P−B Desulfurization: An Enabling Method for Protein Chemical Synthesis and Site‐Specific Deuteration

Abstract

Cysteine‐mediated native chemical ligation is a powerful method for protein chemical synthesis. Herein, we report an unprecedentedly mild system (TCEP/NaBH4 or TCEP/LiBEt3H; TCEP=tris(2‐carboxyethyl)phosphine) for chemoselective peptide desulfurization to achieve effective protein synthesis via the native chemical ligation–desulfurization approach. This method, termed P−B desulfurization, features usage of common reagents, simplicity of operation, robustness, high yields, clean conversion, and versatile functionality compatibility with complex peptides/proteins. In addition, this method can be used for incorporating deuterium into the peptides after cysteine desulfurization by running the reaction in D2O buffer. Moreover, this method enables the clean desulfurization of peptides carrying post‐translational modifications, such as phosphorylation and crotonylation. The effectiveness of this method has been demonstrated by the synthesis of the cyclic peptides dichotomin C and E and synthetic proteins, including ubiquitin, γ‐synuclein, and histone H2A.

A gold mine for drug discovery: Strategies to develop cyclic peptides into therapies

“…Due to the low abundance of Cys in natural cyclopeptides, many desulfurization methods (metal‐based desulfurization, radical‐based desulfurization, P‐B desulfurization, etc) have been developed to convert Cys into alanine (Ala) residues. Through NCL‐desulfurization strategy, many bioactive cyclic peptides have been prepared successfully, such as the antibacterial microcin J25 (Figure ).…”

“…Through NCL‐desulfurization strategy, many bioactive cyclic peptides have been prepared successfully, such as the antibacterial microcin J25 (Figure ). Very recently, Jinn et al reported the total synthesis of two antitumor cyclopeptides, dichotomin C (Figure A) and E (Figure B). By incorporating Cys residues in place of the requisite Ala groups, NCL provided the mutated cyclopeptides successfully.…”

Phosphine-Dependent Photoinitiation of Alkyl Thiols under Near-UV Light Facilitates User-Friendly Peptide Desulfurization

“…This strategy became the go-to method for non-Cys ligation in academic research, and it has remained so for over a decade. Recent radical, photoredox, and phosphorus-boron reductions also employ large excesses of phosphine reagents (typically 30–100 equiv) and require an initiator, photocatalyst, or co-reductant, respectively.…”

Insights into the Mechanism and Catalysis of Peptide Thioester Synthesis by Alkylselenols Provide a New Tool for Chemical Protein Synthesis

“…Chemical protein synthesis is now established as an alternative mode of production of tailored proteins for use in diverse fields of research, primarily biological and medicinal research [ 1 ]. Thioester chemistry is central to the field of chemical protein synthesis by the frequent application of the native chemical ligation (NCL [ 2 , 3 , 4 , 5 , 6 ], Figure 1 A) and desulfurization methods [ 7 , 8 , 9 , 10 ] to the chemoselective formation of peptide junctions to cysteine or alanine in water using unprotected peptide segments as reactants [ 11 ].…”