Hemolysis Assays. We tested and compared the three cyclohexylalanine analogues to D-Thr5-clovibactin in a hemolysis assay to help assess the potential utility of these compounds as intravenous antibiotics (Figure ). D-Thr5-clovibactin showed no hemolytic activity at concentrations as high as 100 μg/mL. Each of the cyclohexylalanine analogues showed some hemolytic activity at the highest concentrations tested, but none at concentrations as high as 12.5 μg/mL. D-Cha2,D-Thr5-clovibactin showed no hemolysis at 25 μg/mL and 1.6% and 6.9% hemolysis at 50 and 100 μg/mL. Cha7,D-Thr5-clovibactin showed 0.8%, 3.4%, and 10.2% hemolysis at 25, 50, and 100 μg/mL. Cha8,D-Thr5-clovibactin was the most hemolytic, with 2.9%, 7.3%, and 15.0 % hemolysis at 25, 50, and 100 μg/mL. These levels of hemolysis suggest that these antibiotics could be administered intravenously at concentrations 10-20 times the MIC values without exhibiting significant hemolytic activity. Nevertheless, the greater hemolytic activity of the cyclohexylalanine analogues over D-Thr5-clovibactin suggests that both antibiotic activity and hemolytic activity increase with increased hydrophobicity and their non-specific interactions with lipid bilayer membranes may be significant.