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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, 2010Automated Microwave-Assisted Peptide Synthesis with aNovel Robotic Synthesizer: Synthesis of Difficult SequencesSøren L. Pedersen 1 , Amit Mehrotra 2 , and Knud J. Jensen 1 *1 University of Copenhagen, Faculty of Life Sciences, IGM, DK-1871, Frederiksberg C,Denmark, E-mail: kjj@life.ku.dk; 2 Biotage AB, SE-753 18, Uppsala, SwedenIntroductionSolid-phase peptide synthesis (SPPS) is still often faced with challenges in the assembly oflong and ‘difficult’ sequences, e.g. due to aggregation and steric hindrance giving rise toincomplete reactions. These problems have only partly been solved by new couplingreagents and solid supports. Precise microwave heating has emerged as one new parameterfor SPPS, in addition to coupling reagents, resins,solvents, etc. [1-5].Recently the first X-Y robotic microwaveassistedpeptide synthesizer was introduced to thepeptide community. The synthesizer is built around aBiotage Initiator and a MultiSynTech Syro I, whichmeans that the instrument has the capability to performa single reaction in the microwave cavity or conventionalroom temperature (RT) peptide synthesis on theparallel reactor block (Figure 1) [4]. The reactor vesselFig. 1. The Biotage Syro Wave TM .is placed in the microwave cavity for the duration ofthe synthesis and mixing is achieved by vortexing.Results and DiscussionThe synthesis of peptides with successive N-methylated amino acids - especially if theyalso are -branched - can be challenging and often needs strong coupling reagents such asPyBOP [6] or triphosgene [7,8] for high coupling yields. As a model peptide, sequence 1was chosen [9]. Here the coupling of the last N-methylated alanine residue onto thepeptidyl bound N-methylated Ile proved to be the major challenge.H-MeAla-MeIle-MeGly-NH 2 (1)Using coupling times of 60 min at RT we were only able to synthesize peptide 1 in verylow purities, even when using coupling reagents such as HATU/HOAt and COMU.Prolonging the coupling time to 24 h using DIC/HOAt led to moderate peptide purity of39% (Table 1) and as predicted the major deletion product was the H-MeIle-MeGly-NH 2dipeptide. Elevating the temperature to 75°C using the Syro Wave TM for 20 min or 2×10min, during the HATU or DIC activated coupling reactions gave the desired tri-peptide inhigh crude purity >75% (Table 1, Figure 2). Interestingly, DIC/HOAt and HATU/HOAtoutperformed COMU which only led to moderate peptide purity.300mAUUV_VIS_1WVL:215 nm200100-50min1.00 2.50 3.75 5.00 6.25 7.50 8.75 10.00Fig. 2. HPLC chromatogram of crude H-MeAla-MeIle-MeGly-NH 2 at 5.3 min (Table 1,Entry 8).174