In this report, we elaborate on two new concepts to activate arginine-rich cell-penetrating peptides (CPPs). Early on, we have argued that repulsion-driven ion-pairing interactions with anionic lipids account for their ability to move across hydrophobic cell membranes and that hydrophobic anions such as pyrenebutyrate can accelerate this process to kinetically outcompete endosomal capture. The original explanation that the high activity of pyrenebutyrate might originate from ionpair-π interactions between CPP and activator implied that replacement of the π-basic pyrene with polarized push-pull aromatics should afford more powerful CPP activators. To elaborate on this hypothesis, we prepared a small collection of anionic amphiphiles that could recognize cations by ionpair-π interactions. Consistent with theoretical predictions, we find that parallel but not antiparallel ionpair-π interactions afford operational CPP activators in model membranes and cells. The alternative suggestion that the high activity of pyrenebutyrate might originate from self-assembly in membranes was explored with perfluorinated fatty acids. Their fluorophilicity was expected to promote self-assembly in membranes, while their high acidity should prevent charge neutralization in response to self-assembly, i.e., generate repulsion-driven ion-pairing interactions. Consistent with these expectations, we find that perfluorinated fatty acids are powerful CPP activators in HeLa cells but not in model membranes. These findings support parallel ionpair-π interactions and repulsion-driven ion pairing with self-assembled fluorophiles as innovative concepts to activate CPPs. These results also add much corroborative support for counterion-mediated uptake as the productive mode of action of arginine-rich CPPs.

In memory of Ronald BreslowThe glycosylation of cell-penetrating poly(disulfide)s (CPDs) is introduced to increase the solubility of classical CPDs and to achieve multifunctional cellular uptake. With the recently developed sidechain engineering, CPDs decorated with a-D-glucose (Glu), b-D-galactose (Gal), D-trehalose (Tre), and triethyleneglycol (TEG) were readily accessible. Confocal laser scanning microscopy images of HeLa Kyoto cells incubated with the new CPDs at 2.5 lM revealed efficient uptake into cytosol and nucleoli of all glycosylated CPDs, whereas the original CPDs and TEGylated CPDs showed much precipitation into fluorescent aggregates at these high concentrations. Flow cytometry analysis identified Glu-CPDs as most active, closely followed by Gal-CPDs and Tre-CPDs, and all clearly more active than nonglycosylated CPDs. In the MTT assay, all glyco-CPDs were non-toxic at concentrations as high as 2.5 lM. Consistent with thiol-mediated uptake, glycosylated CPDs remained dependent on thiols on the cell surface for dynamic covalent exchange, their removal with Ellman's reagent DTNB efficiently inhibited uptake. Multifunctionality was demonstrated by inhibition of Glu-CPDs with D-glucose (IC 50 ca. 20 mM). Insensitivity toward L-glucose and D-galactose and insensitivity of conventional CPDs toward D-glucose supported that glucose-mediated uptake of the multifunctional Glu-CPDs involves selective recognition by glucose receptors at the cell surface. Weaker but significant sensitivity of Gal-CPDs toward D-galactose but not D-glucose was noted (IC 50 ca. 110 mM). Biotinylation of Glu-CPDs resulted in the efficient delivery of streptavidin together with a fluorescent model substrate. Protein delivery with Glu-CPDs was more efficient than with conventional CPDs and remained sensitive to DTNB and D-glucose, i.e., multifunctional.

Cell‐penetrating poly(disulfide)s (CPDs) have been introduced recently to explore new ways to enter into cells. In this report, we disclose a general method to covalently modify the sidechains of CPDs. Compatibility of copper‐catalyzed alkyne‐azide cycloaddition (CuAAC) with the addition of either strained cyclic disulfides of varied ring tension or increasing numbers of guanidinium and phosphonium cations is demonstrated. Reloading CPDs with disulfide ring tension results in an at least 20‐fold increase in activity with preserved sensitivity toward inhibition with the Ellman's reagent. The cumulation of permanent positive charges by sidechain engineering affords Ellman‐insensitive CPDs with similarly increased activity. Co‐localization experiments indicate that the CPDs reach endosomes, cytosol and nucleus, depending on their nature and their concentration. Supported by pertinent controls, these trends confirm that CPDs operate with combination of counterion‐ and thiol‐mediated uptake, and that the balance between the two can be rationally controlled. For the most active CPDs, uptake can be observed at substrate (fluorophore) concentrations as low as 5 nm.

Of 33 seed extracts examined, 12 inhibited protein synthesis in a rabbit reticulocyte lysate. This activity seems to be due to a protein, since (i) it was recovered with the (NH4)2SO4 precipitate, (ii) it was retained by dialysis membranes, and (iii) in all cases but one was destroyed by boiling. Only the extracts from the seeds of Adenia digitata and, to a lower extent, of Euonymus europaeus inhibited protein synthesis in intact cells.

Outperforming cell-penetrating peptides (CPPs), cell-penetrating poly(disulfide)s (CPDs) are attracting increasing interest. CPDs are accessible by ring-opening disulfide-exchange polymerization under mild conditions in neutral water. Initiation of the polymerization with thiols results in quantitative labeling of one CPD terminus with initiators of free choice. In contrast, labeling of the other terminus with iodoacetamides has so far been ineffective because of poor yields and the high excess of reagents needed. In this report, we introduce hypervalent iodine reagents as operational terminators of the synthesis of CPDs, also at high dilution. The power of the approach is exemplified with green-fluorescent initiators and ethynyl benziodoxolone terminators containing additional azides for CuAAC with red-fluorescent alkynes. The absorption spectra of the resulting CPDs demonstrate that stoichiometric application of ethynyl benziodoxolone terminators results in 46% incorporation. FRET between green-fluorescent initiators and red-fluorescent terminators demonstrates significant folding of CPDs in solution; it disappears upon reductive depolymerization. Substrates attached to the new termini are shown to enter into HeLa cells. Moreover, disappearance of FRET in the cytosol corroborated the reductive cleavage of CPDs upon internalization. Beyond the introduction of enthynyl benziodoxolones as operational terminators, these findings thus demonstrate also the compatibility of CuAAC with poly(disulfide)s and the usefulness of doubly-labeled CPDs for structural and mechanistic studies