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polymyxin bicyclic peptide analogues bridged with a bipyridine group in order to introducestructural constraints pertaining to the stabilization of the peptide’s binding conformation,and to incorporate additional hydrogen-bonding acceptor site.Figure 2 shows a general scheme for synthesis of a library of bicyclic peptides withthree amino acids positions available for randomization. The first amino acid Fmoc-Asp-OAllyl was anchored via side chain to the MBHA Rink Amide resin, followed bysubsequential coupling of Fmoc-Lys(Mtt)-OH and one of amino acids. Standard Fmoccouplingconditions including fourfold excess of amino acids and HOBt/HBTU/Nmethylmorpholinewas used throughout the synthesis of this tripeptide precursor. However,coupling of amino acids mixtures in order to randomize the remaining peptide sequencewas shown to be more difficult. Due to the steric effects and different amino acids couplingkinetics, the standard solid-phase synthetic approach had to be optimized. A mixture of 19L-amino acids (Cys was omitted due to the high possibility of an undesired disulfide bridgeformation) was used in 10 fold excess compared to the resins loading capacity, and in molpercent ratios as was outlined in the paper of R. Houghten et al. [2]. Successful couplingwas obtained by using an equimolar amount of HOBt/HBTU/N-methylmorpholinecoupling reagents with the respect to the amount of amino acids in the mixture. Cyclizationof a linear precursor was achieved by using 2 equivalents of PyBOP/HOBt/DIPEA inDMF, and vigorous mixing of the reaction mixture over night. Following the cyclizationstep, ivDde protecting group was removed from Lys side chain with 2% hydrazine in DMF,and coupling of 2,2’-bipyridine-5,5’-dicarboxylic acid to Lys side chain’s amino group wasachieved by PyBOP/HOBt/DIPEA. After removal of Mtt from the peptide, bicycliccompound was prepared using the same synthetic protocol. In all cases coupling efficacywas monitored by the Kaiser test. The final step included removal of the prepared bicyclicpeptides from the resin. The outlined combinatorial chemistry approach allowed us tocreate a quite diverse library of bicyclic peptides for anthrose artificial receptor's bindingoptimization.Lipidic derivative of anthrose was synthesized [3] as a model for B. anthracisexosporium, and lipidic derivative of glucose was synthesized as a control to evaluatespecificity of bicyclic peptide receptor. Our further investigation will include binding assaydevelopment in a 384-well microplate format, and bicyclic peptide library screening forselective anthrose binding.AcknowledgmentsWe gratefully acknowledge the financial support of the NATO Public Diplomacy Division, Science forPeace and Security Programme (SfP 983154) to P.C. and A.J.References1. Daubenspeck, J.M., Zeng, H., Chen, P., Dong, S., Steichen, C.T., Krishana, N.R., Prichard, D.G.,Turnbough, C.L. J. Biol. Chem. 279, 30945-30953 (2004).2. Pinilla, C., Appel, J.R., Houghten, R.A. Methods in Molecular Biology 66, 171-179 (1996).3. Dhénin, S.G.Y., Moreau, V., Morel, N., Nevers, M.C., Volland, H., Créminon, C., Djedaïni-Pilard,F. Carbohydr. Res. 343, 2101-2110 (2008).89