Based on the direct comparison of D22 with D31 and D32, it can be concluded that changes on position 4 from L-threonine to L-alanine or extended L-cysteine residues as described previously, have only minor effects on activity. NMR structure elucidation determined that the L-cysteine in these cases is connected via a disulfide bond to another L-cysteine and lactic acid (Figure S 76-85, Table S 14, S 15). Interestingly, D39, with L-arginine instead of L-lysine as a ring-forming amino acid exhibited stronger antibacterial activity. This finding is contradictory to the 16-fold decrease in activity when comparing DA and DD, but in line with predictions based on the expected binding mode (Figure ), suggesting a different reason for the activity decrease of DD. Our findings regarding SAR of crude extracts are summarised in Figure . While crude extract data should be taken with caution, MIC assays done with purified darobactin derivatives were completely in line with extract data (see below).