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.

  • Review Article
  • Published:

Contemporary approaches to site-selective protein modification

Nature Reviews Chemistry volume 3, pages 147–171 (2019)Cite this article

Subjects

Abstract

Proteins constitute the majority of nature’s worker biomolecules. Designed for specific functions, complex tertiary structures make proteins ideal candidates for analysing natural systems and creating novel biological tools. Owing to both their large size and the need for proper folding, de novo synthesis of proteins has been quite a challenge, leading scientists to focus on modifying protein templates already provided by nature. Recently developed methods for protein modification fall into two broad categories: those that can modify the natural protein template directly and those that require genetic manipulation of the amino acid sequence before modification. The goal of this Review is not only to provide a window through which to view the many opportunities created by novel protein modification techniques‚ but also to act as an initial guide to help scientists find direction and form ideas in an ever-growing field. In addition to highlighting methods reported in the past 5 years, we aim to provide a broader sense of the goals and outcomes of protein modification and bioconjugation in general. While the main body of this paper comprises reactions involving the direct modification of expressed proteins, some further functionalization strategies as well as biological applications are also acknowledged. The discussion concludes by speculating which trends and discoveries will most likely come next in the field.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Juxtaposition of classical and modern protein modification methods.
Fig. 2: N-terminal and C-terminal selective protein modification.
Fig. 3: In-chain endogenous side chain modification.
Fig. 4: Modification methods for engineered Cys residues.
Fig. 5: Insertion of canonical amino acids aside from Cys.
Fig. 6: Insertion of noncanonical amino acids.
Fig. 7: Motif and enzymatic tag insertion.
Fig. 8: Downstream functionalization methods.

Similar content being viewed by others

References

  1. Krall, N., da Cruz, F. P., Boutureira, O. & Bernardes, G. J. L. Site-selective protein-modification chemistry for basic biology and drug development. Nat. Chem. 8, 103–113 (2016).

    Article  CAS  PubMed  Google Scholar 

  2. de Graaf, A. J., Kooijman, M., Hennink, W. E. & Mastrobattista, E. Nonnatural amino acids for site-specific protein conjugation. Bioconjug. Chem. 20, 1281–1295 (2009).

    Article  PubMed  CAS  Google Scholar 

  3. Milczek, E. M. Commercial applications for enzyme-mediated protein conjugation: new developments in enzymatic processes to deliver functionalized proteins on the commercial scale. Chem. Rev. 118, 119–141 (2018).

    Article  CAS  PubMed  Google Scholar 

  4. Takaoka, Y., Ojida, A. & Hamachi, I. Protein organic chemistry and applications for labeling and engineering in live-cell systems. Angew. Chem. Int. Ed. 52, 4088–4106 (2013).

    Article  CAS  Google Scholar 

  5. Boutureira, O. & Bernardes, G. J. L. Advances in chemical protein modification. Chem. Rev. 115, 2174–2195 (2015).

    Article  CAS  PubMed  Google Scholar 

  6. Gunnoo, S. B. & Madder, A. Chemical protein modification through cysteine. ChemBioChem 17, 529–553 (2016).

    Article  CAS  PubMed  Google Scholar 

  7. Koniev, O. & Wagner, A. Developments and recent advancements in the field of endogenous amino acid selective bond forming reactions for bioconjugation. Chem. Soc. Rev. 44, 5495–5551 (2015).

    Article  CAS  PubMed  Google Scholar 

  8. Chalker, J. M., Bernardes, G. J. L. & Davis, B. G. A ‘tag-and-modify’ approach to site-selective protein modification. Acc. Chem. Res. 44, 730–741 (2011).

    Article  CAS  PubMed  Google Scholar 

  9. Boyce, M. & Bertozzi, C. R. Bringing chemistry to life. Nat. Methods 8, 638–642 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  Google Scholar 

  11. Schmidt, M. J. & Summerer, D. A. Need for speed: genetic encoding of rapid cycloaddition chemistries for protein labelling in living cells. ChemBioChem 13, 1553–1557 (2012).

    Article  CAS  PubMed  Google Scholar 

  12. Matos, M. J. et al. Chemo- and regioselective lysine modification on native proteins. J. Am. Chem. Soc. 140, 4004–4017 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Rosen, C. B. & Francis, M. B. Targeting the N terminus for site-selective protein modification. Nat. Chem. Biol. 13, 697–705 (2017).

    Article  CAS  PubMed  Google Scholar 

  14. Hacker, S. M. et al. Global profiling of lysine reactivity and ligandability in the human proteome. Nat. Chem. 9, 1181–1190 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Huang, F., Nie, Y., Ye, F., Zhang, M. & Xia, J. Site selective Azo coupling for peptide cyclization and affinity labeling of an SH3 protein. Bioconjug. Chem. 26, 1613–1622 (2015).

    Article  CAS  PubMed  Google Scholar 

  16. Bloom, S. et al. Decarboxylative alkylation for site-selective bioconjugation of native proteins via oxidation potentials. Nat. Chem. 10, 205–211 (2018).

    Article  CAS  PubMed  Google Scholar 

  17. Zuo, Z. & MacMillan, D. W. C. Decarboxylative arylation of α-amino acids via photoredox catalysis: a one-step conversion of biomass to drug pharmacophore. J. Am. Chem. Soc. 136, 5257–5260 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. McGrath, N. A., Andersen, K. A., Davis, A. K. F., Lomax, J. E. & Raines, R. T. Diazo compounds for the bioreversible esterification of proteins. Chem. Sci. 6, 752–755 (2015).

    Article  CAS  PubMed  Google Scholar 

  19. Totaro, K. A. et al. Systematic investigation of EDC/sNHS-mediated bioconjugation reactions for carboxylated peptide substrates. Bioconjug. Chem. 27, 994–1004 (2016).

