez, S., Peterson, R. T. & Franzini, R. M. Bioorthogonal removal of 3-isocyanopropyl groups enables the controlled release of fluorophores and drugs in vivo. J. Am. Chem. Soc. 140, 8410–8414 (2018).

    Google Scholar 

  333. 333.

    Xu, M., Galindo-Murillo, R., Cheatham, T. & Franzini, R. Dissociative reactions of benzonorbornadienes with tetrazines: scope of leaving groups and mechanistic insights. Org. Biomol. Chem. 15, 9855–9865 (2017).

    Google Scholar 

  334. 334.

    van Onzen, A. H. et al. Bioorthogonal tetrazine carbamate cleavage by highly reactive trans-cyclooctene. J. Am. Chem. Soc. 142, 10955–10963 (2019).

    Google Scholar 

  335. 335.

    Tu, J. et al. Isonitrile-responsive and bioorthogonally removable tetrazine protecting groups. Chem. Sci. 11, 169–179 (2020).

    Google Scholar 

  336. 336.

    Wang, Q. et al. A bioorthogonal system reveals antitumour immune function of pyroptosis. Nature 579, 421–426 (2020).

    ADS  Google Scholar 

  337. 337.

    Völker, T., Dempwolff, F., Graumann, P. L. & Meggers, E. Progress towards bioorthogonal catalysis with organometallic compounds. Angew. Chem. Int. Ed. 53, 10536–10540 (2014).

    Google Scholar 

  338. 338.

    Li, J. et al. Palladium-triggered deprotection chemistry for protein activation in living cells. Nat. Chem. 6, 352–361 (2014).

    Google Scholar 

  339. 339.

    Wang, X. et al. Copper-triggered bioorthogonal cleavage reactions for reversible protein and cell surface modifications. J. Am. Chem. Soc. 141, 17133–17141 (2019).

    Google Scholar 

  340. 340.

    Pérez-López, A. M. et al. Gold-triggered uncaging chemistry in living systems. Angew. Chem. Int. Ed. 56, 12548–12552 (2017).

    Google Scholar 

  341. 341.

    Weiss, J. T. et al. Extracellular palladium-catalysed dealkylation of 5-fluoro-1-propargyl-uracil as a bioorthogonally activated prodrug approach. Nat. Commun. 5, 1–9 (2014).

    ADS  Google Scholar 

  342. 342.

    Wang, J. et al. Palladium-triggered chemical rescue of intracellular proteins via genetically encoded allene-caged tyrosine. J. Am. Chem. Soc. 138, 15118–15121 (2016).

    Google Scholar 

  343. 343.

    Chang, P. V. et al. Copper-free click chemistry in living animals. Proc. Natl Acad. Sci. USA 107, 1821–1826 (2010). This article compares several copper-free chemistries for metabolic labelling in mice.

    ADS  Google Scholar 

  344. 344.

    Ursuegui, S., Recher, M., Krężel, W. & Wagner, A. An in vivo strategy to counteract post-administration anticoagulant activity of azido-warfarin. Nat. Commun. 8, 1–8 (2017).

    Google Scholar 

  345. 345.

    Li, Z. et al. Pretargeting and bioorthogonal click chemistry-mediated endogenous stem cell homing for heart repair. ACS Nano 12, 12193–12200 (2018).

    Google Scholar 

  346. 346.

    Rossin, R. et al. In vivo chemistry for pretargeted tumor imaging in live mice. Angew. Chem. Int. Ed. 49, 3375–3378 (2010). This article presents an initial use of bioorthogonal chemistry for pre-targeted radiochemical imaging in live mice.

    Google Scholar 

  347. 347.

    Zeglis, B. M. et al. A pretargeted PET imaging strategy based on bioorthogonal Diels–Alder click chemistry. J. Nucl. Med. 54, 1389–1396 (2013).

    Google Scholar 

  348. 348.

    Devaraj, N. K., Thurber, G. M., Keliher, E. J., Marinelli, B. & Weissleder, R. Reactive polymer enables efficient in vivo bioorthogonal chemistry. Proc. Natl Acad. Sci. USA 109, 4762–4767 (2012).

    ADS  Google Scholar 

  349. 349.

    Rossin, R. et al. Highly reactive trans-cyclooctene tags with improved stability for Diels–Alder chemistry in living systems. Bioconjugate Chem. 24, 1210–1217 (2013).

    Google Scholar 

  350. 350.

