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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, 2010Post-Synthesis Modification of p-Iodo-Phenylalanine-ContainingPeptides on Solid-Phase via the Palladium-CatalyzedSonogashira ReactionNgoc-Duc Doan, David Chatenet, and Alain FournierInstitut National de la Recherche Scientifique-Institut Armand-Frappier, Université duQuébec, 531 boulevard des Prairies, Ville de Laval, Qc, H7V 1B7, Canada;Laboratoire International Associé Samuel de Champlain (INSERM – INRS)IntroductionPeptides are active regulators and transmitters of key biological functions thus makingthem particularly appealing for drug discovery [1]. As a matter of fact, their high biologicalactivity is often associated with low toxicity and high specificity for their targets. However,due to inherent limitations such as low stability and oral bioavailability, difficult delivery,as well as costly synthesis, the development of peptide-based drugs remains relativelyuncommon. The quest for novel molecular structures with improved biological activityprofiles led to the development of potent small molecules that possess unique propertiesand the ability to modify the activities of larger molecules. Hence, to improve keypharmacological characteristics, structure-activity relationship studies generating librariesof peptide analogs proved to be very helpful to identify new therapeutic peptides. In fact, toimprove the therapeutic potential of peptides, several modifications includinglactamization, “stapling” of peptides, incorporation of non natural amino acids, andpegylation were applied and showed frequently a significant improvement of thepharmacological properties of peptides [2]. In this line of view, we developed an effectivemethod, based on the Sonogashira reaction, to modify on solid phase p-iodo-phenylalaninecontainingpeptides.Results and DiscussionInitially, the Sonogashira cross-coupling reaction was attempted in solution with Boc-piodo-L-phenylalanineand phenylacetylene, in the presence of the palladium-catalystPdCl 2 (PPh 3 ) 2 , copper (I) bromide, triethylamine (TEA) and a phosphine ligand (PCy 3 ), inmethylene chloride (DCM). In this condition, Boc-p-phenylacetylenyl-L-phenylalanine(Boc-Pep-OH) was obtained in good yield (85%) after purification on silica gel (EtOAc:nheptane1:1). Our attention was next turned towards the direct modification of p-iodophenylalanine-containingpeptides on solid support. In this regard, [Ala 1,2,3 , Leu 8 ]Enk(AAAYGGFL) was used as a peptide model to evaluate the feasability of the Sonogashirareaction in solid-phase peptide chemistry.All peptide-resins were synthesized using standard Fmoc chemistry with theN-terminal amino acid being introduced as a Boc derivative. Prior to the Sonogashirareaction, a small amount of peptide was cleaved and analyzed by analytical RP-HPLC andMALDI-TOF MS to ensure the homogeneity and molecular mass of the starting material.The methodology for the Sonogashira solid-phase peptide modification was then optimizedthrough the study of the influence of temperature, base, catalyst, solvent and reaction timeon the reaction yield. It was found that the best results were obtained with PdCl 2 (PPh 3 ) 2 ascatalyst, TEA as base, dimethylformamide (DMF):DCM mixture as solvent andtemperature of 40 o C, overnight.Previous studies suggested that the presence of unprotected amino groups (-NH 2 ) caninteract with Pd and retard the catalytic cycle of metal-catalyzed reaction [3]. The stabilityof Fmoc and Boc protecting groups was assessed and results suggested that both protectinggroups were stable in the optimized reaction conditions, even after 24 hrs; however, longerreaction time (4 days) caused a slight cleavage of the Fmoc moiety. We also observed thatlinkers, such as Wang and Rink Amide, were stable under Sonogashira reaction.Since the conversion of N-α-Boc-[Ala 1,2,3 , Phe(pI) 4 , Leu 8 ]Enk to N-α-Boc-[Ala 1,2,3 ,Pep 4 , Leu 8 ]Enk was successfully achieved using the optimized conditions of theSonogashira reaction described above, we then performed the same modification with morecomplex peptides containing sensitive amino acids such as cysteine, histidine, andtryptophan. Conversion of these iodinated peptides ([Phe(p-I) 13 ]ET(9-21) (KECVF(p-I)FCHLDIIW) and Ac-[Phe(p-I) 6 , Nle 17 ]PACAP27 (Ac-HSDGIF(p-I)TDSYSRYRKQNle-196