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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, 2010A Novel Method to Prepare Cyclic Peptides atNon-Cysteine Sites Using the Auxiliary Group,4,5-Dimethoxy-2-Mercaptobenzyl (Dmmb)Jane SpetzlerNovo Nordisk A/S, Novo Nordisk Park, 2760, Måløv, Copenhagen, DenmarkIntroductionMany cyclic peptides are of therapeutic interest but their preparation is challenging. Thiswork presents a new general cyclization method [1] which applies the auxiliary group,4,5-dimethoxy-2-mercaptobenzyl (Dmmb) to form end-to-end cyclic peptides in aqueoussolution without protection of the side-chains. For native ligation [2] there are severalexamples known using N -(4,5-dimethoxy-2-mercaptobenzyl) (Dmmb) [3-5] peptidefragments to form longer peptides segments at X-Gly or X-Ala sites with C-terminalthioester peptides in the three following steps (1) transthioesterification (2) S to N acyl shiftand (3) removal of the acyl transfer auxiliary group. This strategy has been applied forsynthesis of a cyclic peptide. The peptide was prepared using standard Fmoc/tBu chemistryfollowed by the cyclization reaction. The aim of this study has been to use the strategydescribed above for the synthesis of cyclic peptides.Results and DiscussionThe auxiliary group 4, 5-dimethoxy-2-mercaptobenzylamine (Dmmb) which is similar to1-pheny-2-mercaptoethyl has been extensively applied for native chemical ligation[3,4,6,7]. However, we have exploited the same strategy to form an end-to-end cyclicpeptide from an unprotected linear peptide generated with Fmoc-chemistry as shown inScheme 1. However, other research groups have utilized similar strategy to synthesizecyclic peptides [8-10].The linear peptide precursor contains both N -(Dmmb)-Gly and C -thioester and wasprepared in 2 steps (Scheme 1) by coupling the commercially available building block Boc-(Dmmb(Trt))-Gly-OH [5] to RGDSPA-2-chlorotrityl resin using DIC/HOAt. The protectedpeptide was cleaved from the resin with HOAc/TFE/DCM (1:1:8) and was not purifiedfurther. The peptide thioester 1 was obtained by the method of Von Eggelkraut-Gottanka etal. [11]. The yield of peptide thioester 1 in the second step was 31% and the correct masswas confirmed by LC-MS. The end-to-end cyclization reaction (Scheme 2) was performedin 6M guanidinium chloride at pH 7.5 in the presence of thiophenol and benzyl mercaptan(Scheme 2).Ala-2-chlorotrityl resin1) SPPS (Fmoc-AA)2) Boc-(Dmmb(Trt))-Gly-OH/DIC/HOAt3) HOAc/TFE/DCM (1:1:8)OOS-TrtNBocO(protected-AA)nCH 3N OHO1) 4-Acetamidothiophenol/PyBOP/DIEA2) 95%TFA,TISOOSHNO(AA)n1CH 3N SONHOScheme 1. Synthesis of the linear peptide thioester 1.140