Useful Approaches for Labeling Peptides with Fluorescent Dyes
Due to the high sensitivity, flexibility, selectivity, and environmental stability, fluorescent techniques are broadly applied in biological research. Peptides provide unique possibilities for the development of efficient and selective fluorescent sensors because of their modular nature, synthetic accessibility, and biomolecular recognition potential. Accordingly, labeling peptides with fluorescent dyes become a powerful tool for studying biologically related interactions such as receptor-ligand binding, enzyme activity, and protein structures.
Fluorescence resonance energy transfer (FRET) is a mechanism that describes the energy transfer between two fluorophores. Since FRET efficiency is partly based on the distance between a donor and acceptor molecule, this technique is commonly used for studying enzyme efficiency, protein-protein interactions, or other molecular dynamics. Three strategies are regularly applied to produce fluorescent labeled peptides. On the one hand, researchers can gain ideal labeled peptides through direct modification of isolated peptides in solid-phase synthesis. On the other hand, fluorescence or chromophore labeled amino acids can be added to incorporate labels at specific sites of peptides. Another approach is the use of amine-reactive labels or thiol-reactive protein labels. Examples of frequently used fluorescent dyes include FAM, Cyanines, Fluorescein IsoThioCyanate (FITC), Mca, EDANS, Cy3/5, and so on. Fluorescent dye-labeled peptides can be visualized by fluorescence microscopy or other fluorescence visualization techniques.
Incorporation of the fluorescent tag at the N-terminal is the preferred approach for solid-phase synthesis of fluorescent peptides since it’s not involved in interactions with receptors and its modifications do not influence the biological activity of peptides. N-terminal conjugation of peptides to which fluorescein groups can bind before labeled peptides are removed from other protective groups and released from the resin. However, there is also a need for other methods in some cases to selectively functionalize amino acid residues in peptides without jeopardizing the biological activity of peptides. Fmoc-Lys-OH fluorescent is useful for preparing specifically labeled peptides to use as probes. It allows the incorporation of a fluorescent tag into a peptides chain using standard Fmoc chemistry with Fmoc-protected lysine.
Applications for fluorescent peptides range from the study of peptide-protein interactions, enzyme activity assays, or the development of novel disease models. To be more specific, fluorescent peptides are frequently used as signal peptides for in vivo biomedical imaging, protein binding, and localization studies for instance. Additionally, FRET peptides help study the protease activity, the conformational change of peptides, as well as measurement of the molecular proximity. Peptides can also be fluorescently tagged to measure the proliferation of cell population in FACS which express a ligand of interest as fluorescent probes and to evaluate their biodistribution in vivo during the pre-clinical development.
In comparison to other imaging techniques, fluorescent imaging is particularly advantageous due to its high sensitivity, nonradioactive materials, and ability to be detected by low-cost instruments. Peptides that have fluorescent labeling can provide biological information at the molecular level in living systems, enable the visualization of molecular events occurring in disease processes, and monitor therapeutic effects. Given the increasing interest in the development of chemical tools for biological research, the importance of fluorescent peptides is expected to grow.