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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, 2010Conventional and Microwave-Assisted SPPS Approach: AComparative Study of PTHrP(1-34)NH 2 SynthesisFabio Rizzolo 1,2 , Chiara Testa 1,2,3 , Michael Chorev 4,5 , Mario Chelli 1,2 ,Paolo Rovero 1,6 , and Anna Maria Papini 1,2,31 Laboratory of Peptide & Protein Chemistry & Biology, University of Florence, PoloScientifico e Tecnologico, Sesto Fiorentino (Fi) I-50019, Italy; 2 Department of Chemistry“Ugo Schiff”, Via della Lastruccia 3/13, University of Florence, Polo Scientifico eTecnologico, Sesto Fiorentino (Fi) I-50019, Italy; 3 Laboratoire SOSCO – EA4505Université de Cergy-Pontoise, 5 mail Gay-Lussac, Neuville-sur-Oise, Cergy-Pontoise95031, France; 4 Laboratory for Translational Research, Harvard Medical School,Cambridge, MA, 02139, U.S.A; 5 Department of Medicine, Brigham and Women’s Hospital,Boston, MA, 02115, U.S.A.; 6 Department of Pharmaceutical Science, Via Ugo Schiff 6,University of Florence, Polo Scientifico e Tecnologico, Sesto Fiorentino (Fi), I-50019, ItalyIntroductionParathyroid hormone-related peptide (PTHrP) is an autocrine/paracrine regulator ofendochondral bone development and involved in excessive osteoclastic bone resorptionassociated with humoral hypercalcaemia of malignancy (HHM) [1]. PTHrP is a 139 to 173-amino acid protein having the first thirteen N-terminal amino acids in common withparathyroid hormone (PTH). Like PTH, PTHrP releases calcium from bone and increasessecretion of calcium and phosphate in the distal tubule [2].Similar to PTH the calcium metabolism-related activities are located in the first 34N-terminal residues. Consequently, our synthetic efforts focused on this bioactive sequencethat was the subject of numerous structure-activity-conformation relationship studies [3].Considering the presence of clusters of arginines, sterically hindered and hydrophobicamino acid residues in the 19-28 sequence of PTHrP and the considerable length of thepeptide, the synthesis of PTHrP(1-34)NH 2 is quite challenging. We therefore undertook thesynthesis comparing the conventional (room temperature) Solid-Phase Peptide Synthesis(SPPS) vs. the MicroWave-assisted SPPS [4].Results and DiscussionPTHrP(1-34)NH 2 sequence is: H-Ala 1 -Val-Ser-Glu-His-Gln-Leu-Leu-His-Asp 10 -Lys-Gly-Lys-Ser-Ile-Gln-Asp-Leu-Arg-Arg 20 -Arg-Phe-Phe-Leu-His-His-Leu-Ile-Ala-Glu 30 -Ile-His-Thr-Ala-NH 2 . The peptide was synthesized by SPPS, following Fmoc/tBu strategy, at roomtemperature and by microwave irradiation, using in both cases Liberty peptide synthesizer(CEM) and the same reagents concentration. The synthesis was monitored by LC-MS usingMW-assisted mini-cleavages of the intermediate fragments using Discover single modemicrowave system (CEM).The SPPS was performed using the NovaSyn TGR (500 mg, 0.2 mmol/g), 0.2 Msolution of amino acids in DMF (5 equiv.), 0.5 M solution of TBTU in DMF (5 equiv.), 2M solution of DIPEA in NMP (10 equiv.). Final cleavage of the peptide from the resin andside-chain deprotections were performed with ad hoc cleavage cocktail TFA/water/TISsolution (95:2.5:2.5 v/v/v) for 3 hours at room temperature.The room temperature-SPPS cycle (Liberty, CEM) consisted of two deprotection steps(5 and 10 min respectively) and 20 min for the coupling steps. The MW-SPPS cycle(Liberty, CEM), the deprotection steps were performed at 75 °C using 35 W for 30 sec forthe first one and 60 W for 180 sec for the second one, while the coupling steps wereperformed at 75 °C, using 30 W for 300 sec.MW-assisted mini-cleavage cycles (Discover, CEM) were carried out at 45 °C, using15 W for 15 min with external cooling of the reaction vessel.In this comparative study of PTHrP(1-34)NH 2 SPPS, using the same instrumentationand the same reagents concentration, MW-assisted strategy gave an increased purity of thecrude peptide as compared with the conventional approach. RP-HPLC purification of thecrude peptide was evaluated by LC-MS (ACQUITY UPLC System coupled withMicromass ® Quattro micro API Mass Spectrometer, Waters) yielded 27 mg and 18 mg of>95% pure PTHrP(1-34)NH 2 from the MW-assisted synthesis and SPPS at roomtemperature, respectively (Table 1).188