    Rossin, R., van Duijnhoven, S. M., Läppchen, T., van den Bosch, S. M. & Robillard, M. S. Trans-cyclooctene tag with improved properties for tumor pretargeting with the Diels–Alder reaction. Mol. Pharm. 11, 3090–3096 (2014).

    Google Scholar 

  351. 351.

    Rossin, R., Läppchen, T., Van Den Bosch, S. M., Laforest, R. & Robillard, M. S. Diels–Alder reaction for tumor pretargeting: in vivo chemistry can boost tumor radiation dose compared with directly labeled antibody. J. Nucl. Med. 54, 1989–1995 (2013).

    Google Scholar 

  352. 352.

    Meyer, J.-P. et al. Bioorthogonal masking of circulating antibody–TCO groups using tetrazine-functionalized dextran polymers. Bioconjugate Chem. 29, 538–545 (2018).

    Google Scholar 

  353. 353.

    Keinänen, O. et al. Pretargeting of internalizing trastuzumab and cetuximab with a 18F-tetrazine tracer in xenograft models. EJNMMI Res. 7, 1–12 (2017).

    Google Scholar 

  354. 354.

    Zeglis, B. M. et al. Optimization of a pretargeted strategy for the PET imaging of colorectal carcinoma via the modulation of radioligand pharmacokinetics. Mol. Pharm. 12, 3575–3587 (2015).

    Google Scholar 

  355. 355.

    Poty, S. et al. Leveraging bioorthogonal click chemistry to improve 225Ac-radioimmunotherapy of pancreatic ductal adenocarcinoma. Clin. Cancer Res. 25, 868–880 (2019).

    Google Scholar 

  356. 356.

    Rondon, A. et al. Pretargeted radioimmunotherapy and SPECT imaging of peritoneal carcinomatosis using bioorthogonal click chemistry: probe selection and first proof-of-concept. Theranostics 9, 6706 (2019). This article presents a therapeutic proof of concept of IEDDA-mediated pre-targeted radioimmunotherapy.

    Google Scholar 

  357. 357.

    Rossin, R. et al. Chemically triggered drug release from an antibody–drug conjugate leads to potent antitumour activity in mice. Nat. Commun. 9, 1484 (2018).

    ADS  Google Scholar 

  358. 358.

    Rossin, R. et al. Triggered drug release from an antibody–drug conjugate using fast “click-to-release” chemistry in mice. Bioconjugate Chem. 27, 1697–1706 (2016).

    Google Scholar 

  359. 359.

    Mejia Oneto, J. M., Khan, I., Seebald, L. & Royzen, M. In vivo bioorthogonal chemistry enables local hydrogel and systemic pro-drug to treat soft tissue sarcoma. ACS Cent. Sci. 2, 476–482 (2016).

    Google Scholar 

  360. 360.

    Zhang, G. et al. Bioorthogonal chemical activation of kinases in living systems. ACS Cent. Sci. 2, 325–331 (2016).

    Google Scholar 

  361. 361.

    van der Gracht, A. M. et al. Chemical control over T-cell activation in vivo using deprotection of trans-cyclooctene-modified epitopes. ACS Chem. Biol. 13, 1569–1576 (2018).

    Google Scholar 

  362. 362.

    Li, H., Conde, J., Guerreiro, A. & Bernardes, G. J. Tetrazine carbon nanotubes for pretargeted in vivo ‘click-to-release’ bioorthogonal tumour imaging. Angew. Chem. Int. Ed. 59, 16032 (2020).

    Google Scholar 

  363. 363.

    Yao, Q. et al. Synergistic enzymatic and bioorthogonal reactions for selective prodrug activation in living systems. Nat. Commun. 9, 1–9 (2018).

    ADS  Google Scholar 

  364. 364.

    Xie, X. et al. Bioorthogonal nanosystem for near-infrared fluorescence imaging and prodrug activation in mouse model. ACS Mater. Lett. 1, 549–557 (2019).

    Google Scholar 

  365. 365.

    Miller, M. A. et al. Modular nanoparticulate prodrug design enables efficient treatment of solid tumors using bioorthogonal activation. ACS Nano 12, 12814–12826 (2018).

    Google Scholar 

  366. 366.

    Miller, M. A. et al. Nano-palladium is a cellular catalyst for in vivo chemistry. Nat. Commun. 8, 1–13 (2017).

    Google Scholar 

  367. 367.