    Article  CAS  PubMed  Google Scholar 

  20. Yu, Y. et al. Chemoselective peptide modification via photocatalytic tryptophan β-position conjugation. J. Am. Chem. Soc. 140, 6797–6800 (2018).

    Article  CAS  PubMed  Google Scholar 

  21. Chen, D., Disotuar, M. M., Xiong, X., Wang, Y. & Chou, D. H.-C. Selective N-terminal functionalization of native peptides and proteins. Chem. Sci. 8, 2717–2722 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. 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).

    Article  CAS  PubMed  Google Scholar 

  23. Obermeyer, A. C., Jarman, J. B. & Francis, M. B. N-terminal modification of proteins with O-aminophenols. J. Am. Chem. Soc. 136, 9572–9579 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Casi, G., Huguenin-Dezot, N., Zuberbühler, K., Scheuermann, J. & Neri, D. Site-specific traceless coupling of potent cytotoxic drugs to recombinant antibodies for pharmacodelivery. J. Am. Chem. Soc. 134, 5887–5892 (2012).

    Article  CAS  PubMed  Google Scholar 

  25. Antos, J. M. et al. Site-specific N− and C-terminal labeling of a single polypeptide using sortases of different specificity. J. Am. Chem. Soc. 131, 10800–10801 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Nguyen, G. K. T., Cao, Y., Wang, W., Liu, C. F. & Tam, J. P. Site-specific N-terminal labeling of peptides and proteins using butelase 1 and thiodepsipeptide. Angew. Chem. Int. Ed. 54, 15694–15698 (2015).

    Article  CAS  Google Scholar 

  27. Palla, K. S., Witus, L. S., Mackenzie, K. J., Netirojjanakul, C. & Francis, M. B. Optimization and expansion of a site-selective N-methylpyridinium-4-carboxaldehyde-mediated transamination for bacterially expressed proteins. J. Am. Chem. Soc. 137, 1123–1129 (2015).

    Article  CAS  PubMed  Google Scholar 

  28. Murza, A. et al. C-terminal modifications of apelin-13 significantly change ligand binding, receptor signaling, and hypotensive action. J. Med. Chem. 58, 2431–2440 (2015).

    Article  CAS  PubMed  Google Scholar 

  29. Willwacher, J., Raj, R., Mohammed, S. & Davis, B. G. Selective metal-site-guided arylation of proteins. J. Am. Chem. Soc. 138, 8678–8681 (2016).

    Article  CAS  PubMed  Google Scholar 

  30. Imiolek, M. et al. Selective radical trifluoromethylation of native residues in proteins. J. Am. Chem. Soc. 140, 1568–1571 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Chilamari, M., Purushottam, L. & Rai, V. Site-selective labeling of native proteins by a multicomponent approach. Chemistry 23, 3819–3823 (2017).

    Article  CAS  PubMed  Google Scholar 

  32. Ball, Z. T. Molecular recognition in protein modification with rhodium metallopeptides. Curr. Opin. Chem. Biol. 25, 98–102 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Pentelute, B., Zhang, C., Vinogradova, E., Spokoyny, A. & Buchwald, S. Arylation chemistry for bioconjugation. Angew. Chem. Int. Ed. https://doi.org/10.1002/anie.201806009 (2018).

    Article  Google Scholar 

  34. Lohse, J. et al. Targeted diazotransfer reagents enable selective modification of proteins with azides. Bioconjug. Chem. 28, 913–917 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Vohidov, F., Coughlin, J. M. & Ball, Z. T. Rhodium(II) metallopeptide catalyst design enables fine control in selective functionalization of natural SH3 domains. Angew. Chem. Int. Ed. 54, 4587–4591 (2015).

    Article  CAS  Google Scholar 

  36. Ohata, J. & Ball, Z. T. A. Hexa-rhodium metallopeptide catalyst for site-specific functionalization of natural antibodies. J. Am. Chem. Soc. 139, 12617–12622 (2017).

    Article  CAS  PubMed  Google Scholar 

  37. Yu, C. et al. Proximity-induced site-specific antibody conjugation. Bioconjug. Chem. 29, 3522–3526 (2018).

    Article  CAS  PubMed  Google Scholar 

  38. Adusumalli, S. R. et al. Single-site labeling of native proteins enabled by a chemoselective and site-selective chemical technology. J. Am. Chem. Soc. 140, 15114–15123 (2018).

    Article  CAS  PubMed  Google Scholar 

  39. Kuan, S. L., Wang, T. & Weil, T. Site-selective disulfide modification of proteins: expanding diversity beyond the proteome. Chemistry 22, 17112–17129 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Martinez-Saez, N. et al. Oxetane grafts installed site-selectively on native disulfides to enhance protein stability and activity in vivo. Angew. Chem. Int. Ed. 56, 14963–14967 (2017).

    Article  CAS  Google Scholar 

  41. Badescu, G. et al. Bridging disulfides for stable and defined antibody drug conjugates. Bioconjug. Chem. 25, 1124–1136 (2014).

    Article  CAS  PubMed  Google Scholar 

  42. Wang, T. et al. Water-soluble allyl sulfones for dual site-specific labelling of proteins and cyclic peptides. Chem. Sci. 7, 3234–3239 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Walsh, S. J. et al. A general approach for the site-selective modification of native proteins, enabling the generation of stable and functional antibody–drug conjugates. Chem. Sci. 10, 694–700 (2019).

    Article  CAS  PubMed  Google Scholar 

  44. Lee, M. T. W. et al. Enabling the controlled assembly of antibody conjugates with a loading of two modules without antibody engineering. Chem. Sci. 8, 2056–2060 (2017).

    Article  CAS  PubMed  Google Scholar 

  45. Maruani, A. et al. A plug-and-play approach to antibody-based therapeutics via a chemoselective dual click strategy. Nat. Commun. 6, 6645 (2015).

