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, 2010Jelleine-I Analogues with Improved Antibacterial ActivityPaul R. HansenIGM-Bioorganic Chemistry, Faculty of Life Sciences, University of Copenhagen, DenmarkIntroductionJelleine-I (Figure 1) is an octapeptide amide, H-PFKLSLHL-NH 2 (1) isolated from RoyalJelly of honeybees (Apis mellifera) [1]. The peptide is active against Gram-positive andGram-negative bacteria. However, Jelleine-I is also hemolytic towards human erythrocytes.In the present paper, we report the synthesis and antibacterial activity of 21 analogues ofJelleine I including an Ala-scan and single residue substitutions.Results and DiscussionThe peptides were synthesized using standard Fmoc chemistry, on a TentaGel RAM resin(50 mg, loading 0.24 mmol/g). Activation of the Fmoc amino acids and formation ofpeptide bonds were carried out using DIC and HOBt. All Fmoc amino acids and couplingreagents were used in fourfold excess. Fmoc deprotection was accomplished by treatmentwith 20% piperidine in NMP (1 x 3 min and 1 x 7 minutes). The peptides were cleavedfrom the solid support along with the permanent side chain protection groups usingTFA/H 2 O/TIS/thioanisole (90:5:2.5:2.5: v/v). The crude peptides were purified bypreparative HPLC and characterized by MALDI-TOF-MS. A stock peptide solution in 1%DMSO was prepared and the concentration was determined by amino acid analysis. Thepeptides were tested against the American Type Culture Collection (ATCC) S. aureusATCC 25923 and E. coli ATCC 25922. The MIC of each peptide was determined using amicrotitre broth dilution assay modified from the method of Hancock [2]. Briefly, serialtwofold dilutions of the peptides were made in solutions of 0.2% bovine serum albuminand 0.01% acetic acid in 96-well polypropylene microtiter plates in volumes of 50 µL.To each well was added 50 µL of the test organism in Mueller Hinton Broth to a finalconcentration of approximately 2×10 5 CFU/mL. The plates were incubated overnight at37°C and the MIC of each peptide was read as the lowest concentration of peptide thatinhibited visible growth of the organism. All MIC determinations were performed induplicate, and are the average of three independent determinations.Freshly drawn human erythrocytes in citrate-dextrose-phosphate (CDP) buffer, fromCopenhagen University Hospital were used to determine the hemolytic activity as describedby Ryge and Hansen [3].Alanine positional scanning showed restrictions on 6 out of the 8 residues (data notshown), and a moderate drop in MIC-activity for the [Ala 1 ] and [Ala 5 ] Jelleine-I analogues(Table 1).Based on the above results, two series of analogues were designed and tested. Pro 1was replaced with either Ala or 3-(2 naphthyl)-L-alanine (X) and Ser 5 was replaced withAsn or one of the more hydrophobic amino acids Val, Leu, Ile, Trp, Phe. The MICsobtained for the Jelleine-I analogues are listed in Table 1.Fig. 1. Helical wheel of Jelleine-I.376