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.

Synthesis, delivery and cellular conjugation analysis of differentially phosphorylated ubiquitin probes by parkin E3 ligase during mitophagy.

Modifying cyclic cell‐penetrating deca‐arginine (cR10) peptides with 4‐(4‐dimethylaminophenylazo)benzoic acid (DABCYL) improves the uptake efficiency of synthetic ubiquitin (Ub) cargoes into living cells. To probe the role of the DABCYL moiety, we performed time‐lapse microscopy and fluorescence lifetime imaging microscopy (FLIM) of fluorescent DABCYL‐R10 to evaluate the impact on cell entry by the formation of nucleation zones. Furthermore, we performed a structure–uptake relationship study with 13 DABCYL derivatives coupled to CPP to examine their effect on the cell‐uptake efficiency when conjugated to mono‐Ub through disulfide linkages. Our results show that through structure variations of the DABCYL moiety alone we could reach, at nanomolar concentration, an additional threefold increase in the cytosolic delivery of Ub, which will enable studies on various intracellular processes related to Ub signaling.

A rapid and efficient cyclization of unprotected N-propargylated peptides using the Au(I) organometallic complex is reported. The method relies on the activation of the propargyl functionality using gold(I) to produce a new linkage with the N-terminus amine at the cyclization site. The presented method features a fast reaction rate (within 20 min), mild conditions, chemoselectivity, wide sequence scope, and high yields (up to 87%). The strategy was successfully tested on a wide variety of 30 unprotected peptides having various sequences and lengths, thus providing access to structurally distinct cyclic peptides. The practical usefulness of this method was demonstrated in producing peptides that bind efficiently to Lys48-linked di- and tetra-ubiquitin chains. The new cyclic peptide modulators exhibited high permeability to living cells and promoted apoptosis via binding with the endogenous Lys48-linked ubiquitin chains.

In this study, the live-cell delivery of structurally different synthetic diubiquitin chains was examined.