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

Abstract

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.

Basic Residues at Position 11 of α-Conotoxin LvIA Influence Subtype Selectivity between α3β2 and α3β4 Nicotinic Receptors via an Electrostatic Mechanism

“…Based on our previous work, residues 9, 10, and 11 on LvIA were identified as promising positions to investigate selectivity-altering mutations . Thus, using a recently developed ultrafast de novo synthesis strategy, , we prepared 0.6 to 1.5 mg of wild-type (WT) LvIA, three previously tested controls (D9A, D10A, D11A), and six new α-CTX mutants (N9Y, N9Q, V10Q, V10L, D11R, D11N) (Figure and Synthesis Supplement: Synthetic Methods, HPLC, and MS Spectra) and determined their potencies for α3β2 and α3β4 nAChRs by two-electrode voltage clamp electrophysiology (TEVC) (Table and Figure ). Consistent with the previously reported IC 50 values and 18-fold selectivity, we found that WT LvIA was 25-fold more selective for the α3β2 nAChR over the α3β4 nAChR (Table and Figure ).…”

l-Methionine Selenoxide as an Oxidizing and Deprotection Reagent for the Synthesis of Multiple Disulfide Bonds in Peptides

“…Notably, forming the second disulfide bond was carried out in acidic media, in which a fast reaction rate was achieved, and the disulfide reshuffling could be inhibited. 5 A linear α-conotoxin SI bearing two free Cys residues (Cys2 and Cys7) and two Cys residues with side chains protected by Acm (Cys3 and Cys13) was first treated with MetSeO in a buffer of pH 7.0, followed by treating with HBr to directly generate 1 with two disulfides (Figure 1 and Figures S1 and S2). Therefore, the two Acm protection groups were successfully deprotected by MetSeO assisted by Br − and H + .…”

“…Cysteine (Cys) side chains protected by acetamidomethyl (Acm), 1,3-thiazolidine (Thz), and t -butyl ( t Bu) were often used for chemical syntheses of proteins − and of peptide multiple disulfide bonds through stepwise deprotections followed by oxidations . Recently, one-pot regioselectively synthetic approaches of multiple disulfide bonds in peptides were developed by the Brik group by use of these protected Cys, being more efficient in the synthesis of such kind of peptides. ,,, Deprotection of the Cys protecting groups can be achieved using a variety of reagents such as Hg­(II), Ag­(I), Tl­(III), Pd­(II), Pt­(IV), Cu­(II), I 2 , 2,2 ′ -dithiobis­(5-nitropyridine) (DTNP), triisopropylsilane (TIS), triethylsilane (TES), 2,2-dipyridyl disulfide (DPDS), and 2,2′-dipyridyl diselenide, affording cysteine or cystine peptides. However, some drawbacks still remain, including the oxidation of tryptophan and methionine residues in the peptides, harsh reaction conditions, and over-oxidations, which give some limits for their applications in the formation of disulfide bonds.…”

“…The linear oxytocin remained intact, demonstrating that the tBu cannot be deprotected from Cys(tBu) by MetSeO. Furthermore, a linear conotoxin mr3e 5 bearing two free Cys residues (Cys3 and Cys12), two Acm-protected Cys residues (Cys2 and Cys14), and two Cys modified with tBu (Cys8 and Cys15) as a model peptide was first reacted with MetSeO to construct the first disulfide bond (Cys3/Cys12), and then the pH of the reaction mixture was adjusted to 0.8 by addition of concentration HBr and aged for 20 min; the second disulfide bond (Cys2/Cys14) was formed with loss of the two Acm groups, while the two tBu groups were intact. Therefore, the tBu group was compatible with SeODR.…”

Rapid and Highly Productive Assembly of a Disulfide Bond in Solid-Phase Peptide Macrocyclization

“…Currently, a large number of disulfide-containing peptides are in clinical trials, and the Food and Drug Administration (FDA) has already approved some . Notable efforts have been dedicated to their chemical synthesis, especially establishing regioselective disulfide connection in a disulfide-rich peptide, , yet the developed methods are relatively popular in the solution phase. Single disulfide-driven peptide macrocyclization is well established in the solution phase using diverse reagents .…”