Skip to main content
SpringerLink
Log in

Bioorthogonal chemistry: a covalent strategy for the study of biological systems

  • Reviews
  • Published:
Science China Chemistry Aims and scope Submit manuscript

Abstract

The development of genetically encoded, wavelength-tunable fluorescent proteins has provided a powerful imaging tool to the study of protein dynamics and functions in cellular and organismal biology. However, many biological functions are not directly encoded in the protein primary sequence, e.g., dynamic regulation afforded by protein posttranslational modifications such as phosphorylation. To meet this challenge, an emerging field of bioorthogonal chemistry has promised to offer a versatile strategy to selectively label a biomolecule of interest and track their dynamic regulations in its native habitat. This strategy has been successfully applied to the studies of all classes of biomolecules in living systems, including proteins, nucleic acids, carbohydrates, and lipids. Whereas the incorporation of a bioorthogonal reporter site-selectively into a biomolecule through either genetic or metabolic approaches has been well established, the development of bioorthogonal reactions that allow fast ligation of exogenous chemical probes with the bioorthogonal reporter in living systems remains in its early stage. Here, we review the recent development of bioorthogonal reactions and their applications in various biological systems, with a detailed discussion about our own work—the development of the tetrazole based, photoinducible 1,3-dipolar cycloaddition reaction.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Stockwell BR. Chemical genetics: ligand-based discovery of gene function. Nat Rev Genet, 2000, 1: 116–125

    Article  CAS  Google Scholar 

  2. Prescher JA, Bertozzi CR. Chemistry in living systems. Nat Chem Biol, 2005, 1: 13–21

    Article  CAS  Google Scholar 

  3. van Swieten PF, Leeuwenburgh MA, Kessler BM, Overkleeft HS. Bioorthogonal organic chemistry in living cells: novel strategies for labeling biomolecules. Org Biomol Chem, 2005, 3: 20–27

    Article  Google Scholar 

  4. Barglow KT, Cravatt BF. Activity-based protein profiling for the functional annotation of enzymes. Nat Methods, 2007, 4: 822–827

    Article  CAS  Google Scholar 

  5. Wang L, Brock A, Herberich B, Schultz PG. Expanding the genetic code of Escherichia coli. Science, 2001, 292: 498–500

    Article  CAS  Google Scholar 

  6. Chin JW, Cropp TA, Anderson JC, Mukherji M, Zhang Z, Schultz PG. An expanded eukaryotic genetic code. Science, 2003, 301: 964–967

    Article  CAS  Google Scholar 

  7. Liu W, Brock A, Chen S, Chen S, Schultz PG. Genetic incorporation of unnatural amino acids into proteins in mammalian cell. Nat Methods, 2007, 4: 239–244

    Article  CAS  Google Scholar 

  8. Xie J, Schultz PG. Adding amino acids to the genetic repertoire. Curr Opin Chem Biol, 2005, 9: 548–954

    Article  CAS  Google Scholar 

  9. Schnolzer M, Kent SB. Constructing proteins by dovetailing unprotected synthetic peptides: backbone-engineered HIV protease. Science, 1992, 256: 221–225

    Article  CAS  Google Scholar 

  10. Low DW, Hill MG. Rational fine-tuning of the redox potentials in chemically synthesized rubredoxins. J Am Chem Soc, 1998, 120: 11536–11537

    Article  CAS  Google Scholar 

  11. Muir TW, Sondhi D, Cole PA. Expressed protein ligation: a general method for protein engineering. Proc Natl Acad Sci USA, 1998, 95: 6705–6710

    Article  CAS  Google Scholar 

  12. Cotton GJ, Ayers B, Xu R, Muir TW. Insertion of a synthetic peptide into a recombinant protein framework: a protein biosensor. J Am Chem Soc, 1999, 121: 1100–1101

    Article  CAS  Google Scholar 

  13. Arnold U, Hinderaker MP, Nilsson BL, Huck BR, Gellman SH, Raines RT. Protein prosthesis: a semisynthetic enzyme with a beta-peptide reverse turn. J Am Chem Soc, 2002, 124: 8522–8523

    Article  CAS  Google Scholar 

  14. Pellois JP, Muir TW. Semisynthetic proteins in mechanistic studies: using chemistry to go where nature can’t. Curr Opin Chem Biol, 2006, 10: 487–491

    Article  CAS  Google Scholar 

  15. Mahal LK, Yarema KJ, Bertozzi CR. Engineering chemical reactivity on cell surfaces through oligosaccharide biosynthesis. Science, 1997, 276: 1125–1128

