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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, 2010Novel Peptide Conjugates: Heterocyclic Modifications of theImmunomodulatory Ubiquitin FragmentAlicja Kluczyk 1 , Malgorzata Ratajska 1* , Anna Staszewska 1 ,Marek Cebrat 1 , Hubert Bartosz-Bechowski 1 ,Piotr Stefanowicz 1 , Michal Zimecki 2 , and Zbigniew Szewczuk 11 Faculty of Chemistry, University of Wroclaw, 50-383 Wroclaw, Poland; 2 Institute ofImmunology and Experimental Therapy PAS, 53-114 Wroclaw, Poland;*current address: The Kostrzyca Forest Gene Bank, 58-535 Milkow, PolandIntroductionUbiquitin, a 76-amino acid polypeptide, is one of the pivotal factors in cellular physiologyas a post-translational addition to proteins that targets them for degradation by theproteasome [1]. In addition to protein degradation, ubiquitin is known to activate cellsignals in several pathways: tolerance to DNA damage, inflammatory response, proteintrafficking, and ribosomal protein synthesis. The immunomodulatory properties ofubiquitin were discussed by us recently [2]. We discovered that the peptide I Asp-Gly-Arg-Thr-Leu (52-56) is the shortest active fragment of the immunosuppressory (50-59) loop ofubiquitin [3] and we analyzed the structure of this fragment [4], as well as the activity ofdimeric peptides containing the (50-59) Leu-Glu-Asp-Gly-Arg-Thr-Leu-Ser-Asp-Tyrsequence.Results and DiscussionA series of analogues of peptide I, with the N-terminal Asp residue replaced by novelaromatic nonproteinaceous amino acid residues (Figure 1), were synthesized to investigatetheir physicochemical properties, metal ion affinity and immunosuppressory activity. Aspecial derivative of phenylalanine, Fmoc-β-(4-amino-3-nitrophenyl)-alanine, was used asa substrate to obtain 2-substituted 1H-benzimidazol-5-yl alanine [5] or 2,3-disubstitutedquinoxalin-6-yl alanine [6] analogues of I. The heterocyclic motifs were formed on-resinafter solid phase peptide assembly; the purity and identity of conjugates was confirmedusing HPLC, mass spectrometry and NMR.The interactions with metal ions were investigated in respect to Cu(II) using ESI-MS,circular dichroism and NMR. High resolution mass spectra (Figure 2) reveal formation ofseveral coordination compounds varying in peptide-metal ratio, dimer formation was alsoobserved. The collision induced dissociation experiments suggest that the presence ofcopper ion changes the fragmentation pathway of the conjugate as a result of radicalAnalogues of 52-59 ubiquitinXaa-Gly-Arg-Thr-LeuHNNH 2Peptide IConjugate IIConjugate IIIConjugate IVXaa = Asp(original fragment)Xaa = DpqaXaa = DppaXaa = PbaOHOH 2 NONHHNONHOHONHOCH 3NHOOHNNNH 3 CCH 3NNNNNNNNHH 2 NH 2 NDpqa Dppa PbaOOH 2 NOFig. 1. Peptide conjugates, analogues of 52-59 fragment of ubiquitin, containingsubstituted quinoxaline and benzimidazole heterocyclic motifs.200