Native chemical ligation with N^α acyl transfer auxiliaries

Abstract

Native chemical ligation (NCL) is a simple procedure that enables synthetic access to many proteins and is increasingly harnessed to study protein structure and function. However, the generality of this method is limited by the requirement for cysteine residues suitably positioned throughout the target protein. Auxiliary approaches have been developed to overcome this limitation, wherein a removable group is introduced at the amino terminus of a peptide conveying ligation properties comparable to cysteine. Present auxiliary approaches combine the thioester exchange concept applied first in NCL with a number of acyl transfer reactions first systematically explored by Kemp and coworkers. The current methods for auxiliary mediated ligation appear promising for the synthesis of proteins and in particular post-translational modified proteins.

Building photoswitchable 3,4'-AMPB peptides: Probing chemical ligation methods with reducible azobenzene thioesters

“…In ligation reactions employing the N α -2-mercaptoethyl auxiliary peptide 4 reaction times were doubled for complete turnover (Table 1, entries 2 and 5). These results are in accordance with a chemoselective bimolecular thioester exchange prior to the S- to N-acyl transfer through a five-membered transition state for Cys-peptide 3 , a six-membered transition state for 4,5,6-trimethoxy-2-mercaptobenzyl auxiliary peptide 5 , and a sterically more demanding five-membered transition state for racemic N α -2-mercaptoethyl auxiliary peptide 4 [6]. …”

“…Our purpose was amongst other things (i) to increase flexibility, and (ii) to suppress resonance effects in order to enhance the stability of the diazene unit [4–5]. Since methods based on native chemical ligation (NCL), e.g., NCL with N α -acyl transfer auxiliaries, have gained considerable interest in the past two decades [6], we reasoned that a comparative ligation study with the Boc-protected azobenzene ω-amino acids Boc-3,4'-AMPB 1a and Boc-4,4'-AMPB 2a may result in an in-depth analysis of the redox-stability of these building blocks under the reducing conditions of thiol-based ligation methods. Generally, native chemical ligation allows the coupling of two unprotected peptides in neutral aqueous solution: a C-terminal thioester peptide and either an N-terminal cysteine peptide or N α -auxiliary-capped peptides.…”

“…We explored the conventional cysteine-based NCL with Cys-peptide 3 , and also screened the application of the TFA-cleavable 4,5,6-trimethoxy-2-mercaptobenzyl (Tmb) and 1-(2,4-dimethoxyphenyl)-2-mercaptoethyl auxiliaries by using peptides 4 and 5 (Fig. 1) [6–8], in order to circumvent the need for the presence of a cysteine at the ligation site.…”

“…For the synthetic ligation studies presented herein we had to place the ligation junction next to the third amino acid serine of the SKV binding motif. In the case of the conventional NCL, we used cysteine at the ligation site, and for the auxiliary mediated ligations, we decided to introduce auxiliary-glycine-conjugates at the N-terminus, due to the steric limitations of this type of ligation reaction [6–8]. After ligation, the SKV binding motif in the photoswitchable azopeptides should only be accessible in the dark trans -azo state (Scheme 1), whereas light switching to the cis -azo state should result in unfavorable interactions between the N-terminus, which follows behind the azo-switch, and the side chains that are critical for PDZ recognition (as exemplified in Scheme 1 for the 4,4'-substituted azobenzene system).…”

Ein PEGyliertes, lichtspaltbares Auxiliar für die sequenzielle enzymatische Glykosylierung und native chemische Ligation von Peptiden

“…Die milden Entschützungsbedingungen, in Kombination mit dem häufigen Vorkommen von Glycin in Proteinen, haben bereits zu mehreren Anwendungen in Protein(semi)synthesen geführt. [10] Im Folgenden stellen wir die Synthese und Anwendung eines lichtspaltbaren Auxiliars vor, das die Anbindung eines PEG-Polymers für effiziente enzymatische Glykosylierungen ermçglicht, die funktionellen Gruppen für NCL-Reaktionen bereitstellt und durch UV-Bestrahlung vollständig entfernt werden kann (Schema 1).…”