Abstract: The stability of pairwise tryptophan (Trp) edge-to-face aromatic interactions has been exploited in the design of small tryptophan zipper (Trpzip) peptides. Herein, we report a systematic study of the regiospecific impact of four constitutional isomers of non-natural fluoro-Trp, regarding their incorporation at either edge- or face-position. Single fluorine substituents affect the electron density of the indole moiety and introduce a highly electronegative component while the native geometry of Trp is maintained. We employed a library approach based on the sequence of Trpzip2 and assessed peptide structure and stability using CD, FTIR, and NMR spectroscopy. Global hairpin stability was improved or compromised upon site-specific incorporation of a single monofluoro-Trp regioisomer. Fluorine substitution revealed key CH/π interactions within the Trp/Trp packing and holds potential for the future optimization of aromatic interactions involving Trp. More information can be found in the full paper by B. Koksch et al.