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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, 2010An Efficient MW-Assisted Synthesis of Dicarba-AnaloguesG. Cane, A. Di Cianni, M. Lumini, AM. Papini, and M. GinanneschiLaboratory of Peptide & Protein Chemistry & Biology, University of Florence, PoloScientifico e Tecnologico, Sesto Fiorentino (Fi), I-50019, Italy, and Department ofChemistry ‘‘Ugo Schiff’’, Via della Lastruccia 3/13, University of Florence, Polo Scientificoe Tecnologico, Sesto Fiorentino (Fi), I-50019, ItalyIntroductionA frequent structural motif found in natural peptides and proteins is constituted of thecystine (S-S) group which forms intra- and intermolecular bridges of fundamentalimportance for biomolecules structure and hence their biological activity [1]. Octreotide 1is a cyclic octapeptide somatostatin agonist, showing higher affinity and specificity towardthe somatostatin receptor subtype sst2 and a better resistance to enzymatic degradationcompared to the native somatostatin. It is the first analogue mainly a carrier forradionuclides used in clinical protocols for cancer diagnostics and therapy [2]. It is wellknownthat the disulfide bridge is prone to be attacked by endogenous reducing enzymes orby nucleophilic and basic agents [3]. Therefore, we replaced the cystine bond by thedicarba isostere 2 to increase its metabolic stability. The substitution of the disulfide groupin peptides and proteins by the isosteric, stable, dicarba-bond was easier accessible after thediscovery of the Ru-catalysed mediated Ring-Closing Metathesis (RCM) reaction proposedby Grubbs [4,5]. The difficulty in this synthetic strategy applied to peptides let us toinvestigate the use of microwave (MW) technology. In fact, more and more interestingresults are reporting the use of MW irradiation in organic and also in solid-phase peptidesynthesis. In that case, it is noteworthy that MWs could be useful in reducing peptideaggregation, leading to proper peptide construction and higher crude purity [8,9].OOONHHNH 2NHNHNHONHOHOHOHNNHNHOHO CH 3OHNH 2Results and DiscussionThe key reaction to achieve the target compounds is the ring-closing metathesis (RCM)catalyzed by ruthenium complexes on the linear peptide containing suitably placed, nonproteinogenicL-allylglycine (Hag) residues. The RCM was applied directly on linear hexa-3OClNH 2O-tBuMesNN MesClPhRuClPCy 32 nd generation Grubb's catalyst(7)H 2 NOO O YNH HNHN HHNHOOHO CH 3Reagents and conditions: (a) (i) Fmoc-Hag, HATU/NMM, 40 min, DMF, r.t.; (ii) 20%piperidine in DMF (2 x 10 min); (b) Coupling with 5 aminoacids, DMF; (c) catalyst 11(10% mmol), DCM; (d) (i) 20% piperidine in DMF (2 x 10 min.), Fmoc-D-Phe/HATU/NMM, 40 min, DMF, r.t.; (ii) 20% piperidine in DMF (2 x 10 min.); cleavageof 4 and 2 : [TFA/H 2 O/EDT/Phenol (94:2:2:2)]; 5 and 6: [TFA/H 2 O/EDT/Phenol(70:26:2:2)].a-dO NHOH ON Z NH H4 Y= L-Phe Z= L-Phe2 Y= L-Phe Z= L-Thr5 Y= L-Phe Z= L-Tyr(BzI)6 Y= L-NaI Z= L-Tyr(BzI)Scheme 1. Synthesis of 6-membered ringSomatostatin dicarba-analogues.NHNH 22and octapeptides linked tochlorotrityl resin (3) and Rink amideresin (8) respectively, using bothmicrowaves and normal heating. Thereaction was firstly performed in anoil bath at 50 °C with secondgeneration Grubb’s catalyst undercontrolled experimental conditions,i.e. anhydrous argon atmosphere andlong reaction times [6,7]. Subsequentlywe employed a microwaveassistedstrategy (MW) that emergedin increasing yield in 6-memberedringformation of Somatostatindicarba-analogues (4-6), as well as inshortening reaction times (Scheme 1)[8,9]. This prompted us to evaluate184