    US National Library of Medicine. ClinicalTrials.gov https://clinicaltrials.gov/ct2/show/NCT04106492 (2021).

  368. 368.

    Díaz, D. D. et al. Click chemistry in materials synthesis. 1. Adhesive polymers from copper-catalyzed azide–alkyne cycloaddition. J. Polym. Sci. A Polym. Chem. 42, 4392–4403 (2004).

    ADS  Google Scholar 

  369. 369.

    Arslan, M., Acik, G. & Tasdelen, M. A. The emerging applications of click chemistry reactions in the modification of industrial polymers. Polym. Chem. 10, 3806–3821 (2019).

    Google Scholar 

  370. 370.

    Qin, A., Lam, J. W. Y. & Tang, B. Z. Click polymerization. Chem. Soc. Rev. 39, 2522–2544 (2010).

    Google Scholar 

  371. 371.

    Binder, W. H. & Sachsenhofer, R. ‘Click’ chemistry in polymer and material science: an update. Macromol. Rapid Commun. 29, 952–981 (2008).

    Google Scholar 

  372. 372.

    Hansell, C. F. et al. Additive-free clicking for polymer functionalization and coupling by tetrazine–norbornene chemistry. J. Am. Chem. Soc. 133, 13828–13831 (2011).

    Google Scholar 

  373. 373.

    Zhou, H. et al. Crossover experiments applied to network formation reactions: improved strategies for counting elastically inactive molecular defects in PEG gels and hyperbranched polymers. J. Am. Chem. Soc. 136, 9464–9470 (2014).

    Google Scholar 

  374. 374.

    Arseneault, M., Wafer, C. & Morin, J.-F. Recent advances in click chemistry applied to dendrimer synthesis. Molecules 20, 9263–9294 (2015).

    Google Scholar 

  375. 375.

    Martens, S., Holloway, J. O. & Du Prez, F. E. Click and click-inspired chemistry for the design of sequence-controlled polymers. Macromol. Rapid Commun. 38, 1700469 (2017).

    Google Scholar 

  376. 376.

    Yang, C., Flynn, J. P. & Niu, J. Facile synthesis of sequence-regulated synthetic polymers using orthogonal SuFEx and CuAAC click reactions. Angew. Chem. Int. Ed. 57, 16194–16199 (2018).

    Google Scholar 

  377. 377.

    Cook, B. E., Membreno, R. & Zeglis, B. M. Dendrimer scaffold for the amplification of in vivo pretargeting ligations. Bioconjugate Chem. 29, 2734–2740 (2018).

    Google Scholar 

  378. 378.

    Wang, Q. et al. Bioconjugation by copper(I)-catalyzed azide-alkyne [3 + 2] cycloaddition. J. Am. Chem. Soc. 125, 3192–3193 (2003).

    Google Scholar 

  379. 379.

    Abedin, M. J., Liepold, L., Suci, P., Young, M. & Douglas, T. Synthesis of a cross-linked branched polymer network in the interior of a protein cage. J. Am. Chem. Soc. 131, 4346–4354 (2009).

    Google Scholar 

  380. 380.

    Mellet, C. O., Méndez-Ardoy, A. & Fernández, J. M. G. in Click Chemistry in Glycoscience (eds Witczak, Z. J. & Bielski, R.) 143–182 (Wiley, 2013).

  381. 381.

    Haun, J. B., Devaraj, N. K., Hilderbrand, S. A., Lee, H. & Weissleder, R. Bioorthogonal chemistry amplifies nanoparticle binding and enhances the sensitivity of cell detection. Nat. Nanotechnol. 5, 660–665 (2010).

    ADS  Google Scholar 

  382. 382.

    Collman, J. P., Devaraj, N. K. & Chidsey, C. E. D. “Clicking” functionality onto electrode surfaces. Langmuir 20, 1051–1053 (2004).

    Google Scholar 

  383. 383.

    Escorihuela, J., Marcelis, A. T. M. & Zuilhof, H. Metal-free click chemistry reactions on surfaces. Adv. Mater. Interfaces 2, 1500135 (2015).

    Google Scholar 

  384. 384.

    Azagarsamy, M. A. & Anseth, K. S. Bioorthogonal click chemistry: an indispensable tool to create multifaceted cell culture scaffolds. ACS Macro Lett. 2, 5–9 (2013).

    Google Scholar 

  385. 385.

