Derek Hudson

Orthogonal methods for the synthesis of multiply labeled peptide probes and substrates

Fluorescently labeled peptides have many applications: in cellular uptake and localization studies, for immunological assays, as receptor probes, and as enzyme substrates. An important implementation uses fluorescence resonance energy transfer (FRET) techniques, where the proximity of a dye pair effectively quenches fluorescence, which is then liberated wholly or partially through binding or cleavage. Early work focussed on the use of prederivatized, side-chain modified lysines, generally bearing dabcyl and dansyl groups. This work focuses on methods that allow incorporation of much more chemically sensitive dyes [e.g., fluorescein (FAM) and tetramethylrhodamine (TAMRA) derivatives] onto specific lysine side-chains at any position within a target peptide. This design allows the flexible display of the labels, maximizing their spectral overlap and minimizing the influence of backbone conformation. Gly spacers were used to separate the probe sequence and the fluorophores, reducing any effect the labels might have on binding.