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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.

Aqueous solubility and stability often limit the application of aminophenoxazinones and their sulfur mimics as promising agrochemicals in a sustainable agriculture inspired by allelopathy. This paper presents a solution to the problem using host-guest complexation with cucurbiturils (

Perhalogenated boron clusters derived from B

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.

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., Surprisingly, large peptides (Protamine, 5.1 kDa) can permeate through bacterial outer membrane channels. The use of fluorescence, electrophysiology, and all‐atom modeling allows to quantify the flux. This approach can be transferred to related problems.

Supramolecular hydrogels gained significant attention as shear-thinning, self-healing materials. However, the introduction of non-covalent crosslinks inherently decreases the strength and stability of the hydrogel. In this work, we developed a novel supramolecular hydrogel that undergoes a reversible transformation to the corresponding covalently crosslinked hydrogel upon UV-irradiation. The supramolecular hydrogel was developed based on the ternary host–guest interaction of two anthracene moieties and one large macrocyclic host. Anthracene functionalized poly(N-acryloylmorpholine)s were synthesized by post-polymerization modification of a copolymer consisting of N-acryloylmorpholine and an activated ester comonomer, whereby two different polymers were prepared having either neutral anthracene side-chains or carrying a positive charge next to the anthracene to enhance the interaction with the host. The binding affinity of the anthracene side chains with and without an additional cationic charge were studied with two macrocyclic hosts, namely cucurbit[8]uril and γ-cyclodextrin (γ-CD) by UV-Vis titration revealing a markedly stronger binding in the presence of the cationic charge due to additional ion-dipole one of the macrocyclic hosts, cucurbit[8]uril. Subsequently, the effect of the binding affinity on the hydrogelation was investigated, indicating that the stronger binding affinity facilitated the hydrogel formation at lower concentration. Finally, the reversible transformation of the supramolecular hydrogel to a chemical hydrogel by anthracene dimerization was studied by the UV irradiation of the hydrogel at 365 nm for covalent crosslinking or at 254 nm for decrosslinking. It could be demonstrated that the dynamic nature of the hydrogel, that is responsible for the shear-thinning behavior, was indeed lost upon UV-irradiation indicative of the formation of a covalently crosslinked hydrogel. The capabilities of the formed supramolecular hydrogel that is easily processable and able to reversibly convert to a chemical hydrogel, provides potential applications in applying mechanically robust covalently crosslinked hydrogels in complex shapes and difficult to reach locations making use of the dynamic nature of the supramolecular crosslinks.

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

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 pK

To address a long-standing issue of building molecular electrical circuits heavily relying on anchoring chemistry, we propose an alternative method to immobilize anchor-free molecules in a molecular cavity between metal electrodes. In such a scheme, well-defined conductance distribution of anchor-free molecules was obtained by means of a scanning tunneling microscopy break-junction technique. Density functional theory calculations suggest that effective electronic coupling at the molecule-electrode interface can be achieved through well-defined van der Waals (vdW) interactions when the molecule is confined in a cavity with a defined geometry. This work offers a new paradigm to achieve reliable conductance measurements of molecules via vdW interaction without metal-binding groups.

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.

Peptide flexibility is a determining factor in designing peptide-based drugs and in linker peptides. The flexibility is roughly inversely proportional to the size of the amino acid side chains in a peptide sequence. Glycine homo repeats are, therefore, the most flexible oligopeptides. We synthesized three oligopeptides: a relatively rigid peptide, His-(Arg)4-Trp (1), a flexible peptide, His-(Gly)4-Trp (2), and a “super-flexible” peptide; His-Gly-(GABA)-Gly-Trp (3) in which the central Gly-Gly unit in 2 was substituted by a γ-aminobutyric acid (GABA) linker. The only structural difference between 2 and 3 is that an amide bond in 2 is replaced by –CH2– units in 3. The frequency of end-to-end collisions, which serves as indicator of peptide flexibility, was measured fluorometrically. For comparing peptide flexibility, fluorescence emission spectra of their tryptophan residues were compared. Upon end-to-end collision, the N-terminal histidine residue efficiently quenches the fluorescence emission of the C-terminal tryptophan residue. The quenching rate is directly proportional to the peptide flexibility. The observed strongly increased flexibility in the γ-aminobutyric acid-containing peptide is due to the substitution of a single, rotationally restricted amide bond. Our result demonstrates the importance of amid bonds in limiting peptide dynamics.

We report on the discovery of the first two examples of cationic palladium(II)-oxo clusters (POCs) containing f-metal ions, [Pd

A new set of hybrid guest molecules bearing organic and inorganic residues have been studied for their recognition by cyclodextrins in aqueous solution. The guest molecules consist of nitroanilines linked through their amino group to the dodecahydrido

Compared with primary and local tumor, metastasis is more difficult to resect completely and remain the leading cause of death associated with solid tumors. It is therefore pressing to develop effective strategies to prevent and suppress tumor metastasis in its early stages. Heparanase overexpression is significantly associated with increased malignancy and metastasis. Here, we develop a novel heparanase degradable heparin-based microcarrier, HBMA, for specific staining, targeting, and inhibiting tumor cells with high metastatic potential. HBMA is fabricated through self-assembling of three components, positively charged fluorescent gold nanoclusters (KG-AuNCs), heparin polymers (HP), and antisense miRNA-21 oligonucleotides (AM-21), with each component playing multiple roles. Results of confocal microscopy and flow cytometry show that this agent has an excellent capability to label metastatic tumor cells with high selectivity. On the therapeutic side, the results of cell viability assay, wound healing assay, and plate colony formation assay verify that HBMA induces a significant inhibition effect towards the proliferation and migration of the selectively target tumor cells. The heparanase responsive AM-21 delivery, the specific staining, and the efficient tumor cell activity suppression highlight HBMA as a promising cancer theranostic agent to prevent tumor metastasis.

pK

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.

