Iterative peptide assembly in one-pot using N-selenoethyl cysteine SetCys chemistry with potential application for antifreeze protein synthesis

Les protéines sont les macromolécules naturellement produites par tous les organismes vivants et elles constituent des composants essentiels du monde vivant. Leur fonctions et structure sont le résultat de l'organisation dans l'espace d'une chaîne polypeptidique construite par l'assemblage de 21 acides aminés (AA) différents. Afin d'accéder aux polypeptides par voie chimique, en 1963, Merrifield développa le synthèse peptidique sur support solide (solid phase peptide synthesis, SPPS), une méthode chimique adaptée à l'obtention de peptides et de protéines de petite taille, plutot de moins de 50 AAs. Cette approche assure la haute pureté des produits obtenus et permet de modifier facilement les séquences peptidiques ou de les fonctionnaliser. Afin de faciliter l'accès aux peptides composés d'un nombre d'acide aminés supérieur à 50, une stratégie complémentaire a été développée qui est basée sur assemblage de segments peptidiques non protégés plus courts. Pour rendre cette deuxième approche efficace, les chimistes ont développé quelques réactions de ligation chimique qui permettent de connecter deux peptides non-protégés de manière covalente et chimioselective.Ce manuscrit de thèse se compose de deux parties distinctes. La première partie est dédiée au développement d'une méthode innovante d'assemblage de polypeptides basée sur la réaction de ligation chimique native (NCL) et la N-sélénoéthyl cystéine (SetCys), un acide aminé artificiel dérivé de la cystéine où les groupes amine et thiol sont liés via un pont sélénoéthyle pour former une structure cyclique. Une propriété importante de SetCys est sa capacité à perdre spontanément son bras sélénoéthyle via la rupture de la liaison C-N dans des conditions douces. Je décrirai l'application de la réaction de NCL dans des conditions non standard, les propriétés chimiques de SetCys et le défi de combiner ces chimies pour développer une méthode élégante et chimiosélective en deux étapes permettant la concaténation de segments peptidiques.La deuxième partie définit et décrit les propriétés des protéines capables de s'attacher à la surface de la glace (ice-binding proteins, IBPs) et d'une catégorie particulière appelée protéines antigel (antifreeze proteins, AFPs), qui sont produites par des organismes adaptés au froid dans la nature. Je me concentrerai sur une protéine particulière produite par le coléoptère Tenebrio molitor (Tm), et je décrirai les première tentatives visant à accéder à un analogue par synthèse chimique. En perspective, la méthode innovante décrite dans la première partie du manuscrit pourrait être appliquée àla synthèse de TmAFP.Proteins are macromolecules naturally produced by all living organisms and constitute essential components of organic life. Their functions and structure are the result of spatial organization of a polypeptide chain made of the concatenation of 21 different amino acid (AA) residues. To access polypeptides by chemical synthesis, Merrifield in 1963 introduced the solid phase peptide synthesis (SPPS), a chemical method that is appropriate for the preparation of peptides and small proteins usually with a size less than 50 AAs. This approach guarantees high product purity and allows to easily modify a protein sequence or to functionalize it on demand. To facilitate the access to longer peptide sequences with a size greater than 50 AAs, a complementary strategy was developed based on the concatenation of smaller peptide segments. To make this second approach work, chemists have a few chemical ligation reactions to covalently and chemoselectively connect unprotected peptide segments together.This thesis manuscript consists of two separated parts. The first part is dedicated to the development of an innovative method of polypeptide assembly in one-pot based on native chemical ligation (NCL) reaction and N-selenoethyl cysteine (SetCys), the artificial amino acid where amine and thiol groups are bonded via selenoethyl bridge forming a cyclic structure. The key feature of SetCys is its ability to lose its selenoethyl appendage via C-N bond breaking under mild conditions. I will describe the application of NCL in non-standard conditions, the chemical properties of SetCys and the challenge to combine these chemistries for designing an elegant and chemoselective two-step method enabling peptide segment concatenation.The second part describes the definition and properties of ice binding proteins (IBPs) and their sub-group called antifreeze proteins (AFPs), which are produced by cold coping organisms in nature. I will focus on a particular protein produced by Tenebrio molitor (Tm) beetle, aiming to access its bioinspired analogue by chemical protein synthesis for the first time. In perspective, the innovative method described in the first part of the manuscript would be applied for the synthesis of TmAFP

