Features of Auxiliaries That Enable Native Chemical Ligation beyond Glycine and Cleavage via Radical Fragmentation

Abstract

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 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.

Challenges and Perspectives in Chemical Synthesis of Highly Hydrophobic Peptides

“…Canne et al (1996) introduced another way of circumventing the cysteine moiety by auxiliarated NCL introduced with the N α -(ethanethiol) and N α -(oxyethanethiol)-peptide. In Loibl et al (2018) applied this protocol for cleavage of 2-mercaptoethyl auxiliary group. A recently introduced NCL approach, the diselenide-selenoester ligation (DSL) opens up possibilities for using further amino acids and will be elucidated in the outlook.…”

Ein zur Basenkatalyse befähigtes Ligationsauxiliar ermöglicht die cysteinfreie native chemische Ligation an anspruchsvollen Verknüpfungsstellen

“…Die Verlagerung des Phenylsubstituenten von der α‐ zur β‐Position verringert den sterischen Anspruch in der Nähe des reaktiven Amins, wodurch das MPE‐Auxiliar auch NCL‐Reaktionen an nicht‐glycinhaltigen Verbindungen wie Ala‐Asn, Leu‐Asn erlaubt und die Ligation mit sterisch anspruchsvolleren Aminosäureresten wie Arg, Met und Gln ermöglicht. Nach der Ligation wird das MPE‐Auxiliar durch eine radikalische Fragmentierungsreaktion entfernt [8b] . Jedoch sind sterisch stark gehinderte Verknüpfungen an β‐verzweigten Aminosäuren auch mit diesem Auxiliar nicht zugänglich.…”

Templated chemistry for bioorganic synthesis and chemical biology

“…A suitable ligation template should provide high reactivity for both the thiol exchange and the S → N acyl migratory steps and must lend itself to facile removal under conditions that leave the ligation product unharmed. We systematically analyzed the architecture of the ligation template and found that the substituents at the α‐ and β‐positions in 15′ play key roles . A comparison of the three mercaptoethyl templates ME, MP, and MPE showed, perhaps surprisingly, that the additional β‐substituents in MP and MPE accelerated the ligation.…”

“…However, we discovered a potentially general approach for the removal of N‐amide–linked mercaptoethyl groups. Nuclear magnetic resonance (NMR) analysis of a C 13 ‐labelled ligation template suggested that treatment with phosphine in aqueous morpholine at pH 8.5 triggers a radical‐induced oxidative fragmentation (Figure B) . The investigation led us to the 2‐mercapto‐2‐phenethyl (MPE) template, which is the first ligation auxiliary that enables NCL at ligation sites beyond glycine.…”

“…Typical examples (Figure B) for stand‐alone templates are small molecule scaffolds such as cyclodextrins ( 1 ), calixarenes ( 2 ), and many more as well as larger molecules such as dendrimers ( 3 ), peptides (including oligoproline 4 ), or even entire proteins ( 8 ) or polymers ( 5 ) . In another application scenario, conjugated unimolecular templates are frequently used as reaction scaffolds to facilitate chemical reactions such as ligation auxiliaries in protein synthesis via native chemical ligation (NCL) ( vide infra ) …”