Synthesis and preliminary conformational analysis of TOAC spin-labeled analogues of the medium-length peptaibiotic tylopeptin B

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 a-helix or the 310-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, TOAC8- and TOAC13-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.