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Proceedings of the 31 st European Peptide SymposiumMichal Lebl, Morten Meldal, Knud J. Jensen, Thomas Hoeg-Jensen (Editors)European Peptide Society, 2010Synthesis and Preliminary Conformational Analysis of TOACSpin-Labelled Analogues of the Medium-LengthPeptaibiotic Tylopeptin BMarina Gobbo 1,2 , Barbara Biondi 2 , Marta De Zotti 1 ,Fernando Formaggio 1,2 , and Claudio Toniolo 1,21 Department of Chemical Sciences, University of Padova, Padova, 35131, Italy; 2 CNRInstitute of Biomolecular Chemistry - Padova Unit, Padova, 35131, ItalyIntroductionThe achiral tetrasubstituted α-amino acid 4-amino-1-oxyl-2,2,6,6,-tetramethylpiperidine-4-carboxylic acid (TOAC) has been used as a paramagnetic probe to study, by electron spinresonance (ESR), the mode by which peptides insert into a membrane. In particular,detailed information about the location, orientation and aggregation of membrane-activepeptides in the phospholipid bilayer has been obtained for the TOAC-labeled peptaibioticsalamethicin F50/5 [1,2] and trichogin GA IV [3], in which one or two α-aminoisobutyricacid (Aib) residues in the sequence were replaced by the free radical-containing aminoacid. Considering that the detailed mechanism of membrane permeabilization by mediumlengthpeptaibiotics, e.g. tylopeptin B, is largely unknown, we have planned to synthesize aseries of analogues in which the TOAC amino acid replaces the Aib residue at one of thethree positions (4, 8 or 13) throughout the 14-mer tylopeptin sequence (Figure 1), and toinvestigate their interaction with model membranes by a combination of ESR techniques.Results and DiscussionWe have recently reported the solid-phase synthesis of tylopeptin B based on theFmoc/tBut strategy and the acid sensitive 2-chlorotrityl resin [4]. For the synthesis of theTOAC-containing analogues we decided to avoid any harsh acidic treatment that candestroy the free radical character of the TOAC residue [5], thereby loosing theparamagnetic probe. Consequently, the Gln and Trp residues were introduced without sidechainprotecting groups and as preformed active esters, and the alcoholic function of theSer residue was protected by the nucleofile sensitive tert-butyldimethylsilyl (TBDMS)group. After assembly of peptides on the solid support, the TBDMS group was removed bya 5 min treatment with 0.1 M tetrabutylammonium fluoride in DMF and the peptides werecleaved from the resin upon repeated treatment with 50% hexafluoroisopropanol in DCM.Yields of the TOAC containing analogues of tylopeptin B were in the range 10-65%,depending on the position of the TOAC residue in the peptide sequence. In particular theTOAC residue was difficult to introduce at position 3 or 4. In the course of these syntheseswe observed the formation of several by-products, exceeding those corresponding to thetruncated sequences. After HPLC purification the products were characterized by analyticalHPLC and ESI-MS.1 14Ac-Trp-Val-Aib-Aib-Ala-Gln-Ala-Aib-Ser-Aib-Ala-Leu-Aib-Gln-LolAc-Trp-Val-Aib-Aib-Ala-Gln-Ala-Aib-Ser-Aib-Ala-Leu-TOAC 13 -Gln-LolAc-Trp-Val-Aib-Aib-Ala-Gln-Ala-TOAC 8 -Ser-Aib-Ala-Leu-Aib-Gln-LolAc-Trp-Val-Aib-TOAC 4 -Ala-Gln-Ala-Aib-Ser-Aib-Ala-Leu-Aib-Gln-LolAc-Trp-Val-TOAC 3 -Aib-Ala-Gln-Ala-Aib-Ser-Aib-Ala-Leu-Aib-Gln-LolFig. 1. Amino acid sequences of tylopeptin B and the synthesized TOAC-containing analogues(Lol corresponds to the 1,2-amino alcohol leucinol).382