    Article  CAS  PubMed  Google Scholar 

  46. Greene, M. K. et al. Forming next-generation antibody–nanoparticle conjugates through the oriented installation of non-engineered antibody fragments. Chem. Sci. 9, 79–87 (2018).

    Article  CAS  PubMed  Google Scholar 

  47. Griebenow, N., Dilmaç, A. M., Greven, S. & Bräse, S. Site-specific conjugation of peptides and proteins via rebridging of disulfide bonds using the Thiol–Yne coupling reaction. Bioconjug. Chem. 27, 911–917 (2016).

    Article  CAS  PubMed  Google Scholar 

  48. Cal, P. M. S. D., Bernardes, G. J. L. G. J. L. & Gois, P. M. P. Cysteine-selective reactions for antibody conjugation. Angew. Chem. Int. Ed. 53, 10585–10587 (2014).

    Article  CAS  Google Scholar 

  49. Lyon, R. P. et al. Self-hydrolyzing maleimides improve the stability and pharmacological properties of antibody-drug conjugates. Nat. Biotechnol. 32, 1059–1062 (2014).

    Article  CAS  PubMed  Google Scholar 

  50. Fontaine, S. D., Reid, R., Robinson, L., Ashley, G. W. & Santi, D. V. Long-term stabilization of maleimide-thiol conjugates. Bioconjug. Chem. 26, 145–152 (2015).

    Article  CAS  PubMed  Google Scholar 

  51. Christie, R. et al. Stabilization of cysteine-linked antibody drug conjugates with N-aryl maleimides. J. Control. Release 220, 660–670 (2015).

    Article  CAS  PubMed  Google Scholar 

  52. Kalia, D., Malekar, P. V. & Parthasarathy, M. Exocyclic olefinic maleimides: synthesis and application for stable and thiol-selective bioconjugation. Angew. Chem. Int. Ed. 55, 1432–1435 (2016).

    Article  CAS  Google Scholar 

  53. Tumey, L. N. et al. Mild method for succinimide hydrolysis on ADCs: impact on ADC potency, stability, exposure, and efficacy. Bioconjug. Chem. 25, 1871–1880 (2014).

    Article  CAS  PubMed  Google Scholar 

  54. Morais, M. et al. Optimisation of the dibromomaleimide (DBM) platform for native antibody conjugation by accelerated post-conjugation hydrolysis. Org. Biomol. Chem. 15, 2947–2952 (2017).

    Article  CAS  PubMed  Google Scholar 

  55. Kalia, D., Pawar, S. P. & Thopate, J. S. Stable and rapid thiol bioconjugation by light-triggered thiomaleimide ring hydrolysis. Angew. Chem. Int. Ed. 56, 1885–1889 (2017).

    Article  CAS  Google Scholar 

  56. Boutureira, O. et al. Site-selective modification of proteins with oxetanes. Chemistry 23, 6483–6489 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Lee, B., Sun, S., Jiménez-moreno, E., Neves, A. A. & Bernardes, G. J. L. Site-selective installation of an electrophilic handle on proteins for bioconjugation. Bioorg. Med. Chem. 26, 3060–3064 (2018).

    Article  CAS  PubMed  Google Scholar 

  58. Kolodych, S. et al. CBTF: new amine-to-thiol coupling reagent for preparation of antibody conjugates with increased plasma stability. Bioconjug. Chem. 26, 197–200 (2015).

    Article  CAS  PubMed  Google Scholar 

  59. Koniev, O. et al. Selective irreversible chemical tagging of cysteine with 3-arylpropiolonitriles. Bioconjug. Chem. 25, 202–206 (2014).

    Article  CAS  PubMed  Google Scholar 

  60. Embaby, A. M., Schoffelen, S., Kofoed, C., Meldal, M. & Diness, F. Rational tuning of fluorobenzene probes for cysteine-selective protein modification. Angew. Chem. Int. Ed. 57, 8022–8026 (2018).

    Article  CAS  Google Scholar 

  61. Bernardim, B. et al. Stoichiometric and irreversible cysteine-selective protein modification using carbonylacrylic reagents. Nat. Commun. 7, 13128 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Abbas, A., Xing, B. & Loh, T.-P. Allenamides as orthogonal handles for selective modification of cysteine in peptides and proteins. Angew. Chem. Int. Ed. 53, 7491–7494 (2014).

    Article  CAS  Google Scholar 

  63. Smith, N. J. et al. Fast, irreversible modification of cysteines through strain releasing conjugate additions of cyclopropenyl ketones. Org. Biomol. Chem. 16, 2164–2169 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Canovas, C. et al. Site-specific dual labeling of proteins on cysteine residues with chlorotetrazines. Angew. Chem. Int. Ed. 57, 10646–10650 (2018).

    Article  CAS  Google Scholar 

  65. Ariyasu, S., Hayashi, H., Xing, B. & Chiba, S. Site-specific dual functionalization of cysteine residue in peptides and proteins with 2-azidoacrylates. Bioconjug. Chem. 28, 897–902 (2017).

    Article  CAS  PubMed  Google Scholar 

  66. Vinogradova, E. V., Zhang, C., Spokoyny, A. M., Pentelute, B. L. & Buchwald, S. L. Organometallic palladium reagents for cysteine bioconjugation. Nature 526, 687–691 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Vara, B. A. et al. Scalable thioarylation of unprotected peptides and biomolecules under Ni/photoredox catalysis. Chem. Sci. 9, 336–344 (2018).

