Another approach to darobactin modification is the altering of tryptophans e.g. through halogenation. The tryptophan indole ring positioning is integral for darobactin blocking of the β-barrel lateral gate. We considered the possibility that halogenation of these essential elements could significantly change bioactivity. Five new halogenated derivatives of D9 were indeed produced through feeding (see SI) of halogenated L-tryptophans, two of them harboring 5-Chloro-and three 6-Fluoro-L-tryptophan. However, the initial yields prohibited isolation of all derivatives. The highest yield derivative with 6-Fluoro-L-tryptophan at position 3 of the core-peptide could be purified and its structure was confirmed via NMR spectroscopy (Figure S 86-90; Table ). The corresponding activity analysis of purified, single fluorinated derivatives D9-6F1 and D9-6F7 showed comparable activity to pure D9 with slightly better activity against E. coli and K. pneumoniae and slightly diminished activity against P. aeruginosa. (Figure S 41). In general, modifications to darobactin amino acids, such as halogenation, expand the structural diversity spectrum of new darobactins, potentially influencing binding behavior and pharmacokinetic characteristics as known for fluoroquinolones. This assessment, along with developing a workflow to improve the yield of halogenated darobactins, will be part of a separate study. Thus, for the current phase of this project, we chose to focus on purification and characterisation of new non-halogenated derivatives with promising crude extract activity (Table , Figure ).