    Alge, D. L., Azagarsamy, M. A., Donohue, D. F. & Anseth, K. S. Synthetically tractable click hydrogels for three-dimensional cell culture formed using tetrazine–norbornene chemistry. Biomacromolecules 14, 949–953 (2013).

    Google Scholar 

  386. 386.

    Brown, T. E. & Anseth, K. S. Spatiotemporal hydrogel biomaterials for regenerative medicine. Chem. Soc. Rev. 46, 6532–6552 (2017).

    Google Scholar 

  387. 387.

    Selvanathan, A. et al. Photo-click chemistry strategies for spatiotemporal control of metal-free ligation, labeling, and surface derivatization. Pure Appl. Chem. 85, 1499–1513 (2013).

    Google Scholar 

  388. 388.

    He, M., Li, J., Tan, S., Wang, R. & Zhang, Y. Photodegradable supramolecular hydrogels with fluorescence turn-on reporter for photomodulation of cellular microenvironments. J. Am. Chem. Soc. 135, 18718–18721 (2013).

    Google Scholar 

  389. 389.

    Dietrich, M. et al. Photoclickable surfaces for profluorescent covalent polymer coatings. Adv. Funct. Mater. 22, 304–312 (2012).

    Google Scholar 

  390. 390.

    Hufendiek, A., Carlmark, A., Meier, M. A. R. & Barner-Kowollik, C. Fluorescent covalently cross-linked cellulose networks via light-induced ligation. ACS Macro Lett. 5, 139–143 (2016).

    Google Scholar 

  391. 391.

    Wallin, T. J. et al. Click chemistry stereolithography for soft robots that self-heal. J. Mater. Chem. B 5, 6249–6255 (2017).

    Google Scholar 

  392. 392.

    Diehl, K. L. et al. Click and chemically triggered declick reactions through reversible amine and thiol coupling via a conjugate acceptor. Nat. Chem. 8, 968–973 (2016).

    Google Scholar 

  393. 393.

    Zlitni, A., Janzen, N., Foster, F. S. & Valliant, J. F. Catching bubbles: targeting ultrasound microbubbles using bioorthogonal inverse-electron-demand Diels–Alder reactions. Angew. Chem. Int. Ed. 53, 6459–6463 (2014).

    Google Scholar 

  394. 394.

    Koo, H. et al. Bioorthogonal click chemistry-based synthetic cell glue. Small 11, 6458–6466 (2015).

    Google Scholar 

  395. 395.

    Liu, S. et al. Meter-long multiblock copolymer microfibers via interfacial bioorthogonal polymerization. Adv. Mater. 27, 2783–2790 (2015).

    Google Scholar 

  396. 396.

    Dicker, K. T. et al. Core-shell patterning of synthetic hydrogels via interfacial bioorthogonal chemistry for spatial control of stem cell behavior. Chem. Sci. 9, 5394–5404 (2018).

    Google Scholar 

  397. 397.

    Dicker, K. T. et al. Spatial patterning of molecular cues and vascular cells in fully integrated hydrogel channels via interfacial bioorthogonal cross-linking. ACS Appl. Mater. Interfaces 11, 16402–16411 (2019).

    Google Scholar 

  398. 398.

    Truong, V. X., Ablett, M. P., Richardson, S. M., Hoyland, J. A. & Dove, A. P. Simultaneous orthogonal dual-click approach to tough, in-situ-forming hydrogels for cell encapsulation. J. Am. Chem. Soc. 137, 1618–1622 (2015).

    Google Scholar 

  399. 399.

    Patterson, D. M. & Prescher, J. A. Orthogonal bioorthogonal chemistries. Curr. Opin. Chem. Biol. 28, 141–149 (2015).

    Google Scholar 

  400. 400.

    Westrum, E. F. & Pitzer, K. S. Thermodynamics of the system KHF2–KF–HF, including heat capacities and entropies of KHF2 and KF. The nature of the hydrogen bond in KHF2. J. Am. Chem. Soc. 71, 1940–1949 (1949).

    Google Scholar 

  401. 401.

    Liu, F. et al. Biocompatible SuFEx click chemistry: thionyl tetrafluoride (SOF4)-derived connective hubs for bioconjugation to DNA and proteins. Angew. Chem. Int. Ed. 58, 8029–8033 (2019).

    Google Scholar 

  402. 402.

