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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, 2010Porphyrin-Antimicrobial Peptide Conjugates:Synthesis, Conformational Studies and PreliminaryLight Activated Biocidal ActivityCristiano Tampieri 1 , Barbara Biondi 2 , Sandro Campestrini 1 ,Ryan Dosselli 3 , Elena Reddi 3 , and Marina Gobbo 1,21 Department of Chemical Sciences, University of Padova, Padova, 35131, Italy; 2 CNRInstitute of Biomolecular Chemistry, Padova, 35131, Italy; 3 Department of Biology,University of Padova, Padova, 35131, ItalyIntroductionPhotodynamic therapy (PDT) is a very promising approach for killing bacteria [1]. It is wellestablished that singlet oxygen is produced as the main species responsible for cell death.During PDT multiple cellular targets are damaged and this strongly reduces the probabilityof developing the resistance phenomena, which frequently occur after repeated antibiotictreatments. Porphyrins are commonly used as photosensitizers, that can generate reactiveoxygen species upon exposure to light in the presence of oxygen. Short cationicantimicrobial peptides (CAMP) are components of the innate defense of many organisms[2]. Their overall positive charge ensures accumulationat the poly-anionic microbial cell surfaces.Beyond the presence of several cationic aminoacids, a substantial proportion of hydrophobic aminoacid residues permit most of CAMP to fold into anamphipathic structure, that allows them to insert intothe phospholipid bilayer. After insertion, antimicrobialpeptides act by either disrupting thephysical integrity of the membrane or translocatingacross the membrane to hit bacterial internal targets.By conjugating a porphyrin to an antimicrobialpeptide we can expect to direct the photosensitizeragainst specific bacterial targets and increase theFig. 1. Chemical structure of theTPP-OH photosensitizer.efficacy of PDT. Here, we present the synthesis oftwo conjugates in which a 5(4’-carboxyphenyl)-10,15,20-triphenylporphyrin (TPP-OH, Figure 1)has been covalently linked to the N-terminal end oftwo antimicrobial peptides: apidaecin 1b (GNNRPVYIPQPRPPHPRL) and magainin 2(GIGKFLHSAKKFGKAFVGEIMNS).Results and DiscussionThe synthesis of the photosensitizer, opportunely functionalized for the covalent binding tothe peptide, was carried out according to the Lindsey’s procedure, starting from pyrrole,benzaldehyde and 4-formylbenzoic acid methyl ester [3]. After purification on silica gel,the 5(4’-methoxycarbonylphenyl)-10,15,20-triphenylporphyrin was isolated from themixture of different isomers in 38% yield and, after ester hydrolysis, the photosensitizer(TPP-OH) was characterized by 1 H-NMR, UV-vis and ESI-MS.Peptides were automatically synthesized on solid phase by the standard Fmoc/HBTUprotocol and, after removal of the N-terminal amino protecting group, TPP-OH (2.5 eq)was directly coupled to the peptide chain using diisopropylcarbodiimide/HOBt asactivating agents. TPP-apidaecin was obtained in very good yield (over 75%), whereas thereaction with magainin yielded only about 13% of the corresponding conjugate. Notably,the porphyrin induced also extensive oxidation of the methionine residue in magainin, asdetected by MS analysis. To evade this side-reaction, TPP-OH was reacted with a magaininanalogue in which the Met residue was replaced by Leu, and the TPP-[Leu 21 ]magaininconjugate was obtained in 35% yield. After HPLC purification, both TPP-peptideconjugates were characterized by analytical HPLC, ESI-MS and UV-vis spectroscopy.The conformational preferences of TPP-apidaecin and TPP-[Leu 21 ]magainin werepreliminarily investigated by circular dicroism (CD) and compared to those of the parentpeptides. Magainin and apidaecin belong to different structural classes of antimicrobial384