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.
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.
Molecular iodine-promoted efficient construction of isatins from 2'-aminophenylacetylenes, 2'-aminostyrenes, and 2'-amino-β-ketoesters is developed via oxidative amidation of sp, sp(2), and sp(3) C-H bonds. The reaction involves consecutive iodination, Kornblum oxidation, and intramolecular amidation in a single reactor. The present method meets all of the atom and redox economy principles.
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