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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, 2010Solid-Phase Synthesis of C-Terminus ChloromethylKetone PeptidesWitold A. Neugebauer 1 , Nabil G. Seidah 2 , Delia Susan-Resiga 2 ,Xue Wen Yuan 1 , and Robert Day 11 Institut de pharmacologie de Sherbrooke, Université de Sherbrooke, 3001, 12e AvenueNord, Sherbrooke, J1H 5N4, Québec, Canada; 2 Institut de recherches cliniques deMontréal 110 West Pine Ave, Montreal, H2W 1R7, Québec, CanadaIntroductionHalomethyl ketones and in particular chloromethyl ketones are important tools in enzymeinhibitory studies. Chloromethyl ketones of amino acids, their analogues and peptides arethe most commonly used. There are numerous ways to make halomethyl ketones of aminoacids or peptides in solution. A transformation of molecules such as amino acids to theirchloromethyl ketones in classical organic synthesis is relatively easy. Peptide chloromethylketone preparations give more problems due to peptide solubility, stereochemistry whencouples from C-side of the peptide or peptide direct transformation to chloromethyl ketone.Solid-phase synthesis of peptide chloromethyl ketone would eliminate most of thoseproblems. Classical solid phase synthesis of such chloromethyl ketone peptide could not beapplied due to missing C-terminal carboxyl group. A new strategy, used by Kwon, et al. [1]to build a peptide on special aryl hydrazine resin (Figure 1) and cleave assembled peptidesequence (without the last C-terminal chloromethyl ketone) with hydrochloride ofchloromethyl ketone residue was used. Synthesized peptides: subtilisin/kexin-like isozyme-1 SKI-1 [called also Site-1 protease] inhibitors (Figure 2) were tested on their inhibitorypotency (Figure 3).MethodsPeptide synthesis: Peptides were synthesized (Figure 1) using continuous flow synthesizerwith Fmoc strategy. The protected peptide-resin less C-terminal chloromethyl ketoneresidue was then mildly oxidized to diazene and displaced from the resin with amino acidchloromethyl ketone derivative. Solvent was evaporated and peptides were thendeprotected (TFA/TIPS/water). Peptide salts were precipitated in anhydrous ethyl ether,filtered, dissolved in 25% acetic acid and lyophilized. The crude peptides were finallypurified by C18 column chromatography in acetonitrile gradient in water with 0.1% TFA.Peptide identities were confirmed by MALDI mass spectrometry and their chromatographicpurity verified by analytical HPLC.Source of recombinant hSKI-1: Soluble human SKI-1, lacking the transmembrane domainand cytosolic tail, hSKI-1-BTMD, wasisolated from overnight media of HEK293 cells infected with a recombinantvaccinia virus (VV) vector, VV:hSKI-1-BTMD. Upon collection and cell debrisremoval by centrifugation, the media wasconcentrated 5-fold on an AmiconUltracel 30K (Milipore) and stored at-20°C as a 20% glycerol solution.Overnight media from HEK293 cellsinfected with vv:wild type (WT) that wasprepared as above was used as abackground activity control.In vitro enzymatic assay: Reactions wereperformed at 37°C in 100 ml. Eachreaction mixture consisted of: buffer(25 mM TRIS-HCl, 25 mM MES, 1 mMCaCl 2 , adjusted to pH 7.5), 40 mlFig. 1. Scheme of general peptide-chloromethylketone synthesis.vv:hSKI-1-BTMD or vv:WT, 20 mMinhibitor peptides or DMSO in equivalentvolume, and 200 mM fluorogenic80