    Article  CAS  PubMed  Google Scholar 

  68. Messina, M. S. et al. Organometallic gold(III) reagents for cysteine arylation. J. Am. Chem. Soc. 140, 7065–7069 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Rojas, A. J., Pentelute, B. L. & Buchwald, S. L. Water-soluble palladium reagents for cysteine S-arylation under ambient aqueous conditions. Org. Lett. 19, 4263–4266 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  70. Kubota, K., Dai, P., Pentelute, B. L. & Buchwald, S. L. Palladium oxidative addition complexes for peptide and protein cross-linking. J. Am. Chem. Soc. 140, 3128–3133 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  71. Zhang, Y. et al. Thiol specific and tracelessly removable bioconjugation via Michael addition to 5-methylene pyrrolones. J. Am. Chem. Soc. 139, 6146–6151 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  72. Bandyopadhyay, A. et al. Fast and selective labeling of N-terminal cysteines at neutral pH via thiazolidino boronate formation. Chem. Sci. 7, 4589–4593 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  73. Arumugam, S. et al. Selective and reversible photochemical derivatization of cysteine residues in peptides and proteins. Chem. Sci. 5, 1591–1598 (2014).

    Article  CAS  PubMed  Google Scholar 

  74. Yu, J., Yang, X., Sun, Y. & Yin, Z. Highly reactive and tracelessly cleavable cysteine-specific modification of proteins via 4-substituted cyclopentenone. Angew. Chem. Int. Ed. 57, 11598–11602 (2018).

    Article  CAS  Google Scholar 

  75. Faustino, H., Silva, M. J. S. A., Veiros, L. F., Bernardes, G. J. L. & Gois, P. M. P. Iminoboronates are efficient intermediates for selective, rapid and reversible N-terminal cysteine functionalisation. Chem. Sci. 7, 5052–5058 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  76. Lin, S. et al. Redox-based reagents for chemoselective methionine bioconjugation. Science 355, 597–602 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  77. Seki, Y. et al. Transition metal-free tryptophan-selective bioconjugation of proteins. J. Am. Chem. Soc. 138, 10798–10801 (2016).

    Article  CAS  PubMed  Google Scholar 

  78. Wang, L., Gruzdys, V., Pang, N., Meng, F. & Sun, X.-L. Primary arylamine-based tyrosine-targeted protein modification. RSC Adv. 4, 39446–39452 (2014).

    Article  CAS  Google Scholar 

  79. Wong, C.-Y. et al. A ruthenium(II) complex supported by trithiacyclononane and aromatic diimine ligand as luminescent switch-on probe for biomolecule detection and protein staining. Sci. Rep. 4, 7136 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  80. Solomatina, A. I. et al. Coordination to imidazole ring switches on phosphorescence of platinum cyclometalated complexes: the route to selective labeling of peptides and proteins via histidine residues. Bioconjug. Chem. 28, 426–437 (2017).

    Article  CAS  PubMed  Google Scholar 

  81. Hansen, M. B., Hubálek, F., Skrydstrup, T. & Hoeg-Jensen, T. Chemo- and regioselective ethynylation of tryptophan-containing peptides and proteins. Chemistry 22, 1572–1576 (2015).

    Article  PubMed  CAS  Google Scholar 

  82. Sato, S., Nakamura, K. & Nakamura, H. Tyrosine-specific chemical modification with in situ hemin-activated luminol derivatives. ACS Chem. Biol. 10, 2633–2640 (2015).

    Article  CAS  PubMed  Google Scholar 

  83. Ban, H., Gavrilyuk, J. & Barbas, C. F. Tyrosine bioconjugation through aqueous ene-type reactions: a click-like reaction for tyrosine. J. Am. Chem. Soc. 132, 1523–1525 (2010).

    Article  CAS  PubMed  Google Scholar 

  84. Ohata, J., Miller, M. K., Mountain, C. M., Vohidov, F. & Ball, Z. T. A. Three-component organometallic tyrosine bioconjugation. Angew. Chem. Int. Ed. 57, 2827–2830 (2018).

    Article  CAS  Google Scholar 

  85. Taylor, M. T., Nelson, J. E., Suero, M. G. & Gaunt, M. J. A protein functionalization platform based on selective reactions at methionine residues. Nature 562, 563–568 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  86. Cohen, D. T. et al. A chemoselective strategy for late-stage functionalization of complex small molecules with polypeptides and proteins. Nat. Chem. 11, 78–85 (2018).

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  87. Cui, Z. et al. Combining sense and nonsense codon reassignment for site-selective protein modification with unnatural amino acids. ACS Synth. Biol. 6, 535–544 (2017).

    Article  CAS  PubMed  Google Scholar 

  88. Xie, J. & Schultz, P. G. A chemical toolkit for proteins — an expanded genetic code. Nat. Rev. Mol. Cell. Biol. 7, 775 (2006).

    Article  CAS  PubMed  Google Scholar 

  89. Haney, C. M., Wissner, R. F. & Petersson, E. J. Multiply labeling proteins for studies of folding and stability. Curr. Opin. Chem. Biol. 28, 123–130 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  90. Wang, L., Brock, A., Herberich, B. & Schultz, P. G. Expanding the genetic code of Escherichia coli. Science 292, 498 (2001).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  92. Lee, B. S. et al. Incorporation of unnatural amino acids in response to the AGG codon. ACS Chem. Biol. 10, 1648–1653 (2015).

    Article  CAS  PubMed  Google Scholar 

  93. Iwane, Y. et al. Expanding the amino acid repertoire of ribosomal polypeptide synthesis via the artificial division of codon boxes. Nat. Chem. 8, 317 (2016).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  95. Almhjell, P. J., Boville, C. E. & Arnold, F. H. Engineering enzymes for noncanonical amino acid synthesis. Chem. Soc. Rev. 47, 8980–8997 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  96. Schmidt, M. J. & Summerer, D. Genetic code expansion as a tool to study regulatory processes of transcription. Front. Chem. 2, 7 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  97. Wals, K. & Ovaa, H. Unnatural amino acid incorporation in E. coli: current and future applications in the design of therapeutic proteins. Front. Chem. 2, 15 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  98. Nadal, S., Raj, R., Mohammed, S. & Davis, B. G. Synthetic post-translational modification of histones. Curr. Opin. Chem. Biol. 45, 35–47 (2018).