    Dong, J., Sharpless, K. B., Kwisnek, L., Oakdale, J. S. & Fokin, V. V. SuFEx-based synthesis of polysulfates. Angew. Chem. Int. Ed. 53, 9466–9470 (2014).

    Google Scholar 

  403. 403.

    Jones, L. H. Emerging utility of fluorosulfate chemical probes. ACS Med. Chem. Lett. 9, 584–586 (2018).

    Google Scholar 

  404. 404.

    Guo, T. et al. A new portal to SuFEx click chemistry: a stable fluorosulfuryl imidazolium salt emerging as an “F−SO2+” donor of unprecedented reactivity, selectivity, and scope. Angew. Chem. Int. Ed. 57, 2605–2610 (2018).

    Google Scholar 

  405. 405.

    Zhou, H. et al. Introduction of a crystalline, shelf-stable reagent for the synthesis of sulfur(VI) fluorides. Org. Lett. 20, 812–815 (2018).

    Google Scholar 

Download references

Acknowledgements

J.A.P. acknowledges support over the years from the Alfred P. Sloan Foundation, the Camille & Henry Dreyfus Foundation and the National Institutes of Health (NIH) (R01 GM126226). J.M.F. acknowledges support from the NIH (R01 GM132460), National Science Foundation (NSF) (DMR 1809612 and 2011824) and Pfizer. Q.L. acknowledges support from the NIH (R35 GM130307) and NSF (CHE-1904558). K.L acknowledges support from the German Science Foundation DFG through programmes SFB1035 and SPP1623.

Author information

Affiliations

Authors

Contributions

Introduction (J.M.F., S.L.S., D.A.B., R.H., W.L., S.S.N., M.X., C.W.a.E., M.G.F., K.L., Q.L., J.P.P., J.A.P. and M.S.R.); Experimentation (J.M.F., S.L.S., D.A.B., R.H., W.L., S.S.N., M.X., C.W.a.E., M.G.F., K.L., Q.L., J.P.P., J.A.P. and M.S.R.); Results (J.M.F., S.L.S., D.A.B., R.H., W.L., S.S.N., M.X., C.W.a.E., M.G.F., K.L., Q.L., J.P.P., J.A.P. and M.S.R.); Applications (J.M.F., S.L.S., D.A.B., R.H., W.L., S.S.N., M.X., C.W.a.E., M.G.F., K.L., Q.L., J.P.P., J.A.P. and M.S.R.); Reproducibility and data deposition (J.M.F., S.L.S., D.A.B., R.H., W.L., S.S.N., M.X., C.W.a.E., M.G.F., K.L., Q.L., J.P.P., J.A.P. and M.S.R.); Limitations and optimizations (J.M.F., S.L.S., D.A.B., R.H., W.L., S.S.N., M.X., C.W.a.E., M.G.F., K.L., Q.L., J.P.P., J.A.P. and M.S.R.); Outlook (J.M.F., S.L.S., D.A.B., R.H., W.L., S.S.N., M.X., C.W.a.E., M.G.F., K.L., Q.L., J.P.P., J.A.P. and M.S.R.); Overview of the Primer (J.M.F.).

Corresponding author

Correspondence to Joseph M. Fox.

Ethics declarations

Competing interests

W.L. and C.W.a.E. are employees of Pfizer Inc. M.S.R. is an employee and shareholder of Tagworks Pharmaceuticals. S.L.S., D.A.B., R.H., S.S.N., M.X., M.G.F., K.L., Q.L., J.P.P., J.A.P. and J.M.F. declare no competing interests.

Additional information

Peer review information

Nature Reviews Methods Primers thanks J. Clavadetscher, D. Johnson, M. Vrábel, H. Wu and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Glossary

Chemoselective

A chemical reaction that is selective for a certain functional group even in the presence of differing functional groups. Reaction partners in bioorthogonal chemistry are chemoselective for each other, even in biological settings.

Click chemistry

A concept, coined by K. B. Sharpless and colleagues, describing bond-forming reactions that are thermodynamically driven, highly selective and reliable, and proceed in water without toxic by-products. Click reactions are often, but not strictly, bioorthogonal.

Nucleophilic catalysts

Electron-rich additives that increase the reaction rate for certain polar bioorthogonal chemistries. In general, the most effective catalysts target the rate-limiting step for a given transformation. Catalysts must also adhere to the same strict requirements as bioorthogonal reagents (non-toxic, chemoselective and so on) if used in a biological context.

