Genetically encoded norbornene directs site-specific cellular protein labelling via a rapid bioorthogonal reaction

Journal name:
Nature Chemistry
Volume:
4,
Pages:
298–304
Year published:
DOI:
doi:10.1038/nchem.1250
Received
Accepted
Published online

Abstract

The site-specific incorporation of bioorthogonal groups via genetic code expansion provides a powerful general strategy for site-specifically labelling proteins with any probe. However, the slow reactivity of the bioorthogonal functional groups that can be encoded genetically limits the utility of this strategy. We demonstrate the genetic encoding of a norbornene amino acid using the pyrrolysyl tRNA synthetase/tRNACUA pair in Escherichia coli and mammalian cells. We developed a series of tetrazine-based probes that exhibit ‘turn-on’ fluorescence on their rapid reaction with norbornenes. We demonstrate that the labelling of an encoded norbornene is specific with respect to the entire soluble E. coli proteome and thousands of times faster than established encodable bioorthogonal reactions. We show explicitly the advantages of this approach over state-of-the-art bioorthogonal reactions for protein labelling in vitro and on mammalian cells, and demonstrate the rapid bioorthogonal site-specific labelling of a protein on the mammalian cell surface.

At a glance

Figures

  1. Scheme to label proteins via an inverse electron-demand Diels–Alder cycloaddition, and structural formulae of relevant compounds.
    Figure 1: Scheme to label proteins via an inverse electron-demand Diels–Alder cycloaddition, and structural formulae of relevant compounds.

    a, Genetically encoded norbornenes react rapidly with tetrazines, bearing probes (red star), in aqueous solution at ambient temperature and pressure to site-specifically label proteins. b, Amino acid structures of pyrrolysine (1), Nɛ-5-norbornene-2-yloxycarbonyl-L-lysine (2), Nɛ-tert-butyloxycarbonyl-L-lysine (3) and Nɛ-2-azidoethyloxycarbonyl-L-lysine (4). c, Structures (514) of tetrazines and tetrazine–fluorophores used in this study. TAMRA-X, BODIPY TMR-X and BODIPY-FL are common names for fluorophores: their structural formulae are shown in Supplementary Fig. S4. Red boxes denote parent tetrazines. r.t. = room temperature.

  2. Efficient, genetically-encoded incorporation of 2 using the PylRS/tRNACUA pair in E. coli.
    Figure 2: Efficient, genetically-encoded incorporation of 2 using the PylRS/tRNACUA pair in E. coli.

    a, Amino acid dependent expression of sfGFP that bears an amber codon at position 150 and myoglobin that bears an amber codon at position 4. b, Mass spectrometry characterization of amino acid incorporation. i, sfGFP-2-His6, found: 27,975.5 ± 1.5 Da, calculated: 27,977.5 Da. ii, Myo-2-His6, found: 18,532.5 ± 1.5 Da, calculated: 18,532.2 Da.

  3. Characterization of tetrazine–norbornene reactions.
    Figure 3: Characterization of tetrazine–norbornene reactions.

    a, ‘Turn-on’ fluorescence of tetrazine fluorophores 9 (i) and 13 (ii) on reaction with 5-norbornene-2-ol (Nor). b, Specific and quantitative labelling of sfGFP that bears 2, demonstrated by SDS–PAGE (Coomassie staining and in-gel fluorescence) (i) and mass spectrometry (ii) before bioconjugation (red spectrum, found 27,975.5 ± 1.5 Da, expected 27,977.5 Da) and after bioconjugation (blue spectrum, found 28,783.0 ± 1.5 Da, expected 28,784.4 Da). c, Specificity of labelling 2 in sfGFP versus the E. coli proteome. Lanes 1–5: Coomassie-stained gel showing proteins from E. coli producing sfGFP in the presence of the indicated concentration of unnatural amino acids 2 or 3. Lanes 6–10: The expressed protein was detected in lysates using an anti-His6 antibody. Lanes 11–20: fluorescence images of protein labelled with the indicated fluorophore 12 or 13. d, Labelling of myoglobin that bears 2 at position 4 with fluorophore 12: fluorescence imaging (top) and Coomassie-stained loading control (bottom).