    Article  CAS  Google Scholar 

  16. Bertozzi CR, Kiessling LL. Chemical glycobiology. Science, 2001, 291: 2357–2364

    Article  CAS  Google Scholar 

  17. Prescher JA, Dube DH, Bertozzi CR. Chemical remodelling of cell surfaces in living animals. Nature, 2004, 430: 873–877

    Article  CAS  Google Scholar 

  18. Khidekel N, Ficarro SB, Peters EC, Hsieh-Wilson LC. Exploring the O-GlcNAc proteome: direct identification of O-GlcNAc-modified proteins from the brain. Proc Natl Acad Sci USA, 2004, 101: 13132–13137

    Article  CAS  Google Scholar 

  19. Keppler A, Gendreizig S, Gronemeyer T, Pick H, Vogel H, Johnsson K. A general method for the covalent labeling of fusion proteins with small molecules in vivo. Nat Biotechnol, 2003, 21: 86–89

    Article  CAS  Google Scholar 

  20. Kho Y, Kim SC, Jiang C, Barma D, Kwon SW, Cheng J, Jaunbergs J, Weinbaum C, Tamanoi F, Falck J, Zhao YA. Tagging-via-substrate technology for detection and proteomics of farnesylated proteins. Proc Natl Acad Sci USA, 2004, 101: 12479–12484

    Article  CAS  Google Scholar 

  21. Tai HC, Khidekel N, Ficarro SB, Peters EC, Hsieh-Wilson LC. Parallel identification of O-GlcNAc-modified proteins from cell lysates. J Am Chem Soc, 2004, 126: 10500–10501

    Article  CAS  Google Scholar 

  22. Chen I, Howarth M, Lin W, Ting AY. Site-specific labeling of cell surface proteins with biophysical probes using biotin ligase. Nat Methods, 2005, 2: 99–104

    Article  CAS  Google Scholar 

  23. Lin CW, Ting AY. Transglutaminase-catalyzed site-specific conjugation of small-molecule probes to proteins in vitro and on the surface of living cells. J Am Chem Soc, 2006, 128: 4542–4543

    Article  CAS  Google Scholar 

  24. Carrico IS, Carlson BL, Bertozzi CR. Introducing genetically encoded aldehydes into proteins. Nat Chem Biol, 2007, 3: 321–322

    Article  CAS  Google Scholar 

  25. Rose K. Facile synthesis of homogeneous artificial proteins. J Am Chem Soc, 1994, 116: 30–33

    Article  CAS  Google Scholar 

  26. Canne LE, Ferre-D’Amare AR, Burley SK, Kent SBH. Total chemical synthesis of a unique transcription factor-related protein: cMyc-Max. J Am Chem Soc, 1995, 117: 2998–3007

    Article  CAS  Google Scholar 

  27. Gaertner HF, Rose K, Cotton R, Timms D, Camble R, Offord RE. Construction of protein analogs by site-specific condensation of unprotected fragments. Bioconjugate Chem, 2002, 3: 262–268

    Article  Google Scholar 

  28. Rideout D. Self-assembling cytotoxins. Science, 1986, 233: 561–563

    Article  CAS  Google Scholar 

  29. Zhang Z, Smith BAC, Wang L, Brock A, Cho C, Schultz PG. A new strategy for the site-specific modification of proteins in vivo. Biochemistry, 2003, 42: 6735–6746

    Article  CAS  Google Scholar 

  30. Sadamoto R, Niikura K, Ueda T, Monde K, Fukuhara N, Nishimura S. Control of bacteria adhesion by cell-wall engineering. J Am Chem Soc, 2004, 126, 3755–3761

    Article  CAS  Google Scholar 

  31. Dirksen A, Hackeng TM, Dawson PE. Nucleophilic catalysis of oxime ligation. Angew Chem Int Ed, 2006, 45: 7581–7584

    Article  CAS  Google Scholar 

  32. Zeng Y, Ramya TNC, Dirksen A, Dawson PE, Paulson JC. High-efficiency labeling of sialylated glycoproteins on living cells. Nat Methods, 2009, 6: 207–209

    Article  CAS  Google Scholar 

  33. Kiick KL, Saxon E, Tirrell DA, Bertozzi CR. Incorporation of azides into recombinant proteins for chemoselective modification by the Staudinger ligation. Proc Natl Acad Sci USA, 2002, 99: 19–24