Post-translational modifications

Chemical transformations that occur on reactive side chains (such as lysine, serine and cysteine) of proteins. The identity of the modification can drastically affect the function and activity of the target protein. The modifications can be installed enzymatically or can occur spontaneously in solution.

Solid-phase peptide synthesis

Amino acids are iteratively coupled from the carboxy terminus to the amino terminus on a solid support. Protecting group strategies ensure that only one amide bond is formed at a time, without oligomerization or cross-reactivity with reactive side chains. After cleavage from the solid support, peptides are typically purified through high-performance liquid chromatography.

Schiff base

A subclass of imine compounds characterized by a carbon–nitrogen double bond, with a general formula of R1R2C=NR3, where R3 is not a hydrogen atom. They often arise from the condensation reaction between an amine and a carbonyl, and are classified as secondary ketimines or aldimines.

Reactive oxygen species

Highly reactive forms of oxygen involved in diverse cellular signalling processes, and tightly regulated in cells. For bioorthogonal chemistry, reactive oxygen species arise from the oxidation of copper(I) to copper(II) in water, which generates damaging superoxide or hydroxyl radicals. An accumulation of reactive oxygen species damages nucleic acids, proteins and lipids, and is cytotoxic.

Dienophile

An alkene or alkyne that reacts with a conjugated diene in [4 + 2] cycloadditions. Diels–Alder cycloadditions are enabled by electron-poor dienophiles and electron-rich dienes. Conversely, inverse electron-demand Diels–Alder reactions occur between electron-rich dienophiles and electron-poor dienes.

Photoclick chemistry

Click chemistry in which reactions that are initiated using light as an external stimuli. Photoclick reactions can use light sources ranging from short-wavelength to near-IR light and allow for spatial and temporal control of reactions.

HOMO

(Highest occupied molecular orbital). A molecule’s highest energy molecular orbital containing an electron pair.

LUMO

(Lowest unoccupied molecular orbital). A molecule’s lowest energy molecular orbital not containing an electron. The energies of HOMO and LUMO are related to the reactivity of the molecule and the energy difference between the HOMO and LUMO is termed the HOMO–LUMO gap.

Two-photon upconversion

A molecule is excited from the ground state (S0) to the second excited singlet state (S2) by simultaneous absorption of two photons, via a virtual state. A photon with frequency greater than those of the absorbed photons is emitted upon relaxation from the excited state, that is, two-photon upconversion.

Homogeneous catalysis

The catalyst and reaction mixture are in the same phase.

Heterogeneous catalysis

The catalyst and reaction mixture are in a different phase.

Lewis acid

A chemical species that can accept a pair of non-bonding electrons.

Differential scanning calorimetry

(DSC). A technique that measures heat flow rates to determine phase transitions and quantitative heats of decomposition of a compound of interest. DSC requires only milligram quantities of a sample and provides a rapid measurement of the thermal properties of a compound.

Yoshida correlation

The impact sensitivity and explosive propagation properties of compounds can be derived from differential scanning calorimetry data.

Zymogens

Inactive forms of an enzyme. The enzyme takes its active form following a natural biochemical process such as cleavage, hydrolysis or post-translational modification.

Targeted protein degradation

A technique used for targeting specific proteins for degradation within a cell. Commonly, hetero-bifunctional small-molecule compounds are used for targeting the protein of interest to an E3 ubiquitin ligase protein. This facilitates the polyubiquination and subsequent degradation of the targeted protein.

Self-immolative linker

A class of linker that, when exposed to a certain trigger, is designed to break the payload connecting bonds via an intramolecular process.

Prodrug

A pharmacologically inactive precursor compound that is converted into an active drug through in vivo chemical modification achieved via metabolic/enzymatic processes. Prodrugs are employed to improve pharmacokinetic properties (absorption, distribution, metabolism and elimination) and pharmacodynamics properties (selectivity, reduction of adverse effects) of the active drug molecule.

Sequence-specific polymers

Macromolecules that are monodisperse with defined monomer sequences or block sequences. This requires controlled, sequential addition of subunits using highly efficient bond-forming chemical reactions. Naturally occurring examples of sequence-specific macromolecules include DNA, RNA and protein.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Scinto, S.L., Bilodeau, D.A., Hincapie, R. et al. Bioorthogonal chemistry. Nat Rev Methods Primers 1, 30 (2021). https://doi.org/10.1038/s43586-021-00028-z

Download citation

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing