Boron clusters as broadband membrane carriers.

The membrane translocation of hydrophilic substances constitutes a challenge for their application as therapeutic compounds and labelling probes1-4. To remedy this, charged amphiphilic molecules have been classically used as carriers3,5. However, such amphiphilic carriers may cause aggregation and non-specific membrane lysis6,7. Here we show that globular dodecaborate clusters, and prominently B12Br122-, can function as anionic inorganic membrane carriers for a broad range of hydrophilic cargo molecules (with molecular mass of 146-4,500 Da). We show that cationic and neutral peptides, amino acids, neurotransmitters, vitamins, antibiotics and drugs can be carried across liposomal membranes. Mechanistic transport studies reveal that the carrier activity is related to the superchaotropic nature of these cluster anions8-12. We demonstrate that B12Br122- affects cytosolic uptake of different small bioactive molecules, including the antineoplastic monomethyl auristatin F, the proteolysis targeting chimera dBET1 and the phalloidin toxin, which has been successfully delivered in living cells for cytoskeleton labelling. We anticipate the broad and distinct delivery spectrum of our superchaotropic carriers to be the starting point of conceptually distinct cell-biological, neurobiological, physiological and pharmaceutical studies.

Dispersion Interactions in Condensed Phases and inside Molecular Containers.

ConspectusThe past decade has seen significant progress in the understanding and appreciation of the importance of London dispersion interactions (LDIs) in supramolecular systems and solutions. The Slater-Kirkwood formula relates LDIs to the molecular polarizabilities of the two interacting molecular species (α) and their interaction distance (a dependence of R-6). When advancing arguments related to intermolecular interactions, it is frequently assumed that molecules with larger molecular polarizabilities are more amenable to larger LDIs. However, arguments related to molecular polarizabilities are not always transferable to the condensed phase. In fact, the underlying bulk and molecular polarizabilities of common solvents show opposing trends. The intuitive concept that aromatic molecules are more polarizable than saturated hydrocarbons and that perfluorinated molecules are less polarizable than saturated hydrocarbons applies to the condensed phase only. When treating association phenomena in solution, where LDIs are generally very attenuated, the use of bulk polarizabilities is recommended, which are experimentally accessible through either refractive index measurements or suitable solvatochromic probes. Such probes can also be used to assess polarizabilities inside molecular container compounds, such as cucurbit[n]urils (CBn), cyclodextrins, calixarenes, and hemicarcerands. These macrocyclic cavities can have extreme microenvironments. For example, the inner concave phase of CB7 has been shown to be weakly polarizable, falling in between the gas phase and perfluorohexane; those of ß-cyclodextrin and p-sulfonatocalix[4]arene have been found to be similarly polarizable as water and alkanes, respectively, and the inside of hemicarcerands displays a very large bulk polarizability, exceeding that of diiodomethane. CBn compounds are privileged molecular container compounds, which we exemplify in this Account through case studies. (1) CBn macrocycles are prime water-soluble receptors for hydrocarbons, allowing for the reduction of the binding free energies to two components: the hydrophobic effect and dispersion interactions. To understand hydrocarbon binding, we initiated the HYDROPHOBE challenge, which revealed the shortcomings of both quantum-chemical and molecular dynamics approaches. (2) The smallest CBn receptor, CB5, is uniquely suited to bind the entire noble gas series, where hydrophobic effects and dispersion interactions operate in opposite directions. CB5 was revaled to be a unique synthetic receptor for noble gases, with the dominant driving force being the recovery of the cavitation energies for the hydration of noble gases in aqueous solution. Computational methods that encounter challenges in predicting hydrocarbon affinities and trends for CB6 and CB7 perform well for noble gases binding to CB5. (3) The larger homologue, CB8, allows one to set up intermolecular interaction chambers by the encapsulation of a (first) aromatic guest, thereby tuning LDIs inside the receptor cavity. In this manner, CB8 can be modulated to preferentially bind unsaturated and aromatic rather than saturated hydrocarbons, while the unmodified cavities of the smaller macrocycles CB6 and CB7 show selective binding of saturated hydrocarbons. (4) The (charged) host-guest complexes of CBn hosts are sufficiently stable in the gas phase, allowing for the study of the influence of LDIs on inner-phase chemical reactions. These studies are particularly interesting for the theoretical analysis of isolated host-guest LDIs, as experimental and computational data are directly comparable in the gas phase due to the absence of the solvation effect.

Cellular Uptake of Cell-Penetrating Peptides Activated by Amphiphilic p-Sulfonatocalix[4]arenes.

We report the synthesis of a series of amphiphilic p-sulfonatocalix[4]arenes with varying alkyl chain lengths (CX4-Cn) and their application as efficient counterion activators for membrane transport of cell-penetrating peptides (CPPs). The enhanced membrane activity is confirmed with the carboxyfluorescein (CF) assay in vesicles and by the direct cytosolic delivery of CPPs into CHO-K1, HCT 116, and KTC-1 cells enabling excellent cellular uptake of the CPPs into two cancer cell lines. Intracellular delivery was confirmed by fluorescence microscopy after CPP entry into live cells mediated by CX4-Cn, which was also quantified after cell lysis by fluorescence spectroscopy. The results present the first systematic exploration of structure-activity relationships for calixarene-based counterion activators and show that CX4-Cn are exceptionally effective in cellular delivery of CPPs. The dodecyl derivative, CX4-C12, serves as best activator. A first mechanistic insight is provided by efficient CPP uptake at 4 °C and in the presence of the endocytosis inhibitor dynasore, which indicates a direct translocation of the CPP-counterion complexes into the cytosol and highlights the potential benefits of CX4-Cn for efficient and direct translocation of CPPs and CPP-conjugated cargo molecules into the cytosol of live cells.

Size and Polarizability of Boron Cluster Carriers Modulate Chaotropic Membrane Transport.

Perhalogenated closo-borates represent a new class of membrane carriers. They owe this activity to their chaotropicity, which enables the transport of hydrophilic molecules across model membranes and into living cells. The transport efficiency of this new class of cluster carriers depends on a careful balance between their affinity to membranes and cargo, which varies with chaotropicity. However, the structure-activity parameters that define chaotropic transport remain to be elucidated. Here, we have studied the modulation of chaotropic transport by decoupling the halogen composition from the boron core size. The binding affinity between perhalogenated decaborate and dodecaborate clusters carriers was quantified with different hydrophilic model cargos, namely a neutral and a cationic peptide, phalloidin and (KLAKLAK)2. The transport efficiency, membrane-lytic properties, and cellular toxicity, as obtained from different vesicle and cell assays, increased with the size and polarizability of the clusters. These results validate the chaotropic effect as the driving force behind the membrane transport propensity of boron clusters. This work advances our understanding of the structural features of boron cluster carriers and establishes the first set of rational design principles for chaotropic membrane transporters.

Metallacarborane Cluster Anions of the Cobalt Bisdicarbollide-Type as Chaotropic Carriers for Transmembrane and Intracellular Delivery of Cationic Peptides.

Cobalt bisdicarbollides (COSANs) are inorganic boron-based anions that have been previously reported to permeate by themselves through lipid bilayer membranes, a propensity that is related to their superchaotropic character. We now introduce their use as selective and efficient molecular carriers of otherwise impermeable hydrophilic oligopeptides through both artificial and cellular membranes, without causing membrane lysis or poration at low micromolar carrier concentrations. COSANs transport not only arginine-rich but also lysine-rich peptides, whereas low-molecular-weight analytes such as amino acids as well as neutral and anionic cargos (phalloidin and BSA) are not transported. In addition to the unsubstituted isomers (known as ortho- and meta-COSAN), four derivatives bearing organic substituents or halogen atoms have been evaluated, and all six of them surpass established carriers such as pyrenebutyrate in terms of activity. U-tube experiments and black lipid membrane conductance measurements establish that the transport across model membranes is mediated by a molecular carrier mechanism. Transport experiments in living cells showed that a fluorescent peptide cargo, FITC-Arg8, is delivered into the cytosol.

Large anion binding in water.

Large water-soluble anions with chaotropic character display surprisingly strong supramolecular interactions in water, for example, with macrocyclic receptors, polymers, biomembranes, and other hydrophobic cavities and interfaces. The high affinity is traced back to a hitherto underestimated driving force, the chaotropic effect, which is orthogonal to the common hydrophobic effect. This review focuses on the binding of large anions with water-soluble macrocyclic hosts, including cyclodextrins, cucurbiturils, bambusurils, biotinurils, and other organic receptors. The high affinity of large anions to molecular receptors has been implemented in several lines of new applications, which are highlighted herein.

Stabilization of Guest Molecules inside Cation-Lidded Cucurbiturils Reveals that Hydration of Receptor Sites Can Impede Binding.

Docking of alkali metal ions to water-soluble macrocyclic receptors generally reduces the affinity of guest molecules due to competitive binding. The idea that solvation water molecules could display a larger steric hindrance towards guest binding than cations has not been considered to date. We show that the docking of large cations to cucurbit[5]uril (CB5) unexpectedly increases (by a factor of 5-8) the binding of hydrophobic guests, methane and ethane. This is due to the removal of water molecules from the carbonyl portals of CB5 during cation binding, which frees up space for hydrophobe encapsulation. In contrast, smaller cations like sodium protrude deeply into the cavity of CB5 and cause the expected decrease in binding, such that the rational selection of alkali cations allows for a variation of up to a factor of 20 in binding of methane and ethane. The statistical analysis of crystallographic data shows that the cavity volume of CB5 can be enlarged by placing large alkali ions (Rb+ and Cs+ ) centro-symmetrically at the portals. The results reveal a hitherto elusive steric hindrance of solvation water molecules near receptor binding sites, which is pertinent for the design of supramolecular catalysts and the understanding of biological receptors.

Noncovalent Modulation of Chemoselectivity in the Gas Phase Leads to a Switchover in Reaction Type from Heterolytic to Homolytic to Electrocyclic Cleavage.

In the gas phase, thermal activation of supramolecular assemblies such as host-guest complexes leads commonly to noncovalent dissociation into the individual components. Chemical reactions, for example of encapsulated guest molecules, are only found in exceptional cases. As observed by mass spectrometry, when 1-amino-methyl-2,3-diazabicyclo[2.2.2]oct-2-ene (DBOA) is complexed by the macrocycle ß-cyclodextrin, its protonated complex undergoes collision-induced dissociation into its components, the conventional reaction pathway. Inside the macrocyclic cavity of cucurbit[7]uril (CB7), a competitive chemical reaction of monoprotonated DBOA takes place upon thermal activation, namely a stepwise homolytic covalent bond cleavage with the elimination of N2 , while the doubly protonated CB7⋅DBOA complex undergoes an inner-phase elimination of ethylene, a concerted, electrocyclic ring-opening reaction. These chemical reaction pathways stand in contrast to the gas-phase chemistry of uncomplexed monoprotonated DBOA, for which an elimination of NH3 predominates upon collision-induced activation, as a heterolytic bond cleavage reaction. The combined results, which can be rationalized in terms of organic-chemical reaction mechanisms and density-function theoretical calculations, demonstrate that chemical reactions in the gas phase can be steered chemoselectively through noncovalent interactions.

Electrostatically induced pKa shifts in oligopeptides: the upshot of neighboring side chains.

pKa values of homorepeat hexapeptides with a 2,3-diazabicyclo[2.2.2]oct-2-ene (DBO) chromophore attached at the peptide C termini, through an asparagine derivative (Dbo), namely His6-Dbo (H6), Lys6-Dbo (K6), and Arg6-Dbo (R6), were determined by a novel fluorescence-based method. The fluorescence lifetime of Dbo in the peptides (τ) was measured as a function of pH. The side chains collide with Dbo intramolecularly and quench it efficiently only when they are deprotonated (i.e., pH ≥ side chain pKa). The pKa values of the H6, K6, and R6 peptides, attributable to side chain ionization, were found to be depressed compared to the pKa values of the His, Lys, and Arg residues in their free amino acid forms. We further looked into the structural changes of the peptides by molecular dynamics (MD) simulations; the peptides were structurally more expanded when their side chains are protonated. The structural expansion of the peptides reflects an electrostatic repulsion between the protonated side chain residues, which also accounts for the observed decrease in pKa values, which corresponds to a facilitated deprotonation, assisted by electrostatic repulsion.

Proton-Gradient-Driven Sensitivity Enhancement of Liposome-Encapsulated Supramolecular Chemosensors.

An overarching challenge in the development of supramolecular sensor systems is to enhance their sensitivity, which commonly involves the synthesis of refined receptors with increased affinity to the analyte. We show that a dramatic sensitivity increase by 1-2 orders of magnitude can be achieved by encapsulating supramolecular chemosensors inside liposomes and exposing them to a pH gradient across the lipid bilayer membrane. This causes an imbalance of the influx and efflux rates of basic and acidic analytes leading to a significantly increased concentration of the analyte in the liposome interior. The utility of our liposome-enhanced sensors was demonstrated with various host-dye reporter pairs and sensing mechanisms, and we could easily increase the sensitivity towards multiple biologically relevant analytes, including the neurotransmitters serotonin and tryptamine.

Dynamic Interconversions of Single Molecules Probed by Recognition Tunneling at Cucurbit[7]uril-Functionalized Supramolecular Junctions.

We introduce a versatile recognition tunneling technique using doubly cucurbit[7]uril-functionalized electrodes to form supramolecular junctions that capture analytes dynamically by host-guest complexation. This results in characteristic changes in their single-molecule conductance. For structurally related drug molecules (camptothecin, sanguinarine, chelerythrine, and berberine) and mixtures thereof, we observed distinct current switching signals related to their intrinsic conductance properties as well as pH-dependent effects which can be traced back to their different states (protonated versus neutral). The conductance variation of a single molecule with pH shows a sigmoidal distribution, allowing us to extract a pKa value for reversible protonation, which is consistent with the reported macroscopic results. The new electronic method allows the characterization of unmodified drug molecules and showcases the transfer of dynamic supramolecular chemistry principles to single molecules.

Discrete, Cationic Palladium(II)-Oxo Clusters via f-Metal Ion Incorporation and their Macrocyclic Host-Guest Interactions with Sulfonatocalixarenes.

We report on the discovery of the first two examples of cationic palladium(II)-oxo clusters (POCs) containing f-metal ions, [PdII6 O12 M8 {(CH3 )2 AsO2 }16 (H2 O)8 ]4+ (M=CeIV , ThIV ), and their physicochemical characterization in the solid state, in solution and in the gas phase. The molecular structure of the two novel POCs comprises an octahedral {Pd6 O12 }12- core that is capped by eight MIV ions, resulting in a cationic, cubic assembly {Pd6 O12 MIV8 }20+ , which is coordinated by a total of 16 terminal dimethylarsinate and eight water ligands, resulting in the mixed PdII -CeIV /ThIV oxo-clusters [PdII6 O12 M8 {(CH3 )2 AsO2 }16 (H2 O)8 ]4+ (M=Ce, Pd6 Ce8 ; Th, Pd6 Th8 ). We have also studied the formation of host-guest inclusion complexes of Pd6 Ce8 and Pd6 Th8 with anionic 4-sulfocalix[n]arenes (n=4, 6, 8), resulting in the first examples of discrete, enthalpically-driven supramolecular assemblies between large metal-oxo clusters and calixarene-based macrocycles. The POCs were also found to be useful as pre-catalysts for electrocatalytic CO2 -reduction and HCOOH-oxidation.

