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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, 2010Convergent Syntheses of HuPrP106-126 (Difficult Sequence)Using Native Chemical Ligation andDesulfurization/DeselenizationJaroslav Šebestík, Martin Šafařík, Zbigniew Zawada, andJan Hlaváček*Institute of Organic Chemistry and Biochemistry, vvi, Academy of Sciences, CR,Flemingovo n. 2, 166 10, Prague, Czech RepublicIntroductionPrion proteins are causative agents of neurodegenerative diseases such as scrapie, CJD,mad-cow disease (nvCJD), GSS, etc. [1]. The Prion-derived peptide HuPrP106-126 wasfound as a difficult sequence for the Fmoc approach [2]. We have described its synthesisusing a divergent approach in 9% yield [3]. In order to improve the yield of this peptide byFmoc approach, we employed 2- and 3-segment convergent approaches using a nativechemical ligation with subsequent desulfurization/deselenization [4] of peptide precursors.Results and DiscussionTwo splitting places of the difficult sequence of HuPrP106-126 were designed. The firstone between Gly 114 -Ala 115 residues and the second one between Gly 119 -Ala 120 . This choicedemanded the syntheses of peptide thioesters with C-terminal Gly residue and thus avoidedracemization. Retro syntheticaly functional group introduction was applied on N-terminalalanine residue i.e. Ala to Cys transformation was carried out. Syntheses of segments with12 and 14 residues were non-competitive to divergent solid phase approach (comparison ofitems 4, 6, 8, 10, and 12 at Table 1 with item 2). Competitive could be solely synthesis ofselenocysteine (U) derivative (item 3), and synthesis of peptide thioester by Beck-Sickinger's [5] method (item 11). Limited availability of difficult peptide segments led us tothe synthesis of HuPrP106-126 using Kent's modular approach [6]. Here, we used bothspliting sites in one synthetic scheme. The middle part of peptide was prepared by Beck-Sickinger's [5] method with protection of N-terminal Cys as 4-thiazolidine carboxylic acid.This method provided two peptides (items 13 and 14) in very competitive yields todivergent peptide synthesis (item 2). When the HuPrP106-126 was prepared by consecutivechemical ligation from the peptides (item 3, 11, and 14) the yield limiting step was thesynthesis of N-terminal peptide thioester (item 3).MethodsA: Synthesis of peptides on Wang polystyrene resin by Fmoc/tBu strategy. Cleavage ofpeptides was achived by TFA/TIS/H 2 O/EDT mixture (90:2.5:2.5:5).B: Synthesis of peptides on Wang polystyrene resin by Fmoc/tBu strategy. Cleavage byHilvert's method [7] with AlMe 3 /EtSH, followed by TFA/TIS/H 2 O/EDT mixture(90:2.5:2.5:5). Unfortunatelly, Asn residues were converted to aspartimide (Table 1,underlined).C: Synthesis of peptides on Ellman's sulfonamide resin [8]. The resin was activated withTMS-CHN 2 and cleaved with Mpa-OnBu/DMF (55 o C, several days). The side protectiongroups were removed with TFA/TIS/H 2 O/EDT mixture (90:2.5:2.5:5).D: Syntheses of protected peptides on chlorotritylchloride resin [5]. Protected peptide wascleaved with HFIP/DCM (1:3) 5 min, and coupling with DIC/DMAP/EtSH followed.Protected peptide thioester was deprotected with TFA/TIS/H 2 O/EDT mixture(90:2.5:2.5:5).204