Organic chemistry allows for the modification and chemical preparation of protein analogues for various studies. The thiolate side chain of the Cys residue has been a key functionality in these ventures. In order to generate complex molecular targets, there is a particular need to incorporate orthogonal protecting groups of the thiolated amino acids to control the directionality of synthesis and modification site. Here, we demonstrate the tuning of palladium chemoselectivity in aqueous medium for on-demand deprotection of several Cys-protecting groups that are useful in protein synthesis and modification. These tools allow the preparation of highly complex analogues as we demonstrate in the synthesis of the copper storage protein and selectively modified peptides with multiple Cys residues. We also report the synthesis of an activity-based probe comprising ubiquitinated histone H2A and its incorporation into nucleosomes and demonstrate its reactivity with deubiquitinating enzyme to generate a covalent nucleosome–enzyme complex.

Chemical protein synthesis and biorthogonal modification chemistries allow production of unique proteins for arange of biological studies.B ond-forming reactions for siteselective protein labeling are commonly used in these endeavors.Selective bond-cleavage reactions,however,are muchless explored and still pose ag reat challenge.I na ddition, most of studies with modified proteins prepared by either total synthesis or semisynthesis have been applied mainly for in vitro experiments with very limited extension to live cells.Reported here is an approach for studying uniquely modified proteins containing at raceless cell delivery unit and palladium-based cleavable element for chemical activation, and monitoring the effect of these proteins in live cells.T his approach is demonstrated for the synthesis of ac aged ubiquitin-aldehyde, which was decaged for the inhibition of deubiquitinases in live cells.

Live-cell delivery of afully synthetic protein having selectivity towards aparticular target is ap romising approach with potential applications for basic researchand therapeutics. Cell-penetrating peptides (CPPs) allowthe cellular delivery of proteins but mostly result in endosomal entrapment, leading to lacko fb ioavailability.H erein, we report the design and synthesis of aC PP fused to 4-((4-(dimethylamino)phenyl)azo)benzoica cid (DABCYL) to enhance cellular uptake of fluorescently labelled synthetic protein analogues in low micromolar concentration. The attachment of cyclic decaarginine (cR10) modified with as ingle lysine linked to DABCYL to synthetic ubiquitin (Ub) and small ubiquitin-like modifier-2 (SUMO-2) scaffolds resulted in at hreefold higher uptake efficacy in live cells compared to the unmodified cR10. We could also achieve cR10DABCYL-assisted delivery of Ub and aU bv ariant (Ubv) based activity-based probes for functional studies of deubiquitinases in live cells.

Reversible attachment of solubilizing tags to hydrophobic peptides to facilitate their purification and ligation is an essential yet challenging task in chemical protein synthesis. The efficient palladium-assisted removal of the solubilizing tag linked to the Cys side chain is reported. The strategy was applied for the efficient preparation of histone protein H4 from two fragments via one-pot operation of ligation, removal of the solubilizing tag, and desulfurization.

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.