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Table 1. Antimicrobial and hemolytic activity of lasiocepsin (LAS) and its two analogousside products differing in the pattern of disulfide bridges (LAS 8-27,17-25 and LAS 8-17,25-27) compared to other antimicrobial compoundsPeptideMw[Da]Lasiocepsin primary sequence determined by Edman degradation and pattern of disulfidebridges (Cys8-Cys25, Cys17-Cys27) determined by MS analysis of the fragments resultingfrom trypsin digestion of the peptide, is shown on Figure 1.The linear peptide was synthesized manually using a solid-phase method in a 5 mLpolypropylene syringe with a bottom Teflon filter. The synthesis was done using standardprotocol of N α -Fmoc chemistry on 2-chlorotrityl chloride resin. All Cys side chains wereprotected with trityl groups. The crude peptide showing HPLC profile dominated by thepeak of required product was purified by HPLC. The purified linear peptide was thenoxidized by stirring the peptide in the 0.1 M ammonium acetate buffer pH 7.8 in the openair. This oxidative folding resulted however in the mixture of three peptides differing in thepattern of disulfide bridges. The amount of lasiocepsin in the mixture represented roughlyhalf of the distribution. Synthetic lasiocepsin (LAS) showed potent antimicrobial activityagainst both Gram-positive and -negative bacteria. Especially interesting is its high potencyagainst Pseudomonas aeruginosa and no hemolytic activity against rat and humanerythrocytes. Lasiocepsin also possess antifungal activity against Candida albicans. CDspectra of lasiocepsin and of its analog having wrong pattern of disulfide bridges bothshowed significant portion of α-helical structure (35% for the natural LAS, lower – 26%proportion for the non natural analog LAS 8-27,17-25). Upon addition of 2,2,2-trifluoroethanol as a helix promoting solvent both peptides adopted similar conformationcontaining about 40% of α-helical structure although dynamics of this change was fasterfor the natural LAS. This conformational behavior correlates with differences inantibacterial activity and supports the idea of the significance of α-helical conformation asa part of the lasiocepsin molecule for the biological process.AcknowledgmentsThis work was supported by the Czech Science Foundation, grant No. 203/08/0536 and the researchproject No. Z40550506 of the Institute of Organic Chemistry and Biochemistry, Academy of Sciencesof the Czech Republic.References1. Čeřovský, V., et al. ChemBioChem 10, 2089-2099 (2009).Antimicrobial activity MIC [μM] aB.s. E.c. S.a. P.a.HemolyticactivityLC 50 [μM]LAS 2891.7 0.4 8.6 93 15 > 200LAS 8-27,17-25 2891.7 0.8 41.7 > 100 > 80 > 200LAS 8-17,25-27 2891.7 6.0 > 25 > 100 > 25 > 200LL-I 1722.1 0.8 1.7 14.3 15.8 > 200Indolicidin 1906.3 1.0 > 100 13.0 > 100 > 200Tetracycline 444.4 12.5 0.4 1.5 75.7 > 200a B.s., Bacillus subtilis; E.c., Escherichia coli; S.a., Staphylococcus aureus; P.a.,Pseudomonas aeruginosa407