Enabling Cysteine‐Free Native Chemical Ligation at Challenging Junctions with a Ligation Auxiliary Capable of Base Catalysis

Abstract

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.

Dissecting the role of protein phosphorylation: a chemical biology toolbox

“…173 Most recently, we introduced the 2-mercapto-2-(pyridin-2-yl)ethyl (MPyE) group, which is the first auxiliary designed to aid ligation by intramolecular base catalysis. 174 The MPyE auxiliary enables ligation at sterically hindered junctions, including proline or β-branched amino acids. At the time this review was drafted, auxiliaries have found little use so far in phosphoprotein synthesis.…”

Enhancing Auxiliary‐Mediated Native Chemical Ligation at Challenging Junctions with Pyridine Scaffolds

“…Interestingly, the thioester-linked intermediate 42AN*, formed by the reaction of the selenoester with the Chemistry-A European Journal auxiliary thiol, was still detectable via UPLC analysis. This intermediate could not be observed with the MPyE auxiliary (see Figure 3 in [6]), indicating again, that the additional methoxy group moderately lowers the rate of S,N-acyl transfer. Nevertheless, complete conversion to ligated Ala-Asn peptide was observed in less than 24 h. We also tested the MMPyE auxiliary in a Leu-Val ligation using selenoesters.…”

“…LYRAA-SePh (22A): Selenoester 22A was synthesized according to the published procedure. [6] UPLC-MS: t R = 5.31 min (3-30 % B in 6 min); m/z = 733.…”

A versatileo-aminoanilide linker for native chemical ligation

“…1,2 The chemospecic nature of this reaction prevents the need for the side chain protection of peptides, and thus it can be used just as effectively in recombinant protein-COSR obtained by splicing of protein-intein fusions. 3 Further elaborations of the NCL extended the reaction to other N-terminal residues, [4][5][6][7] peptide selenoesters 8 and selenoester-diselenide ligations. 9 Since Fmoc-solid phase peptide synthesis (Fmoc-SPPS) is currently the most adopted methodology for peptide synthesis, the direct preparation of peptide thioesters is poorly efficient, necessitating other approaches that overcome the piperidine-mediated aminolysis of the peptide-COSR.…”