Anion-π catalysis on carbon allotropes.

Anion-π catalysis, introduced in 2013, stands for the stabilization of anionic transition states on π-acidic aromatic surfaces. Anion-π catalysis on carbon allotropes is particularly attractive because high polarizability promises access to really strong anion-π interactions. With these expectations, anion-π catalysis on fullerenes has been introduced in 2017, followed by carbon nanotubes in 2019. Consistent with expectations from theory, anion-π catalysis on carbon allotropes generally increases with polarizability. Realized examples reach from enolate addition chemistry to asymmetric Diels-Alder reactions and autocatalytic ether cyclizations. Currently, anion-π catalysis on carbon allotropes gains momentum because the combination with electric-field-assisted catalysis promises transformative impact on organic synthesis.

The Dynamic Range of Acidity: Tracking Rules for the Unidirectional Penetration of Cellular Compartments.

Labeled ammonium cations with pKa ∼7.4 accumulate in acidic organelles because they can be neutralized transiently to cross the membrane at cytosolic pH 7.2 but not at their internal pH<5.5. Retention in early endosomes with less acidic internal pH was achieved recently using weaker acids of up to pKa 9.8. We report here that primary ammonium cations with higher pKa 10.6, label early endosomes more efficiently. This maximized early endosome tracking coincides with increasing labeling of Golgi networks with similarly weak internal acidity. Guanidinium cations with pKa 13.5 cannot cross the plasma membrane in monomeric form and label the plasma membrane with selectivity for vesicles embarking into endocytosis. Self-assembled into micelles, guanidinium cations enter cells like arginine-rich cell-penetrating peptides and, driven by their membrane potential, penetrate mitochondria unidirectionally despite their high inner pH. The resulting tracking rules with an approximated dynamic range of pKa change ∼3.5 are expected to be generally valid, thus enabling the design of chemistry tools for biology research in the broadest sense. From a practical point of view, most relevant are two complementary fluorescent flipper probes that can be used to image the mechanics at the very beginning of endocytosis.

Photocleavable Fluorescent Membrane Tension Probes: Fast Release with Spatiotemporal Control in Inner Leaflets of Plasma Membrane, Nuclear Envelope, and Secretory Pathway.

Mechanosensitive flipper probes are attracting interest as fluorescent reporters of membrane order and tension in biological systems. We introduce PhotoFlippers, which contain a photocleavable linker and an ultralong tether between mechanophore and various targeting motifs. Upon irradiation, the original probe is released and labels the most ordered membrane that is accessible by intermembrane transfer. Spatiotemporal control from photocleavable flippers is essential to access open, dynamic or elusive membrane motifs without chemical or physical interference. For instance, fast release with light is shown to place the original small-molecule probes into the innermost leaflet of the nuclear envelope to image changes in membrane tension, at specific points in time of membrane trafficking along the secretory pathway, or in the inner leaflet of the plasma membrane to explore membrane asymmetry. These results identify PhotoFlippers as useful chemistry tools to enable research in biology.

Oligomers of Cyclic Oligochalcogenides for Enhanced Cellular Uptake.

Monomeric cyclic oligochalcogenides (COCs) are emerging as attractive transporters to deliver substrates of interest into the cytosol through thiol-mediated uptake. The objective of this study was to explore COC oligomers. We report a systematic evaluation of monomers, dimers, and trimers of asparagusic, lipoic, and diselenolipoic acid as well as their supramolecular monomers, dimers, trimers, and tetramers. COC dimers were more than twice as active as the monomers on both the covalent and noncovalent levels, whereas COC trimers were not much better than dimers. These trends might suggest that thiol-mediated uptake of COCs is synergistic over both short and long distances, that is, it involves more than two COCs and more than one membrane protein, although other interpretations cannot be excluded at this level of complexity. These results thus provide attractive perspectives for structural evolution as well as imminent use in practice. Moreover, they validate automated HC-CAPA as an invaluable method to collect comprehensive data on cytosolic delivery within a reasonable time at a level of confidence that is otherwise inconceivable.

Flipper Probes for the Community.

This article describes four fluorescent membrane tension probes that have been designed, synthesized, evaluated, commercialized and applied to current biology challenges in the context of the NCCR Chemical Biology. Their names are Flipper-TR®, ER Flipper-TR®, Lyso Flipper-TR®, and Mito Flipper-TR®. They are available from Spirochrome.

