How do amphiphiles form ion-conducting channels in membranes? Lessons from linear oligoesters.

The X-ray crystal structures of biological ion channels are exquisitely complex, but not all natural products capable of forming ion-conducting channels are equally elaborate. Examples such as the peptides gramicidin or alamethicin or the polyene antibiotics amphotericin and nystatin clearly form well-defined channels without requiring a massive protein superstructure. These molecules form the starting point for a supramolecular chemistry challenge: how to create synthetic compounds and systems that catalyze the translocation of ionic species across bilayer membranes mimicking naturally occurring channels. Over the past three decades, supramolecular chemists have developed numerous examples of systems with transport rates and efficiencies that rival natural channels. As the field developed, researchers discovered many compounds that are functional for ion transport but bear very little resemblance to any imagined architectures of ion channels. We and others have followed these lead compounds extensively in a quest to focus on the mechanisms such simple compounds use to achieve their function. These compounds show all the hallmarks of ion channels including high activity, ion specificity, regular time-dependent conductance changes, and in some cases higher-order phenomena such as voltage-dependent activity. In this Account, we summarize experimental evidence derived from an extensive class of oligoester bolaamphiphiles that illustrates how amphiphilic molecules can form ion-conducting channels in membranes. Examination of increasingly simple compounds over the past two decades has shifted the focus away from biological paradigms towards alternative modes for transmembrane ion transport. We have developed new tools to move beyond simple on-off channel openings to complex bursts of high activity. From the perspective of flux, the highly conducting bursts clearly move ions more efficiently than simple on-off openings. High and sustained conductance, whatever its structural origin, has direct applications in amplification of chemical signals or membrane-disrupting biological activity. These results ensure that simple transporters will continue to fascinate and puzzle for a long time to come.

Chemical-genetic identification of the biochemical targets of polyalkyl guanidinium biocides.

Alkylated guanidinium compounds exhibit microbiocidal activity in marine environments, yet the mode of action of these compounds has not been defined. A comprehensive chemical-genetic approach in budding yeast was used to define the biological processes affected by these compounds. N-Butyl-N'-decylguanidinium and N-hexyl-N'-(3-hydroxypropyl)-N''-octylguanidinium chlorides were shown to prevent yeast growth in a dose-dependent manner. All non-essential genes required for tolerance of sub-lethal amounts of these biocides were identified. These unbiased and systematic screens reveal the two related guanidinium compounds have a non-overlapping spectrum of targets in vivo. A functional tryptophan biosynthetic pathway is essential for tolerance of both biocides, which identifies tryptophan amino acid import as one process affected by these compounds. Further analysis of hypersensitive gene lists demonstrates that the substitutions on alkylated guanidiums confer important functional differences in vivo: one derivative renders the ability to generate acidic vacuoles essential, while the other is synthetically lethal with mutants in the transcriptional response to chemical stress. Altogether the results define the common and distinct biological processes affected by biocidal alkylated guanidinium salts.

Ionic conductance of synthetic channels: analysis, lessons, and recommendations.

Synthetic ion channels have been known for nearly three decades, but it is only in the past decade that analysis of the currents these ionic conductors carry has become a standard technique. A broad range of structural types have been explored and these reports have produced a very diverse collection of ion channel conductance behaviours. In this critical review we describe a notational method to extract salient information from reported ion channel experiments. We use an activity grid to represent quantitative information on conductance and opening duration with a five-level colour code to represent qualitative information on the nature of the conductance-time profile. Analysis of the cumulative dataset suggests that the reported conductance data can reflect the structural features of the compounds prepared, but does also reflect the energetic landscape of the bilayer membrane in which synthetic ion channels function (143 references).

Mechanism of ion transport by fluorescent oligoester channels.

The synthesis and membrane activity of a suite of linear oligoesters containing a common diphenylacetylene unit core and differing in the hydroxyl terminus are reported. Active compounds formed high-conductance channels efficiently in both vesicle and planar bilayers, with one compound showing a very unusual slow loss of transport activity over a 20-30 min period. Steady-state and time-resolved fluorescence studies establish the rapid partition of active compounds to the bilayer and identify at least three types of membrane-associated species by their differing fluorescence lifetimes. The change in the distribution of species is correlated with the slow loss of activity. The results are interpreted in terms of an aggregate within a single bilayer leaflet that is nonetheless competent to transport ionic species through the bilayer. The properties of such structures, revealed by these compounds, appear to be consistent with commonly observed behaviors of other synthetic ion channels.

Testing specificity and sensitivity of wastewater-based epidemiology for detecting SARS-CoV-2 in four communities on Vancouver Island, Canada.

