Fluorescent Amino Acid Initiated de novo Cyclic Peptides for the Label‐Free Assessment of Cell Permeability

The major obstacle in applying peptides to intracellular targets is their low inherent cell permeability. Standard approaches to attach a fluorophore (e. g. FITC, TAMRA) can change the physicochemical properties of the parent peptide and influence their ability to penetrate and localize in cells. We report a label‐free strategy for evaluating the cell permeability of cyclic peptide leads. Fluorescent tryptophan analogues 4‐cyanotryptophan (4CNW) and β‐(1‐azulenyl)‐L‐alanine (AzAla) were incorporated into in vitro translated macrocyclic peptides by initiator reprogramming. We then demonstrate these efficient blue fluorescent emitters are good tools for monitoring peptide penetration into cells. Without a trace: Fluorescent amino acids ClAc−4CNW, ClAc−AzAla were incorporated into in vitro translated macrocyclic peptides by genetic code reprogramming. The minimally perturbing dyes were found to be good tools for tracking peptide penetration into cells by fluorescence imaging and flow cytometry.