Cysteine Promoted C‐Terminal Hydrazinolysis of Native Peptides and Proteins

Abstract

Tagging the terminus: N→S acyl transfer in native peptides and proteins can be reliably intercepted with hydrazine. The method allows selective labeling and ligation, without recourse to the use of protein‐splicing elements. NCL=native chemical ligation.

Expanding the scope of N → S acyl transfer in native peptide sequences

“…51 The combination of carboxamide-terminated precursor and acidic solvents led us to the conclusion that the reaction was likely only to be practically useful across Gly-Cys, His-Cys, and Cys-Cys motifs. 53 Consequently thioester formation is often conducted at 60 o C when this temperature is well tolerated, but we have also reported several successful reactions at temperatures ranging from 45-50 o C. [54][55][56][57] Additionally, we have demonstrated that peptide hydrazides, which can be formed as "shelf-stable" thioester equivalents can also be formed at lower temperature and are more stable to prolonged reaction times than the corresponding thioester. The first is that the thioester product has limited stability under the reaction conditions and so thioester formation does not profit from a protracted reaction time.…”

“…52 In fact NS acyl transfer in native peptides bearing a Cterminal Gly-Cys carboxylate (Gly-Cys-OH) motif occurs to an observable extent at room temperature, this process is currently impractical in light of at least two further influencing factors. 57 An advantage of using a single native amino acid residue to facilitate thioester synthesis is its operational simplicity, requiring only standard procedures, and readily available resins and amino acids. The second is that a mildly reducing environment must be maintained throughout the process and this is more difficult to achieve in a user friendly manner over a longer duration.…”

From protein total synthesis to peptide transamidation and metathesis: playing with the reversibility of N,S-acyl or N,Se-acyl migration reactions

“…The cleavage of the peptide bond to cysteine proceeded significantly faster for a C-terminal cysteine residue than for internal ones. Recent studies also showed that peptides or proteins featuring a Cterminal cysteine residue can be converted to peptide hydrazides when heated in the presence of excess hydrazine [36,37]. These reactions show that, under forcing conditions, the amide bond to cysteine can be reversible and exploited for useful synthetic transformations such as thioester synthesis and peptide cyclization [38,39].…”

Invited review GPCR structural characterization: Using fragments as building blocks to determine a complete structure

“…Our work using native chemical ligation and those of others have resulted in the preparation of only small pieces of GPCRs and/or quantitatively low yields, and the preparation of the thioesters of these extremely hydrophobic peptides needed for the ligation reactions has proved to be extremely challenging. From this perspective, the recent generation of thioesters in situ from chemically synthesized peptide hydrazides using the acyl azide method is promising and should be further explored.…”