A Type of Auxiliary for Native Chemical Peptide Ligation beyond Cysteine and Glycine Junctions

Abstract

Native chemical ligation enables the chemical synthesis of proteins. Previously, thiol-containing auxiliary groups have been used to extend the reaction scope beyond N-terminal cysteine residues. However, the N-benzyl-type auxiliaries used so far result in rather low reaction rates. Herein, a new N(α) -auxiliary is presented. Consideration of a radical fragmentation for cleavage led to the design of a new auxiliary group which is selectively removed under mildly basic conditions (pH 8.5) in the presence of TCEP and morpholine. Most importantly and in contrast to previously described auxiliaries, the 2-mercapto-2-phenethyl auxiliary is not limited to Gly-containing sites and ligations succeed at sterically demanding junctions. The auxiliary is introduced in high yield by on-resin reductive amination with commercially available amino acid building blocks. The synthetic utility of the method is demonstrated by the synthesis of two antimicrobial proteins, DCD-1L and opistoporin-2.

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

“…As shown previously, the MPE scaffold is the first auxiliary to enable native chemical ligation beyond glycine . We were interested to elucidate the role of the β‐aryl substituent and examined ligation with the unsubstituted 2‐mercaptoethyl auxiliary.…”

“…We sought to i) improve ligation rates by avoiding steric bulk at the ligation site and ii) prevent cleavage of the formed N ‐alkyl amides by getting around the acidic cleavage conditions that typically induce S→N acyl shift reactions. This analysis guided us to the development of new auxiliary scaffolds such as the 2‐mercapto‐2‐phenethyl (MPE) auxiliary, which is the first auxiliary that escapes the need for glycine at the ligation junction and permits auxiliary removal under mild basic conditions when S→N acyl shifts are favored over N→S acyl shifts. Previous reports stressed the importance of native chemical ligation‐type reactions proceeding through 5‐membered ring transition states .…”

Parallel Chemical Protein Synthesis on a Surface Enables the Rapid Analysis of the Phosphoregulation of SH3 Domains

“…[12,27] One limitation we foresee is to assure protein folding on the surface,a lthough this was not ap roblem for the tested SH3 domains.…”

“…TheN CL was performed in solution prior to immobilization because of the sensitivity of the Ni-histidine complex to thiol additives.Solution steps are labor intensive.The weakness of the Ni-His linkage was also the reason why extended NCL based on the removal of ligation auxiliaries or desulfurization [12,13] was not explored. This limited the synthesis to SH3 domains,inw hich cysteineoccurs naturally.…”

Enzyme-catalyzed expressed protein ligation

“…Cys is one of the least frequently encoded residues in proteins 7 , and this sharply confines the flexibility in the ligation position or requires the introduction of Cys at unnatural locations, sometimes perturbing function. Methods to overcome the Cys requirement 8,9 in EPL are under development but have not yet proved robust.…”