    Article  CAS  Google Scholar 

  34. Lemieux GA, De Graffenried CL, Bertozzi CR. A fluorogenic dye activated by the Staudinger ligation. J Am Chem Soc, 2003, 125: 4708–4709

    Article  CAS  Google Scholar 

  35. Staudinger H, Meyer J. Uber neue organische phosphoverbindungen III Phosphinmethlenderivate und phosphiniimine. Helv Chim Acta 1919, 2: 635–646

    Article  CAS  Google Scholar 

  36. Gololobov YG, Kasukhin LF. Recent advances in the Staudinger reaction. Tetrahedron, 1992, 48, 1353–1406

    Article  CAS  Google Scholar 

  37. Saxon E, Bertozzi CR. Cell surface engineering by a modified Staudinger reaction. Science, 2000, 287: 2007–2010

    Article  CAS  Google Scholar 

  38. Kohn M, Breinbauer R. The Staudinger ligation-a gift to chemical biology. Angew Chem Int Ed, 2004, 43: 3106–3116

    Article  Google Scholar 

  39. Nilsson BL, Kiessling LL, Raines RT. Staudinger ligation: a peptide from a thioester and azide. Org Lett, 2000, 2: 1939–1941

    Article  CAS  Google Scholar 

  40. Saxon E, Armstrong JI, Bertozzi CR. A “traceless” Staudinger ligation for the chemoselective synthesis of amide bonds. Org Lett, 2000, 2: 2141–2143

    Article  CAS  Google Scholar 

  41. Vocadlo DJ, Hang HC, Kim EJ, Hanover JA, Bertozzi CR. A chemical approach for identifying O-GlcNAc-modified proteins in cells. Proc Natl Acad Sci USA, 2003, 100: 9116–9121

    Article  CAS  Google Scholar 

  42. Hang HC, Yu C, Kato DL, Bertozzi CR. A metabolic labeling approach toward proteomic analysis of mucin-type O-linked glycosylation. Proc Natl Acad Sci USA, 2003, 100: 14846–14851

    Article  CAS  Google Scholar 

  43. Luchansky SJ, Argade S, Hayes BK, Bertozzi CR. Metabolic functionalization of recombinant glycoproteins. Biochemistry, 2004, 43: 12358–12366

    Article  CAS  Google Scholar 

  44. Ovaa H, van Swieten PF, Kessler BM, Leeuwenburgh MA, Fiebiger E, van den Nieuwendijk AM, Galardy PJ, van der Marel GA, Ploegh HL, Overkleeft HS. Chemistry in living cells: detection of active proteasomes by a two-step labeling strategy. Angew Chem Int Ed, 2003, 42: 3626–3629

    Article  CAS  Google Scholar 

  45. Hangauer MJ, Bertozzi CR. A FRET-based fluorogenic phosphine for live-cell imaging with the Staudinger ligation. Angew Chem Int Ed, 2008, 47: 2394–2397

    Article  CAS  Google Scholar 

  46. Huisgen R. 1,3-Dipolar cycloaddition. Angew Chem Int Ed, 1963, 2: 565–598

    Article  Google Scholar 

  47. Rostovtsev VV, Green LG, Fokin VV, Sharpless KB. A stepwise Huisgen cycloaddition process: Copper(I)-catalyzed regioselective “ligation” of azides and terminal alkynes. Angew Chem Int Ed, 2002, 41: 2596–2599

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  49. Wang Q, Chan TR, Hilgraf R, Fokin VV, Sharpless KB, Finn MG. Bioconjugation by copper(I)-catalyzed azide-alkyne [3+2] cycloaddition. J Am Chem Soc, 2003, 125: 3192–3193

    Article  CAS  Google Scholar 

  50. Link AJ, Tirrell DA. Cell surface labeling of Escherichia coli via copper(I)-catalyzed [3+2] cycloaddition. J Am Chem Soc, 2003, 125: 11164–11165

    Article  CAS  Google Scholar 

  51. Gierlich J, Burley GA, Gramlich PME, Hammond DM, Carell T. Click chemistry as a reliable method for the high-density postsynthetic functionalization of alkyne-modified DNA. Org Lett, 2006, 8: 3639–3642

    Article  CAS  Google Scholar 

  52. Seo TS, Bai X, Ruparel H, Li Z, Turro NJ, Ju J. Photocleavable fluorescent nucleotides for DNA sequencing on a chip constructed by site-specific coupling chemistry. Proc Natl Acad Sci USA, 2004, 101: 5488–5493

