1. Peptoid analogs of the antibacterial peptide anoplin Introduction Karin Meinike a,b and Paul R. Hansen a a IGM-Bioorganic Chemistry, Faculty of Life Sciences, University of Copenhagen, Denmark b Present Address: Department of Clinical Biochemistry and Immunology, Statens Serum Institut, Copenhagen, Denmark. References 1.Konno, K. et al. Biochim Biophys Acta, 2001, 1550, 70-80. 2.Ifrah, D., Doisy, X., Ryge, T.S., and Hansen, P.R. J Pept Sci., 2005, 11,113-21. 3.Nielsen, S.L., Frimodt-Møller, N., Kragelund B.B. and Hansen, P.R. Protein Science, 2007, 16, 1969-1976. Acknowledgements We thank Ms. Jette Petersen for excellent technical assistance. This work was supported by the Danish Research Council grant number 9900234, the Augustinus Foundation, the Frimodt–Heineke Foundation, the Family Hede Nielsen Foundation, the JS Foundation and the Aase & Ejnar Danielsens Foundation. Anoplin (H-GLLKRIKTLL-NH 2 ) is a recently discovered antibacterial peptide amide found in the venom sac of the spider wasp Anoplius Samariensis [1]. Anoplin is active against both Gram-positive and Gram-negative bacteria, including Staphylococcus aureus ATCC 25923 and Eschericia coli ATCC 25922. Furthermore, the hemolytic activity against human erythrocytes is very low. We have previously reported a structure-activity study of anoplin [2] based on 37 analogs. The substitutions were designed using helical wheel considerations (figure 1). We found that substitution of Arg 5 with Val, Leu, Ile, Phe,Trp or Lys resulted in analogs with MIC-values in the range of 2-22  M. Unfortunately, the analogs were very hemolytic against human erythrocytes. In the present study, we have synthezised nine peptoid analogs of anoplin. In each analog, Arg 5 was substituted with a peptoid monomer X (H-GLLKXIKTLL-NH 2 ) resulting in a side chain either identical to (Val, Leu, Ile or Phe), resembling (Trp) or different from a natural amino acid (analogues 1-4, 5 and 6-9, respectively). Structures are shown in figure 2. Figure 2: Peptoid building blocks used in this study Conclusion In conclusion, we have shown that Arg 5 of anoplin may be substituted with either N-(2,2-diphenylethyl)Gly or N-(1-naphthalenemethyl)Gly, resulting in peptoid analogs with a hemolytic activity and antibacterial activity which are comparable to anoplin, however with reversed selectivity. Since peptoid residues are stable to proteolytic breakdown, this study indicates that peptoid analogs of anoplin are promising lead-structures for the development of new antibacterial agents. Results and Discussion The peptoid analogs of anoplin were synthesized using a combination of Fmoc SPPS and the submonomer approach. A purity of >95% was obtained by preparative and analytical HPLC and the identity of the analogs was confirmed by MALDI-TOF MS. The antibacterial activity of the analogs was tested against the Gram-positive bacterium S.aureus and the Gram-negative bacterium E.coli using a micro-dilution assay described previously [3]. Table 1 lists the MICs obtained for the nine peptoid analogs and anoplin. Of the analogs with a peptoid monomer yielding a side chain identical to that of a natural amino acid or resembling one, only analogs 4 and 5 showed antibacterial activity in the concentration range used with MICs ranging from 25-100  M. The best candidates were 7-9 with MICs of 12.5-25  M against E.coli and 25-50  M against S.aureus. Analogs 5 and 7-9 showed a clear selectivity towards E.coli, with analogs 7 and 8 having MICs comparable to those of anoplin (23 µM and 11 µM against E.coli and S.aureus, respectively). However the selectivity was reversed. The hemolytic activity of the analogs against human erythrocytes was determined as described previously [3]. The analogs showed very low hemolytic activity ranging from 4 to 9 percent. The lowest hemolytic activity was observed for the analogs with the highest MICs ranging from 4- 7%. The analogs with aromatic or ring structured side-chains had the highest hemolytic values ranging from 6 to 9 percent, though a value of 9% still might be considered fairly low. SequenceS.aureus E.coli% hemolysis anoplin 11 23 - 1[N-(2-propyl)Gly 5 ] anoplin >100 4 2[N-(2-methylpropyl)Gly 5 ]anoplin >100 >100 7 3[N-(1-methylpropyl)Gly 5 ]anoplin >100 4 4[N-(benzyl)Gly 5 ]anoplin >100 50-100 6 5[N-(2-(1H-Indol-3-yl)ethyl)Gly 5 ]anoplin 50-100 25 8 6 [N-(butyl)Gly 5 ]anoplin >100 100 5 7[N-(2,2-diphenylethyl)Gly 5 ]anoplin 25 12.5 7 8[N-(1-naphthalenemethyl)Gly 5 ]anoplin 25 12.5 9 9[N-(cyclohexylmethyl)Gly 5 ]anoplin 50 25 9 Figure 1: Helical wheel of anoplin Table: Minimum inibitory concentration (  M) and hemolytic activity (50  M) of the analogs