Design and Synthesis of Cross-Link-Dense Peptides by Manipulating Regioselective Bisthioether Cross-Linking and Orthogonal Disulfide Pairing

Abstract

Existing disulfide-rich peptides, both naturally occurring and de novo designed, only represent a tiny amount of the possible sequence space because natural evolution and de novo design only keep sequences that are structurally approachable by correct disulfide pairings. To bypass this limitation for designing new peptide scaffolds beyond the natural sequence space, we dedicate to developing novel disulfide-rich peptides with predefined disulfide pairing patterns irrelevant to primary sequences. However, most of these designed peptides still suffer from disulfide rearrangements to at least one to three possible isomers. Here, we report a general and reliable strategy for the design and synthesis of a range of structurally diverse cross-link-dense peptide (CDP) scaffolds with two orthogonal disulfide bonds and a bisthioether bridge that are not subject to disulfide isomerizations. Altering the pattern of cysteine and penicillamine generates hundreds of different CDP scaffolds tolerant to extensive sequence manipulations. This work thus provides many useful scaffolds for the design of functional molecules such as protein binders with improved proteolytic stability (e.g., designed by epitope grafting).

Design and Synthesis of Disulfide-Rich Peptides with Orthogonal Disulfide Pairing Motifs

“…The expected fold can be obtained as a major product after the oxidative folding (Figures S10 and S11). Its binding affinity to the Keap1 Kelch domain was then evaluated using a previously validated fluorescence polarization assay (Figure b) . Tricyclic 10 can bind to the target with an IC 50 of 0.34 ± 0.07 μM, albeit with a slightly weaker affinity compared to its linear counterpart (i.e., the fully reduced peptide).…”

“…Recently, great progress toward this direction has been made by developing orthogonal disulfide pairing chemistry, which builds up of the specific pairing of cysteine (C) with penicillamine (Pen) and the unique ring-opening and dimerization properties of the CXPen (and the inverted PenXC; X can be any amino acid residues) motifs (Figure a) . Many C/Pen-based DRP scaffolds with two to four disulfide bonds have been designed and used for epitope grafting applications, , in which isolated CXPen and/or PenXC motifs were exploited to direct the oxidative folding. We anticipated that novel C/Pen-DRPs with fewer number of isomers can be designed using double or triple (i.e., tandem) CXPen/PenXC motifs.…”

Constitutionally Isomeric Aromatic Tripeptides: Self‐Assembly and Metal‐Ion‐Modulated Transformations

“…Though noncovalent interactions are primary stabilizing forces for self‐assembled structures but covalent crosslinking such as disulfide linkage and metal ion coordination with side chain also plays an important role in the formation, controlling and transformation of supramolecular nanostructures . Transition metal ions frequently utilized for conformational and morphological transformation due to their unique electronic structure and coordinating sites .…”

“…Interestingly the addition of Zn(II) ions increases β‐sheet component in both tripeptides (Supporting Information). Hence the presence of metal ions triggering the transformation of secondary structures and therefore can be used to inhibit the formation of amyloid‐like structures by using such model peptides …”

A Shelf Stable Fmoc Hydrazine Resin for the Synthesis of Peptide Hydrazides

“…17 This method avoids the need for strong oxidizing conditions, enabling broader compatibility with common synthetic peptide functionalities, and has been widely adopted in a broad range of applications. [20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37] The increased use of C-terminal α-hydrazides in peptide chemistry creates a demand for robust synthetic tools for accessing these moieties. A number of methods have been published to allow access to C-terminal hydrazides on both synthetic 15,16,38 and expressed 15,39,40 peptides and proteins.…”