Thiol Catalysis of Selenosulfide Bond Cleavage by a Triarylphosphine

International audienceThe arylthiol 4-mercaptophenylacetic acid (MPAA) is a powerful catalyst of selenosulfide bond reduction by the triarylphosphine 3,3′,3″-phosphanetriyltris(benzenesulfonic acid) trisodium salt (TPPTS). Both reagents are water-soluble at neutral pH and are particularly adapted for working with unprotected peptidic substrates. Contrary to trialkylphosphines such as tris(2-carboxyethyl)phosphine hydrochloride (TCEP), TPPTS has the advantage of not inducing deselenization reactions. We believe that the work reported here will be of value for those manipulating selenosulfide bonds in peptidic or protein molecules

Thiol catalysis of selenosulfide bond cleavage by a triarylphosphine

The arylthiol 4-mercaptophenylacetic acid (MPAA) is a powerful catalyst of selenosulfide bond reduction by the triarylphosphine 3,3′,3′′-phosphanetriyltris(benzenesulfonic acid) trisodium salt (TPPTS). Both reagents are water-soluble at neutral pH and are particularly adapted for working with unprotected peptidic substrates. Contrary to trialkylphosphines such as tris(2-carboxyethyl)phosphine hydrochloride (TCEP), TPPTS has the advantage of not inducing deselenization reactions. We believe that the work reported here will be of value for those manipulating selenosulfide bonds in peptidic or protein molecules

A selenium-based cysteine surrogate for protein chemical synthesis

We provide in this protocol detailed procedures for the synthesis of SetCys cysteine surrogate and its use for chemical synthesis of proteins through the redox-controlled assembly of three peptide segments in one-pot

A Selenium-based Cysteine Surrogate for Protein Chemical Synthesis

International audienceN-selenoethyl cysteine (SetCys) in the form of its cyclic selenosulfide is a cysteine surrogate, whose reactivity depends on the reducing power of the medium. SetCys does not interfere with the native chemical ligation reaction under mild reducing conditions, that is in the absence of tris(2-carboxyethyl)phosphine (TCEP). In contrast, subjecting SetCys to TCEP results in the spontaneous loss of its N-selenoethyl appendage and thus to its conversion into a Cys residue. Therefore, SetCys can be used for the redox-controlled assembly of peptide segments using NCL. We provide in this protocol detailed procedures for the synthesis of Fmoc-protected SetCys residue and for its incorporation into peptides using standard solid-phase peptide synthesis protocols. We also describe its use for the chemical synthesis of proteins through the redox-controlled assembly of three peptide segments in one-pot

Interest of novel N-alkylpyridinium-indolizine hybrids in the field of Alzheimer's disease: synthesis, characterization and evaluation of antioxidant activity, cholinesterase inhibition, and amyloid fibrillation interference

International audienceA small library of molecules combining indolizine and N-alkyl pyridinium was synthesized and evaluated in a multi-target-directed-ligand strategy for Alzheimer's disease (AD) treatment. The new compounds were classified in three series depending on the number of methylene residues linking the two heterocycles (Ind-PyCx with x = 0, 2 or 3). The molecules were synthesized from the corresponding bis-pyridines by two-step formation of the indolizine core including mono-alkylation of pyridine and 1,3-dipolar cycloaddition with an alkylpropiolate. Their activities against AD's key-targets were evaluated in vitro: acetyl-and butyrylcholinesterase (AChE and BChE) inhibition, antioxidant properties and inhibition of amyloid fibril formation (ThT assays). None of the three series showed significant activities against all the targets. The Ind-PyC2 and Ind-PyC3 series are relatively active on eeAChE and hAChE (µM IC50 values). Except compound 10 and 15, charged molecules appeared also active against eqBChE with micromolar IC50, while they lost their activity on hBChE. Comparative molecular modeling of 13 and 15 docked in hAChE and hBChE highlighted the importance of the substituent pmethoxybenzoyl or methyloxycarbonyl located on the indolizine part for the binding. The molecules displayed antioxidant activity at the highest tested concentration, up to 95% inhibition of DPPH radical scavenging for 10. Most of the Ind-PyC2 and Ind-PyC3 hybrids were able also to interact with amyloid fibers even if the observed inhibitory effect on fibrillation process was observed at a high 100 µM concentration. For the Ind-PyC0 series, despite the fact that their spectroscopic properties did not allow their evaluation with ThT assay, they showed interesting features in the presence of preformed fibers. In particular, the sharp increase of the fluorescence of 3 in the presence of amyloid fibers is a promising indication of the potential use of this series as fluorescent amyloid fibrillation reporter