Despite six decades of efforts to synthesize peptides and proteins bearing multiple disulfide bonds, this synthetic challenge remains an unsolved problem in most targets (e.g., knotted mini proteins). Here we show a de novo general synthetic strategy for the ultrafast, high-yielding formation of two and three disulfide bonds in peptides and proteins. We develop an approach based on the combination of a small molecule, ultraviolet-light, and palladium for chemo- and regio-selective activation of cysteine, which enables the one-pot formation of multiple disulfide bonds in various peptides and proteins. We prepare bioactive targets of high therapeutic potential, including conotoxin, RANTES, EETI-II, and plectasin peptides and the linaclotide drug. We anticipate that this strategy will be a game-changer in preparing millions of inaccessible targets for drug discovery.

Disulfide-rich peptides and proteins are among the most fascinating bioactive molecules.T he difficulties associated with the preparation of these targets have prompted the development of various chemical strategies.N evertheless,t he production of these targets remains very challenging or elusive. Recently,w ei ntroduced as trategy for one-pot disulfide bond formation, tackling most of the previous limitations.However, the effect of the order of oxidation remained an underexplored issue.Herein we report on the complete synthetic flexibility of the approach with respect to the order of oxidation of three disulfide bonds in targets that lack the knot motif.Incontrast, our study reveals an essential order of disulfide bond formation in the EETI-II knotted miniprotein. This synthetic strategy was applied for the synthesis of novel analogues of the plectasin antimicrobial peptide with enhanced activities against methicillin-resistant Staphylococcus aureus (MRSA), an otorious human pathogen.

<p><b>Despite six decades of efforts to synthesize peptides and proteins bearing multiple disulfide bonds, this synthetic challenge remains an unsolved problem in most targets (e.g. knotted mini proteins). Here we show a de novo general synthetic strategy for the ultrafast, high-yielding formation of two and three disulfide bonds in peptides and proteins. We developed an approach based on the combination of a small molecule, UV-light, and palladium for chemo- and regio-selective activation of Cys, which enables the one-pot formation of multiple disulfide bonds in various peptides and proteins. We prepared bioactive targets of high therapeutic potential, including conotoxin, RANTES, EETI-II, and plectasin peptides and the linaclotide drug. We anticipate that this strategy will be a game-changer in preparing millions of inaccessible targets for drug discovery.</b><br></p>

<p><b>Despite six decades of efforts to synthesize peptides and proteins bearing multiple disulfide bonds, this synthetic challenge remains an unsolved problem in most targets (e.g. knotted mini proteins). Here we show a de novo general synthetic strategy for the ultrafast, high-yielding formation of two and three disulfide bonds in peptides and proteins. We developed an approach based on the combination of a small molecule, UV-light, and palladium for chemo- and regio-selective activation of Cys, which enables the one-pot formation of multiple disulfide bonds in various peptides and proteins. We prepared bioactive targets of high therapeutic potential, including conotoxin, RANTES, EETI-II, and plectasin peptides and the linaclotide drug. We anticipate that this strategy will be a game-changer in preparing millions of inaccessible targets for drug discovery.</b><br></p>

Disulfide-rich peptides and proteins are among the most fascinating bioactive molecules.T he difficulties associated with the preparation of these targets have prompted the development of various chemical strategies.N evertheless,t he production of these targets remains very challenging or elusive. Recently,w ei ntroduced as trategy for one-pot disulfide bond formation, tackling most of the previous limitations.However, the effect of the order of oxidation remained an underexplored issue.Herein we report on the complete synthetic flexibility of the approach with respect to the order of oxidation of three disulfide bonds in targets that lack the knot motif.Incontrast, our study reveals an essential order of disulfide bond formation in the EETI-II knotted miniprotein. This synthetic strategy was applied for the synthesis of novel analogues of the plectasin antimicrobial peptide with enhanced activities against methicillin-resistant Staphylococcus aureus (MRSA), an otorious human pathogen.