Bakanovych I. Synthesis and research of peptides involved in the transport of nanoparticles and biologically active compounds across lipid membranes.

The thesis is devoted to study of structure and transport properties of transmembrane protein bilitranslocase, conjugates of cell-penetrating peptides with nanoparticles which were obtained by electrochemical anodation of polycrystallic slilcon carbide, and also study of photosensitive stapled peptides. This work devoted to establishing main principles of cell-penetrating peptides usage for transport of bilogical active organic substances and nanoparticels into eykariotic cells. Forth segment of bilitranslocase, TM4 was studied by NMR, its structure and properties in model micellar media was determined. By NMR data it was found that TM4 fragment contains two proline residues near N-terminus, in positions 254 and 258 respectively. In two different micellar media, the X-Pro peptide bonds are in trans-conformation. It was found by molecular dynamics calculations that secondary structure motif of TM4 peptide is a stable α-helix in both SDS and DPC micelles. It was also found by RDF data analysis that central α-helix of TM4 fragment has different character in different media: hydrophobic in anionic SDS and amphiphilic in zwitterionic DPC micellar media. Cell uptake of SiC nanoparticles was studied. It was proposed and proved by experiment that SAP-derived cell-penetrating peptides allow nanoparticles cell internalization into both proliferating and non-proliferating cells. For each SiC-nanoparticles, modification corresponding the CPP was chosen in the way that the surface functional groups of SiC-NP form non-covalent (supramolecular) bonds with complementary CPP side chains. Fluorescent microscopy was used to establish that treatment of SiC NP(-) with SAP peptide and SiC-NPs(+) with SAP-E peptide facilitates cell internalization of nanoparticles. It was proven that successful nanoparticle delivery into cells strongly depends on peptide carrier, surface functional groups of nanoparticles, its ionization state and complemention between nanoparticle and peptide side chains charge. Light-sensitive staples peptides – inhibitors of р53-MDM2 interaction – were designed and synthesized by the double-click reaction. For this, three diarylethene linkers with alkyne groups were synthesized. A linear peptide precursor pDI-E was used as the design template, which is known as effective MDM2 binder. It was modified by introduction of non-natural aminoacid – azidoornitine into proper positions of peptide chain. With the aid of azide-alkyne [3+2]-cycloaddition reaction the library of photosensitive stapled peptides was synthesized. These peptidomimetics were obtained in both open and closed photoforms and their photophysical and biological properties were studied by CD spectroscopy and competitive fluorescent polarization assay. It was found that introduction of the photoswitch in the peptide molecules allows regulation of binding affinity of stapled peptide with target MDM2 protein. The best achieved open:closed photoform binding affinity ratio is 8,3. To improve the cell penetrating ability of peptidomimetics, starting linear precursor was modified by arginine residues at six different amino asid positions. It was found that one of arginine variants is cell-permeable and increase p53 concentration inside T22 cells more then 100-fold. These results show that such an approach is suitable for increasing cell permeability of diarylethene-based stapled pDI-E derived peptides. It is an important result for future development of photocontrolled drug candidates for cancer treatment. Keywords: membrane, transmembrane protein, cell-penetrating peptide, nanoparticles, stapled peptide.