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In this paper, the first semi-synthesis of the Alzheimer-relevant tau protein carrying an O-GlcNAcylation is demonstrated by using sequential chemoselective ligation. The 52-amino acid C-terminus of tau was obtained by native chemical ligation between two synthetic peptide fragments, one carrying the O-GlcNAc moiety on Ser400, which has recently been demonstrated to inhibit tau phosphorylation and to hinder tau oligomerization, and the other equipped with a photocleavable biotin handle. After desulfurization to deliver a native alanine at the ligation junction, the N-terminal cysteine was unmasked, and the peptide was further used for expressed protein ligation to generate the full-length tau protein, which was purified by a photocleavable biotin tag. We thus provide a synthetic route to obtain a homogenous tag-free O-GlcNAcylated tau protein that can further help to elucidate the significance of posttranslational modification on the tau protein and pave the way for evaluating possible drug targets in Alzheimer's disease. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

A thiofunctional thiazolidine is introduced as a new low-molar-mass building block for the introduction of cysteine residues via a thiol-ene reaction. Allyl-functional polyglycidol (PG) is used as a model polymer to demonstrate polymer-analogue functionalization through reaction with the unsaturated side-chains. A modified trinitrobenzenesulfonic acid (TNBSA) assay is used for the redox-insensitive quantification and a precise final cysteine content can be predetermined at the polymerization stage. Native chemical ligation at cysteine-functional PG is performed as a model reaction for a chemoselective peptide modification of this polymer. The three-step synthesis of the thiofunctional thiazolidine reactant, together with the standard thiol-ene coupling and the robust quantification assay, broadens the toolbox for thiol-ene chemistry and offers a generic and straightforward approach to cysteine-functional materials. [ABSTRACT FROM AUTHOR]

Wir präsentieren eine neue Strategie zur spurlosen Aufreinigung und synthetischen Modifikation von durch native chemische Ligation erzeugten Peptiden und Proteinen. Die Strategie umfasst die Immobilisierung eines photospaltbaren semisynthetischen Biotin ‐ Protein ‐ Konjugats an Streptavidin ‐ Agarosepartikel, wodurch aufwendiges Umpuffern vermieden und überschüssiges Peptid und Additive entfernt werden können. Eine Desulfurierung der immobilisierten Peptide und Proteine liefert nach Abspaltung die gewünschten Tag ‐ freien Produkte. Mit dieser Methode konnte Alzheimer ‐ relevantes Tau ‐ Protein aus einer komplexen EPL ‐ Mischung und dreifach phosphoryliertes C ‐ terminales Tau ‐ Peptid isoliert werden. Fischen nach Ligationsprodukten: Bei einer neuen Methode zur spurlosen Ligation, Desulfurierung und Aufreinigung werden synthetische Peptide mit photospaltbarem Biotin in der nativen chemischen Ligation und der Ligation an exprimierte Proteine eingesetzt. Die Methode liefert reine, Tag ‐ freie Ligationsprodukte unter geringem Zeitaufwand und wurde zur NCL ‐ basierten Synthese des vollständigen Tau ‐ Proteins genutzt. [ABSTRACT FROM AUTHOR]

We present a novel strategy for the traceless purification and synthetic modification of peptides and proteins obtained by native chemical ligation. The strategy involves immobilization of a photocleavable semisynthetic biotin-protein conjugate on streptavidin-coated agarose beads, which eliminates the need for tedious rebuffering steps and allows the rapid removal of excess peptides and additives. On-bead desulfurization is followed by delivery of the final tag-free protein product. The strategy is demonstrated in the isolation of a tag-free Alzheimer's disease related human tau protein from a complex EPL mixture as well as a triphosphorylated peptide derived from the C-terminus of tau. [ABSTRACT FROM AUTHOR]

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