Palladium-Triggered Chemical Rescue of Intracellular Proteins via Genetically Encoded Allene-Caged Tyrosine

Abstract

Chemical de-caging has emerged as an attractive strategy for gain-of-function study of proteins via small-molecule reagents. The previously reported chemical de-caging reactions have been largely centered on liberating the side chain of lysine on a given protein. Herein, we developed an allene-based caging moiety and the corresponding palladium de-caging reagents for chemical rescue of tyrosine (Tyr) activity on intracellular proteins. This bioorthogonal de-caging pair has been successfully applied to unmask enzymatic Tyr sites (e.g., Y671 on Taq polymerase and Y728 on Anthrax lethal factor) as well as the post-translational Tyr modification site (Y416 on Src kinase) in vitro and in living cells. Our strategy provides a general platform for chemical rescue of Tyr-dependent protein activity inside cells.

Palladium‐Mediated Cleavage of Proteins with Thiazolidine‐Modified Backbone in Live Cells

“…Early mechanistic studies have identified Ubal as the first Ub activity based probe,w hich forms hemithioacetals with the active site Cys of DUBs in presence of complex biological molecules. [29][30][31][32] Inspired by this work, Thzc leavage was achieved in cell lysates for the labeling of as ide-chain aldehyde. We have recently demonstrated the cleavage of peptides,bearing backbone Thz, using [PdCl(allyl)] 2 , [25,28] which is known to be compatible with reactions in live cells because of its reported low toxicity.…”

Phosphine‐Activated Lysine Analogues for Fast Chemical Control of Protein Subcellular Localization and Protein SUMOylation

“…The UAA mutagenesis approach requires minimal engineering of the protein of interest; when the proper orthogonal translational machinery is provided, UAAs are incorporated simply by mutating the codon at the desired position to the amber stop codon . UAAs allow investigators to site‐specifically insert new chemical functionalities into proteins, thereby enabling small‐molecule control in live biological systems . For example, palladium complexes can trigger protein function using propargylated or allylated lysine and allenylated tyrosine .…”

“…UAAs allow investigators to site‐specifically insert new chemical functionalities into proteins, thereby enabling small‐molecule control in live biological systems . For example, palladium complexes can trigger protein function using propargylated or allylated lysine and allenylated tyrosine . Additionally, tetrazine‐mediated triggering of UAAs masked with trans ‐cyclooctene (TCO) moieties provides another clever approach to conditional control over protein function .…”

“…Additionally, tetrazine‐mediated triggering of UAAs masked with trans ‐cyclooctene (TCO) moieties provides another clever approach to conditional control over protein function . While Pd‐catalyzed depropargylation and deallylation is generalizable and highly bioorthogonal, modest protein deprotection yields (≤50 %) and slow kinetics (2–3 h) limit usability for certain applications . Tetrazine‐mediated deprotection of TCOs is significantly faster, with rate constants exceeding 10 5 to 10 6 m −1 s −1 for optimized reagents.…”

Palladium in the Chemical Synthesis and Modification of Proteins

“…[22] Specifically,t he discovery of palladium-mediated carbon-carbon bond formation and carbon-oxygen bond cleavage,which revolutionized organic synthesis,isnow being successfully applied to protein modification. [24] Theunique catalytic properties of palladium in C À Cbond formation and C À Ob ond cleavage is due to the presence of as quare-planar geometry of palladium(II) complexes (d 8 ), with extra coordination sites at the axial positions which can accommodate various ligands for different chemistries.Moreover the unique ground-state structure of palladium (4d 10 5s 0 ), Pd 0 ,w here the filled dorbital can combine with the empty frontier sorbitals,a lso provides important properties to palladium catalysis. [23] Furthermore,less toxic palladium reagents are being used to modulate the activity of enzymes for ag ain of function by decaging catalytic residues.…”