    Article  CAS  PubMed  Google Scholar 

  99. Xiao, H., Xuan, W., Shao, S., Liu, T. & Schultz, P. G. Genetic incorporation of ε-N-2-hydroxyisobutyryl-lysine into recombinant histones. ACS Chem. Biol. 10, 1599–1603 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  100. Hoppmann, C. et al. Site-specific incorporation of phosphotyrosine using an expanded genetic code. Nat. Chem. Biol. 13, 842 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  101. Wang, Z. A. et al. A Genetically encoded allysine for the synthesis of proteins with site-specific lysine dimethylation. Angew. Chem. Int. Ed. 56, 212–216 (2016).

    Article  CAS  Google Scholar 

  102. Yang, T., Li, X.-M., Bao, X., Fung, Y. M. E. & Li, X. D. Photo-lysine captures proteins that bind lysine post-translational modifications. Nat. Chem. Biol. 12, 70 (2015).

    Article  PubMed  CAS  Google Scholar 

  103. Xuan, W., Shao, S. & Schultz, P. G. Protein crosslinking by genetically encoded noncanonical amino acids with reactive aryl carbamate side chains. Angew. Chem. Int. Ed. 56, 5096–5100 (2017).

    Article  CAS  Google Scholar 

  104. Xuan, W., Li, J., Luo, X. & Schultz, P. G. Genetic incorporation of a reactive isothiocyanate group into proteins. Angew. Chem. Int. Ed. 55, 10065–10068 (2016).

    Article  CAS  Google Scholar 

  105. Xuan, W. et al. Site-specific incorporation of a thioester containing amino acid into proteins. ACS Chem. Biol. 13, 578–581 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  106. Tian, Y. et al. Genetically encoded 2-aryl-5-carboxytetrazoles for site-selective protein photo-cross-linking. J. Am. Chem. Soc. 139, 6078–6081 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  107. Yamaguchi, A. et al. Incorporation of a doubly functionalized synthetic amino acid into proteins for creating chemical and light-induced conjugates. Bioconjug. Chem. 27, 198–206 (2016).

    Article  CAS  PubMed  Google Scholar 

  108. Hoppmann, C. et al. Genetically encoding photoswitchable click amino acids in Escherichia coli and mammalian cells. Angew. Chem. Int. Ed. 53, 3932–3936 (2014).

    Article  CAS  Google Scholar 

  109. Rashidian, M., Dozier, J. K. & Distefano, M. D. Enzymatic labeling of proteins: techniques and approaches. Bioconjug. Chem. 24, 1277–1294 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  110. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  111. Li, C. & Wang, L.-X. Chemoenzymatic methods for the synthesis of glycoproteins. Chem. Rev. 118, 8359–8413 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  112. Plaks, J. G., Falatach, R., Kastantin, M., Berberich, J. A. & Kaar, J. L. Multisite clickable modification of proteins using lipoic acid ligase. Bioconjug. Chem. 26, 1104–1112 (2015).

    Article  CAS  PubMed  Google Scholar 

  113. Schumacher, D. et al. Versatile and efficient site-specific protein functionalization by tubulin tyrosine ligase. Angew. Chem. Int. Ed. 54, 13787–13791 (2015).

    Article  CAS  Google Scholar 

  114. Patterson, J. T. et al. Human serum albumin domain I fusion protein for antibody conjugation. Bioconjug. Chem. 27, 2271–2275 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  115. Sunbul, M., Nacheva, L. & Jäschke, A. Proximity-induced covalent labeling of proteins with a reactive fluorophore-binding peptide tag. Bioconjug. Chem. 26, 1466–1469 (2015).

    Article  CAS  PubMed  Google Scholar 

  116. Meyer, C., Liebscher, S. & Bordusa, F. Selective coupling of click anchors to proteins via trypsiligase. Bioconjug. Chem. 27, 47–53 (2016).

    Article  CAS  PubMed  Google Scholar 

  117. Liebscher, S. et al. N-terminal protein modification by substrate-activated reverse proteolysis. Angew. Chem. Int. Ed. 53, 3024–3028 (2014).

    Article  CAS  Google Scholar 

  118. Zhang, C. et al. π-Clamp-mediated cysteine conjugation. Nat. Chem. 8, 120–128 (2016).

    Article  PubMed  CAS  Google Scholar 

  119. Zhang, C., Dai, P., Vinogradov, A. A., Gates, Z. P. & Pentelute, B. L. Site-selective cysteine–cyclooctyne conjugation. Angew. Chem. Int. Ed. 57, 6459–6463 (2018).

    Article  CAS  Google Scholar 

  120. Ramil, C. P., An, P., Yu, Z. & Lin, Q. Sequence-specific 2-cyanobenzothiazole ligation. J. Am. Chem. Soc. 138, 5499–5502 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  121. Agrawalla, B. K. et al. Chemoselective dual labeling of native and recombinant proteins. Bioconjug. Chem. 29, 29–34 (2018).

    Article  CAS  PubMed  Google Scholar 

  122. Bellucci, J. J., Bhattacharyya, J. & Chilkoti, A. A. Noncanonical function of sortase enables site-specific conjugation of small molecules to lysine residues in proteins. Angew. Chem. Int. Ed. 54, 441–445 (2015).

    CAS  Google Scholar 

  123. Peciak, K., Laurine, E., Tommasi, R., Choi, J.-w & Brocchini, S. Site-selective protein conjugation at histidine. Chem. Sci. 10, 427–439 (2019).