Carbon Dot Blinking Enables Accurate Molecular Counting at Nanoscale Resolution.

Accurate counting of single molecules at nanoscale resolution is essential for the study of molecular interactions and distribution in subcellular fractions. By using small-sized carbon dots (CDs), we have now developed a quantitative single-molecule localization microscopy technique (qSMLM) based on spontaneous blinking to count single molecules with a localization precision of 10 nm, which can be accomplished on conventional microscopes without sophisticated laser control. We explore and adapt the blinking of CDs with diverse structures and demonstrate a counting accuracy of >97% at a molecular density of 500 per µm2. When applied to G-protein coupled receptors on a cell membrane, we discriminated receptor oligomerization and clustering and revealed ligand-regulated receptor distribution patterns. This is the first example of adapting nanoparticle self-blinking for molecular counting, and this demonstrates the power of CDs as SMLM probes to reliably decipher sub-diffraction structures that mediate crucial biological functions.

Efficient Hydro- and Organogelation by Minimalistic Diketopiperazines Containing a Highly Insoluble Aggregation-Induced, Blue-Shifted Emission Luminophore*.

We report the synthesis, gelation abilities and aggregation-induced, blue-shifted emission (AIBSE) properties of two minimalistic diketopiperazine-based gelators. Despite containing a highly insoluble luminophore that makes up more than half of their respective molecular masses, efficient hydrogelation by multiple stimuli for one and efficient organogelation for the other compound are reported. Insights into the aggregation and gelation properties were gained through examination of the photophysical and material properties of selected gels, which are representative of the different modes of gelation. The synthesis of the gelators is highly modular and based on readily available amino acid building blocks, allowing the efficient and rapid diversification of these core structures and fine-tuning of gel properties.

A reference scale of cucurbit[7]uril binding affinities.

The accurate determination of ultra-high binding affinities in supramolecular host-guest chemistry is a challenging endeavour because direct binding titrations are generally limited to affinities <106 M-1 due to sensitivity constraints of common titration methods. To determine higher affinities, competitive titrations are usually performed, in which one compound with a well established binding affinity serves as a reference. Herein, we propose a reference scale for such competitive titrations with the host cucurbit[7]uril (CB7) comprising binding affinities in the range from 103 to 1015 M-1. The suggested reference compounds are commercially available and will aid in the future determination of CB7 binding affinities for stimuli-responsive host-guest systems.

Large-Peptide Permeation Through a Membrane Channel: Understanding Protamine Translocation Through CymA from Klebsiella Oxytoca*.

Quantifying the passage of the large peptide protamine (Ptm) across CymA, a passive channel for cyclodextrin uptake, is in the focus of this study. Using a reporter-pair-based fluorescence membrane assay we detected the entry of Ptm into liposomes containing CymA. The kinetics of the Ptm entry was independent of its concentration suggesting that the permeation through CymA is the rate-limiting factor. Furthermore, we reconstituted single CymA channels into planar lipid bilayers and recorded the ion current fluctuations in the presence of Ptm. To this end, we were able to resolve the voltage-dependent entry of single Ptm peptide molecules into the channel. Extrapolation to zero voltage revealed about 1-2 events per second and long dwell times, in agreement with the liposome study. Applied-field and steered molecular dynamics simulations added an atomistic view of the permeation events. It can be concluded that a concentration gradient of 1 µm Ptm leads to a translocation rate of about one molecule per second and per channel.

An Amphiphilic Sulfonatocalix[5]arene as an Activator for Membrane Transport of Lysine-rich Peptides and Proteins.

