Fluorescent Amino Acid Initiated <i>de novo</i> Cyclic Peptides for the Label‐Free Assessment of Cell Permeability**

Abstract

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.