Tylopeptin B

The medium-length peptide Tylopeptin B possesses activity against Gram-positive bacteria. It binds to bacterial membranes altering their mechanical properties and increasing their permeability. This action is commonly related with peptide self-assembling, resulting in the formation of membrane channels. Here, pulsed double electron-electron resonance (DEER) data for spin-labeled Tylopeptin B in palmitoyl-oleoyl-glycero-phosphocholine (POPC) model membrane reveal that peptide self-assembling starts at concentration as low as 0.1 mol%; above 0.2 mol% it attains a saturation-like dependence with a mean number of peptides in the cluster = 3.3. Using the electron spin echo envelope modulation (ESEEM) technique, Tylopeptin B molecules are found to possess a planar orientation in the membrane. In the peptide concentration range between 0.1 and 0.2 mol%, DEER data show that the peptide clusters have tendency of mutual repulsion, with a circle of inaccessibility of radius around 20 nm. It may be proposed that within this radius the peptides destabilize the membrane, providing so the peptide antimicrobial activity. Exploiting spin-labeled stearic acids as a model for free fatty acids (FFA), we found that at concentrations of 0.1-0.2 mol% the peptide promotes formation of lipid-mediated FFA clusters; further increase in peptide concentration results in dissipation of these clusters.

A set of analogues of the 14-residue peptaibol tylopeptin B, containing the stable free-radical 4-amino-1-oxyl-2,2,6,6,-tetramethylpiperidine-4-carboxylic acid (TOAC) at one or two selected positions, was synthesized by the solid-phase methodology. A solution conformational analysis performed by FTIR absorption and CD suggests that, in membrane-mimicking solvents, the labeled tylopeptin B analogues preserve the helical propensity of the parent peptide, with a preference for the α-helix or the 3(10) -helix type depending upon the nature of the solvent. In aqueous environment, the spin-labeled analogues present a higher content of helical conformation as a consequence of the strong helix promoter effect of the conformationally constrained TOAC residue. We observed a progressive increase of the quenching effect of the nitroxyl radical on the fluorescence of the N-terminal tryptophan as TOAC replaces the Aib residue at positions 13, 8, and 4, respectively. A membrane permeabilization assay performed on two selected analogues, TOAC(8) - and TOAC(13) -tylopeptin B, showed that the labeled peptides exhibit membrane-modifying properties comparable with those of the natural peptaibiotic. We conclude that our TOAC paramagnetic analogues of tylopeptin B are good models for a detailed ESR investigation of the mechanism of membrane permeabilization induced by medium-length peptaibiotics.

The Chilean Sepedonium aff. chalcipori strain KSH 883, isolated from the endemic Boletus loyo Philippi, was studied in a polythetic approach based on chemical, molecular, and biological data. A taxonomic study of the strain using molecular data of the ITS, EF1-α, and RPB2 barcoding genes confirmed the position of the isolated strain within the S. chalcipori clade, but also suggested the separation of this clade into three different species. Two new linear 15-residue peptaibols, named chilenopeptins A (1) and B (2), together with the known peptaibols tylopeptins A (3) and B (4) were isolated from the semisolid culture of strain KSH 883. The structures of 1 and 2 were elucidated on the basis of HRESIMS(n) experiments in conjunction with comprehensive 1D and 2D NMR analysis. Thus, the sequence of chilenopeptin A (1) was identified as Ac-Aib(1)-Ser(2)-Trp(3)-Aib(4)-Pro(5)-Leu(6)-Aib(7)-Aib(8)-Gln(9)-Aib(10)-Aib(11)-Gln(12)-Aib(13)-Leu(14)-Pheol(15), while chilenopeptin B (2) differs from 1 by the replacement of Trp(3) by Phe(3). Additionally, the total synthesis of 1 and 2 was accomplished by a solid-phase approach, confirming the absolute configuration of all chiral amino acids as l. Both the chilenopeptins (1 and 2) and tylopeptins (3 and 4) were evaluated for their potential to inhibit the growth of phytopathogenic organisms.