Two-Step
Protein Labeling Utilizing Lipoic Acid Ligase
and Sonogashira Cross-Coupling
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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 <i>p</i>-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 <i>p</i>-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