Cell-Penetrating Streptavidin: A General Tool for Bifunctional Delivery with Spatiotemporal Control, Mediated by Transport Systems Such as Adaptive Benzopolysulfane Networks.

In this report, cell-penetrating streptavidin (CPS) is introduced to exploit the full power of streptavidin-biotin biotechnology in cellular uptake. For this purpose, transporters, here cyclic oligochalcogenides (COCs), are covalently attached to lysines of wild-type streptavidin. This leaves all four biotin binding sites free for at least bifunctional delivery. To maximize the standards of the quantitative evaluation of cytosolic delivery, the recent chloroalkane penetration assay (CAPA) is coupled with automated high content (HC) imaging, a technique that combines the advantages of fluorescence microscopy and flow cytometry. According to the resulting HC-CAPA, cytosolic delivery of CPS equipped with four benzopolysulfanes was the best among all tested CPSs, also better than the much smaller TAT peptide, the original cell-penetrating peptide from HIV. HaloTag-GFP fusion proteins expressed on mitochondria were successfully targeted using CPS carrying two different biotinylated ligands, HaloTag substrates or anti-GFP nanobodies, interfaced with peptide nucleic acids, flipper force probes, or fluorescent substrates. The delivered substrates could be released from CPS into the cytosol through desthiobiotin-biotin exchange. These results validate CPS as a general tool which enables unrestricted use of streptavidin-biotin biotechnology in cellular uptake.

Automated high-content imaging for cellular uptake, from the Schmuck cation to the latest cyclic oligochalcogenides.

Recent progress with chemistry tools to deliver into living cells has seen a shift of attention from counterion-mediated uptake of cell-penetrating peptides (CPPs) and their mimics, particularly the Schmuck cation, toward thiol-mediated uptake with cell-penetrating poly(disulfide)s (CPDs) and cyclic oligochalcogenides (COCs), here exemplified by asparagusic acid. A persistent challenge in this evolution is the simultaneous and quantitative detection of cytosolic delivery and cytotoxicity in a high-throughput format. Here, we show that the combination of the HaloTag-based chloroalkane penetration assay (CAPA) with automated high-content (HC) microscopy can satisfy this need. The automated imaging of thousands of cells per condition in multiwell plates allows us to obtain quantitative data on not only the fluorescence intensity but also on the localization in a very short time. Quantitative and statistically relevant results can be obtained from dose-response curves of the targeted delivery to selected cells and the cytotoxicity in the same experiment, even with poorly optimized cellular systems.

The Emergence of Anion-π Catalysis.

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.

Cell-Penetrating Dynamic-Covalent Benzopolysulfane Networks.

Cyclic oligochalcogenides (COCs) are emerging as promising systems to penetrate cells. Clearly better than and different to the reported diselenolanes and epidithiodiketopiperazines, we introduce the benzopolysulfanes (BPS), which show efficient delivery, insensitivity to inhibitors of endocytosis, and compatibility with substrates as large as proteins. This high activity coincides with high reactivity, selectively toward thiols, exceeding exchange rates of disulfides under tension. The result is a dynamic-covalent network of extreme sulfur species, including cyclic oligomers, from dimers to heptamers, with up to nineteen sulfurs in the ring. Selection from this unfolding adaptive network then yields the reactivities and selectivities needed to access new uptake pathways. Contrary to other COCs, BPS show high retention on thiol affinity columns. The identification of new modes of cell penetration is important because they promise new solutions to challenges in delivery and beyond.

Anion-π Catalysis on Carbon Nanotubes.

Induced π acidity from polarizability is emerging as the most effective way to stabilize anionic transition states on aromatic π surfaces, that is, anion-π catalysis. To access extreme polarizability, we propose a shift from homogeneous toward heterogeneous anion-π catalysis on higher carbon allotropes. According to benchmark enolate addition chemistry, multi-walled carbon nanotubes equipped with tertiary amine bases outperform single-walled carbon nanotubes. This is consistent with the polarizability of the former not only along but also between the tubes. Inactivation by π-basic aromatics and saturation with increasing catalyst concentration support that catalysis occurs on the π surface of the tubes. Increasing rate and selectivity of existing anion-π catalysts on the surface of unmodified nanotubes is consistent with transition-state stabilization by electron sharing into the tubes, i.e., induced anion-π interactions. On pristine tubes, anion-π catalysis is realized by non-covalent interfacing with π-basic pyrenes.

