Abstracts Biological Sciences

Synthesis and Biological Evaluation of Artificial Metalloproteases Based on Amphiphilic Cyclen Derivatives

by Chrischani Perera-Bobusch

Numerous diseases are caused by the misfolding of specific proteins and the subsequent formation of aggregates, for example Parkinson's disease, type II diabetes and Alzheimer's disease (AD). In the last years the scientific interest in AD has grown remarkably due to new insights into the development of this complex disorder. Numerous therapeutic strategies have been developed for the treatment of AD. Cleavage of pathogenic misfolded proteins and their aggregates with small metal complexes acting as artificial proteases is one of them. Cu(II) and Co(III) complexes of the macrocycle cyclen were shown to cleave proteins and numerous derivatives have already been synthesized to improve the proteolytic activity. In the scope of the present thesis monoalkylated derivatives of cyclen and its oxa and dioxa analoga were synthesized. Amphiphilic metal complexes of these ligands were generated to investigate the influence of micelle formation on the proteolytic activity of cyclen. Five ligands and their Cu(II) complexes as well as two Co(III) complexes were synthesized. The protein cleavage ability was investigated via SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) with various model proteins. An increase of at least two orders of magnitude was observed with all seven complexes compared to the corresponding cyclen compounds. A comparison of the cyclen complexes of varying alkyl chain length could show that the proteolytic activity depends on the number of CH2 groups in the chain with the longest alkyl moiety being the most effective cleavage agent. The highest activity of all complexes could be detected with the Co(III) complex of 1-hexadecylcyclen, even though an improvement of the activity was expected for the oxa analoga of the same chain length. Remarkably, the ligands alone exhibited a comparable proteolytic activity to the complexes. Further investigations are necessary to exclude traces of metal as origin for this activity. An interesting finding was the decrease of the proteolytic activity above a certain complex concentration towards heme proteins, whereas non-heme protein cleavage increases consistently with the complex concentration. Molecular modeling studies are underway to further investigate these results. Fragments of the cleavage process were detected by MALDI-TOF (Matrix-assisted laser desorption/ionization time-of-flight) mass spectrometry. One of the fragments found in these studies reflects a specific cleavage site in proximity to the heme group of myoglobin already established in the literature as initial cleavage site of another cyclen derivative. These findings suggest a key role for the heme group in the cleavage process of heme proteins. Cytotoxicity studies via MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay showed a drastic increase of the cytotoxicity for the complexes of 1-dodecylcyclen compared to cyclen. However, in comparison with other compounds currently under investigation for the treatment of AD…