Efficient intracellular drug delivery and target specificity are often hampered by the presence of biological barriers. Thus, compounds that efficiently cross cell membranes are the key to improving the therapeutic value and on-target specificity of non-permeable drugs. The discovery of cell-penetrating peptides (CPPs) and the early design approaches through mimicking the natural penetration domains used by viruses have led to greater efficiency of intracellular delivery. Following these nature-inspired examples, a number of rationally designed CPPs has been developed. In this review, a variety of CPP designs will be described, including linear and flexible, positively charged and often amphipathic CPPs, and more rigid versions comprising cyclic, stapled, or dimeric and/or multivalent, self-assembled peptides or peptido-mimetics. The application of distinct design strategies to known physico-chemical properties of CPPs offers the opportunity to improve their penetration efficiency and/or internalization kinetics. This led to increased design complexity of new CPPs that does not always result in greater CPP activity. Therefore, the transition of CPPs to a clinical setting remains a challenge also due to the concomitant involvement of various internalization routes and heterogeneity of cells used in the in vitro studies.

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Cell-Penetrating Peptides: Design Strategies beyond Primary Structure and Amphipathicity.

The use of dithiolane-containing polymers to construct responsive and dynamic networks is an attractive strategy in material design. Here, we provide a detailed mechanistic study on the self-assembly and gelation behavior of a class of ABA triblock copolymers containing a central poly(ethylene oxide) block and terminal polycarbonate blocks with pendant 1,2-dithiolane functionalities. In aqueous solution, these amphiphilic block copolymers self-assemble into bridged flower micelles at high concentrations. The addition of a thiol initiates the reversible ring-opening polymerizations of dithiolanes in the micellar cores to induce the cross-linking and gelation of the micellar network. The properties of the resulting hydrogels depend sensitively on the structures of 1,2-dithiolanes. While the methyl asparagusic acid-derived hydrogels are highly dynamic, adaptable, and self-healing, those derived from lipoic acid are rigid, resilient, and brittle. The thermodynamics and kinetics of ring-opening polymerization ...

1,2-Dithiolane-Derived Dynamic, Covalent Materials: Cooperative Self-Assembly and Reversible Cross-Linking

The objective of this Account is to summarize the first five years of anion-π catalysis. The general idea of anion-π catalysis is to stabilize anionic transition states on aromatic surfaces. This is complementary to the stabilization of cationic transition states on aromatic surfaces, a mode of action that occurs in nature and is increasingly used in chemistry. Anion-π catalysis, however, rarely occurs in nature and has been unexplored in chemistry. Probably because the attraction of anions to π surfaces as such is counterintuitive, anion-π interactions in general are much younger than cation-π interactions and remain under-recognized until today. Anion-π catalysis has emerged from early findings that anion-π interactions can mediate the transport of anions across lipid bilayer membranes. With this evidence for stabilization in the ground state secured, there was no reason to believe that anion-π interactions could not also stabilize anionic transition states. As an attractive reaction to develop anion-π catalysis, the addition of malonic acid half thioesters to enolate acceptors was selected. This choice was also made because without enzymes decarboxylation is preferred and anion-π interactions promised to catalyze selectively the disfavored but relevant enolate addition. Concerning anion-π catalysts, we started with naphthalene diimides (NDIs) because their intrinsic quadrupole moment is highly positive. The NDI scaffold was used to address questions such as the positioning of substrates on the catalytic π surface or the dependence of activity on the π acidity of this π surface. With the basics in place, the next milestone was the creation of anion-π enzymes, that is, enzymes that operate with an interaction rarely used in biology, at least on intrinsically π-acidic or highly polarizable aromatic amino-acid side chains. Electric-field-assisted anion-π catalysis addresses topics such as heterogeneous catalysis on electrodes and remote control of activity by voltage. On π-stacked foldamers, anion-(π) n-π catalysis was discovered. Fullerenes emerged as the scaffold of choice to explore contributions from polarizability. On fullerenes, anionic transition states are stabilized by large macrodipoles that appear only in response to their presence. With this growing collection of anion-π catalysts, several reactions beyond enolate addition have been explored so far. Initial efforts focused on asymmetric anion-π catalysis. Increasing enantioselectivity with increasing π acidity of the active π surface has been exemplified for enamine and iminium chemistry and for anion-π transaminase mimics. However, the delocalized nature of anion-π interactions calls for the stabilization of charge displacements over longer distances. The first step in this direction was the formation of cyclohexane rings with five stereogenic centers from achiral acyclic substrates on π-acidic surfaces. Moreover, the intrinsically disfavored exo transition state of anionic Diels-Alder reactions is stabilized selectively on π-acidic surfaces; endo products and otherwise preferred Michael addition products are completely suppressed. Taken together, we hope that these results on catalyst design and reaction scope will establish anion-π catalysis as a general principle in catalysis in the broadest sense.

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The Emergence of Anion−π Catalysis

In this review, the multifunctionality of dithieno[3,2-b:2′,3′-d]thiophenes (DTTs) is covered comprehensively. This is of interest because all involved research is very recent and emphasizes timely...

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Dithienothiophenes at Work: Access to Mechanosensitive Fluorescent Probes, Chalcogen-Bonding Catalysis, and Beyond.

Anion receptor chemistry: Highlights from 2016

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 o...

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Activation of Cell-Penetrating Peptides with Ionpair−π Interactions and Fluorophiles

The 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 α-d-glucose (Glu), β-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 μm 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 non-glycosylated CPDs. In the MTT assay, all glyco-CPDs were non-toxic at concentrations as high as 2.5 μm. 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 (IC50 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 (IC50 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.

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Glycosylated Cell‐Penetrating Poly(disulfide)s: Multifunctional Cellular Uptake at High Solubility

https://pubs.rsc.org/en/content/articlepdf/2016/py/c6py00562d

Ethynyl benziodoxolones: functional terminators for cell-penetrating poly(disulfide)s

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 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.

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Paola Morelli

Papers

Activation of Cell-Penetrating Peptides with Ionpair−π Interactions and Fluorophiles

Glycosylated Cell‐Penetrating Poly(disulfide)s: Multifunctional Cellular Uptake at High Solubility

Ethynyl benziodoxolones: functional terminators for cell-penetrating poly(disulfide)s

Sidechain Engineering in Cell-Penetrating Poly(disulfide)s