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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, 2010The Microwave Revolution: Recent Advances in MicrowaveAssisted Peptide SynthesisSandeep K. Singh, Alicia D. Douglas, Eric J. Williamson, andGrace S. Vanier*CEM Corporation, Bioscience Division, PO Box 200, 3100 Smith Farm Road, Matthews,NC, 28106, U.S.A.IntroductionIn solid phase peptide synthesis (SPPS), while certain peptide sequences are synthesizedrelatively easily, some sequences are much more difficult. Efficient couplings occur within afully solvated peptide-polymer matrix, where reagent penetration is rapid and unhindered.Sudden decreases in reaction rates and incomplete couplings have been attributed to peptideaggregation resulting in poor salvation [1]. Microwave energy represents a fast and efficientway to enhance both the deprotection and coupling reactions hindered by aggregation. TheN-terminal amino group and peptide backbone are polar and they may constantly try to alignwith the alternating electric field of the microwave, and assist in breaking up the chainaggregation. The application of microwave energy has proved to be a major enabling tool forenhancing slow and difficult chemical reactions [2]. Unlike conventional heating, microwaveenergy could selectively activate any molecule with a dipole moment and thereby allow forrapid heating at the molecular level. Microwave assisted SPPS has been successfully appliedto and shown useful for the synthesis of a range of difficult peptides [3]. Microwave peptidesynthesis routinely shows substantial improvements in crude purity with reduced synthesistime compared to conventional SPPS. Previous studies have investigated the effects ofmicrowave on aspartimide formation and epimerization, and offered optimized conditions forsusceptible sequences to these well-known side reactions [4]. We now report our recent resultson the development of microwave assisted N-terminal modifications and head-to-tail on-resincyclization.Results and DiscussionN-terminal modifications. Fatty acid acylation on the N-terminus of a peptide increases its cellpermeability and affinity, and is a common post-translational modification for a wide varietyof viral, bacterial and eukaryotic proteins and peptides [5]. Biotin labeled peptides havenumerous biochemical and microbiological applications [6]. Under conventional conditions,these modifications often require coupling reactions of 24 h or more due to poor solubility andreactivity of the fatty acid or biotinylating reagents. We envisaged that sluggish reactionkinetics in these couplings could be overcome by the application of microwave irradiation.The acyl carrier protein, ACP-(65-74) sequence (Val-Gln-Ala-Ala-Ile-Asp-Tyr-Ile-Asn-Gly) was selected as the test peptide for the present study. ACP-(65-74) was synthesized onFmoc-Gly Wang resin (0.61 mmol/g) in less than 5 h using the CEM Liberty automatedmicrowave peptide synthesizer. Fmoc deprotection with 20% piperidine in DMF for 0.5 minand 3 min at 75 o C and coupling with Fmoc-AA-OH/HBTU/DIEA for 5 min at 75 o C gavethe ACP-(65-74) with a crude purity of more than 95% (Figure 1a). Double coupling ofn-hexanoic acid to ACP for 5 min at 75 o C using HCTU/DIEA activation gave the N-cappedpeptide in 80% crude purity (Figure 1b). Similarly, microwave coupling of biotin-LC to ACPusing HATU/DIEA for 5 min at 75 o C gave the biotin labeled peptide in 93% crude purity(Figure 1c). Thus, N-terminal modifications with a fatty acid or biotin were completed inFig. 1. HPLC crude chromatograms of (a) ACP-(65-74), (b) n-Hexanoic-ACP-(65-74), (c)Biotin-LC- ACP-(65-74).168