Koniev S. Synthesis of fluorescently-labeled amino acids and biologically active peptides based on them

The thesis is devoted to the study of spectroscopic properties of fluorescent amino acids based on 3-hydroxychromone, their influence on the structure of the peptide to which they are included, as well as the synthesis of new derivatives of fluorescent amino acids based on 3-hydroxychromone. The literature review, which contains 89 references and 10 figures, describes the properties and uses of fluorescent amino acids of different classes. A review of the literature has shown that most of the examples described relating to amino acids with conjugated chromophores, and the fluorescent amino acids that could be included by the solid-phase peptide synthesis (SPPS) method are relatively limited. Of particular interest are the FlAA demonstrating intramolecular proton transfer (ESIPT). In the first step was to study the fluorescent analogs of the antimicrobial peptide cWFW simultaneously for their structure and function, by individually replacing aromatic amino acids with fluorescent amino acids (FHC). The first step was the label validation using CD spectropolarimetry and biological activity of fluorescently labeled peptides. The cRW-F2/FHC spectrum was the least similar to the cWFW spectrum. In contrast, both peptides labeled with the FHC had similar spectra to the cWFW spectra. All labeled peptides showed the same (within experimental errors) bacteriostatic activity, which was relatively high and differed from the MIC of the parent cWFW by two dilutions. Hemolysis analysis, in contrast, revealed increased activity of all three labeled peptides compared to cWFW. The cRW-F2/FHC peptide was the most hemolytic but was less polar than both cRW-Trp analogs. To assess cell permeability, cWFW fluorescent analogs were incubated with HeLa cell culture and the ability of the peptides to penetrate the cells was assessed by confocal fluorescence microscopy. FHC-labeled peptides entered the cells, demonstrating both cytosolic and microsomal penetration. No differences were found between the three cWFW analogs. From the results of the studies, it was concluded that FHC does not significantly impair the structure and function of cWFW compared to the native unlabeled peptide and that among the three analogs, cRW-W3/FHC is closest to the unlabeled peptide. The final step was to study the biodistribution of fluorescently labeled peptides in vivo using zebrafish embryos. No signs of peptide toxicity against embryos were detected within 3 hours of imaging. There was also no significant difference between the peptides. The fluorescence associated with blood vessels was observed for all peptides. This observation indicates that cWFW analogs were not soluble in plasma. The final stage of this work was the synthesis of new fluorescent amino acids based on 3-hydroxychromone. It was decided to synthesize three new N-Fmoc protected amino acids for inclusion in the peptides under study by the SPPS method: thiophene-containing analog of FHC (THC) and N-methylpyrrole-containing analog (NMPHC) and a dimethylamino group–containing analog (DMAr). The synthesis was carried out according to the scheme used for similar structures with a key stage of cyclization of chalcones to the corresponding flavones by the Algar-Flynn-Oyamadi reaction (AFO). In terms of its optical properties, Boc-THC is very close to Boc-FHC. The emission maximum of the N-methylpyrrole derivative had a bathochromic shift compared to the emission maximum of the furane-containing analog (~25 nm) and had only an N *-form peak, which may be the result of a deviation from planarity of the chromophore system. The optical properties of Boc-DMAr were like those of DMA. To validate the new amino acids, their incorporation by the SPPS method into an inverted analog of gramicidin S (GS) was used. In addition to the new amino acids, GS analogs with FHC and DMA were also synthesized for comparison. Conformational changes of the synthesized fluorescent peptides in different media were studied by CD spectropolarimetry comparing the CD spectra of labeled peptides with the corresponding spectra of natural GS and its stereoinverted analog GS-inv. From the obtained results it was concluded that inverted analogs have the same secondary structure as the natural GS - beta-hairpin.