  4. Site-specific incorporation of 2 into proteins in mammalian cells and the specific labelling of EGFR-GFP on the cell surface with 9.
    Figure 4: Site-specific incorporation of 2 into proteins in mammalian cells and the specific labelling of EGFR-GFP on the cell surface with 9.

    a, Cells that contain the PylRS/tRNACUA pair and the mCherry(TAG)eGFP-HA reporter produced GFP only in the presence of 2. b, Western blots confirm that the expression of full length mCherry(TAG)eGFP-HA is dependent on the presence of 2. c, Specific and rapid labelling of a cell surface protein in live mammalian cells. EGFR-GFP that bears 2 or 3 at position 128 is visible as green fluorescence at the membrane of transfected cells (left panels). Treatment of cells with 9 (200 nM) leads to selective labelling of EGFR that contains 2 (middle panels). Right panels show merged green and red fluorescence images, DIC = differential interference contrast. Cells were imaged four hours after the addition of 9.

Compounds

23 compounds View all compounds
  1. (S)-2-Amino-6-((2R,3R)-3-methyl-3,4-dihydro-2H-pyrrole-2-carboxamido)hexanoic acid
    Compound 1 (S)-2-Amino-6-((2R,3R)-3-methyl-3,4-dihydro-2H-pyrrole-2-carboxamido)hexanoic acid
  2. (S)-2-Amino-6-((((1R,4R)-bicyclo[2.2.1]hept-5-en-2-yloxy)carbonyl)amino)hexanoic acid
    Compound 2 (S)-2-Amino-6-((((1R,4R)-bicyclo[2.2.1]hept-5-en-2-yloxy)carbonyl)amino)hexanoic acid
  3. (S)-2-Amino-6-((tert-butoxycarbonyl)amino)hexanoic acid
    Compound 3 (S)-2-Amino-6-((tert-butoxycarbonyl)amino)hexanoic acid
  4. (S)-2-Amino-6-(((2-azidoethoxy)carbonyl)amino)hexanoic acid
    Compound 4 (S)-2-Amino-6-(((2-azidoethoxy)carbonyl)amino)hexanoic acid
  5. tert-Butyl (2-oxo-2-((6-(6-(pyridin-2-yl)-1,2,4,5-tetrazin-3-yl)pyridin-3-yl)amino)ethyl)carbamate
    Compound 5 tert-Butyl (2-oxo-2-((6-(6-(pyridin-2-yl)-1,2,4,5-tetrazin-3-yl)pyridin-3-yl)amino)ethyl)carbamate
  6. tert-Butyl (2-oxo-2-((6-(6-(pyrimidin-2-yl)-1,2,4,5-tetrazin-3-yl)pyridin-3-yl)amino)ethyl)carbamate
    Compound 6 tert-Butyl (2-oxo-2-((6-(6-(pyrimidin-2-yl)-1,2,4,5-tetrazin-3-yl)pyridin-3-yl)amino)ethyl)carbamate
  7. tert-Butyl (2-(6-(6-methyl-1,2,4,5-tetrazin-3-yl)nicotinamido)ethyl)carbamate
    Compound 7 tert-Butyl (2-(6-(6-methyl-1,2,4,5-tetrazin-3-yl)nicotinamido)ethyl)carbamate
  8. tert-Butyl (2-(6-(6-(pyrimidin-2-yl)-1,2,4,5-tetrazin-3-yl)nicotinamido)ethyl)carbamate
    Compound 8 tert-Butyl (2-(6-(6-(pyrimidin-2-yl)-1,2,4,5-tetrazin-3-yl)nicotinamido)ethyl)carbamate
  9. 5,5-Difluoro-7-(4-methoxyphenyl)-1,3-dimethyl-2-(3-oxo-3-((6-oxo-6-((2-oxo-2-((6-(6-(pyridin-2-yl)-1,2,4,5-tetrazin-3-yl)pyridin-3-yl)amino)ethyl)amino)hexyl)amino)propyl)-5H-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinin-4-ium-5-uide
    Compound 11 5,5-Difluoro-7-(4-methoxyphenyl)-1,3-dimethyl-2-(3-oxo-3-((6-oxo-6-((2-oxo-2-((6-(6-(pyridin-2-yl)-1,2,4,5-tetrazin-3-yl)pyridin-3-yl)amino)ethyl)amino)hexyl)amino)propyl)-5H-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinin-4-ium-5-uide