    Article  CAS  Google Scholar 

  53. Deiters A, Cropp T A, Mukherji M, Chin JW, Anderson C, Schultz PG. Adding amino acids with novel reactivity to the genetic code of Saccharomyces cerevisiae. J Am Chem Soc, 2003, 125: 11782–11783

    Article  CAS  Google Scholar 

  54. Speers AE, Adam GC, Cravatt BF. Activity-based protein profiling in vivo using a copper(I)-catalyzed azide-alkyne [3 + 2] cycloaddition. J Am Chem Soc, 2003, 125: 4686–4687

    Article  CAS  Google Scholar 

  55. Agard NJ, Baskin JM, Prescher JA, Lo A, Bertozzi CR. A comparative study of bioorthogonal reactions with azides. ACS Chem Biol, 2006, 1: 644–648

    Article  CAS  Google Scholar 

  56. Speers AE, Cravatt BF. Profiling enzyme activities in vivo using click chemistry methods. Chem Biol, 2004, 11: 535–546

    Article  CAS  Google Scholar 

  57. Becer CR, Hoogenboom R, Schubert US. Click chemistry beyond metal-catalyzed cycloaddition. Angew Chem Int Ed, 2009, 48: 4900–4908

    Article  CAS  Google Scholar 

  58. Turner RB, Jarrett AD, Goebel P, Mallon BJ. Heat of hydrogenation. IX. Cyclic acetylenes and some micellaneous olefins. J Am Chem Soc, 1973, 95: 790–792

    Article  CAS  Google Scholar 

  59. Agard NJ, Prescher JA, Bertozzi CR. A strain-promoted [3 + 2] azide-alkyne cycloaddition for covalent modification of biomolecules in living systems. J Am Chem Soc, 2004, 126: 15046–15047

    Article  CAS  Google Scholar 

  60. Lin FL, Hoyt HM, van Halbeek H, Bergman RG, Bertozzi CR. Mechanistic investigation of the Staudinger ligation. J Am Chem Soc, 2005, 127: 2686–2695

    Article  CAS  Google Scholar 

  61. Baskin JM, Prescher JA, Laughlin ST, Agard NJ, Chang PV, Miller IA, Lo A, Codelli JA, Bertozzi CR. Copper-free click chemistry for dynamic in vivo imaging. Proc Natl Acad Sci USA, 2007, 104, 16793–16797

    Article  CAS  Google Scholar 

  62. Laughlin ST, Baskin JM, Amacher SL, Bertozzi CR. In vivo imaging of membrane-associated glycans in developing zebrafish. Science, 2008, 320: 664–667

    Article  CAS  Google Scholar 

  63. Codelli JA, Baskin JM, Agard NJ, Bertozzi CR. Second-generation difluorinated cyclooctynes for copper-free click chemistry. J Am Chem Soc, 2008, 130: 11486–11493

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  65. Lin YA, Chalker JM, Floyd N, Bernardes GJ, Davis BG. Allyl sulfides are privileged substrates in aqueous cross-metathesis: Application to site-selective protein modification. J Am Chem Soc, 2008, 130, 9642–9643

    Article  CAS  Google Scholar 

  66. Garber SB, Kingsbury JS, Gray BL, Hoveyda AH. Efficient and recyclable monomeric and dendritic Ru-based metathesis catalysts. J Am Chem Soc, 2000, 122: 8168–8179

    Article  CAS  Google Scholar 

  67. Breslow R, Rideout DC. Hydrophobic acceleration of Diels-Alder reactions. J Am Chem Soc, 1980, 102: 7816–7817

    Article  Google Scholar 

  68. Otto S, Engberts JBFN. Diels-Alder reactions in water. Pure Appl Chem, 2000, 72: 1365–1372

    Article  CAS  Google Scholar 

  69. Seelig B, Jäschke A. Site-specific modification of enzymatically synthesized RNA: transcription initiation and Diels-Alder reaction. Tetrahedron Lett, 1997, 38: 7729–7732

    Article  CAS  Google Scholar 

  70. Yousaf MN, Mrksich M. Diels-Alder reaction for the selective immobilization of protein to electroactive self-assembled monolayers. J Am Chem Soc, 1999, 121: 4286–4287

    Article  CAS  Google Scholar 

  71. Latham-Timmons HA, Wolter A, Roach JS, Giare R, Leuck M. Novel method for the covalent immobilization of oligonucleotides via Diels-Alder bioconjugation. Nucleos Nucleot Nucl Acids, 2003, 22: 1495–1497

