Michurin O. Synthesis and application of conformationally restricted cyclobutane-derived analogues of arginine, lysine and ornithine.

The thesis is devoted to the synthesis and application of conformationally restricted analogues of amino acids with positively charged side chains – arginine, lysine and ornithine. Synthetic approach for new cyclobutane-derived analogues of amino acids with fluorine-containing substituents, and synthetic protocols for the preparation of difluoromethyl derivatives were developed. The practical value of fluorine-containing analogues of lysine and arginine for structural investigations of membrane-active peptides has been proved. The result of work is the development of a practical synthetic approach for getting a number of conformationally constrained analogues of arginine, lysine and ornithine. The developed synthetic protocols allowed us to obtain cyclobutane analogs of arginine, lysine and ornithine in gram quantities. The structure of the compounds was unequivocally proved by 2D-spectroscopy and structural X-ray analysis. The designed amino acid isosteres were used, for the first time, to study antimicrobial and membrane-active peptides. The effect of a conformational restriction of arginine side chains on the biological activity of antimicrobial cyclic hexapeptide was studied. It has been shown that the conformational restriction of arginine side chain does not affect significantly the antimicrobial activity of cyclic hexapeptide c-WFW. Practical application of fluorine-containing conformationally-restricted analogues of arginine and lysine for studying of membrane-active peptides by 19F-ssNMR was experimentally demonstrated. Six 19F-labeled analogues of the membrane-active peptide PGla(Nle) were synthesized using trans- and cis-isosters of lysine with trifluoromethyl substituents (trans-/cis-TCBLys). To assess the applicability of newly designed 19F-labels, the structural impact of Lys to trans-/cis-TCBLys substitution was evaluated using circular dichroism spectroscopy (CD). Minimum inhibitory concentrations (MIC) values for 19F-labeled and non-labeled PGla(Nle) were taken and compared. Based on these data, we concluded that the introduction of lysine-based 19F-labels (trans-/cis-TCBLys) does not cause dramatic structural and functional changes in PGla(Nle). PGla(Nle) orientation in model lipid bilayer membranes was confirmed by the 19F-ssNMR. The simultaneous use of non-polar and charged 19F-labels has been shown to significantly increase the accuracy of the structural and orientational analysis of membrane-active peptides using the 19F-ssNMR method.