We report wastewater surveillance of the spread of SARS-CoV-2 based upon 24-h composite influent samples taken weekly from four wastewater treatment plants (WWTP) on Vancouver Island, BC, Canada between January 3, 2021 and July 10, 2021. Samples were analyzed by reverse transcription quantitative polymerase chain reaction targeting the N1 and N2 gene fragments of SARS-CoV-2 and a region of the replication associate protein of the pepper mottle mosaic virus (PMMoV) serving as endemic control. Only a small proportion of samples had quantifiable levels of N1 or N2. Overall case rates are weakly correlated with the concentration (gene copies/L) and with the flux of viral material influent to the WWTP (gene copies/day); the latter accounts for influent flow variations. Poisson multimodal rank correlation accounts for differences between the four WWTP and shows a significant correlation with a significant positive intercept. Receiver operator characteristics (ROC) analysis confirms a cut-off of cases based on amplified/not-amplified experimental data. At the optimal cut point of 19 (N1) or 17 (N2) cases/week/100,000 the sensitivity and specificity is about 75% for N1 and 67% for N2.

Synthesis and ion transport activity of oligoesters containing an environment-sensitive fluorophore.

A series of oligoesters based on a rigid triphenyl-diyne core is described. The molecules were readily synthesized from key intermediates, and retained good solubility properties. One of the compounds displayed modest ion transport activity in vesicles, was capable of forming highly conducting single channels in planar bilayers and exhibited an irregular non-linear current-voltage response. All the reported molecules had minimal aqueous fluorescence while being highly fluorescent in less-polar media including lipid vesicles; their partitioning into the membrane could be monitored by a significant blue-shift and increase in fluorescence intensity, as well as a decreased extent of quenching in vesicles over that in water. The combined data indicated that the compounds are highly aggregated in aqueous solution, which limits their membrane partitioning and ion transport activity, in agreement with mechanisms proposed for other 'simple' oligoester channels.

Planar-bilayer activities of linear oligoester bolaamphiphiles.

Voltage-clamp experiments of eight oligoester bolaamphiphiles in two subclasses are described. Syntheses of three new terephthalate-based compounds were achieved in three linear steps. Together with five previously described, related compounds, the ion transport activity was assessed by means of the voltage-clamp technique. All of the compounds show multiple types of conductance behavior in planar bilayers, a subset of which was exponentially voltage-dependent. The varied and irregular activities were summarized with the aid of a recently developed "activity-grid" method.

Synthesis, transport activity, membrane localization, and dynamics of oligoester ion channels containing diphenylacetylene units.

Four new linear oligoesters containing a diphenylacetylene unit were prepared by fragment coupling sequences and the ion channel forming ability of the compounds was investigated. Activity in vesicles was very strongly controlled by overall length; the longest compound was effectively inactive. Planar bilayer studies established that all compounds are able to form channels, but that regular step changes in conductance depend on the location of the diphenylacetylene unit within the oligoester and on the electrolyte. The intrinsic fluorescence of the diphenylacetylene unit was used to probe aggregation and membrane localization. Both monomer (320 nm) and excimer (380 nm) emissions are quenched by copper ions; quenching of the excimer emission from an aqueous aggregate is very efficient. Time-dependent changes in the intensities of monomer and excimer emission show slow transfer of diphenylacetylene units from an aqueous aggregate to a membrane-bound monomer with subsequent growth of emission from a membrane-bound excimer. The latter species is not quenched by aqueous copper ions. The implications of these species and processes for the mechanism of ion channel formation by simple oligoesters are discussed.

Fluorescent boron bis(phenolate) with association response to chloride and dissociation response to fluoride.

Addition of chloride ions to boron bis(phenolate) 5 in dichloromethane solution produces a selective fluorescence decrease. The fluorescence change is believed to be caused by associative hydrogen bonding between the chloride ion and two boronic acid groups. While addition of fluoride ions to bis(phenolate) 5 generates a purple colorimetric response, the colorimetric response is caused by fluoride induced B-O bond cleavage and air oxidation of the phenolate anion formed by this dissociation.

A ditopic fluorescent sensor for potassium fluoride.

The addition of potassium fluoride 'switches on' the fluorescence of sensors and while potassium chloride and bromide cause no fluorescence change; the fluorescence can be 'switched off' by removing the potassium cation from the benzocrown ether receptors of sensors and through the addition of [2.2.2]-cryptand and restored by the addition of the potassium cation as potassium chloride.

Synthesis and structural characterisation of the first bis(bora)calixarene: a selective, bidentate, fluorescent fluoride sensor.

A bis(bora)calixarene 3, the first lower-rim boron derivatised calixarene to be structurally characterised, is synthesised by the reaction of PhBCl2 with 4-tert-butylcalix[4]arene, and is demonstrated to be a sensitive and selective fluorescent fluoride sensor.

Aspects of nonviral gene therapy: correlation of molecular parameters with lipoplex structure and transfection efficacy in pyridinium-based cationic lipids.

