title: Diels-Alder cycloaddition for fluorophore targeting to specific proteins inside living cells.
abstract: The inverse-electron-demand Diels-Alder cycloaddition between trans-cyclooctenes and tetrazines is biocompatible and exceptionally fast. We utilized this chemistry for site-specific fluorescence labeling of proteins on the cell surface and inside living mammalian cells by a two-step protocol. Escherichia coli lipoic acid ligase site-specifically ligates a trans-cyclooctene derivative onto a protein of interest in the first step, followed by chemoselective derivatization with a tetrazine-fluorophore conjugate in the second step. On the cell surface, this labeling was fluorogenic and highly sensitive. Inside the cell, we achieved specific labeling of cytoskeletal proteins with green and red fluorophores. By incorporating the Diels-Alder cycloaddition, we have broadened the panel of fluorophores that can be targeted by lipoic acid ligase.
authors.affiliation: Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA.
authors.forename: Daniel S
authors.initials: DS
authors.lastname: Liu
authors.forename: Anupong
authors.initials: A
authors.lastname: Tangpeerachaikul
authors.forename: Ramajeyam
authors.initials: R
authors.lastname: Selvaraj
authors.forename: Michael T
authors.initials: MT
authors.lastname: Taylor
authors.forename: Joseph M
authors.initials: JM
authors.lastname: Fox
authors.forename: Alice Y
authors.initials: AY
authors.lastname: Ting
mesh_terms: D016503:Drug Delivery Systems; D004926:Escherichia coli / Q000201:enzymology; D005456:Fluorescent Dyes / Q000008:administration & dosage* / Q000737:chemistry*; D057809:HEK293 Cells; D006801:Humans; D008025:Ligases / Q000737:chemistry / Q000378:metabolism*; D015394:Molecular Structure; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; D011506:Proteins; D008025:Ligases