  10. 5,5-Difluoro-7,9-dimethyl-3-(3-oxo-3-((2-(6-(6-(pyrimidin-2-yl)-1,2,4,5-tetrazin-3-yl)nicotinamido)ethyl)amino)propyl)-5H-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinin-4-ium-5-uide
    Compound 13 5,5-Difluoro-7,9-dimethyl-3-(3-oxo-3-((2-(6-(6-(pyrimidin-2-yl)-1,2,4,5-tetrazin-3-yl)nicotinamido)ethyl)amino)propyl)-5H-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinin-4-ium-5-uide
  11. (1R,4R)-Bicyclo[2.2.1]hept-5-en-2-yl (2,5-dioxopyrrolidin-1-yl) carbonoperoxoate
    Compound S2a (1R,4R)-Bicyclo[2.2.1]hept-5-en-2-yl (2,5-dioxopyrrolidin-1-yl) carbonoperoxoate
  12. (S)-6-((((1R,4R)-Bicyclo[2.2.1]hept-5-en-2-yloxy)carbonyl)amino)-2-((tert-butoxycarbonyl)amino)hexanoic acid
    Compound S2b (S)-6-((((1R,4R)-Bicyclo[2.2.1]hept-5-en-2-yloxy)carbonyl)amino)-2-((tert-butoxycarbonyl)amino)hexanoic acid
  13. 6-(6-(Pyridin-2-yl)-1,4-dihydro-1,2,4,5-tetrazin-3-yl)pyridin-3-amine
    Compound S5a 6-(6-(Pyridin-2-yl)-1,4-dihydro-1,2,4,5-tetrazin-3-yl)pyridin-3-amine
  14. tert-Butyl (2-oxo-2-((6-(6-(pyridin-2-yl)-1,4-dihydro-1,2,4,5-tetrazin-3-yl)pyridin-3-yl)amino)ethyl)carbamate
    Compound S5b tert-Butyl (2-oxo-2-((6-(6-(pyridin-2-yl)-1,4-dihydro-1,2,4,5-tetrazin-3-yl)pyridin-3-yl)amino)ethyl)carbamate
  15. 2-Oxo-2-((6-(6-(pyridin-2-yl)-1,2,4,5-tetrazin-3-yl)pyridin-3-yl)amino)ethanaminium chloride
    Compound S5c 2-Oxo-2-((6-(6-(pyridin-2-yl)-1,2,4,5-tetrazin-3-yl)pyridin-3-yl)amino)ethanaminium chloride
  16. 6-(6-(Pyrimidin-2-yl)-1,4-dihydro-1,2,4,5-tetrazin-3-yl)pyridin-3-amine
    Compound S6a 6-(6-(Pyrimidin-2-yl)-1,4-dihydro-1,2,4,5-tetrazin-3-yl)pyridin-3-amine
  17. tert-Butyl (2-oxo-2-((6-(6-(pyrimidin-2-yl)-1,4-dihydro-1,2,4,5-tetrazin-3-yl)pyridin-3-yl)amino)ethyl)carbamate
    Compound S6b tert-Butyl (2-oxo-2-((6-(6-(pyrimidin-2-yl)-1,4-dihydro-1,2,4,5-tetrazin-3-yl)pyridin-3-yl)amino)ethyl)carbamate
  18. 2-Oxo-2-((6-(6-(pyrimidin-2-yl)-1,2,4,5-tetrazin-3-yl)pyridin-3-yl)amino)ethanaminium chloride
    Compound S6c 2-Oxo-2-((6-(6-(pyrimidin-2-yl)-1,2,4,5-tetrazin-3-yl)pyridin-3-yl)amino)ethanaminium chloride
  19. tert-Butyl (2-(6-cyanonicotinamido)ethyl)carbamate
    Compound S7a tert-Butyl (2-(6-cyanonicotinamido)ethyl)carbamate
  20. tert-Butyl (2-(6-(6-methyl-1,4-dihydro-1,2,4,5-tetrazin-3-yl)nicotinamido)ethyl)carbamate
    Compound S7b tert-Butyl (2-(6-(6-methyl-1,4-dihydro-1,2,4,5-tetrazin-3-yl)nicotinamido)ethyl)carbamate
  21. tert-Butyl (2-(6-(6-(pyrimidin-2-yl)-1,4-dihydro-1,2,4,5-tetrazin-3-yl)nicotinamido)ethyl)carbamate
    Compound S8b tert-Butyl (2-(6-(6-(pyrimidin-2-yl)-1,4-dihydro-1,2,4,5-tetrazin-3-yl)nicotinamido)ethyl)carbamate
  22. 2-(6-(6-(Pyrimidin-2-yl)-1,2,4,5-tetrazin-3-yl)nicotinamido)ethanaminium chloride
    Compound S8c 2-(6-(6-(Pyrimidin-2-yl)-1,2,4,5-tetrazin-3-yl)nicotinamido)ethanaminium chloride
  23. N-{2-[3',6'-Bis(dimethylamino)-3-oxo-3H-spiro[2-benzofuran-1,9'-xanthene]-5-ylformamido]ethyl}-2-{tricyclo[10.4.0.04,9]hexadeca-1(16),4,6,8,12,14-hexaen-10-yn-2-yloxy}acetamide
    Compound S17 N-{2-[3',6'-Bis(dimethylamino)-3-oxo-3H-spiro[2-benzofuran-1,9'-xanthene]-5-ylformamido]ethyl}-2-{tricyclo[10.4.0.04,9]hexadeca-1(16),4,6,8,12,14-hexaen-10-yn-2-yloxy}acetamide