This study seeks correlations between the molecular structures of cationic and neutral lipids, the lipid phase behavior of the mixed-lipid lipoplexes they form with plasmid DNA, and the transfection efficacy of the lipoplexes. Synthetic cationic pyridinium lipids were co-formulated (1:1) with the cationic lipid 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (EPC), and these lipids were co-formulated (3:2) with the neutral lipids 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) or cholesterol. All lipoplex formulations exhibited plasmid DNA binding and a level of protection from DNase I degradation. Composition-dependent transfection (beta-galactosidase and GFP) and cytotoxicity was observed in Chinese hamster ovarian-K1 cells. The most active formulations containing the pyridinium lipids were less cytotoxic but of comparable activity to a Lipofectamine 2000™ control. Molecular structure parameters and partition coefficients were calculated for all lipids using fragment additive methods. The derived shape parameter values correctly correlated with observed hexagonal lipid phase behavior of lipoplexes as derived from small-angle X-ray scattering experiments. A transfection index applicable to hexagonal phase lipoplexes derived from calculated parameters of the lipid mixture (partition coefficient, shape parameter, lipoplex packing) produced a direct correlation with transfection efficiency.

Geometric attributes of retaining glycosyltransferase enzymes favor an orthogonal mechanism.

Retaining glycosyltransferase enzymes retain the stereochemistry of the donor glycosidic linkage after transfer to an acceptor molecule. The mechanism these enzymes utilize to achieve retention of the anomeric stereochemistry has been a matter of much debate. Re-analysis of previously released structural data from retaining and inverting glycosyltransferases allows competing mechanistic proposals to be evaluated. The binding of metal-nucleotide-sugars between inverting and retaining enzymes is conformationally unique and requires the donor substrate to occupy two different orientations in the two types of glycosyltransferases. The available structures of retaining glycosyltransferases lack appropriately positioned enzymatic dipolar residues to initiate or stabilize the intermediates of a dissociative mechanism. Further, available structures show that the acceptor nucleophile and anomeric carbon of the donor sugar are in close proximity. Structural features support orthogonal (front-side) attack from a position lying ≤ 90° from the C1-O phosphate bond for retaining enzymes. These structural conclusions are consistent with the geometric conclusions of recent kinetic and computational studies.

Carrier-mediated electrodialysis.

Supported liquid membranes containing valinomycin or a calix[4]arene carrier can support electrodialysis under an imposed transmembrane potential. Under optimal conditions both transmembrane flux and carrier-based cation selectivity are enhanced relative to simple dialysis mediated by the same carriers.

Apparent fractal distribution of open durations in cyclodextrin-based ion channels.

Cyclodextrin-based ion channels are readily prepared via a "click" reaction and exhibit a range of membrane activities consistent with the formation of ion channels in planar bilayer membranes. The durations of irregular and transient conductance events appear to follow a power-law scaling.

Carbohydrate sensing using a fluorescent molecular tweezer.

A fluorescent molecular tweezer for carbohydrates has been prepared which utilises two boronic acid receptor groups.

Synthetic ion channels in bilayer membranes.

Natural ion channels are large protein complexes that regulate key functions of cells. Supramolecular chemists have been able to take hints from Nature to design and prepare completely synthetic ion channel systems that reproduce many of the fundamental functions of natural channels. This tutorial review introduces the field to non-specialists. It examines the design, synthesis, incorporation, and characterization of synthetic ion channels in bilayer membranes, and points to potential applications of synthetic ion channels.

Simple bis-thiocarbono-hydrazones as sensitive, selective, colorimetric, and switch-on fluorescent chemosensors for fluoride anions.

Bis-thiocarbono-hydrazones are found to be a class of sensitive, selective, ratiometric, and colorimetric chemosensors for anions such as fluoride (F(-)) or acetate (Ac(-)). The sensitivities, or the binding constants of the sensors with anions, were found to be strongly dependent on the substituents appended on the pi-conjugation framework, the delocalization bridge CH==N, the aromatic moiety, and the hetero atom in the C==X group (X=O, S) of the sensors. Single-crystal structures and (1)H NMR titration analysis shows that the --CH==N-- moiety is a hydrogen-bond donor, and it is proposed that an additional CHF hydrogen bond is formed for the sensors in the presence F(-). A sensor bearing anthracenyl groups is demonstrated as a switch-on fluorescent chemosensor for F(-) and Ac(-). The recognition of F(-) in acetonitrile (MeCN) by a sensor with nitrophenyl substituents is tolerant to MeOH (MeCN/MeOH=10:1, v/v) and water (MeCN/H(2)O=30:1, v/v); at these solvent ratios the absorption intensity of the sensor-F(-) complex solution at maximal absorption wavelength was attenuated to half of the original value in pure MeCN.

Solid-phase synthesis of oligoester ion channels.

A simple synthesis of oligoesters from TBDMS-protected-beta- and THP-protected-omega-hydroxycarboxylic acids using a solid-phase synthesis protocol is reported. Procedures were optimized for the efficient production of ion channel candidates in high purity with minimal purification. The product oligoesters were evaluated for ion transport activity using a fluorescent dye/vesicle assay. Oligoesters produced by this strategy show structure-dependent activity; the most active compounds are closely comparable to previously known oligoesters, but are available at a fraction of the synthetic effort.