Estudos de síntese de glicotriazol-peptídeos derivados do peptídeo antimicrobiano Hilaseptina 1 e suas interações com membranas

Abstract

This work is related to the synthesis of two novel glycotriazole-peptides, namely 1,5-GtP[(p-OAc)Glc-1,5-trz-A1 ]HSP1-NH2 e 1,4-GtP[Glc-1,4-trz-A1 ]HSP1-NH2, which are glycosylated derivatives of hylaseptin 1 (HSP1), a peptide that presents moderate antimicrobial activity. According to the proposed methodologies, the peptide backbone is to be obtained by solid phase peptide synthesis (SPPS), using the 9-Fluorenylmethoxycarbonyl (Fmoc) strategy. To link the glycotriazole and polypeptide moieties, it was initially proposed the incorporation of a Fmoc-propargylglycine residue at the last step of the SPPS to give the alkyne-decorated peptidyl-resin, which would then be used in 1,3-dipolar cycloaddition reactions with azide derivatives of glucose to provide the respective GtPs. For the synthesis of 1,5-GtP[(p-OAc)Glc1,5-trz-A1 ]HSP1-NH2, the per-O-acetylated azide derivative of glucose was used together with Cp*RuCl(PPh3)2, which is supposed lead to 1,5-triazoles (RuAAC reaction – Rutheniumcatalyzed Alkyne Azide Cycloaddition reaction). Several attempts and reaction conditions were investigated but, despite these efforts, the desired 1,5-GtP was not obtained. Therefore, the synthesis strategy was modified to firstly obtain the Fmoc-1,5-glycotriazol-amino acid by reacting the per-O-acetylated azide derivative of glucose with Fmoc-propargylglycine under ruthenium catalysis, inasmuch as this product could be incorporated into the peptide backbone during the SPPS. Although the synthesis of the Fmoc-1,5-glycotriazol-amino acid was successfully confirmed, very small amounts were obtained, which has obliterated any possibility of carrying on the synthesis of the desired 1,5-GtP. Nevertheless, after several attempts the viability of the RuAAC reaction to obtain the Fmoc-1,5-glycotriazol-amino acid derivative was confirmed, which means that higher amounts can be prepared and used in the synthesis of the respective 1,5-GtPs. On the other hand, the Copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction between the alkyne-decorated peptidyl-resin and the non-acetylated azide derivative of glucose was successful and, up to our knowledge, this is the first report of the synthesis of a 1,4-GtP peptide, which carries a non-acetylated saccharide ring. Actually, this cycloaddition reaction afforded a couple of isomers, namely 1,4-GtP[Glc-1,4-trz-A 1]HSP1-NH2 A e B. This was confirmed by MALDI-TOF mass spectrometry, high performance liquid chromatography (HPLC) analyses and nuclear magnetic resonance (NMR) spectroscopy. Besides, circular dichroism (CD) and NMR spectroscopies indicated that both isomers present helical conformations in membrane mimetic environments, although B presents higher helicity when compared to A. The biological activities of these isomers were investigated against three fungal strains and they showed similar antifungal potentials. Interestingly, this pair of 1,4-GtPs showed significantly stronger activities in comparison to the peptide HSP1-NH2, which indicates that the glycotriazole moiety enhances the antifungal potential of these molecules. However, these two 1,4-GtPs were less active in comparison to the derivative containing the per-O-acetylated glucose, which indicates that acetylation is important to the activity of the GtP derivatives of hylaseptin 1. Nevertheless, it is well-known that peptide-membrane interactions, which is essential for antifungal and antibacterial activities, depend on the presence of hydrophilic and hydrophobic residues at specific sites of the primary structure, which means that it might be interesting for some glycotriazole-peptide sequences the incorporation of non-acetylated saccharide rings.