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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, 2010Convenient Synthesis of C-Terminal Glycopeptide Conjugatesvia Click ChemistryJean-Philippe Ebran, Nabil Dendane, and Oleg Melnyk*CNRS UMR 8161, University of Lille Nord de France, Institut Pasteur de Lille, IFR 142,Institut de Biologie de Lille, 1 rue du Pr. Calmette, 59021, Lille, France ; http://csb.ibl.frIntroductionGlycoproteins are involved in many biological processes such as immune defense, cellgrowth or inflammation. Access to natural glycoproteins and glycopeptides are usuallyachieved by recombinant techniques or, by total or semisynthetic chemical ligationstrategies. Another approach relies on the assembly of glycopeptides mimetics containingunnatural linkages between the peptide and oligosaccharide moieties using chemicalligation. To that purpose, the regioselective Cu-Catalyzed Azide-Alkyne Cycloaddition(CuAAC) developed independently by Meldal [1] and Sharpless [2] proved to be a veryefficient and versatile tool. Indeed the chemoselective 1,3-dipolar cycloaddition between anorganic azide and a terminal alkyne lead to the 1,4-disubstituted triazole linkage. Previousworks [3,4] have reported the CuAAC ligation of unprotected azide carbohydrates andalkyne side chain peptides. Recently our research focused on the design of a novel access tounprotected C-terminal azide linker peptides and their application to copper catalyzedcycloaddition with oligosaccharide-based acetylenes.Results and DiscussionA new reliable and efficient approach regarding the synthesis of 3-azidopropyl-amine [5]salts is depicted herein (Figure 1). A two steps synthesis from phtalimide bromidederivative, via an azide phtalimideintermediate, afforded the expectedhydrochloride or tosylate salts with82 and 85% overall yield,respectively. Fmoc/tBu solid phaseFig. 1. Synthesis of amine-azide salts.FmocHNNOHONH1. SPPS2. Cu(OAc) 2, Pyridine,DCM, O 2, DIPEACl - + H 3NAc-I-L-K-E-P-V-Y-X-NH1 G 23N 3N 3%2 A 29 %3. TFA/H 2O/TIS 95/2.5/2.53 S 12 %4 V 14 %Fig. 2. Synthesis of C-terminal azide linkerpeptides.Ac-I-L-K-E-P-V-Y-A-NH N 3 +Ph Cu AAC ConditionsAc-I-L-K-E-P-V-Y-A-NH25 °C2 5peptide synthesis (SPPS) wascarried on 4-Fmoc hydrazinobenzoylAM resin. The aminolysiscleavage [6] from the solid supportwas achieved by oxidation withCu(II) in aerobic media usingazidopropylamine as nucleophilicamine. The released peptides weredeprotected by trifluoroacetic acidtreatment giving the correspondingC-terminal azide linker peptideswith yields from 12 to 29%NN NEntry Catalyst Solvent Time Conversion b1 10 mol % CuI5 eq DIPEADMF 20 h < 5%2 10 mol % CuSO4-5H2O100 mol % NaAscCH3CN/H2O 1:1 20 h < 1%3 10 mol % CuSO4-5H2O100 mol % NaAsctBuOH/H2O 1:1 4 h 99% (89 %) c4 5 mol % CuSO4-5H2O50 mol % NaAsctBuOH/H2O 1:1 20 h 93 %5 10 mol % CuSO4-5H2O100 mol % Cu(0)tBuOH/H2O 1:1 20 h 14 %6 10 mol % CuSO4-5H2O100 mol % NaAscH2O 20 h < 5%a Reactions were performed on 3 mM concentration of peptide with a ratio Azide to Alkyne of 1:2. bConversion determined by HPLC integration on azide consumption. c Isolated yieldFig. 3. Cu(I)-catalyzed cycloaddition optimization.Ph(Figure 2). Copperoxidative cleavagewas found to be thesuitable methodsince low yield(