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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, 2010Repetitive Cleavage of Aib-Peptides by Trifluoroacetic AcidHans Brückner 1 , Christoph Theis 1 , Thomas Degenkolb 1,2 ,Renate Gessmann 3 , and Michael Kokkinidis 31 Research Center for BioSystems, Land Use and Nutrition (IFZ), Department of FoodSciences, University of Giessen, Giessen, 35392, Germany; 2 Department of Entomology(IFZ); 3 Institute of Molecular Biology and Biotechnology (IMBB), FORTH,Heraklion, 71110, Crete, GreeceIntroductionCrystalline, synthetic homo-Aib-peptides of the general structure Z-(Aib) n -OtBu (n = 3–11)form consecutive -turns of type III resulting in full turns of (3 10 )-helices for n = 5, 8, and11 (Aib, -aminoisobutyric acid, 2-methylalanine) [1]. Native peptaibols are helical,fungal peptides containing Aib as well as proteinogenic amino acids and a C-terminal 2-amino alcohol. Natural peptides lacking the amino alcohol are named peptaibiotics [2,3].Peptide bonds formed by proteinogenic amino acids are commonly stable against TFA atmoderate temperature, whereas those formed by Aib are less resistant. We present a studyand propose a mechanism concerning the cleavage of synthetic Aib-peptides by anhydrousTFA.Results and DiscussionAnhydrous TFA (100 l) was added to 0.1 mg of each peptide (Figure 1). Solutions werekept at 37 °C for 0.5 – 26 h. Dried residues were dissolved in MeOH, and the resultingcleavage products were analyzed by RP-HPLC and online ESI-CID-MS. Details of thecrystal structures and synthesis of peptides are described in references [1c] and [4],respectively. For the crystal structures of Z-(Aib) 10 -OtBu and Z-(Aib) 7 -OtBu see Figure 2.Release of regular series of Z-(Aib) 10-5 -OH from synthetic Z-(Aib) 10 -OtBu within0.5 h was recognized (Figure 1). Concomitant formation of H-(Aib) 10-3 -OH was alsoobserved. After 3 h, a regular series Z-(Aib) 7-3 -OH from Z-(Aib) 7 -OtBu was formed.However, cleavage of the Z-groups also occurred. From Ac-(Aib) 10 -OtBu C-terminal Aibresidueswere cleaved but N-terminal Ac-(Aib) 5 -OH was still observed after 8 h. Based onthese data, a repetitive cleavage mechanism of homo-Aib-peptides via formation ofC-terminal oxazolones has been proposed [5] and is presented (Figure 2). Relatedmechanisms have beendiscussed in theZ-(Aib) 10 -OtBu Z __ Aib 1__ Aib 2__ Aib 3__ Aib 4__ Aib 5__ Aib 6__ Aib 7__ Aib 8__ Aib 9__ Aib 10__ OtBuliterature [6]. In nativepeptaibols very fastcleavage of the Aib-Probonds was observed aswell as scission ofAc-(Aib) 10 -OtBuAc __ Aib 1__ Aib 2__ Aib 3__ Aib 4__ Aib 5__ Aib 6__ Aib 7__ Aib 8__ Aib 9__ Aib 10__ OtBucertain Aib-Aaa (Aaa =Gly, Ala, Gln) and Aib-Aib bonds. Considerableagreement was noticedwith the formation ofZ-(Aib) Z __ Aib 1__ Aib 2__ Aib 3__ Aib 4__ Aib 5__ Aib 6__ Aib 7__ 7 -OtBuOtBuregular b-series of0.5 h3 h3 hFig. 1. Structures of homo-Aib-peptides analyzed. Preferredcleavages of peptide bonds at certain time intervals areindicated.1 h3 h0.5 h8 h0.5 h0.5 h0.5 h3 h0.5 h3 h0.5 h0.5 h0.5 h0.5 h0.5 h0.5 h0.5 hpositive acylium oroxazolonium ionsresulting from ESI-MS(Figure 2).58