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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, 2010Efficient Microwave-Assisted Synthesis of Mitochondrial SignalPeptide Using Cltr-Cl and Wang ResinMaria-Eleni Androutsou 1 , Panagiotis Plotas 2 , George Agelis 1 , andJohn Matsoukas 1,21 Department of Chemistry, University of Patras, Patras, 26500, Greece;2 Eldrug S.A., Patras Science Park, Rio, 26504, GreeceIntroductionMitochondrial targeting peptide plays an important role in transporting all the necessaryinformation of proteins into mitochondria. A signal peptide is a short (3-60 amino acidslong) peptide chain that directs the transport of a protein. The signal peptide that directs tothe mitochondrial matrix has a sequence consisting of an alternating pattern (amphipathichelix) with a few hydrophobic amino acids and a few positively charged amino acids at theN terminus [1]. In this study the effect of two different resins (Cltr-Cl and Wang) wascompared using microwave energy for the synthesis of a 18-mer signal peptide (H-Met-Leu-Ser-Leu-Arg-Gln-Ser-Ile-Arg-Phe-Phe-Lys-Pro-Ala-Thr-Arg-Thr-Leu-OH). Microwaveenergy represents a fast and efficient way to enhance both the deprotection andcoupling reactions hindered by aggregation. In peptides, the N-terminal amine group andpeptide backbone are polar, causing them to constantly try to align with the alternatingelectric field of the microwave. During peptide synthesis this can help break up the chainaggregation owing to intra and interchain association and allow for easier access to thesolid phase reaction matrix [2].Results and Discussion1. Materials: The 18-mer signal peptide was synthesized by an automated microwavepeptide synthesizer (Liberty, CEM) using Fmoc strategy. Cltr-Cl resin and protected aminoacids were obtained from CBL Patras, Greece. Wang resin was obtained from Sigma-Aldrich. Purification of final products was performed with Waters Preparative HPLC(Waters Prep LC Controller) using SunFire Prep C18 column (50x100 mm) with 5 μmpacking material. Purity was determined by Alliance HPLC of Waters 2695 Separationmodule equipped with Empower operating system using a Waters 2996 diode array UV/Visdetector and XBridge C18 column (4.6x150 mm) with 3.5 μm packing material.Identification was determined by Waters Acquity UPLC/MS System equipped withMassLynx operating system.2. Esterification of Fmoc-Leu-OH on Cltr-Cl resin: 2-Chlorotritylchloride resin (0.36 g, 0.6mmol Cl-/g of resin) was left to swell in dry DCM (5 mL) for 30 min, N-Fmoc-Leu-OH(0.338 g, 0.36 mmol), DIPEA (0.9 mmol) were added and stirred at RT. After 1 h, thesuspension was filtered, washed successively with DCM/MeOH/DIPEA (17:2:1, v/v) (2 xFig. 1. Analytical RP-HPLC of final crude peptide using Cltr-Cl resin (A), Wang resin (B),final product (C) and ESI-MS of Mitochondrial Signal Peptide (D).100