    Article  CAS  PubMed  Google Scholar 

  124. Grünewald, J. et al. Optimization of an enzymatic antibody–drug conjugation approach based on coenzyme A analogs. Bioconjug. Chem. 28, 1906–1915 (2017).

    Article  PubMed  CAS  Google Scholar 

  125. Grünewald, J. et al. Efficient preparation of site-specific antibody–drug conjugates using phosphopantetheinyl transferases. Bioconjug. Chem. 26, 2554–2562 (2015).

    Article  PubMed  CAS  Google Scholar 

  126. Montanari, E. et al. Tyrosinase-mediated bioconjugation. A versatile approach to chimeric macromolecules. Bioconjug. Chem. 29, 2550–2560 (2018).

    Article  CAS  PubMed  Google Scholar 

  127. Siegmund, V. et al. Locked by design: a conformationally constrained transglutaminase tag enables efficient site-specific conjugation. Angew. Chem. Int. Ed. 54, 13420–13424 (2015).

    Article  CAS  Google Scholar 

  128. Wang, H. H., Altun, B., Nwe, K. & Tsourkas, A. Proximity-based sortase-mediated ligation. Angew. Chem. Int. Ed. 56, 5349–5352 (2017).

    Article  CAS  Google Scholar 

  129. Struck, A.-W. et al. An enzyme cascade for selective modification of tyrosine residues in structurally diverse peptides and proteins. J. Am. Chem. Soc. 138, 3038–3045 (2016).

    Article  CAS  PubMed  Google Scholar 

  130. Zhang, Y. et al. Simultaneous site-specific dual protein labeling using protein prenyltransferases. Bioconjug. Chem. 26, 2542–2553 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  131. King, M. & Wagner, A. Developments in the field of bioorthogonal bond forming reactions—past and present trends. Bioconjug. Chem. 25, 825–839 (2014).

    Article  CAS  PubMed  Google Scholar 

  132. Haldon, E., Nicasio, M. C. & Perez, P. J. Copper-catalysed azide-alkyne cycloadditions (CuAAC): an update. Org. Biomol. Chem. 13, 9528–9550 (2015).

    Article  CAS  PubMed  Google Scholar 

  133. Finbloom, J. A., Han, K., Slack, C. C., Furst, A. L. & Francis, M. B. Cucurbit[6]uril-promoted click chemistry for protein modification. J. Am. Chem. Soc. 139, 9691–9697 (2017).

    Article  CAS  PubMed  Google Scholar 

  134. Yang, M., Li, J. & Chen, P. R. Transition metal-mediated bioorthogonal protein chemistry in living cells. Chem. Soc. Rev. 43, 6511–6526 (2014).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  136. Darko, A. et al. Conformationally strained trans-cyclooctene with improved stability and excellent reactivity in tetrazine ligation. Chem. Sci. 5, 3770–3776 (2014).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  138. Borrmann, A. et al. Strain-promoted oxidation-controlled cyclooctyne−1,2-quinone cycloaddition (SPOCQ) for fast and activatable protein conjugation. Bioconjug. Chem. 26, 257–261 (2015).

    Article  CAS  PubMed  Google Scholar 

  139. Lampkowski, J. S., Villa, J. K., Young, T. S. & Young, D. D. Development and optimization of Glaser–Hay bioconjugations. Angew. Chem. Int. Ed. 54, 9343–9346 (2015).

    Article  CAS  Google Scholar 

  140. Kwan, T. T. L. et al. Protein modification via alkyne hydrosilylation using a substoichiometric amount of ruthenium(II) catalyst. Chem. Sci. 8, 3871–3878 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  142. Spears, R. J. et al. Site-selective C–C modification of proteins at neutral pH using organocatalyst-mediated cross aldol ligations. Chem. Sci. 9, 5585–5593 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  143. Kudirka, R. et al. Generating site-specifically modified proteins via a versatile and stable nucleophilic carbon ligation. Chem. Biol. 22, 293–298 (2015).

    Article  CAS  PubMed  Google Scholar 

  144. Kudirka, R. A. et al. Site-specific Tandem Knoevenagel condensation-Michael addition to generate antibody-drug conjugates. ACS Med. Chem. Lett. 7, 994–998 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  145. Wang, P. et al. Site-specific chemical modification of peptide and protein by thiazolidinediones. Org. Lett. 17, 1361–1364 (2015).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  147. Tomlin, F. M. et al. Site-specific incorporation of quadricyclane into a protein and photocleavage of the quadricyclane ligation adduct. Bioorg. Med. Chem. 26, 5280–5290 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  148. 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).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  150. Bernardes, G. J. L., Chalker, J. M., Errey, J. C. & Davis, B. G. Facile conversion of cysteine and alkyl cysteines to dehydroalanine on protein surfaces: versatile and switchable access to functionalized proteins. J. Am. Chem. Soc. 130, 5052–5053 (2008).

    Article  CAS  PubMed  Google Scholar 

  151. Wright, T. H. et al. Posttranslational mutagenesis: A chemical strategy for exploring protein side-chain diversity. Science 354, 597 (2016).

    Article  CAS  Google Scholar 

  152. Spicer, C. D. & Davis, B. G. Selective chemical protein modification. Nat. Commun. 5, 4740 (2014).

    Article  CAS  PubMed  Google Scholar 

  153. Freedy, A. M. et al. Chemoselective installation of amine bonds on proteins through Aza-Michael ligation. J. Am. Chem. Soc. 139, 18365–18375 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  154. Dadova, J. et al. Precise probing of residue roles by post-translational β, γ-C,N aza-Michael mutagenesis in enzyme active sites. ACS Cent. Sci. 3, 1168–1173 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  155. Yang, A. et al. A chemical biology route to site-specific authentic protein modifications. Science 354, 623–626 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  156. Galan, S. R. G. et al. Post-translational site-selective protein backbone α-deuteration. Nat. Chem. Biol. 14, 955–963 (2018).