References

  1. Chalfie, M., Tu, Y., Euskirchen, G., Ward, W. W. & Prasher, D. C. Green fluorescent protein as a marker for gene expression. Science 263, 802805 (1994).
  2. Heim, R., Prasher, D. C. & Tsien, R. Y. Wavelength mutations and posttranslational autoxidation of green fluorescent protein. Proc. Natl Acad. Sci. USA 91, 1250112504 (1994).
  3. Giepmans, B. N., Adams, S. R., Ellisman, M. H. & Tsien, R. Y. The fluorescent toolbox for assessing protein location and function. Science 312, 217224 (2006).
  4. Shaner, N. C., Steinbach, P. A. & Tsien, R. Y. A guide to choosing fluorescent proteins. Nature Methods 2, 905909 (2005).
  5. Los, G. V. et al. HaloTag: a novel protein labeling technology for cell imaging and protein analysis. ACS Chem. Biol. 3, 373382 (2008).
  6. Keppler, A. et al. A general method for the covalent labeling of fusion proteins with small molecules in vivo. Nature Biotechnol. 21, 8689 (2003).
  7. Kosaka, N. et al. In vivo stable tumor-specific painting in various colors using dehalogenase-based protein-tag fluorescent ligands. Bioconjug. Chem. 20, 13671374 (2009).
  8. Gautier, A. et al. An engineered protein tag for multiprotein labeling in living cells. Chem. Biol. 15, 128136 (2008).
  9. George, N., Pick, H., Vogel, H., Johnsson, N. & Johnsson, K. Specific labeling of cell surface proteins with chemically diverse compounds. J. Am. Chem. Soc. 126, 88968897 (2004).
  10. Zhou, Z., Koglin, A., Wang, Y., McMahon, A. P. & Walsh, C. T. An eight residue fragment of an acyl carrier protein suffices for post-translational introduction of fluorescent pantetheinyl arms in protein modification in vitro and in vivo. J. Am. Chem. Soc. 130, 99259930 (2008).
  11. Yin, J. et al. Genetically encoded short peptide tag for versatile protein labeling by Sfp phosphopantetheinyl transferase. Proc. Natl Acad. Sci. USA 102, 15815158120 (2005).
  12. Fernandez-Suarez, M. et al. Redirecting lipoic acid ligase for cell surface protein labeling with small-molecule probes. Nature Biotechnol. 25, 14831487 (2007).
  13. Uttamapinant, C. et al. A fluorophore ligase for site-specific protein labeling inside living cells. Proc. Natl Acad. Sci. USA 107, 1091410919 (2010).
  14. Popp, M. W., Antos, J. M., Grotenbreg, G. M., Spooner, E. & Ploegh, H. L. Sortagging: a versatile method for protein labeling. Nature Chem. Biol. 3, 70778 (2007).
  15. 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, 1080010801 (2009).
  16. Griffin, B. A., Adams, S. R. & Tsien, R. Y. Specific covalent labeling of recombinant protein molecules inside live cells. Science 281, 269272 (1998).
  17. Halo, T. L., Appelbaum, J., Hobert, E. M., Balkin, D. M. & Schepartz, A. Selective recognition of protein tetraserine motifs with a cell-permeable, pro-fluorescent bis-boronic acid. J. Am. Chem. Soc. 131, 438439 (2009).
