Switching Between Bicyclic and Linear Peptides — The Sulfhydryl-Specific Linker TPSMB Enables Reversible Cyclization of Peptides

Abstract

Phage display-selected bicyclic peptides have already shown their great potential for the development as bioactive modulators of therapeutic targets. They can provide enhanced proteolytic stability and improved membrane permeability. Molecular design of new linker molecules has led to a variety of new synthetic approaches for the generation of chemically constrained cyclic peptides. This diversity can be useful for the development of novel peptide-based therapeutic, diagnostic, and scientific tools. Herein, we introduce 1,3,5-tris((pyridin-2-yldisulfanyl)methyl)benzene (TPSMB) as a planar, trivalent, sulfhydryl-specific linker that facilitates reversible cyclization and linearization via disulfide bond formation and cleavage of bicyclic peptides of the format CXnCXnC, where X is any proteinogenic amino acid except cysteine. The rapid and highly sulfhydryl-specific reaction of TPSMB under physiological conditions is demonstrated by selecting bicyclic peptide binders against c-Jun N-terminal kinase 3 (JNK3) as a model target. While model peptides remain stably cyclized for several hours in presence of typical blood levels of glutathione in vitro, high cytosolic concentrations of glutathione linearize these peptides completely within 1 h. We propose that reversible linkers can be useful tools for several technical applications where target affinity depends on the bicyclic structure of the peptide.

Phage Display-Derived Peptide for the Specific Binding of SARS-CoV-2

“…These cyclic peptides represent a large class of bioactive molecules, 35 which usually have a higher binding affinity to the target proteins than their linear forms. 36 In another scenario, this peptide may form intermolecular disulfides that result in peptide homodimerization or forms polymers, which usually will improve the binding ability to the target proteins by the potential conformational changes with higher binding affinity and/or increasing binding avidity. 37,38 Indeed, once we blocked the first cysteine in peptide 1 (peptide 6), the affinity constant (K d ) dropped 3.5 folds from 5.8 to 19 μM.…”

Peptide–Bismuth Bicycles: In Situ Access to Stable Constrained Peptides with Superior Bioactivity

“…Conventional scaffolds like TBMB bind irreversibly and can hence modify other reactive peptide residues, even if only used in slight excess [4] . Nonspecific alkylation and cross‐linking of cysteine or lysine residues in proteins may also occur and has, for example, been observed in phage display [3a] .…”

“…Nonspecific alkylation and cross‐linking of cysteine or lysine residues in proteins may also occur and has, for example, been observed in phage display [3a] . Alternative, reversible linkers that form disulfide bonds between peptide cysteines and scaffolds offer better selectivity but suffer from redox sensitivity [4] . Although scaffolds like TATA and TBAB were specifically developed to stabilize peptide conformations by promoting hydrogen bond networks, [3b] all conventional scaffolds have flexible bonds that ultimately compromise the full potential for rigidification of peptide bicycles.…”

“…[3] Conventional scaffolds like TBMB bind irreversibly and can hence modify other reactive peptide residues, even if only used in slight excess. [4] Nonspecific alkylation and crosslinking of cysteine or lysine residues in proteins may also occur and has, for example, been observed in phage display. [3a] Alternative, reversible linkers that form disulfide bonds between peptide cysteines and scaffolds offer better selectivity but suffer from redox sensitivity.…”

Biocompatible and Selective Generation of Bicyclic Peptides**

“…[6] Irrespective of the outstanding success of these reagents in phage display and other encoded chemical libraries, they are neither fully selective nor biocompatible as they may modify other nucleophilic residues in peptides or target proteins. [7] Alternative late-stage modifications such as C-H activation between tryptophan and phenylalanine/tyrosine residues (Scheme 1b) require metal catalysts and organic solvents, rendering them bioincompatible. [8] We present a peptide bicyclization technique that is catalyst-free, biocompatible, and orthogonal to all canonical amino acids including cysteine (Scheme 1c).…”