title: Two-step protein labeling utilizing lipoic acid ligase and Sonogashira cross-coupling.
abstract: Labeling proteins in their natural settings with fluorescent proteins or protein tags often leads to problems. Despite the high specificity, these methods influence the natural functions due to the rather large size of the proteins used. Here we present a two-step labeling procedure for the attachment of various fluorescent probes to a small peptide sequence (13 amino acids) using enzyme-mediated peptide labeling in combination with palladium-catalyzed Sonogashira cross-coupling. We identified p-iodophenyl derivatives from a small library that can be covalently attached to a lysine residue within a specific 13-amino-acid peptide sequence by Escherichia coli lipoic acid ligase A (LplA). The derivatization with p-iodophenyl subsequently served as a reactive handle for bioorthogonal transition metal-catalyzed Sonogashira cross-coupling with alkyne-functionalized fluorophores on both the peptide as well as on the protein level. Our two-step labeling strategy combines high selectivity of enzyme-mediated labeling with the chemoselectivity of palladium-catalyzed Sonogashira cross-coupling.
authors.affiliation: Institute for Pharmacy and Molecular Biotechnology, Heidelberg University , Im Neuenheimer Feld 364, 69120 Heidelberg, Germany.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Hauke
authors.forename: Marcel
authors.initials: M
authors.lastname: Best
authors.forename: Tobias T
authors.initials: TT
authors.lastname: Schmidt
authors.forename: Mathis
authors.initials: M
authors.lastname: Baalmann
authors.forename: André
authors.initials: A
authors.lastname: Krause
authors.forename: Richard
authors.initials: R
authors.lastname: Wombacher
mesh_terms: D000595:Amino Acid Sequence; D002264:Carboxylic Acids / Q000737:chemistry; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology; D019793:Fluorescein / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D008025:Ligases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation; D010165:Palladium / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism*; D011487:Protein Conformation; D015202:Protein Engineering; D013194:Staining and Labeling / Q000379:methods*; D013379:Substrate Specificity; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism; D008063:Thioctic Acid / Q000378:metabolism*; D014867:Water / Q000737:chemistry
chemical_list: D002264:Carboxylic Acids; D005456:Fluorescent Dyes; D010446:Peptide Fragments; D014867:Water; D010165:Palladium; D008063:Thioctic Acid; D013762:Tetrahydrofolate Dehydrogenase; D008025:Ligases; D019793:Fluorescein