Lysine (K) is an important target residue for protein and peptide delivery across membranes. K is the most frequently exposed residue in proteins, leading to high demand for the development of K-compatible transport activators. However, designing activators for K-rich peptides and proteins is more challenging than for arginine-rich species because of the kosmotropic nature of K and its recognition difficulty. In this study, we designed a new amphiphilic sulfonatocalix[5]arene (sCx5-6C) as a K-compatible transport activator. sCx5-6C was tailored with two key elements, recognition of K and the ability to embed into membranes. We measured the membrane transport efficiencies of α-poly-l-lysine, heptalysine, and histones across artificial membranes and of α-poly-l-lysine into live cells, activated by sCx5-6C. The results demonstrate that sCx5-6C acts as an efficient activator for translocating K-rich peptides and proteins, which cannot be achieved by known arginine-compatible activators.

Face-Fusion of Icosahedral Boron Hydride Increases Affinity to γ-Cyclodextrin: closo,closo-[B21 H18 ]- as an Anion with Very Low Free Energy of Dehydration.

The supramolecular recognition of closo,closo-[B21 H18 ]- by cyclodextrins (CDs) has been studied in aqueous solution by isothermal titration calorimetry and nuclear magnetic resonance spectroscopy. These solution studies follow up on previous mass-spectrometric measurements and computations, which indicated the formation and stability of CD ⋅ B21 H18- complexes in the gas phase. The thermodynamic signature of solution-phase binding is exceptional, the association constant for the γ-CD complex with B21 H18- reaches 1.8×106  M-1 , which is on the same order of magnitude as the so far highest observed value for the complex between γ-CD and a metallacarborane. The nature of the intermolecular interaction is also examined by quantum-mechanical computational protocols. These suggest that the desolvation penalty, which is particularly low for the B21 H18- anion, is the decisive factor for its high binding strength. The results further suggest that the elliptical macropolyhedral boron hydride is another example of a CD binder, whose extraordinary binding affinity is driven by the chaotropic effect, which describes the intrinsic affinity of large polarizable and weakly solvated chaotropic anions to hydrophobic cavities and surfaces in aqueous solution.

Encapsulation of ionic liquids inside cucurbiturils.

Cucurbit[n]urils (CBn, n = 6-8) serve as molecular receptors for imidazolium-based ionic liquids (ILs) in aqueous solution. The amphiphilic nature of 1-alkyl-3-methylimidazolium guests (Cnmim), with a cationic imidazolium residue and a hydrophobic alkyl chain, enabled their complexation with CBn through a combination of the hydrophobic effect and ion-dipole interactions. 1H NMR experiments revealed that the cavity of CBn can host the hydrophobic chain of the ILs, while one of the carbonyl rims served as a docking site for the imidazolium ring. The structure of the complexes was further analyzed by molecular dynamics (MD) simulations, which indicated that the cavity of CB6 can accommodate up to 5 carbon atoms, while the larger cavity of CB7 and CB8 can encapsulate longer alkyl chains in folded conformations. Isothermal titration calorimetry (ITC) experiments provided up to micromolar affinity of ILs to CBn in aqueous solution, which was independently quantified by indicator displacement titrations.

Fluorescence Monitoring of Peptide Transport Pathways into Large and Giant Vesicles by Supramolecular Host-Dye Reporter Pairs.

The membrane transport mechanisms of cell-penetrating peptides (CPPs) are still controversial, and reliable assays to report on their internalization in model membranes are required. Herein, we introduce a label-free, fluorescence-based method to monitor membrane transport of peptides in real time. For this purpose, a macrocyclic host and a fluorescent dye forming a host-dye reporter pair are encapsulated inside phospholipid vesicles. Internalization of peptides, which can bind to the supramolecular host, leads to displacement of the dye from the host, resulting in a fluorescence change that signals the peptide uptake and, thus, provides unambiguous evidence for their transport through the membrane. The method was successfully validated with various established CPPs, including the elusive peptide TP2, in the presence of counterion activators of CPPs, and with a calixarene-based supramolecular membrane transport system. In addition, transport experiments with encapsulated host-dye reporter pairs are not limited to large unilamellar vesicles (LUVs) but can also be used with giant unilamellar vesicles (GUVs) and fluorescence microscopy imaging.