Primary Anion-π Catalysis and Autocatalysis.

Epoxide-opening ether cyclizations are shown to occur on π-acidic aromatic surfaces without the need of additional activating groups and with autocatalytic amplification. Increasing activity with the intrinsic π acidity of benzenes, naphthalenediimides (NDIs) and perylenediimides (PDIs) support that anion-π interactions account for function. Rate enhancements maximize at 270 for anion-π catalysis on fullerenes and at 5100 M-1 for autocatalysis. The occurrence of anion-π autocatalysis is confirmed with increasing initial rates in the presence of additional product. Computational studies on autocatalysis reveal transition state and product forming a hydrogen-bonded noncovalent macrocycle, like holding their hands and dancing on the active π surface, with epoxide opening and nucleophile being activated by anion-π interactions and hydrogen bonds to the product, respectively.

Tuning Optoelectronic and Chiroptic Properties of Peptide-Based Materials by Controlling the Pathway Complexity.

Supramolecular chemistry has evolved from the traditional focus on thermodynamic on-pathways to the complex study of kinetic off-pathways, which are strongly dependent on environmental conditions. Moreover, the control over pathway complexity allows nanostructures to be obtained that are inaccessible through spontaneous thermodynamic processes. Herein, we present a family of peptide-based π-extended tetrathiafulvalene (exTTF) molecules that show two self-assembly pathways leading to two distinct J-aggregates, namely metastable (M) and thermodynamic (T), with different spectroscopic, chiroptical, and electrochemical behavior. Moreover, cryo-transmission electron microscopy (cryo-TEM) reveals a different morphology for both aggregates and a direct observation of the morphological transformations from tapes to twisted ribbons.

Remote Control of Anion-π Catalysis on Fullerene-Centered Catalytic Triads.

The design, synthesis and evaluation of catalytic triads composed of a central C60 fullerene with an amine base on one side and polarizability enhancers on the other side are reported. According to an enolate addition benchmark reaction, fullerene-fullerene-amine triads display the highest selectivity in anion-π catalysis observed so far, whereas NDI-fullerene-amine triads are not much better than fullerene-amine controls (NDI=naphthalenediimide). These large differences in activity are in conflict with the small differences in intrinsic π acidity, that is, LUMO energy levels and π holes on the central fullerene. However, they are in agreement with the high polarizability of fullerene-fullerene-amine triads. Activation and deactivation of the fullerene-centered triads by intercalators and computational data on anion binding further indicate that for functional relevance, intrinsic π acidity is less important than induced π acidity, that is, the size of the oriented macrodipole of polarizable π systems that emerges only in response to the interaction with anions and anionic transition states. The resulting transformation is thus self-induced, the anionic intermediates and transition states create their own anion-π catalyst.

Anion-π Catalysis on Fullerenes.

Anion-π interactions on fullerenes are about as poorly explored as the use of fullerenes in catalysis. However, strong exchange-correlation contributions and the localized π holes on their surface promise unique selectivities. To elaborate on this promise, tertiary amines are attached nearby. Dependent on their positioning, the resulting stabilization of anionic transition states on fullerenes is shown to accelerate disfavored enolate addition and exo Diels-Alder reactions enantioselectively. The found selectivities are consistent with computational simulations, particularly concerning the discrimination of differently planarized and charge-delocalized enolate tautomers by anion-π interactions. Enolate-π interactions on fullerenes are much shorter than standard π-π interactions and anion-π interactions on planar surfaces, and alternative cation-π interactions are not observed. These findings open new perspectives with regard to anion-π interactions in general and the use of carbon allotropes in catalysis.

Catalysis with Chalcogen Bonds.

Herein, we introduce catalysts that operate with chalcogen bonds. Compared to conventional hydrogen bonds, chalcogen bonds are similar in strength but more directional and hydrophobic, thus ideal for precision catalysis in apolar solvents. For the transfer hydrogenation of quinolines and imines, rate enhancements well beyond a factor of 1000 are obtained with chalcogen bonds. Better activities with deeper σ holes and wider bite angles, chloride inhibition and correlation with computed anion binding energies are consistent with operational chalcogen bonds. Comparable to classics, such as 2,2'-bipyrroles or 2,2'-bipyridines, dithieno[3,2-b;2',3'-d]thiophenes (DTTs), particularly their diimides, but also wide-angle cyclopentadithiazole-4-ones are identified as privileged motifs to stabilize transition states in the focal point of the σ holes on their two co-facial endocyclic sulfur atoms.

