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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, 2010Microwave-Assisted Solid-Phase Peptide Synthesis of the 60-110Domain of Human Pleiotrophin (hPTN) on CLTR-Cl ResinIrene Friligou 1 , Evangelia Papadimitriou 2 , Dimitrios Gatos 1 ,John Matsoukas 1 , and Theodore Tselios 11 Department of Chemistry, University of Patras, GR-265 04, Patras, Greece;2 Laboratory of Molecular Pharmacology, Department of Pharmacy,University of Patras, GR-265 04, Patras, GreeceIntroductionHuman pleiotrophin (hPTN) is a heparin-binding growth factor with diverse biologicalactivities, the most studied being those related to the nervous system, tumor growth andangiogenesis. It is interesting to determine which regions of hPTN are responsible for itsdiverse functions, in order to identify the molecular mechanisms involved and to identifypossible therapeutic targets or/and agents. The binding of hPTN to heparin is mediated bythe two central regions that are homologous to the thrombospondin type I repeat (TSR-1),with the carboxyl terminal TSR-1 domain (60-110) being the main heparin-binding site ofPTN [1,2]. Here we report the Microwave Enhanced Solid Phase Peptide Synthesis(MW-SPPS) of the C-terminal 60-110 domain of hPTN composed of 51 amino acids usingthe Fmoc/tBu methodology [3,4].Results and DiscussionThe linear protected peptide was synthesized using the Liberty TM Microwave PeptideSynthesizer (CEM) on 2-chlorotrityl chloride resin (CLTR-Cl) [3]. Fmoc deprotection wasachieved with 20% piperidine in DMF, while for the coupling reactions HOBt/DIC in DMFwere used (Figure 1). Moreover, this domain is supposed to contain two disulfide bonds,one between 67-99 residues and one between 77-109 residues [2]. In order to achieveselective formation of these disulfide bonds, the Cys(Trt) at 77, 109 positions andCys(Acm) at 67, 99 positions were used. After cleavage of the protected peptide from theresin and removal of the side chain protecting groups [except for Cys(Acm) 67 andCys(Acm) 99 ], the first disulfide bond was formed by dimethyl sulfoxide (DMSO) [4]. Thesecond disulfide bridge was formed simultaneously with the Acm group removal usingiodine. The desired linear peptide was obtained in only 30h of total processing time and in60% crude yield [4]. The products were checked for their purity by analytical HPLC(Waters Alliance 2695 Separations Module combined with Waters 2996 photodiode arraydetector) using a C-8 Purospher column (5 μm, 250 × 4 mm) at 214 nm and 254 nm,separation was achieved by gradient elution of 5% to 100% solvent B (solvent A=0.08%TFA in H 2 O; solvent B=0.08% TFA in ACN) over 30 min at a flow rate of 1ml/min(Figure 2), and they were identified by ESI-MS [4]. In this report, we demonstrated thatmicrowave energy can also be applied in the case of the solid-phase synthesis of largepeptides utilizing the acid sensitive CLTR-Cl resin.Fig. 1. Synthetic procedure of [Cys(Acm) 67,99 ]-hPTN 60-110 .170