    Article  CAS  PubMed  Google Scholar 

  157. Addy, P. S., Erickson, S. B., Italia, J. S. & Chatterjee, A. A. Chemoselective rapid Azo-coupling reaction (CRACR) for unclickable bioconjugation. J. Am. Chem. Soc. 139, 11670–11673 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  158. Zegota, M. M. et al. “Tag and modify” protein conjugation with dynamic covalent chemistry. Bioconjug. Chem. 29, 2665–2670 (2018).

    Article  CAS  PubMed  Google Scholar 

  159. Row, R. D. & Prescher, J. A. Constructing new bioorthogonal reagents and reactions. Acc. Chem. Res. 51, 1073–1081 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  160. Beck, A., Goetsch, L., Dumontet, C. & Corvaia, N. Strategies and challenges for the next generation of antibody-drug conjugates. Nat. Rev. Drug Discov. 16, 315–337 (2017).

    Article  CAS  PubMed  Google Scholar 

  161. Chudasama, V., Maruani, A. & Caddick, S. Recent advances in the construction of antibody-drug conjugates. Nat. Chem. 8, 114–119 (2016).

    Article  CAS  PubMed  Google Scholar 

  162. Akkapeddi, P. et al. Construction of homogeneous antibody-drug conjugates using site-selective protein chemistry. Chem. Sci. 7, 2954–2963 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  163. Kelkar, S. S. & Reineke, T. M. Theranostics: combining imaging and therapy. Bioconjug. Chem. 22, 1879–1903 (2011).

    Article  CAS  PubMed  Google Scholar 

  164. Li, X. et al. Site-specific dual antibody conjugation via engineered cysteine and selenocysteine residues. Bioconjug. Chem. 26, 2243–2248 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  165. Pelegri-O’Day, E. M., Lin, E.-W. & Maynard, H. D. Therapeutic protein–polymer conjugates: advancing beyond PEGylation. J. Am. Chem. Soc. 136, 14323–14332 (2014).

    Article  PubMed  CAS  Google Scholar 

  166. Cini, E. et al. Antibody drug conjugates (ADCs) charged with HDAC inhibitor for targeted epigenetic modulation. Chem. Sci. 9, 6490–6496 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  167. Lee, B.-C. et al. FRET reagent reveals the intracellular processing of peptide-linked antibody–drug conjugates. Bioconjug. Chem. 29, 2468–2477 (2018).

    Article  CAS  PubMed  Google Scholar 

  168. Wang, R. E. et al. An immunosuppressive antibody-drug conjugate. J. Am. Chem. Soc. 137, 3229–3232 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  169. Lehar, S. M. et al. Novel antibody-antibiotic conjugate eliminates intracellular S. aureus. Nature 527, 323–328 (2015).

    Article  CAS  PubMed  Google Scholar 

  170. Balintová, J., Welter, M. & Marx, A. Antibody–nucleotide conjugate as a substrate for DNA polymerases. Chem. Sci. 9, 7122–7125 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  171. Adumeau, P., Davydova, M. & Zeglis, B. M. Thiol-reactive bifunctional chelators for the creation of site-selectively modified radioimmunoconjugates with improved stability. Bioconjug. Chem. 29, 1364–1372 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  172. Lerchen, H.-G. et al. Antibody–drug conjugates with pyrrole-based KSP inhibitors as the payload class. Angew. Chem. Int. Ed. 57, 15243–15247 (2018).

    Article  CAS  Google Scholar 

  173. Kelemen, R. E. et al. A precise chemical strategy to alter the receptor specificity of the adeno-associated virus. Angew. Chem. Int. Ed. 55, 10645–10649 (2016).

    Article  CAS  Google Scholar 

  174. Greineder, C. F. et al. Site-specific modification of single-chain antibody fragments for bioconjugation and vascular immunotargeting. Bioconjug. Chem. 29, 56–66 (2018).

    Article  CAS  PubMed  Google Scholar 

  175. Wu, T. et al. Transglutaminase mediated PEGylation of nanobodies for targeted nano-drug delivery. J. Mater. Chem. B 6, 1011–1017 (2018).

    Article  CAS  PubMed  Google Scholar 

  176. Brasino, M. et al. Anti-EGFR Affibodies with site-specific photo-cross-linker incorporation show both directed target-specific photoconjugation and increased retention in tumors. J. Am. Chem. Soc. 140, 11820–11828 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  177. Kuan, S. L. et al. Boosting antitumor drug efficacy with chemically engineered multidomain proteins. Adv. Sci. 5, 1701036 (2018).

    Article  CAS  Google Scholar 

  178. Jones, L. H. Recent advances in the molecular design of synthetic vaccines. Nat. Chem. 7, 952 (2015).

    Article  CAS  PubMed  Google Scholar 

  179. Schoonen, L., Pille, J., Borrmann, A., Nolte, R. J. M. & van Hest, J. C. M. Sortase A-mediated N-terminal modification of Cowpea Chlorotic Mottle virus for highly efficient cargo loading. Bioconjug. Chem. 26, 2429–2434 (2015).

    Article  CAS  PubMed  Google Scholar 

  180. Kobayashi, T., Hoppmann, C., Yang, B. & Wang, L. Using protein-confined proximity to determine chemical reactivity. J. Am. Chem. Soc. 138, 14832–14835 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  181. Strumillo, M. & Beltrao, P. Towards the computational design of protein post-translational regulation. Bioorg. Med. Chem. 23, 2877–2882 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  182. Wagner, J. A., Mercadante, D., Nikic, I., Lemke, E. A. & Gräter, F. Origin of orthogonality of strain-promoted click reactions. Chemistry 21, 12431–12435 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  183. Narayanam, M. K., Liang, Y., Houk, K. N. & Murphy, J. M. Discovery of new mutually orthogonal bioorthogonal cycloaddition pairs through computational screening. Chem. Sci. 7, 1257–1261 (2016).

