1. Enabling Cysteine-Free Native Chemical Ligation at Challenging Junctions with a Ligation Auxiliary Capable of Base Catalysis
Olaf Fuchs, Sebastian Trunschke, Hendrik Hanebrink, Marc Reimann, Oliver Seitz Angew Chem Int Ed Engl. 2021 Aug 23;60(35):19483-19490. doi: 10.1002/anie.202107158. Epub 2021 Jul 29.
Ligation auxiliaries are used in chemical protein synthesis to extend the scope of native chemical ligation (NCL) beyond cysteine. However, auxiliary-mediated ligations at sterically demanding junctions have been difficult. Often the thioester intermediate formed in the thiol exchange step of NCL accumulates because the subsequent S→N acyl transfer is extremely slow. Here we introduce the 2-mercapto-2-(pyridin-2-yl)ethyl (MPyE) group as the first auxiliary designed to aid the ligation reaction by catalysis. Notably, the MPyE auxiliary provides useful rates even for junctions containing proline or a β-branched amino acid. Quantum chemical calculations suggest that the pyridine nitrogen acts as an intramolecular base in a rate-determining proton transfer step. The auxiliary is prepared in two steps and conveniently introduced by reductive alkylation. Auxiliary cleavage is induced upon treatment with TCEP/morpholine in presence of a MnII complex as radical starter. The synthesis of a de novo designed 99mer peptide and an 80 aa long MUC1 peptide demonstrates the usefulness of the MPyE auxiliary.
2. Features of Auxiliaries That Enable Native Chemical Ligation beyond Glycine and Cleavage via Radical Fragmentation
Simon F Loibl, Andre Dallmann, Kathleen Hennig, Carmen Juds, Oliver Seitz Chemistry. 2018 Mar 7;24(14):3623-3633. doi: 10.1002/chem.201705927. Epub 2018 Feb 12.
Native chemical ligation (NCL) is an invaluable tool in the total chemical synthesis of proteins. Ligation auxiliaries overcome the requirement for cysteine. However, the reported auxiliaries remained limited to glycine-containing ligation sites and the acidic conditions applied for cleavage of the typically applied N-benzyl-type linkages promote side reactions. With the aim to improve upon both ligation and cleavage, we systematically investigated alternative ligation scaffolds that challenge the N-benzyl dogma. The study revealed that auxiliary-mediated peptide couplings are fastest when the ligation proceeds via 5-membered rather than 6-membered rings. Substituents in α-position of the amine shall be avoided. We observed, perhaps surprisingly, that additional β-substituents accelerated the ligation conferred by the β-mercaptoethyl scaffold. We also describe a potentially general means to remove ligation auxiliaries by treatment with an aqueous solution of triscarboxyethylphosphine (TCEP) and morpholine at pH 8.5. NMR analysis of a 13 C-labeled auxiliary showed that cleavage most likely proceeds through a radical-triggered oxidative fragmentation. High ligation rates provided by β-substituted 2-mercaptoethyl scaffolds, their facile introduction as well as the mildness of the cleavage reaction are attractive features for protein synthesis beyond cysteine and glycine ligation sites.
3. Native chemical ligation at a base-labile 4-mercaptobutyrate N(α)-auxiliary
Ziv Harpaz, Simon Loibl, Oliver Seitz Bioorg Med Chem Lett. 2016 Mar 1;26(5):1434-7. doi: 10.1016/j.bmcl.2016.01.060. Epub 2016 Jan 22.
Native chemical ligation (NCL) proceeds via a S-N acyl shift and, therefore, requires N-terminal cysteine. N(α)-auxiliaries have long been used to enable NCL beyond cysteine. However, the reversibility of the S-N acyl shift under the acidic conditions used to remove the commonly applied N-benzyl auxiliaries limits the scope of this reaction. Herein, we introduce a new class of N(α)-auxiliary which is designed for removal under mild basic conditions. The 3-N-linked 4-mercaptobutyrate auxiliary is readily synthesized in a single step and enables introduction on solid phase by means of reductive amination. The usefulness of the new auxiliary was demonstrated in the synthesis of the anti-microbial C-terminal domain of Dermicidine-1L.
