Synthesis of 7‐Aminocoumarin by Buchwald–Hartwig Cross Coupling for Specific Protein Labeling in Living Cells

Abstract

To enable minimally invasive studies of proteins in their native context, it is desirable to tag proteins with small, bright reporter groups. Recently, our lab described PRIME (probe incorporation mediated by enzymes) technology for such tagging.[1–3] An engineered variant of Escherichia coli lipoic acid ligase (LplA) is used to covalently attach a fluorescent substrate, such as 7-hydroxycoumarin, onto a 13-residue peptide-recognition sequence (called LAP, for ligase acceptor peptide) that is genetically fused to a protein of interest (POI; Scheme 1A). The targeting specificity is derived from the extremely high natural sequence specificity of LplA.[4] PRIME was used to label and visualize various LAP-tagged cytoskeletal and adhesion proteins in living mammalian cells.