(N-Hydroxy)amide-Mediated Ligation of Peptides in Chemical Protein Synthesis
Abstract
Chemical protein synthesis enables the production of synthetic proteins with precisely controlled structures. Some chemoselective methods for ligation of peptides in water have been developed thus far, and a variety of target proteins were successfully prepared, in which some specific amino acids such as N-terminal Cys, Ser/Thr or surrogates containing different auxiliaries are usually required at the ligation sites. Therefore, it is highly desirable to develop a chemoselective peptide ligation that is not limited by the specific amino acids at the ligation sites for chemical protein synthesis. In this paper, we have developed an efficient chemoselective ligation between C-terminal peptidyl (N-methoxy)aminoacyl hydrazides and N-terminal aminoacyl (N-hydroxy)peptides in water. In this protocol, nitrite oxidation of our prepared C-terminal peptidyl (N-methoxy)aminoacyl hydrazides at pH 3.0 at-15 oC afforded the corresponding peptidyl (N-methoxy)aminoacyl azides, intermolecular N,O-exchange of the in-situ formed peptide (N-methoxy)aminoacyl azides with N-terminal aminoacyl (N-hydroxy)peptides at pH 8.0 at room temperature afforded O-acyl peptides, and intramolecular O,N-acyl transfer of the O-acyl peptides across a six-membered ring gave the corresponding ligated N-methoxy (N'-hydoxy)peptides. Reduction of the N-methoxy (N'-hydoxy)peptides with zinc in water (pH 3.0) at 37 oC produced the ligated long peptides with native amide bonds. This peptide ligation strategy was successfully used in the total synthesis of a long peptide and a protein with biological activities. Interestingly, introduction of the N-methoxy group in C-terminal peptidyl (N-methoxy)aminoacyl hydrazides inhibited epimerization of the (N-methoxy)amino acid residues during the ligation of peptides. The present peptide ligation shows some advantages including no requirement for specific amino acid residues at the ligation sites, rapid ligation (completion within 60 minutes) even at the sterically hindered junctions, almost no epimerization, high yields and avoiding use of thiophenols that are prone to oxidation and have a foul smell in the native chemical ligation. Therefore, this (N-hydroxy)amide-mediated ligation of peptides provides a novel, efficient and versatile strategy for chemical protein synthesis.
Related
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- Immobilized Thiol Catalysts for Native Chemical Ligation in Flow
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BibTeX
@misc{wang2026nhydroxyamidemediated,
title = {{(N-Hydroxy)amide-Mediated Ligation of Peptides in Chemical Protein Synthesis}},
author = {Weifeng Wang and Yue Zhang and Haijun Yang and Hua Fu},
howpublished = {ChemRxiv},
year = {2026},
month = sep,
doi = {10.26434/chemrxiv.15009475/v1},
url = {https://chemrxiv.org/doi/full/10.26434/chemrxiv.15009475/v1},
note = {CC BY},
}
RIS
TY - UNPB
TI - (N-Hydroxy)amide-Mediated Ligation of Peptides in Chemical Protein Synthesis
AU - Weifeng Wang
AU - Yue Zhang
AU - Haijun Yang
AU - Hua Fu
PY - 2026
DA - 2026/09/25
PB - ChemRxiv
DO - 10.26434/chemrxiv.15009475/v1
UR - https://chemrxiv.org/doi/full/10.26434/chemrxiv.15009475/v1
AB - Chemical protein synthesis enables the production of synthetic proteins with precisely controlled structures. Some chemoselective methods for ligation of peptides in water have been developed thus far, and a variety of target proteins were successfully prepared, in which some specific amino acids such as N-terminal Cys, Ser/Thr or surrogates containing different auxiliaries are usually required at the ligation sites. Therefore, it is highly desirable to develop a chemoselective peptide ligation that is not limited by the specific amino acids at the ligation sites for chemical protein synthesis. In this paper, we have developed an efficient chemoselective ligation between C-terminal peptidyl (N-methoxy)aminoacyl hydrazides and N-terminal aminoacyl (N-hydroxy)peptides in water. In this protocol, nitrite oxidation of our prepared C-terminal peptidyl (N-methoxy)aminoacyl hydrazides at pH 3.0 at-15 oC afforded the corresponding peptidyl (N-methoxy)aminoacyl azides, intermolecular N,O-exchange of the in-situ formed peptide (N-methoxy)aminoacyl azides with N-terminal aminoacyl (N-hydroxy)peptides at pH 8.0 at room temperature afforded O-acyl peptides, and intramolecular O,N-acyl transfer of the O-acyl peptides across a six-membered ring gave the corresponding ligated N-methoxy (N'-hydoxy)peptides. Reduction of the N-methoxy (N'-hydoxy)peptides with zinc in water (pH 3.0) at 37 oC produced the ligated long peptides with native amide bonds. This peptide ligation strategy was successfully used in the total synthesis of a long peptide and a protein with biological activities. Interestingly, introduction of the N-methoxy group in C-terminal peptidyl (N-methoxy)aminoacyl hydrazides inhibited epimerization of the (N-methoxy)amino acid residues during the ligation of peptides. The present peptide ligation shows some advantages including no requirement for specific amino acid residues at the ligation sites, rapid ligation (completion within 60 minutes) even at the sterically hindered junctions, almost no epimerization, high yields and avoiding use of thiophenols that are prone to oxidation and have a foul smell in the native chemical ligation. Therefore, this (N-hydroxy)amide-mediated ligation of peptides provides a novel, efficient and versatile strategy for chemical protein synthesis.
ER -