Genetic code expansion is commonly used to introduce bioorthogonal reactive functional groups onto proteins for labeling. In recent years, the inverse electron demand Diels-Alder reaction between tetrazines and strained trans-cyclooctenes has increased in popularity as a bioorthogonal ligation for protein labeling due to its fast reaction rate and high in vivo stability. We provide methods for the facile synthesis of a tetrazine containing amino acid, Tet-v2.0, and the site-specific incorporation of Tet-v2.0 into proteins via genetic code expansion. Furthermore, we demonstrate that proteins containing Tet-v2.0 can be quickly and efficiently reacted with strained alkene labels at low concentrations. This chemistry has enabled the labeling of protein surfaces with fluorophores, inhibitors, or common posttranslational modifications such as glycosylation or lipidation.
Keywords: Bioorthogonal ligations; Fluorescent labeling; Genetic code expansion; Inverse electron demand Diels-Alder; Protein labeling; Tetrazine; Trans-cyclooctene.