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

Journal name:
Nature Chemistry
Year published:
Published online


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


  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.


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[,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[,9]hexadeca-1(16),4,6,8,12,14-hexaen-10-yn-2-yloxy}acetamide


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Author information

  1. These authors contributed equally to this work

    • Kathrin Lang &
    • Lloyd Davis


  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


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.

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