On-Demand Detachment of Succinimides on Cysteine to Facilitate (Semi)Synthesis of Challenging Proteins

Abstract

The maleimide group is a widely used reagent for bioconjugation of peptides, proteins, and oligonucleotides employing Michael addition and Diels–Alder cycloaddition reactions. However, the utility of this functionality in chemical synthesis of peptides and proteins remains unexplored. We report, for the first time that Pd II complexes can mediate the efficient removal of various succinimide derivatives in aqueous conditions. Succinimide removal by Pd II was applied for the synthesis of two ubiquitin activity-based probes (Ub-ABPs) employing solid phase chemical ligation (SPCL). SPCL was achieved through a sequential three segment ligation on a polymer support via a maleimide anchor. The obtained probes successfully formed the expected covalent complexes with deubiquitinating enzymes (DUBs) USP2 and USP7, highlighting the use of our new method for efficient preparation of unique synthetic proteins. Importantly, we demonstrate the advantages of our newly developed method for the protection and deprotection of native cysteine with a succinimide group in a peptide fragment derived from thioredoxin-1 (Trx-1) obtained via intein based expression to enable ligation/desulfurization and subsequent disulfide bond formation in a one-pot process.

Cysteine protecting groups: applications in peptide and protein science

“…29a) for use in protein semisynthesis. 136 Suc undergoes deprotection using PdCl 2 similar to tBu and Acm, with deprotection significantly accelerated by addition of MgCl 2 (giving total Suc deprotection within 45 min as opposed to 4 h). Additionally, Cys(Suc) remains stable to treatment with [Pd(allyl)Cl 2 ], giving a degree of orthogonality to Thz (Thz is partially labile to PdCl 2 treatment).…”

“…Maleimides for Suc protection of Cys can also be functionalised for further applications; for example, a Suc-based protecting group could act both as a protecting group and a linker to a solid phase resin (PEGA resin) in the synthesis of ubiquitin activity-based probes. 136 5.8 N-terminal cysteine protecting groups 5.8.1 Thiazolidine (Thz). The thiazolidine group (Thz) was introduced as far back as 1937, where it was noted that formaldehyde reacts with Cys residues to form thiazolidinecarboxylic acids.…”

Surface Functionalization of PLGA Nanoparticles to Increase Transport across the BBB for Alzheimer’s Disease

“…Despite their wide application, there are still some limitations regarding the stability of maleimide-based linkers [135]. Indeed, it has been demonstrated that under certain conditions the Michael addition reaction is reversible [136][137][138].…”

Traceless enzymatic protein synthesis without ligation sites constraint

“…Having veri ed the PALME platform's broad application scope, we next attempted to apply it to the currently intractable targets. Recombinant proteins bearing multiple adjacent Cys residues are tricky to handle because cysteine-based chemical methods require pretreatment of the native proteins to reduce disul de bonds 40 . When applying thiol-dependent chemical methods to synthesize these targets, native Cys residues were often mutated or protected to avoid side reactions.…”