  18. Hinner, M. J. & Johnsson, K. How to obtain labeled proteins and what to do with them. Curr. Opin. Biotechnol. 21, 766776 (2010).
  19. Chin, J. W. et al. Addition of p-azido-L-phenylalanine to the genetic code of Escherichia coli. J. Am. Chem. Soc. 124, 90269027 (2002).
  20. Zhang, Z., Wang, L., Brock, A. & Schultz, P. G. The selective incorporation of alkenes into proteins in Escherichia coli. Angew. Chem. Int. Ed. 41, 28402842 (2002).
  21. Chin, J. W. et al. An expanded eukaryotic genetic code. Science 301, 964967 (2003).
  22. Deiters, A. et al. Adding amino acids with novel reactivity to the genetic code of Saccharomyces cerevisiae. J. Am. Chem. Soc. 125, 1178211783 (2003).
  23. Deiters, A., Cropp, T. A., Summerer, D., Mukherji, M. & Schultz, P. G. Site-specific PEGylation of proteins containing unnatural amino acids. Bioorg. Med. Chem. Lett. 14, 57435745 (2004).
  24. Mehl, R. A. et al. Generation of a bacterium with a 21 amino acid genetic code. J. Am. Chem. Soc. 125, 935939 (2003).
  25. Wang, L., Zhang, Z., Brock, A. & Schultz, P. G. Addition of the keto functional group to the genetic code of Escherichia coli. Proc. Natl Acad. Sci. USA 100, 5661 (2003).
  26. Carrico, Z. M., Romanini, D. W., Mehl, R. A. & Francis, M. B. Oxidative coupling of peptides to a virus capsid containing unnatural amino acids. Chem. Commun. 12051207 (2008).
  27. Fekner, T., Li, X., Lee, M. M. & Chan, M. K. A pyrrolysine analogue for protein click chemistry. Angew. Chem. Int. Ed. 48, 16331635 (2009).
  28. Nguyen, D. P. et al. Genetic encoding and labeling of aliphatic azides and alkynes in recombinant proteins via a pyrrolysyl-tRNA synthetase/tRNA(CUA) pair and click chemistry. J. Am. Chem. Soc. 131, 87208721 (2009).
  29. 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, 53305333 (2009).
  30. Agard, N. J., Baskin, J. M., Prescher, J. A., Lo, A. & Bertozzi, C. R. A comparative study of bioorthogonal reactions with azides. ACS Chem. Biol. 1, 644648 (2006).
  31. Wang, J. et al. A biosynthetic route to photoclick chemistry on proteins. J. Am. Chem. Soc. 132, 1481214818 (2010).
  32. Nguyen, D. P., Elliott, T., Holt, M., Muir, T. W. & Chin, J. W. Genetically encoded 1,2-aminothiols facilitate rapid and site-specific protein labeling via a bio-orthogonal cyanobenzothiazole condensation. J. Am. Chem. Soc. 133, 1141811421 (2011).
  33. Laughlin, S. T. & Bertozzi, C. R. Imaging the glycome. Proc. Natl Acad. Sci. USA 106, 1217 (2009).
  34. Prescher, J. A. & Bertozzi, C. R. Chemical technologies for probing glycans. Cell 126, 851854 (2006).
  35. Johnson, J. A., Lu, Y. Y., Van Deventer, J. A. & Tirrell, D. A. Residue-specific incorporation of non-canonical amino acids into proteins: recent developments and applications. Curr. Opin. Biotechnol. 14, 774780 (2010).
  36. 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, 1351813519 (2008).
