Peptides and glycotriazole-peptides: synthesis and solid-state NMR investigations of their membrane interactions

• Autor(a) principal: José María Muñoz López
• Data de Publicação: 2020
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Institucional da UFMG
• Texto Completo: http://hdl.handle.net/1843/33978
• topic: Antimicrobial peptides; Solid-phase synthesis; Solid-State NMR; Biophysics; Supramolecular chemistry; Organic chemistry; Peptídeos antimicrobianos; RMN em estado sólido; Síntese em fase sólida; Biofísica; Química Supramolecular; Química Orgânica; Atividade antifúngica; Ressonância magnética nuclear; Peptídios - Síntese; Biofísica; Química supramolecular; Química orgânica; Testes microbiológicos; Dinâmica molecular

On the verge of a post-antibiotic era due to the rapidly increasing resistance that countless pathogens are developing, antimicrobial peptides are in the limelight for research as an alternative to conventional antibiotics. Besides, several studies have revealed that their mechanism of action is through the cell membrane disruption, which hampers microbial resistance. Ocellatins are a family of antimicrobial peptides which has been isolated from the skin secretions of anurans of the Leptodactylus genus. Three of these peptides, namely ocellatin-LB1, -LB2 and -F1 present a primary structure identical from residues 1 to 23 (Oce-LB1 sequence), whereas Oce-LB2 carries and extra Asn and Oce-F1 extra Asn-Lys-Leu residues at their C-termini. In spite of having similar primary structures, these extra amino acids ensure different membrane interactions as well as different antimicrobial potentials (-F1>>, -LB1 ≥ -LB2), as proved by several biophysical studies and biological assays. In addition, the synthetic modification of natural peptides, such as hylaseptin-P1 (HSP1) and phylloseptin-2 (PS-2), by adding a glycotriazole residue led to enhanced antifungal activities of the respective glycotriazole-peptides (GtPs). Thus, in order to gain extra insight into how the subtle differences in the primary sequence of ocellatins at the C-termini and how the glycotriazole moiety increases both antimicrobial and antifungal peptides activities, we decided to investigate the membrane topologies of these peptides by static solid-state NMR spectroscopy. To this end, peptides were selectively labeled at single positions with 15N-Leu or 15N-Ala and 3,3,3-2H3-Ala amino acid analogs during the peptide synthesis, which followed the Fmoc solid-phase strategy. Ocellatins were yielded as (3,3,3-2H3-Ala-10, 15N-Leu-17)-Oce (-LB1, -LB2 and -F1). Not least, the synthetic modification of labelled HSP1 and PS-2 peptides was carried out by the “click” reaction between the azide per-O-acetylate-glycose derivate and the propargyl glycine residue contained in their respective peptidyl-resin derivatives, yielding (3,3,3-2H3-Ala-8, 15N-Ala-10)-[pOAcGlc-Trz-A1]-HSP1-NH2 and (3,3,3-2H3-Ala-10, 15N-Leu-15)-[pOAcGlc-Trz-A14]-PS-2. Thus, labelled peptides were reconstituted into oriented phospholipid bilayers. The phospholipid bilayers encompassing peptides or GtPs were monitored by proton-decoupled 31P solid-state NMR spectroscopy. On the other hand, peptides’ both tilt and pitch angles were studied by combined proton-decoupled 15N and 2H solid-state NMR spectroscopy along with simulations of both 15N chemical shift and 2H quadrupolar splitting. To our knowledge, this work investigates in an unprecedent way the membrane topologies of both glycotriazole-peptides and ocellatins. The membrane interactions of the ocellatin peptides were further investigated by isothermal titration calorimetry, surface plasmon resonance spectroscopy and computational simulations of molecular dynamics.