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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, 2010Fmoc Deprotection by tert-Butylamine in Solution and in theSynthesis of Cyclic Part of Oxytocin-Like PeptidesMartin Flegel 1,3 , Zuzana Flegelová 2 , Petr Maloň 3 , Sixtus Hynie 1 ,and Věra Klenerová 11 1st. Faculty of Medicine, Charles University in Prague, Prague 2, Albertov 4, 12800,Czech Republic; 2 Vidia spol.s.r.o, Nad Safinou II. 365. Jesenice u Prahy-Vestec, 25242,Czech Republic; 3 IOCB, Flemingovo n. 2 Prague 6, 16610, Czech RepublicIntroductionThe Fmoc protecting group is usually cleaved at basic conditions by 20% piperidine inDMF [1]. For deprotection of difficult sequences diazabicycloundecene (2 % DBU inDMF) is often used. Both commonly used agents are unfortunately not very suitable in thealternative liquid phase synthesis. Therefore there is a permanent effort and search for theagents easily removable during inevitable intermediates isolation [2]. The cyclic part ofoxytocin H-c(Cys-Tyr(I 2 )-Ile-Gln-Asn-Cys)-NH 2 was manually synthesized on the solidphase. For the Fmoc group removal both 20% piperidine in DMF and 30% of tertbutylamine(TBA) in DMF were compared. TBA in the mixture with 1-octadecanethiol(C18-SH) was then used for deprotection of some Fmoc-AA derivatives to study thecleavage condition affordable for liquid phase. The choice of the model peptide wasmotivated by its possible exploitation either for CD spectral measurement and/or forbiological effects in CNS during the study of stress. It was also desirable to develop asimple method for the iodination of Tyr residue.Results and DiscussionFmoc-Cys(Trt)-Tyr(tBu)-Ile-Gln(Trt)-Asn(Trt)-Cys(Trt)-Rink-resin was synthesized in twobatches employing the usual scheme. Fmoc derivatives were used in 3 eq. excess,DIC/HOBt was used for coupling and deprotection was carried out for 5 and 20 min in 20%piperidine /DMF solution. The analogous scheme was applied in the second batch, where30% TBA in DMF with 0.1% dithiothreitol (DTT) for 5 and 40 min was used for the Fmocremoval (Table 1).The peptides were then detached from the resin in the cocktail of 95%TFA, 3% anisoleand 2% water in 3 hours, precipitated by diethylether, dissolved in methanol, acidified byAcOH (pH 4.5, 1 mg/ml), and oxidized by 1% iodine in MeOH to create a disulfide bridge.After 30 min the yellowish solution was decolorized by ascorbic acid, and the solutiongently evaporated at 40 o C in vacuum. The residue was dissolved in water, desalted andpurified on HPLC column. The peptides were then freeze dried. In both batches the peptideiodinated in the Tyr aromatic ring was surprisingly found as the main product.Some Fmoc derivatives of Tyr(tBu), Trp, Gln(Trt), Nal, and Cys(Trt) were chosen forthe study of the cleavage reaction in solution. For all of them the Fmoc group was fullyremoved with TBA in 30-60 min. To scavenge dibenzofulvene (DBF), DTT [6] or C18-SHwas added in 5-10 molar excess to 30% solution of TBA in ACN or in 100%TBA.The isolation process in solution approach using TBA was found to be easier. DBF, asthe primary product of the Fmoc elimination is a very reactive intermediate, which in thereversible Michael addition reacts with nucleophiles to form adducts with piperidine,primary or secondary amines, or preferably with the soft nucleophiles such as thiols. In thesolid phase, large excess of the base guarantee full conversion of DBF to piperidine adduct.In the liquid phase other methods can serve this purpose. Easy and friendly reactionconditions and isolation of the pure intermediate are of importance.Table 1. Monitoring of peptide synthesismmol/g(FmocRelease)Piperidine 0.60 0.61 0.61 0.61 0.64 0.68Sequence C Y I Q N Ctert-Butylamine 0.61 0.63 0.64 0.69 0.73 0.7354