  37. Devaraj, N. K., Weissleder, R. & Hilderbrand, S. A. Tetrazine-based cycloadditions: application to pretargeted live cell imaging. Bioconjug. Chem. 19, 22972299 (2008).
  38. Devaraj, N. K. & Weissleder, R. Biomedical applications of tetrazine cycloadditions. Acc. Chem. Res. 44, 816827 (2011).
  39. Mukai, T. et al. Adding L-lysine derivatives to the genetic code of mammalian cells with engineered pyrrolysyl-tRNA synthetases. Biochem. Biophys. Res. Commun. 371, 818822 (2008).
  40. Neumann, H., Peak-Chew, S. Y. & Chin, J. W. Genetically encoding N(epsilon)-acetyllysine in recombinant proteins. Nature Chem. Biol. 4, 232234 (2008).
  41. Hancock, S. M., Uprety, R., Deiters, A. & Chin, J. W. Expanding the genetic code of yeast for incorporation of diverse unnatural amino acids via a pyrrolysyl-tRNA synthetase/tRNA pair. J. Am. Chem. Soc. 132, 1481914824 (2010).
  42. Greiss, S. & Chin, J. W. Expanding the genetic code of an animal. J. Am. Chem. Soc. 133, 1419614199 (2011).
  43. Polycarpo, C. R. et al. Pyrrolysine analogues as substrates for pyrrolysyl-tRNA synthetase. FEBS Lett. 580, 66956700 (2006).
  44. Li, X., Fekner, T., Ottesen, J. J. & Chan, M. K. A pyrrolysine analogue for site-specific protein ubiquitination. Angew. Chem. Int. Ed. 48, 91849187 (2009).
  45. Nguyen, D. P., Garcia Alai, M. M., Kapadnis, P. B., Neumann, H. & Chin, J. W. Genetically encoding N(epsilon)-methyl-L-lysine in recombinant histones. J. Am. Chem. Soc. 131, 1419414195 (2009).
  46. Gautier, A. et al. Genetically encoded photocontrol of protein localization in mammalian cells. J. Am. Chem. Soc. 132, 40864088 (2010).
  47. Wijinen, J. W., Zavarise, S., Engberts, J. B. F. N, Cahrton, Ml. J. Substituent effects on an inverse electron demand hetero Diels–Alder reaction in aqueous solution and organic solvents: cycloaddition of substituted styrenes to di(2-pyridyl)-1,2,4,5-tetrazine. J. Org. Chem. 61, 20012005 (1996).
  48. 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, 28692872 (2010).

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Author information

  1. These authors contributed equally to this work

    • Kathrin Lang &
    • Lloyd Davis

Affiliations

  1. Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, UK

    • Kathrin Lang,
    • Lloyd Davis &
    • Jason W. Chin
  2. Department of Chemistry, North Carolina State University, Raleigh, North Carolina 27695-8204, USA

    • Jessica Torres-Kolbus,
    • Chungjung Chou &
    • Alexander Deiters

Contributions

K.L, L.D. J.T.K., C.C., A.D. & J.W.C. designed the research and analysed the data. K.L, L.D., J.T.K. and C.C. performed the experiments. K.L. and J.W.C co-wrote the paper with input from the co-workers.

Competing financial interests

The authors declare no competing financial interests.

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