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

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 PdII
complexes can mediate the efficient removal of various succinimide
derivatives in aqueous conditions. Succinimide removal by PdII 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.