Fluorescence Labeling of Peptides: Finding the Optimal Protocol for Coupling Various Dyes to ATCUN-like Structures

Labeling of peptides and proteins with fluorescent dyes is a key step in functionalizing these structures for a wide array of biological assays. However, coupling strategies of such dyes have not been optimized for the most common compounds, while this step is typically the most precious and costly of the whole synthesis. We searched for the best conditions for attachment of the most widely used fluorescent dyes such as 6-carboxyfluorescein, Rhodamine B, and BODIPY-FL to peptides, where amino terminal Cu(II) and Ni(II) binding site (ATCUN) peptides were used as a model system. Surprisingly, conventional methods of dye attachment proved to not be satisfactory and yielded poor efficiency results. We have discovered that when labeling primary amines on peptides, the uncommon synthesis of activated pentafluorophenol (PFP) esters is the most efficient strategy, expedited by microwave irradiation. Coupling to secondary amines is achieved most efficiently through conventional coupling reagents such as HATU and PyBOP. Furthermore, we have employed our fluorescently labeled ATCUN peptides in studies for Cu(II) and Ni(II) sensing, showing that changing the fluorophore does not significantly affect the fluorescence quenching process and discovering the optimal linker length between the ATCUN core and the dye, expanding the repertoire of fluorophores that can be used in this application.