The increasing number of infections caused by multidrug-resistant bacteria requires an intensified search for new antibiotics. Pep19-4LF is a synthetic antimicrobial peptide (GKKYRRFRWKFKGKLFLFG) that was previously designed with the main focus on high antimicrobial activity. The hydrophobic motif, LFLFG, was found to be essential for antimicrobial activity. However, this motif shows several limitations such as aggregation in biological media, low solubility, and small yields in peptide synthesis. In order to obtain more appropriate peptide characteristics, the hydrophobic motif was replaced with fatty acids. For this purpose, a shortened variant of Pep19-4LF (Pep19-short; GKKYRRFRWKFKGK) was synthesized and covalently linked to saturated fatty acids of different chain lengths. The peptide conjugates were tested with respect to their antibacterial activity by microdilution experiments on different bacterial strains. The length of the fatty acid was found to be directly correlated to the antimicrobial activity up to an ideal chain length (undecanoic acid, C11:0). This conjugate showed high antimicrobial activity in absence of toxicity. Time–kill studies revealed a fast and bactericidal mode of action. Furthermore, the first in vivo experiments of the conjugate in rodents demonstrated pharmacokinetics appropriate for application as a drug. These results clearly indicate that the hydrophobic motif of the peptide can be replaced by a single fatty acid of medium length, simplifying the design of this antimicrobial peptide while retaining high antimicrobial activity in the absence of toxicity.

Sepsis is the number one cause of death in intensive care units. This life-threatening condition is caused by bacterial infections and triggered by endotoxins of Gram-negative bacteria that leads to an overreaction of the immune system. The synthetic anti-lipopolysaccharide peptide Pep19-2.5 is a promising candidate for the treatment of sepsis as it binds sepsis-inducing lipopolysaccharides and thus prevents initiation of septic shock. For clinical evaluation precise quantification of the peptide in blood and tissue is required. As the peptide is not extractable from biological samples by commonly used methods there is a need for a new analysis method that does not rely on extraction of the peptide. In order to quantify the peptide by mass spectrometry, the peptide was synthesized containing ¹³C₉,¹⁵N₁-labeled phenylalanine residues. This modification offers high stability during acidic hydrolysis. Following acidic hydrolysis of the samples, the concentration of ¹³C₉,¹⁵N₁-labeled phenylalanine determined by LC-MS could be unambiguously correlated to the content of Pep19-2.5. Further experiments validated the accuracy of the data. Moreover, the quantification of Pep19-2.5 in different tissues (as studied in Wistar rats) was shown to provide comparable results to the results obtained with radioactively-labeled (¹⁴C) Pep19-2.5- Radioactive labeling is considered as the gold standard for quantification of compounds that refrain from reliable extraction methods. This novel method represents a valuable procedure for the determination of Pep19-2.5 and sticky peptides with unpredictable extraction properties in general.