Synthesis of Disulfide Surrogate Peptides Incorporating Large‐Span Surrogate Bridges Through a Native‐Chemical‐Ligation‐Assisted Diaminodiacid Strategy

Abstract

The use of synthetic bridges as surrogates for disulfide bonds has emerged as a practical strategy to obviate the poor stability of some disulfide‐containing peptides. However, peptides incorporating large‐span synthetic bridges are still beyond the reach of existing methods. Herein, we report a native chemical ligation (NCL)‐assisted diaminodiacid (DADA) strategy that enables the robust generation of disulfide surrogate peptides incorporating surrogate bridges up to 50 amino acids in length. This strategy provides access to some highly desirable but otherwise impossible‐to‐obtain disulfide surrogates of bioactive peptide. The bioactivities and structures of the synthetic disulfide surrogates were verified by voltage clamp assays, NMR, and X‐ray crystallography; and stability studies established that the disulfide replacements effectively overcame the problems of disulfide reduction and scrambling that often plague these pharmacologically important peptides.

A Diaminodiacid (DADA) Strategy for the Development of Disulfide Surrogate Peptides

“…[24] Recently, we reported a hydrazide-based NCL (native chemical ligation) assisted DADA strategy that may help synthesis of disulfide surrogate peptides incorporating synthetic bridges of up to 50 amino acids in length (Figure 9). [25] This strategy enabled the preparation of some functionally interesting but otherwise difficult-to-obtain peptide disulfide surrogates, such as mimics of μ-conotoxin KIIIA (a potent inhibitor of voltage-gated Na channels), mimics of Hm-3 (a spider toxin containing disulfide bridges that span large rings), and calcicludine (a snake toxin that incorporates a disulfide bridge spanning 50 amino acid residues).…”

“…Taking the synthesis of Cys7-Cys57 disulfide mimics of calcicludine as an example, Gly39-Cys40 was selected as the intramolecular cyclization site (Figure 10). [25] Using diaminodiacid 3 as the key building block, the synthesis of linear peptide hydrazide 38 was conducted using 2-Cl-Trt-NHNH 2 resin in 6% isolated yield. [9,26] Subsequently, hydrazide-based NCL was employed to achieve the intramolecular ligation of 38 at a concentration of 0.1 mM, giving product 39 within 5 h with 52% isolated yield.…”

“…Through stability studies, it was found that the disulfide-to-thioether substitutions can overcome the problems of SÀ S reduction and misfolding that may plague disulfide-containing peptides. [25] For example, the solutions of native KIIIA and its disulfide surrogates were treated with glutathione (0.25 mM, 1 equivalent). About 15% destruction of native KIIIA was detected after 0.5 h by RP-HPLC and ESI-MS analysis.…”

“…On the contrary, little destruction of disulfide surrogates was detected after 6 h treatment of glutathione. [25] Because most disulfide-rich peptides contain four or more Cys residues, it is usually not difficult to identify the appropriate Cys residue with which to implement the intramolecular NCLassisted DADA strategy. By expanding access to disulfide surrogate peptides with desired structures and enhanced stability, the NCL-assisted DADA strategy could facilitate the development of disulfide-containing peptides as diagnostic and therapeutic agents.…”

Chemical methods for studying the crosstalk between histone H2B ubiquitylation and H3 methylation

“…14,[20][21][22][23][24][25][26] Protein chemistry also enables the flexible alteration of protein at the histone and/or modifications for comprehensive mechanistic investigation. [27][28][29][30][31][32][33][34][35][36][37][38][39] Moreover, mechanism-based chemical probes can be used for trapping specific reaction states of the PTM enzymes for structural determination. These efforts have greatly expanded our understanding of the underlying molecular mechanisms of histone PTM crosstalks.…”