    Article  CAS  Google Scholar 

  72. de AraÚjo AD, Palomo JM, Cramer J, Köhn M, Schröder H, Wacker R, Niemeyer C, Alexandrov K, Waldmann H. Diels-Alder ligation and surface immobilization of proteins. Angew Chem Int Ed, 2006, 45: 296–301

    Article  Google Scholar 

  73. Blackman ML, Royzen M, Fox JM. Tetrazine ligation: fast bioconjugation based on inverse-electron-demand Diels-Alder reactivity. J Am Chem Soc, 2008, 130: 13518–13519

    Article  CAS  Google Scholar 

  74. Boger DL. Diels-Alder reactions of heterocyclic aza dienes Scope and applications. Chem Rev, 1986, 86: 781–793

    Article  CAS  Google Scholar 

  75. Devaraj NK, Weissleder R, Hilderbrand SA. Tetrazine-based cycloadditions: application to pretargeted live cell imaging. Bioconjugate Chem, 2008, 19: 2297–2299

    Article  CAS  Google Scholar 

  76. Clovis JS, Eckell A, Huisgen R, Sustmann R. 1,3-Dipolare cycloadditionen, XXV. Der Nachweis des freien diphenylnitrilimins als zwischenstufe bei cycloadditionen. Chem Ber, 1967, 100: 60–70

    Article  CAS  Google Scholar 

  77. Wang Y, Rivera Vera CI, Lin Q. Convenient synthesis of highly functionalized pyrazolines via mild, photoactivated 1,3-dipolar cycloaddition. Org Lett, 2007, 9: 4155–4158

    Article  CAS  Google Scholar 

  78. Song W, Wang Y, Qu J, Madden MM, Lin Q. A photoinducible 1,3-dipolar cycloaddition reaction for rapid, selective modification of tetrazole-containing proteins. Angew Chem Int Ed, 2008, 47: 2832–2835

    Article  CAS  Google Scholar 

  79. Wang Y, Hu WJ, Song W, Lim RKV, Lin Q. Discovery of long-wavelength photoactivatable diaryltetrazoles for bioorthogonal 1,3-dipolar cycloaddition reactions. Org Lett, 2008, 10: 3725–3728

    Article  CAS  Google Scholar 

  80. 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, 2008 130: 9654–9655

    Article  CAS  Google Scholar 

  81. Zhang Z, Wang L, Brock A, Schultz PG. The selective incorporation of alkenes into proteins in Escherichia coli. Angew Chem Int Ed, 2002, 41: 2840–2842

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  83. Ito S, Tanaka Y, Kakehi A, Kondo K. Facile synthesis of 2,5-disubstituted tetrazoles by reaction of phenylsulfonylhydrazones with arenediazonium salts. Bull Chem Soc Jpn,1976, 49: 1920–1923

    Article  CAS  Google Scholar 

  84. Wang Y, Lin Q. Synthesis and evaluation of photoreactive tetrazole amino acids. Org Lett, 2009, 10: 3570–3573

    Article  Google Scholar 

  85. van Kasteren SI, Kramer HB, Jensen HH, Campbell SJ, Kirkpatrick J, Oldham NJ, Anthony DC, Davis BG. Expanding the diversity of chemical protein modification allows post-translational mimicry. Nature, 2007, 446: 1105–1109

    Article  Google Scholar 

  86. Serwa R, Wilkening I, Del Signore G, Muhlberg M, Claussnitzer I, Weise C, Gerrits M, Hackenberger CP. Chemoselective staudinger-phosphite reaction of azides for the phosphorylation of proteins. Angew Chem Int Ed, 2009, 48: 8234–8239

    Article  CAS  Google Scholar 

  87. Benner SA, Sismour AM. Synthetic biology. Nat Rev Genet, 2005, 6: 533–543

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Chemistry, State University of New York at Buffalo, Buffalo, New York, 14260-3000, USA

    Reyna K. V. Lim & Qing Lin

Authors
  1. Reyna K. V. Lim
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Qing Lin
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Qing Lin.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Lim, R.K.V., Lin, Q. Bioorthogonal chemistry: a covalent strategy for the study of biological systems. Sci. China Chem. 53, 61–70 (2010). https://doi.org/10.1007/s11426-010-0020-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11426-010-0020-4

Keywords

Search

Navigation