Proceedings book download - 5Z.com

Proceedings of the 31 st European Peptide SymposiumMichal Lebl, Morten Meldal, Knud J. Jensen, Thomas Hoeg-Jensen (Editors)European Peptide Society, 2010Synthesis and Antimicrobial Evaluation of Temporin LAnalogues Containing D-Amino AcidsAlfonso Carotenuto 1 , Maria Luisa Mangoni 2 , Ludovica MarcelliniHercolani Gaddi 2 , Maria Rosaria Saviello 1 , Salvatore Di Maro 1 ,Pietro Campiglia 3 , Isabel Monterrey-Gomez 1 , Luigia Auriemma 1 ,Ettore Novellino 1 , and Paolo Grieco 11 Dept. Chimica Farmaceutica e Toss., University of Naples, “Federico II”, Napoli, 80131,Italy; 2 Dept. Biochemical Science, University of Rome, “La Sapienza”, Rome, 00185, Italy;3 Dept. Pharmaceutical Science, University of Salerno, Fisciano, 84084, ItalyIntroductionTemporins A and L are antimicrobial peptides isolated from the skin of Red European frog“Rana temporaria”. Actually, they are the smallest natural antimicrobial amide-peptidescharacterized by short sequence (10-14 residues) with a net positive charge at neutral pHvalue. Temporins are active against a broad spectrum of microorganism: Temporin A (TA)(FLPLIGRVLSGIL-NH 2 ) is preferentially active against Gram-positive bacterial strains;Temporin L (TL) (FVQWFSKFLGRIL-NH 2 ) has the highest activity among all temporinsagainst fungi, and bacteria, including resistant Gram-negative strains, but it showshemolytic activity too. They have the ability to bind and permeate both artificial andbiological membranes. We have recently investigated two members of this AMP familyTemporin L and Temporin A [1-3]. At the same time, we developed new analogues of thesepeptides, among which Pro3TL (FVPWFSKFLGRILNH 2 ) exhibiting a higherantimicrobial activity and a lower hemolytic activity than the native peptide TL. Toelucidate the molecular basis of the interaction of the native TL with bacterial membraneand to develop new potent analogues with improved activity, but without haemolyticactivity, we synthesized new analogues of Pro3TL, where C-terminal residues werereplaced one-by-one by D-amino acid. Here we report the preliminary results of this study.Results and DiscussionThe temporin L and their analogues were synthesized in solid phase by the classicalstrategy via Fmoc using Rink-Amide resin. Finally, the peptide chains were cleaved fromthe resin using TFA 95% TIS 2.5% H 2 O 2.5% mixture. The purification was achievedusing a semipreparative RP-HPLC C-18 bonded silica column (Vydac 218TP1010). Thepurified peptides were 98% pure as determined by analytical RP-HPLC. The correctmolecular weight of the peptide was confirmed by mass spectrometry. The structures ofsynthesized peptides are showed in Table 1 [4]. In Figure 1, we report the NMR structuresin DPC of Pro 3 -Temporin L and Pro 3 -dLeu 9 -Temporin. In particular, main differences arelocalized in the C-terminal region of the peptides, where the TL-D3 is missing of helicalcharacter observed in Pro 3 -Temporin L. This behaviour could be responsible of a reductionTable 1. Peptides synthesizedTemporin L FVQWFSKFLGRIL-NH 2TL-D1 FVPWFSdKFLGRIL-NH 2TL-D2 FVQWFSKdFLGRIL-NH 2TL-D3 FVPWFSKFdLGRIL-NH 2TL-D4 FVPWFSKFLGdRIL-NH 2TL-D5 FVPWFSKFLGRdIL-NH 2TL-D6 FVPWFSKFLGRIdL-NH 2TL-D7 FVPWFSKFLdPRIL-NH 2of hemolytic activity of peptide TL-D3,without significant difference in antimicrobialactivity. The antimicrobialactivity of these peptides was evaluatedas the ability to inhibit the growth ofGram-positive, Gram-negative andfungi strains, using the microdilutionbroth method. Antimicrobial activitieswere expressed as the minimalinhibitory concentration (MIC), theconcentration of peptide at which100% inhibition of microbial growth isobserved after 18–20 h of incubation.The results are shown in Table 2.360