Síntese de peptídeos: cinética de dimerização via formação de ligação de dissulfeto, obtenção e estudos biofísicos de glicotriazol-peptídeos

Abstract

In face of the enormous biological potential of peptides, the detailed knowledge of the chemistry involved in the preparation of these compounds has acquired high relevance and it has reached over the years a high degree of maturity. This thesis is related to the chemical synthesis of antimicrobial peptides, which is a relevant class of these compounds. This work addresses two main topics: the kinetics of peptide dimerization through disulfide bond formation and the preparation and biophysical studies of glycotriazole-peptides. Firstly, the synthesis of homotarsinin (Htr), a rare homodimeric peptide originally isolated from the three-frog Phyllomedusa tarsius, is investigated in detail. Several dimerization reactions of the peptide monomeric chain Htr-M have been performed in different media and at different experimental conditions, in order to elucidate the kinetics of disulfide bond formation and to figure out the mechanistic aspects of this reaction. A synthetic approach for obtaining glycotriazole-peptides is also presented and it takes the advantage of two particularly interesting methodologies: the copper (I)-catalyzed azide alkyne cycloaddition (CuAAC) reaction and the Fmoc solid-phase peptide synthesis. Glycotriazole derivatives of the antimicrobial peptides phylloseptin-1, -2, -3 and hylaseptin-P1 have been prepared. The three phylloseptins have originally been isolated from the three-frog Phyllomedusa hypochondrialis, whereas hilaseptina 1 have been isolated from the three-frog Hyla punctata. In the case of the three phylloseptins and their glycoforms, some preliminary investigations of their membrane interactions have been performed by circular dichroism spectroscopy and isothermal titration calorimetry. The membrane interactions of hilaseptina 1, a triazole derivative and two glycoforms have been investigated in more details by different biophysical approaches, namely: dynamic light scattering, fluorescence, plasmon surface resonance and circular dichroism spectroscopies. The antibacterial and antifungal activities of these compounds were also investigated along with their potential of inhibiting ergosterol biosynthesis. In conclusion, the increased fungicidal activity of the glycotriazole-peptides seems to be the result of (A) more pronounced membrane-disruptive properties, which is related to presence of a saccharide ring, together with (B) the inhibition of ergosterol biosynthesis, which seems to be related to the presence of both the monosaccharide and the triazole rings.