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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, 2010Amino Acid Coupling Reactions in Aqueous Environment UsingMicrowave Assistance HeatingAthanassios S. Galanis 1 , Morten Grøtli 2 , and Fernando Albericio 31 Department of Pharmacy, University of Patras, Patra, 26504, Greece; 2 Department ofChemistry, University of Gothenburg, Gothenburg, 41296, Sweden; 3 Institute for Researchin Biomedicine, Barcelona Science Park, Barcelona, 08028, SpainIntroductionThe solid-phase method is the principal method for peptide synthesis, but it requires a largeamount of organic solvent. As the safe disposal of organic solvent waste is an importantenvironmental issue, as well as the increase of the synthesis cost, a method for peptidesynthesis in water would be desirable. Thus, the aim of this project is to replace costlyorganic solvents for peptide synthesis with inexpensive, environmentally-benign solvents,such as water. We previously reported the solid phase peptide synthesis in aqueousenvironment using microwave heating, Boc-derivatives and the mixture of HONB/EDC ascoupling reagents [1,2]. In the following, we present the studies of the twenty native aminoacids coupling reaction using commercial available Boc-derivatives. Moreover, comparisonamong the microwave heating efficiency and the conventional thermal heating method isalso reported.Results and DiscussionThe concept of this work was the use of microwave influence for SPPS in water.Obviously, the major issue was the amino acids coupling reaction efficiency in water. Inorder to develop a strategy that could be easily applied in every laboratory or in industry,the evaluation of commercial available amino acid derivatives and reagents or derivativesthat could be easily synthesized, was performed. Thus, the Fmoc and Boc amino acids wereevaluated, even though those groups are hydrophobic. Furthermore synthesis andevaluation of several azido acids was carried out, as azido group is an hydrophilic group,can be used as alternative N α -protecting group, and azido acids derivatives can be easilysynthesized. Regarding the solid support, the poly (ethylenglycol) type resins were used, asthey are more hydrophilic compared to polystyrene resins. Several coupling reagents wereevaluated, starting with the N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) that isan excellent water soluble carbodiimide, following by studies of several additive reagentsefficiency. In coupling reaction experiments, Tentagel and ChemMatrix resins were used assolid support with a pre-attached Rink amide linker, EDC carbodiimide with or without thepresence of additive reagents, applying microwave irradiation to elevate the temperature upto 80 degrees for 10 to 30 min. The coupling reactions of all experiments were performedon solid phase and each amino acid derivative was reacted to a pre-synthesized dipeptide ofGly-Phe bound onto the RAM-TentaGel/ChemMatrix resin (Figure 1).Among the amino acid derivatives that were studied, Boc-amino acids provided by farbetter results compared to Fmoc- and azido acid derivatives. However, active estersstability studies, showed that the esters are not stable in water at elevated temperature.Thus, short reaction time (up to 10 min) were essential, in order to avoid byproductsdetected when extended time was applied. Among the coupling reagents that were studied,the mixture of EDC / HONB (N-hydroxy-5-norbornene-2,3-dicarboxylic acid imide)provided the better results as the reactions were completed and traces of byproducts wereonly detected in some cases. After optimization of coupling conditions, the reactions ofeach Boc-amino acid derivative was performed using microwave assisted heating at 70 o Cfor 7min (two couplings; Table 1). Except for the derivatives in which the Bzl group isFig. 1. Coupling reaction experiments. Each derivative was coupled onto a Gly-Phe boundon resin dipeptides and the targeted tripeptide product was analyzed by HPLC after thecleavage from the resin.10