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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, 2010Antimicrobial Activity of Small 3-(2-Naphthyl)-L-AlanineContaining PeptidesPaul R. Hansen 1,3 and Niels-Frimodt Møller 2,31 IGM-Bioorganic Chemistry, Faculty of Life Sciences, University of Copenhagen,Denmark; 2 National Center for Antimicrobials and Infection Control, SSI, Denmark;3 Danish National Center for Antibiotic Research and Development, DenmarkIntroductionAntibiotic-resistant pathogens have become a very serious problem world-wide. Mostalarming is the discovery of Gram-negative Enterobacteriaceae containing New Delhimetallo- -lactamase 1 (NDM-1) which are highly resistant to many antibiotic classes,including -lactams, fluoroquinolones, and aminoglycosides—the main antibiotic classesfor the treatment of Gram-negative infections [1].Antimicrobial peptides (AMPs) are a part of the innate immunity in all multicellularorganisms and are promising lead structures for developing new antimicrobial agents [2].However, AMPs often display undesirable pharmaceutical properties, such as susceptibilityto proteolytic breakdown. This may be circumvented by incorporation of non-standardamino acids into the peptide, such as 3-(2-naphthyl)-L-alanine.In the present study, we investigated the antimicrobial activities of 6 peptidescontaining 3-(2-naphthyl)-L-alanine: H-XFXLKKK-NH 2 (1), H-FXLKKK-NH 2 (2), H-XFLKKK-NH 2 (3), H-XKFKXKLKK-NH 2 (4), H-KFKXKLKK-NH 2 (5), H-XKFKKLKK-NH 2 (6), X being the non-proteinogenic amino acid.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 by preparative HPLC and characterized byMALDI-TOF-MS. A stock peptide solution in 1% DMSO was prepared and theconcentration was determined by amino acid analysis. The peptides were tested againstclinically relevant bacteria and fungi as described previously [3].The MIC of each peptide was determined using a microtitre broth dilution assaymodified from the method of Hancock [4]. Briefly, serial twofold dilutions of the peptideswere made in solutions of 0.2% bovine serum albumin and 0.01% acetic acid in 96-wellpolypropylene microtiter plates in volumes of 50 µL. To each well was added 50 µL of thetest organism in Mueller Hinton Broth to a final concentration of approximately 2×10 5CFU/mL. The plates were incubated overnight at 37°C (two days for the fungi) and theMIC of each peptide was read as the lowest concentration of peptide that inhibited visiblegrowth of the organism. All MIC determinations were performed in duplicate, and are theaverage 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 [5].378