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.

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.

Supramolecular Chemistry in the Biomembrane.

The combination of supramolecular functional systems with biomolecular chemistry has been a fruitful exercise for decades, leading to a greater understanding of biomolecules and to a great variety of applications, for example, in drug delivery and sensing. Within these developments, the phospholipid bilayer membrane, surrounding live cells, with all its functions has also intrigued supramolecular chemists. Herein, recent efforts from the supramolecular chemistry community to mimic natural functions of lipid membranes, such as sensing, molecular recognition, membrane fusion, signal transduction, and gated transport, are reviewed.

High-Affinity Binding of Metallacarborane Cobalt Bis(dicarbollide) Anions to Cyclodextrins and Application to Membrane Translocation.

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.

Characterization of mixed-ligand shells on gold nanoparticles by transition metal and supramolecular surface probes.

We report herein two methods to characterize the surface of mixed-ligand shell gold nanoparticles, which was explored with gold nanoparticles containing varying molar ratios of 3-mercaptopropionic acid (MPA) and 3-mercapto-1-propanesulfonate (MPS) or 11-mercaptoundecanoic acid (MUA) and triethylene glycol mono-11-mercaptoundecyl ether (TEG) in their ligand shell. Incubation of gold nanoparticles with a solution containing the transition metal cation Ni2+ allows the extraction of Ni2+ depending on the number of negatively charged surface groups and the reaction of surface carboxylic acid groups with an aminomethyladamantane derivative allows the extraction of the supramolecular host molecule cucurbit[7]uril (CB7) depending on the number of reactive surface groups. In both the methods, the remaining surface probes in the supernatant could be conveniently quantified in a homogeneous solution after a simple centrifugation step. An excellent linear correlation between the amount of Ni2+ extracted and the ligand density of MPA and MPS in MPA/MPS gold nanoparticles or MUA in MUA/TEG gold nanoparticles afforded a simple and reliable assay method to determine the number of negatively charged surface groups. The supramolecular CB7 assay enabled the determination of the accessible ligand density of reactive surface carboxylic acid groups and revealed a striking difference in the number of surface groups that can be reacted with MPA/MPS gold nanoparticles or MUA/TEG gold nanoparticles, which suggests a simple method to probe the surface structure of mixed monolayer gold nanoparticles.

Rational design of boron-dipyrromethene (BODIPY) reporter dyes for cucurbit[7]uril.

We introduce herein boron-dipyrromethene (BODIPY) dyes as a new class of fluorophores for the design of reporter dyes for supramolecular host-guest complex formation with cucurbit[7]uril (CB7). The BODIPYs contain a protonatable aniline nitrogen in the meso-position of the BODIPY chromophore, which was functionalized with known binding motifs for CB7. The unprotonated dyes show low fluorescence due to photoinduced electron transfer (PET), whereas the protonated dyes are highly fluorescent. Encapsulation of the binding motif inside CB7 positions the aniline nitrogen at the carbonyl rim of CB7, which affects the pKa value, and leads to a host-induced protonation and thus to a fluorescence increase. The possibility to tune binding affinities and pKa values is demonstrated and it is shown that, in combination with the beneficial photophysical properties of BODIPYs, several new applications of host-dye reporter pairs can be implemented. This includes indicator displacement assays with favourable absorption and emission wavelengths in the visible spectral region, fluorescence correlation spectroscopy, and noncovalent surface functionalization with fluorophores.

Dual-Color Real-Time Chemosensing of a Compartmentalized Reaction Network Involving Enzyme-Induced Membrane Permeation of Peptides.

The design of synthetic systems with interrelated reaction sequences that model incipient biological complexity is limited by physicochemical tools that allow the direct monitoring of the individual processes in real-time. To mimic a simple digestion-resorption sequence, the authors have designed compartmentalized liposomal systems that incorporate extra- and intravesicular chemosensing ensembles. The extravesicular reporter pair consists of cucurbit[7]uril and methylene blue to monitor the enzymatic cleavage of short enkephalin-related peptides by thermolysin through a switch-off fluorescence response ("digestion"). Because the substrate is membrane-impermeable, but the dipeptide product is permeable, uptake of the latter into the pre-formed liposomes occurs as a follow-up process. The intravesicular chemosensing ensemble consists of i) cucurbit[8]uril, 2-anilinonaphthalene-6-sulfonic acid, and methyl viologen or ii) cucurbit[7]uril and berberine to monitor the uptake ("resorption") of the enzymatic products through the liposomal membranes by i) a switch-on or ii) a switch-off fluorescence response. The dyes are designed to allow selective optical excitation and read-out of the extra- and intravesicular dyes, which allow for dual-color chemosensing and, therefore, kinetic discrimination of the two sequential reactions.

Enzyme assays with supramolecular chemosensors - the label-free approach.

