Spirohexene-Tetrazine Ligation Enables Bioorthogonal Labeling of Class B G Protein-Coupled Receptors in Live Cells

Carlo P. Ramil, Maoqing Dong, Peng An, Tracey M. Lewandowski, Zhipeng Yu, Laurence J. Miller, Qing Lin

Research output: Contribution to journalArticlepeer-review

39 Scopus citations


A new bioorthogonal reactant pair, spiro[2.3]hex-1-ene (Sph) and 3,6-di(2-pyridyl)-s-tetrazine (DpTz), for the strain-promoted inverse electron-demand Diels-Alder cycloaddition, that is, tetrazine ligation, is reported. As compared to the previously reported strained alkenes such as trans-cyclooctene (TCO) and 1,3-disubstituted cyclopropene, Sph exhibits balanced reactivity and stability in tetrazine ligation with the protein substrates. A lysine derivative of Sph, SphK, was site-selectively incorporated into the extracellular loop regions (ECLs) of GCGR and GLP-1R, two members of class B G protein-coupled receptors (GPCRs) in mammalian cells with the incorporation efficiency dependent on the location. Subsequent bioorthogonal reactions with the fluorophore-conjugated DpTz reagents afforded the fluorescently labeled GCGR and GLP-1R ECL mutants with labeling yield as high as 68%. A multitude of functional assays were performed with these GPCR mutants, including ligand binding, ligand-induced receptor internalization, and ligand-stimulated intracellular cAMP accumulation. Several positions in the ECL3s of GCGR and GLP-1R were identified that tolerate SphK mutagenesis and subsequent bioorthogonal labeling. The generation of functional, fluorescently labeled ECL3 mutants of GCGR and GLP-1R should allow biophysical studies of conformation dynamics of this important class of GPCRs in their native environment in live cells.

Original languageEnglish (US)
Pages (from-to)13376-13386
Number of pages11
JournalJournal of the American Chemical Society
Issue number38
StatePublished - Sep 27 2017

ASJC Scopus subject areas

  • Catalysis
  • General Chemistry
  • Biochemistry
  • Colloid and Surface Chemistry


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