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, 134 (25), 10317-20

Genetic Encoding of Bicyclononynes and Trans-Cyclooctenes for Site-Specific Protein Labeling in Vitro and in Live Mammalian Cells via Rapid Fluorogenic Diels-Alder Reactions


Genetic Encoding of Bicyclononynes and Trans-Cyclooctenes for Site-Specific Protein Labeling in Vitro and in Live Mammalian Cells via Rapid Fluorogenic Diels-Alder Reactions

Kathrin Lang et al. J Am Chem Soc.


Rapid, site-specific labeling of proteins with diverse probes remains an outstanding challenge for chemical biologists. Enzyme-mediated labeling approaches may be rapid but use protein or peptide fusions that introduce perturbations into the protein under study and may limit the sites that can be labeled, while many "bioorthogonal" reactions for which a component can be genetically encoded are too slow to effect quantitative site-specific labeling of proteins on a time scale that is useful for studying many biological processes. We report a fluorogenic reaction between bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN) and tetrazines that is 3-7 orders of magnitude faster than many bioorthogonal reactions. Unlike the reactions of strained alkenes, including trans-cyclooctenes and norbornenes, with tetrazines, the BCN-tetrazine reaction gives a single product of defined stereochemistry. We have discovered aminoacyl-tRNA synthetase/tRNA pairs for the efficient site-specific incorporation of a BCN-containing amino acid, 1, and a trans-cyclooctene-containing amino acid 2 (which also reacts extremely rapidly with tetrazines) into proteins expressed in Escherichia coli and mammalian cells. We demonstrate the rapid fluorogenic labeling of proteins containing 1 and 2 in vitro, in E. coli , and in live mammalian cells. These approaches may be extended to site-specific protein labeling in animals, and we anticipate that they will have a broad impact on labeling and imaging studies.


Scheme 1
Scheme 1. Genetic Encoding and Fluorogenic Labeling of Unnatural Amino Acids 1 and 2
Figure 1
Figure 1
Structures of unnatural amino acids 15 and tetrazine derivatives 617 used in this study. For the structures of TAMRA-X, Bodipy TMR-X, Bodipy-FL, and CFDA, see Figure S4.
Figure 2
Figure 2
Characterization of the reaction of BCN with 7. (a) Stopped-flow kinetics of the reaction. The inset shows the conjugation of 7 to 5-norbornene-2-ol (Nor); the different time scales should be noted. Conditions: c7 = 0.05 mM and cBCN = cNor = 5 mM in 55:45 MeOH/H2O at 25 °C. (b) Determination of the second-order rate constant k for the reaction of 7 and BCN. (c) Fluorogenic reaction of 11 with BCN.
Figure 3
Figure 3
Efficient genetically encoded incorporation of unnatural amino acids in E. coli. (a) Amino acid-dependent overexpression of sfGFP-His6 bearing an amber codon at position 150. The expressed protein was detected in lysates using an anti-His6 antibody and Coomassie staining. (b) Coomassie-stained gel showing purified proteins. (c–e) ESI-MS data for amino acid incorporation. For sfGFP-1-His6: found, 28017.54 Da; calcd, 28017.62 Da. For sfGFP-2-His6: found, 27993.36 Da; calcd, 27992.82 Da. For sfGFP-His6 produced with 3 as described: found, 28019.34 Da; calcd, 28019.63 Da. The minor peaks in the mass spectra correspond to loss of the N-terminal methionine.
Figure 4
Figure 4
Rapid and specific labeling of recombinant proteins with tetrazine–fluorophore conjugates. (a) Specific labeling of sfGFP bearing 1, 2, or 4 with 11 (10 equiv) demonstrated by SDS-PAGE and in-gel fluorescence. (b) Quantitative labeling of sfGFP-1 with 11 demonstrated by ESI-MS. Before bioconjugation (blue): found, 28018.1 Da; calcd, 28017.6 Da. After bioconjugation (red): found, 28824.2 Da; calcd, 28823.2 Da. (c) Quantitative labeling of sfGFP-2 with 11 demonstrated by ESI-MS. Before bioconjugation (blue): found, 27993.2 Da; calcd, 27992.8 Da. After bioconjugation (red): found, 28799.4 Da; calcd, 28799.1 Da. (d) No labeling with 11 of sfGFP-His6 expressed in the presence of 3 could be detected by MS. (e) Very rapid labeling of proteins containing 1 or 2.
Figure 5
Figure 5
Site-specific incorporation of 1 and 2 into proteins in mammalian cells and their rapid and specific labeling with tetrazine fluorophores. (a) Western blots demonstrate that the expression of full-length mCherry(TAG)eGFP-HA is dependent on the presence of 1 or 2 and tRNACUA. BCNRS and TCORS were FLAG-tagged. (b) Specific and ultrarapid labeling of a cell-surface protein in live mammalian cells. Left: EGFR-GFP bearing 1, 2, or 5 at position 128 is visible as green fluorescence at the membrane of transfected cells. Middle: treatment of cells with 11 (400 nM) selectively labels EGFR containing 1 or 2. Right: merged green and red fluorescence images with differential interference contrast (DIC). Cells were imaged 2 min after the addition of 11. (c) Specific and rapid labeling of a nuclear protein in live mammalian cells. Left: jun-1-mCherry and jun-5-mCherry are visible as red fluorescence in the nuclei of transfected cells. Middle: Selective labeling of jun-1-mCherry with 17 (200 nM). Right: merged red and green fluorescence with DIC. No labeling was observed for cells bearing jun-5-mCherry.

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