Enzyme activity measurements are essential for many research areas, e.g., for the identification of inhibitors in drug discovery, in bioengineering of enzyme mutants for biotechnological applications, or in bioanalytical chemistry as parts of biosensors. In particular in high-throughput screening (HTS), sensitive optical detection is most preferred and numerous absorption and fluorescence spectroscopy-based enzyme assays have been developed, which most frequently require time-consuming fluorescent labelling that may interfere with biological recognition. The use of supramolecular chemosensors, which can specifically signal analytes with fluorescence-based read-out methods, affords an attractive and label-free alternative to more established enzyme assays. We provide herein a comprehensive review that summarizes the current state-of-the-art of supramolecular enzyme assays ranging from early examples with covalent chemosensors to the most recent applications of supramolecular tandem enzyme assays, which utilize common and often commercially available combinations of macrocyclic host molecules (e.g. cyclodextrins, calixarenes, and cucurbiturils) and fluorescent dyes as self-assembled reporter pairs for assaying enzyme activity.

Membrane Permeability and Its Activation Energies in Dependence on Analyte, Lipid, and Phase Type Obtained by the Fluorescent Artificial Receptor Membrane Assay.

Time-resolved monitoring of the permeability of analytes is of utmost importance in membrane research. Existing methods are restricted to single-point determinations or flat synthetic membranes, limiting access to biologically relevant kinetic parameters (permeation rate constant, permeation coefficients). We now use the recently introduced fluorescent artificial receptor membrane assay (FARMA) as a method to monitor, in real time, the permeation of indole derivatives through liposomal membranes of different lipid compositions. This method is based on the liposomal encapsulation of a chemosensing ensemble or "fluorescent artificial receptor", consisting of 2,7-dimethyldiazapyrenium as a fluorescent dye and cucurbit[8]uril as the macrocyclic receptor, that responds to the complexation of a permeating aromatic analyte by fluorescence quenching. FARMA does not require a fluorescent labeling of the analytes and allows access to permeability coefficients in the range from 10-8 to 10-4 cm s-1. The effect of temperature on the permeation rate of a series of indole derivatives across the phospholipid membranes was studied. The activation energies for permeation through POPC/POPS phospholipid membranes were in the range of 28-96 kJ mol-1. To study the effect of different lipid phases on the membrane permeability, we performed experiments with DPPC/DOPS vesicles, which showed a phase transition from a gel phase to a liquid-crystalline phase, where the activation energies for the permeation process were expected to show a dramatic change. Accordingly, for the permeation of the indole derivatives into the DPPC/DOPS liposomes, discontinuities were observed in the Arrhenius plots, from which the permeation activation energies for the distinct phases could be determined, for example, for tryptamine 245 kJ mol-1 in the gel phase and 47 kJ mol-1 in the liquid-crystalline phase.

Precise supramolecular control of surface coverage densities on polymer micro- and nanoparticles.

We report herein the controlled surface functionalization of micro- and nanoparticles by supramolecular host-guest interactions. Our idea is to exploit the competition of two high-affinity guests for binding to the surface-bound supramolecular host cucurbit[7]uril (CB7). To establish our strategy, surface azide groups were introduced to hard-sphere (poly)methylmethacrylate particles with a grafted layer of poly(acrylic acid), and a propargyl derivative of CB7 was coupled to the surface by click chemistry. The amount of surface-bound CB7 was quantified with the high-affinity guest aminomethyladamantane (AMADA), which revealed CB7 surface coverage densities around 0.3 nmol cm-2 indicative of a 3D layer of CB7 binding sites on the surface. The potential for surface functionalization was demonstrated with an aminoadamantane-labeled rhodamine (Ada-Rho) as a second high-affinity guest. Simultaneous incubation of CB7-functionalized particles with both high-affinity guests, AMADA and Ada-Rho, revealed a simple linear relationship between the resulting surface coverage densities of the model fluorescent dye and the mole fraction of Ada-Rho in the incubation mixture. This suggests a highly modular supramolecular strategy for the stable immobilization of application-relevant molecules on particle surfaces and a precise control of their surface coverage densities.

A Label-Free Continuous Fluorescence-Based Assay for Monitoring Ornithine Decarboxylase Activity with a Synthetic Putrescine Receptor.

Polyamines play an important role in cell growth, differentiation, and cancer development, and the biosynthetic pathway of polyamines is established as a drug target for the treatment of parasitic diseases, neoplasia, and cancer chemoprevention. The key enzyme in polyamine biosynthesis is ornithine decarboxylase (ODC). We report herein an analytical method for the continuous fluorescence monitoring of ODC activity based on the supramolecular receptor cucurbit[6]uril (CB6) and the fluorescent dye trans-4-[4-(dimethylamino)styryl]-1-methylpyridinium iodide (DSMI). CB6 has a significantly higher binding constant to the ODC product putrescine (>107 M-1) than to the substrate L-ornithine (340 M-1). This enables real-time monitoring of the enzymatic reaction through a continuous fluorescence change caused by dye displacement from the macrocycle by the formed product, which allowed a straightforward determination of enzyme kinetic parameters ( kcat = 0.12 s-1 and KM = 24 µM) and inhibition constants of the two ODC inhibitors α-difluoromethylornithine (DFMO) and epigallocatechin gallate (EGCG). The potential for high-throughput screening (HTS) was demonstrated by excellent Z' factors (>0.9) in a microplate reader format, and the sensitivity of the assay is comparable to or better than most established complementary methods, which invariably have the disadvantage of not being compatible with direct implementation and upscaling to HTS format in the drug discovery process.