A first aim of this work was to develop novel antimicrobial peptidomimetics based on PG-1 in order to improve the activity against Gram-negative bacteria and to decrease toxic effects for human red blood cells. As described before, several peptidomimetics based on the ß- hairpin structure of PG-1 have been developed with potent antimicrobial activity in the nanomolar range including L27-11, POL7001 and POL7080. Furthermore, they show no haemolytic activity against human red blood cells. Yet, they act only on a narrow spectrum of pathogens, namely Pseudomonas sp. On the other hand, they interact with LptD, an antimicrobial target, which has not yet been targeted by existing approved antibiotics. Given the success of this novel class of mimetics, a question that arose was whether similar peptidomimetics can be found that target LptD or related OM proteins in other Gram-negative bacteria. It was envisioned here that new scaffolds for the development of broad spectrumantimicrobial mimetics could be created. For this reason, a first part of this work was a stepwise optimisation of the compound L23-67, which was found earlier during the development of L27-11. Important residues were identified by an Ala scan. A further focus was on libraries with hydrophobic and basic residues. Furthermore, to examine the influence of various different amino acids several single and multiple substitutions were performed.
During earlier work, a method based on the principle of photoaffinity labeling was used in order to determine whether the previously mentioned compound L27-11 binds to LptD. These photoaffinity labeling experiments were based on a photoprobe containing L-4,4- diazirinylproline instead of L-proline. In the case of an interaction between the substrate and a protein, upon irradiation with UV light, a covalent linkage should be formed. The question then arose, whether similar photoprobes could be made to test a possible binding site for new mimetics having a broad spectrum antimicrobial activity. For this reason, it was necessary to synthesize a related photoprobe. In order to detect the binding interaction after photolabeling a biotin residue was also incorporated. This was accomplished by using a glutamate side chain containing a polyethylene glycol (PEG) linker with a biotin moiety attached to the linker. The photolabeling studies focussed solely on OM proteins. Any labeled proteins
should then be identified by proteomic MS-analyses. Thus, in this part of the project, the purpose was to identify the labeled proteins and to compare the different photolabeling patterns.