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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, 20103TS: Thiazolidine-Triggered Thioester SynthesisJulien Dheur, Nathalie Ollivier, Annick Blanpain, and Oleg MelnykCNRS UMR 8161, Univ Lille Nord de France, Institut Pasteur de Lille, IFR 142,Institut de Biologie de Lille, 1 rue du Pr. Calmette, 59021, Lille, France;CSB platform (http://csb.ibl.fr)IntroductionNowadays, chemical synthesis of moderate size proteins is principally achieved by use ofNative Chemical Ligation (NCL) developed by Dawson et al. in the middle of the 90’s [1].The principal constraint on NCL widespread application is the chemical synthesis ofunprotected α-thioester fragments by the widely used Fmoc-Solid Phase Peptide Synthesis(Fmoc-SPPS) method. Based on both well-known N to S-acyl shift [2] and thiazolidinechemistry, we describe herein the efficient synthesis of C-term thioester peptides bycombining supported and solution peptide chemistry. First, Fmoc / t-Bu SPPS is used toobtain highly stable amide peptides derivatives, featuring a bis-β-amino thiol arm at theC-terminus. In acidic medium, these peptides undergo a N to S-acyl-transfer-mediated stepand the presence of an aldehyde such as glyoxylic acid affords the 3-(2-sulfanylethyl)-thiazolidine-2-carboxylic acid (SETCA) functionality in good yields.Results and DiscussionRacemization-free synthesis of peptides (1-4a) featuring a bis-β-aminothiol arm at theC-terminus was achieved by Fmoc/t-Bu SPPS based on an innovative solid supportcompatible with classical elongation/deprotection/cleavage conditions (Figure 1).Reduction of (1-4a) disulfide bridge induced a pH dependent N,S acyl shift equilibriumbetween peptide amide (1-4b) and peptide thioester (1-4c). The later form was the majorspecie at pH below 2.8 (Figure 1).H peptide Aa N1-4aSSReductionH peptide Aa NAmide form 1-4bSHSHFmoc-SPPS synthesis of peptides 1-4a :+ H +N to S acyl shifta Isolated yields. b Determined by chiral GC-MSH peptide Aa SThioester form 1-4cFig. 1. Study of the N,S acyl shift for peptides (1-4b).NH 2SHStudy of the amide / thioester rearrangement=f(pH)([H-ILKEPVHGA-dithiazepane] 1 mM, TCEP 80 mM,t = 6 h, 20°C).To displace the amide / thioester equilibrium toward peptide thioesters, we investigated theusefulness of the widespread thiazolidine chemistry. Indeed, the intramolecularrearrangement of the bis-β-aminothiol leads to a free aminothiol moiety which could beengaged in the formation of a thiazolidine ring with an external aldehyde functionality. Forthis, dithiazepane peptides (1-4a) were first reduced with 5 equivalents of zinc dust inacidic medium (1% TFA aqueous solution) to afford quantitatively and rapidly the desiredpeptides 1-4b [3]. Then, excess zinc was removed by centrifugation and the supernatantwas recovered and engaged without further treatment in the thiazolidine formation step.SETCA-peptides formation took place efficiently in the 1% TFA solution (pH ~ 1) used forpeptide (1-4a) reduction. Five equivalents of glyoxylic acid at 37°C led to the efficientformation of SETCA-peptides within 24 h, except for bulky amino acids like Valine whichrequired longer reaction times. SETCA-peptides appeared to be very stable to RP-HPLCpurification conditions and compounds were isolated with good yields (Figure 2).214