Highly ordered n/p-co-assembled materials with remarkable charge mobilities.

Controlling self-organization and morphology of chemical architectures is an essential challenge in the search for higher energy-conversion efficiencies in a variety of optoelectronic devices. Here, we report a highly ordered donor/acceptor functional material, which has been obtained using the principle of ionic self-assembly. Initially, an electron donor π-extended tetrathiafulvalene and an electron-acceptor perylene-bisimide were self-organized separately obtaining n- and p-nanofibers at the same scale. These complementary n- and p-nanofibers are endowed with ionic groups with opposite charges on their surfaces. The synergic interactions establish periodic alignments between both nanofibers resulting in a material with alternately segregated donor/acceptor nanodomains. Photoconductivity measurements show values for these n/p-co-assembled materials up to 0.8 cm(2) V(-1) s(-1), confirming the effectiveness in the design of these heterojunction structures. This easy methodology offers great possibilities to achieve highly ordered n/p-materials for potential applications in different areas such as optoelectonics and photovoltaics.

A collection of fullerenes for synthetic access toward oriented charge-transfer cascades in triple-channel photosystems.

The development of synthetic methods to build complex functional systems is a central and current challenge in organic chemistry. This goal is important because supramolecular architectures of highest sophistication account for function in nature, and synthetic organic chemistry, contrary to high standards with small molecules, fails to deliver functional systems of similar complexity. In this report, we introduce a collection of fullerenes that is compatible with the construction of multicomponent charge-transfer cascades and can be placed in triple-channel architectures next to stacks of oligothiophenes and naphthalenediimides. For the creation of this collection, modern fullerene chemistry-methanofullerenes and 1,4-diarylfullerenes-is combined with classical Nierengarten-Diederich-Bingel approaches.

Tutorial: fluorescence lifetime microscopy of membrane mechanosensitive Flipper probes.

Measuring forces within living cells remains a technical challenge. In this Tutorial, we cover the development of hydrophobic mechanosensing fluorescent probes called Flippers, whose fluorescence lifetime depends on lipid packing. Flipper probes can therefore be used as reporters for membrane tension via the measurement of changes in their fluorescence lifetime. We describe the technical optimization of the probe for imaging and provide working examples for their characterizations in a variety of biological and in vitro systems. We further provide a guideline to measure biophysical parameters of cellular membranes by fluorescence lifetime imaging microscopy using Flipper probes, providing evidence that flippers can report long range forces in cells, tissues and organs.

Concave versus planar geometries for the hierarchical organization of mesoscopic 3D helical fibers.

Chromophore-peptide systems: a study on a series of pentapeptides covalently connected to planar π systems (1 a and 1 b) or to a curved π system (1 c) showed the influence of the concave shape on the efficient chiral transmission at nano- and mesoscales. Control over the hierarchical growth by H bonding, π-π, and solvophobic interactions made possible the efficient generation of electroactive 3D helical fibers.

HydroFlipper membrane tension probes: imaging membrane hydration and mechanical compression simultaneously in living cells.

HydroFlippers are introduced as the first fluorescent membrane tension probes that report simultaneously on membrane compression and hydration. The probe design is centered around a sensing cycle that couples the mechanical planarization of twisted push-pull fluorophores with the dynamic covalent hydration of their exocyclic acceptor. In FLIM images of living cells, tension-induced deplanarization is reported as a decrease in fluorescence lifetime of the dehydrated mechanophore. Membrane hydration is reported as the ratio of the photon counts associated to the hydrated and dehydrated mechanophores in reconvoluted lifetime frequency histograms. Trends for tension-induced decompression and hydration of cellular membranes of interest (MOIs) covering plasma membrane, lysosomes, mitochondria, ER, and Golgi are found not to be the same. Tension-induced changes in mechanical compression are rather independent of the nature of the MOI, while the responsiveness to changes in hydration are highly dependent on the intrinsic order of the MOI. These results confirm the mechanical planarization of push-pull probes in the ground state as most robust mechanism to routinely image membrane tension in living cells, while the availability of simultaneous information on membrane hydration will open new perspectives in mechanobiology.