    Article  CAS  PubMed  Google Scholar 

  184. Knall, A.-C., Hollauf, M., Saf, R. & Slugovc, C. A trifunctional linker suitable for conducting three orthogonal click chemistries in one pot. Org. Biomol. Chem. 14, 10576–10580 (2016).

  185. Bryden, F. et al. Assembly of high-potency photosensitizer–antibody conjugates through application of dendron multiplier technology. Bioconjug. Chem. 29, 176–181 (2018).

    Article  CAS  PubMed  Google Scholar 

  186. Cazzamalli, S., Dal Corso, A., Widmayer, F. & Neri, D. Chemically defined antibody– and small molecule–drug conjugates for in vivo tumor targeting applications: a comparative analysis. J. Am. Chem. Soc. 140, 1617–1621 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

The authors thank the Foundation for Science and Technology (FCT) Portugal (FCT Investigator to G.J.L.B., IF/00624/2015 and Postdoctoral Fellowship to P.M.S.D.C.), the Herchel Smith Fund (PhD Studentship to E.A.H.) and the European Union Horizon 2020 programme (Marie Skłodowska-Curie ITN grant agreement no. 675007 to G.J.L.B. and Marie Skłodowska-Curie IEF grant agreement no. 702574 to B.L.O.) for funding. G.J.L.B. is a Royal Society University Research Fellow (UF110046 and URF\R\180019) and the recipient of a European Research Council Starting Grant (TagIt, grant agreement no. 676832).

Author information

Authors and Affiliations

  1. Department of Chemistry, University of Cambridge, Cambridge, UK

    Emily A. Hoyt, Bruno L. Oliveira & Gonçalo J. L. Bernardes

  2. Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisbon, Portugal

    Pedro M. S. D. Cal, Bruno L. Oliveira & Gonçalo J. L. Bernardes

Authors
  1. Emily A. Hoyt
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Pedro M. S. D. Cal
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Bruno L. Oliveira
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Gonçalo J. L. Bernardes
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

All authors made substantial contributions to the discussion and organization of the content as well as reviewed and/or edited the manuscript before submission. Additionally, E.A.H. conducted the research, wrote the main body of the paper, edited the figures and put the complete manuscript together; P.M.S.D.C. played an integral role in writing the introduction, researching and providing content guidance at all stages of manuscript preparation; and B.L.O. designed and created the figures. G.J.L.B. coordinated the research and writing.

Corresponding author

Correspondence to Gonçalo J. L. Bernardes.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note

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

Glossary

Post-translational modification

(PTM). A post-translational, covalent protein modification that has critical roles in cell signalling and control of protein activation or function.

Site-selective

Referring to modification methods that target a certain residue over other types of amino acids.

Homogeneous product

A single product — type and position of modification — is formed. Conversely, heterogeneous products are those in which different and multiple modifications have occurred, including on different amino acids or at various occurrences of the same amino acid type.

Site-specific

Referring to modification methods that target a single occurrence of a particular type of amino acid.

Protein microenvironment

The manipulation of amino acid side chain properties (for example, steric or electric characteristics) and reactivity based on the identity of surrounding amino acids in the protein sequence.

Human insulin

A protein that is made up of two separate chains of amino acids labelled A and B, bound together by two disulfide bridges.

Canonical amino acids

The standard 20 amino acid types encoded and inserted naturally by the genetic code and by native protein biosynthesis systems.

SH3-domain proteins

Proteins that contain SH3 domains for the regulation of cytoplasmic signalling pathways.

Antibodies

Proteins that are composed of two main regions: Fc regions (constant regions for the support and stability of the antibody) and Fab regions (variable regions of the antibody that must be preserved in order to retain affinity and specificity for a corresponding antigen).

Disulfide rebridging

A process by which two Cys residues, revealed by disulfide reduction, reform the disrupted disulfide either through the construction of a mixed disulfide or through the introduction of a synthetic stapling molecule to connect the two residues.

Conjugate payload

The chemical linker and added functionality (for example, fluorophore or cytotoxic drug) in a protein conjugate.

Noncanonical amino acids

(ncAAs). Amino acids that are most often synthesized and non-proteinogenic (with the exception of Sec and Pyl) and can be inserted either residue-specifically or site-specifically into protein sequences.

Endogenous residues

Amino acid residues that are synthesized by the host organism rather than by artificial synthesis.

Click chemistry

Chemical reactions that can be defined by high reaction and conversion rate, green solvent systems, low by-product levels and broad functional group applicability.

Bioorthogonal reactions

Chemical reactions that can be executed in the complex environment of living systems (that is, in the presence of many nucleophiles, reductants and so on) without altering or affecting native processes.

Orthogonal aminoacyl-tRNA synthetase–tRNA pairs

These orthogonal pairs can use native protein biosynthesis machinery for the site-specific insertion of noncanonical amino acids and require that no native RNA synthetase be able to aminoacylate the incorporated tRNA and no native tRNA be modified by the incorporated RS.

Fusion proteins

Proteins that are produced by combining parts from different proteins or proteins with smaller amino acid sequences/tags to create one expressed entity.

Upconversion nanoparticles

Nanoscale particles that allow for photon upconversion (the absorption of two lower-energy photons to create one higher-energy, emitted photon) for imaging and sensors in deep tissue environments.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Hoyt, E.A., Cal, P.M.S.D., Oliveira, B.L. et al. Contemporary approaches to site-selective protein modification. Nat Rev Chem 3, 147–171 (2019). https://doi.org/10.1038/s41570-019-0079-1

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41570-019-0079-1

This article is cited by

Search

Advanced 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