General synthetic strategy for regioselective ultrafast formation of disulfide bonds in peptides and proteins

Abstract

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.

Cysteine protecting groups: applications in peptide and protein science

“…20d). 215 In addition to Pd(II) chemistry, deprotection of Cys(Acm) using CuSO 4 has been reported. 73 Initially, it was noted that CuSO 4 -mediated deprotection of Cys(Thz) would also lead to deprotection of Cys(Acm) in the same peptide scaffold if ascorbate was not added, but would leave Acm intact if ascorbate was added.…”

“…31c). 215 The oNB protecting group is, however, a poor chromophore and displays low two-photon sensitivity. Coumarin-based protecting groups have since been described in an attempt to address some of these issues.…”

Insight on the Order of Regioselective Ultrafast Formation of Disulfide Bonds in (Antimicrobial) Peptides and Miniproteins

“…We have previously shown the efficient synthesis of various targets such as the a-conotoxin SI peptide,composed of 13 AAs and two disulfide bonds. [13] In order to probe the effect of the order of disulfide bonds formation in this system, we prepared by Fmoc solid-phase peptide synthesis (SPPS), a-conotoxin peptide,b earing two Cys residues (3 and 13) protected with Acm, and the two Cys residues (2 and 7) protected with 2-nitrobenzyl (NBzl;F igure S1). This structural design has the opposite protection scheme in comparison to our previous study.…”

“…This structural design has the opposite protection scheme in comparison to our previous study. [13] We dissolved the peptide in 6M Gn•HCl and exposed it to PdCl 2 (10 equiv) for 5min in pH 1, at 37 8 8C. Subsequently,w ea dded diethyldithiocarbamate (DTC;2 0equiv) to quench and precipitate Pd.…”

“…[13] Our strategy relies on the combination of disulfiram (DSF), [14] ultraviolet (UV) light, and Pd II , [15] where all synthetic steps are performed in one pot and completed within 5or13min for two or three disulfides, respectively. [13] While our strategy tackled most of the limitations of both oxidative folding and traditional stepwise oxidation, the effect of the order of disulfide bonds formation on the process remained unexplored. [13] In general, in the stepwise approach the order of formation of disulfide bonds plays akey role in the successful synthesis,y et the prediction of the exact order remains enigmatic in correlation with the long standing protein folding problem.…”