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

Abstract

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, i.e., tetrazine ligation, is reported. Compared with 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.

Protein Spin Labeling with a Photocaged Nitroxide Using Diels–Alder Chemistry

“…Both model proteins exhibit native cysteine residues; thus providing the possibility to test for orthogonal labeling without affecting cysteine. The first ncAA that could undergo a DA inv reaction, a lysine‐derived cyclooctyne, was introduced for the copper‐free click reaction in 2011, before a tetrazine, norbornenes, TCOs, and a spirohexene were also genetically encoded. The rationally designed Methanosarcina mazei mutant tRNA Pyl /PylRS AF (Y306A, Y384F) possesses an enlarged binding pocket that is suitable for incorporating these bulky ncAAs in response to the amber stop codon .…”

Achieving Controlled Biomolecule–Biomaterial Conjugation

“… 250 − 252 Recently developed spirohexene reactive handles offer a potential alternative, combining sufficient stability with fast reaction rates. 253 …”

Highly Strained Unsaturated Carbocycles

“…More recently, the same research group incorporated the above mentioned lysine derivative containing the spiro [2.3]hex-1-ene framework into the extracellular loop regions (ECL) of GCGR and GLP1R, two members of class B G protein-coupled receptors (GPCRs) in mammalian cells. [83] Subsequent bioorthogonal reactions with the fluorophore-conjugated tetrazine reagent afforded the fluorescently labeled GCGR and GLP1R ECL mutants with very high labeling yield. The generation of functional, fluorescently labeled ECL3 mutants of GCGR and GLP1R is important for biophysical studies of conformation dynamics of this class of GPCRs in their native environment in live cells.…”