Proceedings book download - 5Z.com

Proceedings of the 31 st European Peptide SymposiumMichal Lebl, Morten Meldal, Knud J. Jensen, Thomas Hoeg-Jensen (Editors)European Peptide Society, 2010Mimicking of Disulfide Bonds by TriazolesKai Holland-Nell 1,2 and Morten Meldal 11 Carlsberg Laboratory, Valby, 2500, Denmark; 2 Leibniz-Institute of MolecularPharmacology, Berlin, 13125, GermanyIntroductionDisulfide bonds stabilize peptide and protein structures to maintain biological functions butpresents rather unstable bonds and replacements with thioethers [1], amides [2] as well ascarbon based bridges [3] and diselenides [4] have been investigated to overcome theproblems associated with disulfides. We aimed at investigating the potential of triazoles asdisulfide mimetics. Stability and formation by orthogonal click chemistry [5] favor triazolesas an improved disulfide substitute. The stabilitity of triazoles towards oxidative andreducing reagents is complemented by a remarkable biological stability. The triazole motif- usually not occurring in natural peptides and proteins - is not effected by any isomerases,hydrolases, and proteases. Reaction of azides to alkynes by CuAAC facilitates triazoleformation under very mild conditions.Results and DiscussionThe approach has been applied to two model peptides: tachyplesin-I and conotoxinXen2174. Tachyplesin-I is a 17 aa peptide isolated from the Japanese horseshoe crabTachypleus tridentatus. It shows an interesting antimicrobial activity and antitumor activityhas also been reported recently [6]. Conotoxin Xen2174 is an MrIA conotoxin analog,which selectively blocks the norepinephrine transporter and interferes with pain signalingin the human body [7]. This property made the peptide a promising analgesic drugcandidate which is currently studied in clinical phase IIb. Both peptides exhibit a -hairpinfold structure which is stabilized by two disulfide bridges. The focus of this work wasplaced on the substitution of the two disulfide bonds and the impact on the biologicalactivity of those peptides.In the first step, different tachyplesin and conotoxin analogs were synthesized bystandard SPPS using Fmoc/ t Bu-strategy. Thereby, the four Cys in each peptide werereplaced by appropriate alkyno and azido amino acids. Propargylglycine was selected as analkyno component while -azido-alanine, -azido-homoalanine, and -azido-norvaline allwere investigated as azido components.In the second step, the peptides were subjected to an on-resin cyclization on PEGAresin applying click chemistry conditions. Due to the high orthogonality of this reaction,the peptides can be cyclized in unprotected form. The hydroxymethyl-benzoic acid linkerallowed the selective deprotection while maintaining resin attachment. Several cyclizationconditions were evaluated. Neither CuI or CuBr in DMF nor CuSO 4 /NaAsc in H 2 O formedthe desired products. Moreover, in the case of -azido-alanine, a -elimination of the azideto yield dehydro-alanine was observed. Finally, the formation of the triazoles and thecyclization of the peptides succeed with the application of CuSO 4 /TCEP in water underargon. We took advantage of the excellent swelling properties of Versamatrix TM PEGA 1900resins in water.The presence of two triazole moieties allowed the formation of two different reactionproducts: a globular and a correctly folded -hairpin product. We observed the formation oftwo products exhibiting the same molecular weight but showing different HPLC retentiontime compared to the starting material. Ratios of 1:2 (tachyplesin) and 1:7 (conotoxin) infavor of the -hairpin were observed. Notably, no linear or cyclic dimers were formedunder these conditions. The reaction time could be reduced from 16h to 1h by applyingmicrowave conditions. However, at the raised temperature most of the selectivity forformation of the correctly folded -hairpin was lost.Several experiments were performed in order to prove the cyclic nature of theproducts. Simple reduction experiments with dithiothreitol showed the absence of azidogroups in the products and suggest the formation of triazoles. MS/MS-fragmentationpatterns exhibited tremendous differences between the cyclic and linear peptides. Almostall fragments of the y- and b-series could be detected in case of the linear peptides. Incontrast, the cyclic peptides showed only fragments of amino acids located outside thebicyclic peptide region.146