The combination of the macrocyclic hosts p‐sulfonatocalix[4]arene and cucurbit[7]uril with the fluorescent dyes lucigenin and berberine affords two label‐free enzyme assays for the detection of kinase and phosphatase activity by fluorescence monitoring. In contrast to established assays, no substrate labeling is required. Since phosphorylation is one of the most important regulatory mechanisms in biological signal transduction, the assays should be useful for identification of inhibitors and activators in high‐throughput screening (HTS) format for drug discovery., Monitoring enzyme activity: Reporter pairs composed of macrocyclic hosts and dyes afford kinase and phosphatase assays suitable for drug discovery

A convenient supramolecular strategy for constructing a ratiometric fluorescent chemosensing ensemble, consisting of a macrocyclic host (cucurbit[8]uril CB[8]), and a pyrene‐tagged amphiphilic peptide beacon (AP 1), is reported. AP 1 unfolds upon encapsulation of the pyrene termini into the hydrophobic CB[8] cavity. This changes pyrene excimer to monomer emission. Substrates with higher affinity for the CB[8] cavity can displace AP 1 from the ensemble. The released AP 1 folds again to form a pyrene excimer, which allows for the ratiometric fluorescence monitoring of the substrate. In this report, the ensemble capacity for ratiometric fluorescence monitoring of biological substrates, such as amino acid derivatives, specific peptides, and proteins, in aqueous media is demonstrated., A switching chemosensing ensemble was obtained by combining a macrocyclic host, cucurbit[8]uril (CB[8]), and a pyrene‐tagged amphiphilic peptide beacon (AP 1) for excellent ratiometric fluorescent recognition of biological substrates in aqueous media. The pyrene termini of the flexible AP 1 compete with substrates for encapsulation into the CB[8] cavity which changes pyrene excimer/monomer emission.

Metallacarboranes are a class of inorganic boron clusters that have recently been recognized as biologically active compounds. Herein, we report on the host-guest complexation of several cobalt bis(1,2-dicarbollide) anions (COSANs) with cyclodextrins (CDs) in aqueous solution. The binding affinities reach micromolar values, which are among the highest known values for native CDs, and exceed those for neutral hydrophobic organic guest molecules. The entrapment of the COSANs inside the cavity of CDs was confirmed using NMR and UV-visible spectroscopy, mass spectrometry, cyclic voltammetry, and isothermal titration calorimetry. Complexation by CDs greatly influences the photophysical and electrochemical properties of COSANs. In combination with indicator displacement assays, a label-free fluorescence-based method was developed to allow real-time monitoring of the translocation of COSANs through lipid bilayer membranes.

In this study, we investigate the influence of γ-cyclodextrin (γCD) or octakis-(2,3,6-tri-O-methyl)-γ-cyclodextrin (TMe-γCD) encapsulation on the solubility, thermal, optical, electrochemical, morphological, and electrical properties of an aromatic polyazomethine (PA). PA was prepared by oxidative C C coupling of pyrenyl groups from the monomer MA using FeCl3 as catalyst in DMF. After threading of γCD or TMe-γCD macrocycles onto the PA chains, the 1H NMR spectra were in line with the formation of the respective inclusion complexes, PA·γCD and PA·TMe-γCD, which showed distinct improvements of their solubility in common organic solvents, higher thermal stability, as well as better film-forming ability. Fluorescence spectroscopy revealed that the emission maximum shifted from 388 nm for PA to 425 nm and 428 nm for PA·γCD and PA·TMe-γCD, which is in good agreement with encapsulation of PA backbones into the hydrophobic cavities. Fluorescence lifetime measurements indicated multiexponential decays for all compounds, which is in accordance with different conformers in the excited state. Atomic force microscopy indicated that the surfaces of PA·γCD and PA·TMe-γCD in the solid state showed needle-shaped morphologies with widths of 105 ± 25 nm and 46 ± 13 nm, respectively, whereas the surface of the naked PA polymer displayed an irregular morphology with embedded particles and width of 158 ± 29 nm. The electrochemical band gaps (ΔEg) of PA·γCD and PA·TMe-γCD were lower than that of non-encapsulated PA. The electrical conductivities of the investigated compounds were about 10−11 S cm−1, which is in the range of insulating materials.

A putrescine derivative of aminomethyladamantane is established as a ditopic guest with two mutually exclusive binding sites for cucurbit[6]uril and cucurbit[7]uril. A mixture containing both hosts, the ditopic guest, and two fluorescent dyes affords a relay system with a ratiometric fluorescence response and enables a kinetic analysis of the switching mechanism.

A supramolecular host-guest FRET pair based on a carboxyfluorescein-labelled cucurbit[7]uril (CB7-CF, as acceptor) and the fluorescent dye 4',6-diamidino-2-phenylindole (DAPI, as donor) is developed for sensing of DNA. In comparison to the commercial DNA staining dye SYBR Green I, the new chemosensing ensemble offers dual-emission signals, which allows a linear ratiometric response over a wide concentration range.

In this manuscript we describe a 2,5-diketopiperazine (DKP) which is further functionalized with a carbazol-luminophore. Gelation properties as well as optical properties of produced organo- and hydrogels based on this small molecule are described.

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.

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 across 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 provided an atomistic view on the permeation. It can be concluded that a concentration gradient of 1 M Ptm leads to a translocation rate of about 1 molecule per second and per channel.