Assessing the mechanism of facilitated proton transport across GUVs trapped in a microfluidic device.

Proton transport across lipid membranes is one of the most fundamental reactions that make up living organisms. In vitro, however, the study of proton transport reactions can be very challenging due to limitations imposed by proton concentrations, compartment size, and unstirred layers as well as buffer exchange and buffer capacity. In this study, we have developed a proton permeation assay based on the microfluidic trapping of giant vesicles enclosing the pH-sensitive dye pyranine to address some of these challenges. Time-resolved fluorescence imaging upon a rapid pH shift enabled us to investigate the facilitated H+ permeation mediated by either a channel or a carrier. Specifically, we compared the proton transport rates as a function of different proton gradients of the channel gramicidin D and the proton carrier carbonyl cyanide-m-chlorophenyl hydrazone. Our results demonstrate the efficacy of the assay in monitoring proton transport reactions and distinguishing between a channel-like and a carrier-like mechanism. This groundbreaking result enabled us to elucidate the enigmatic mode of the proton permeation mechanism of the recently discovered natural fibupeptide lugdunin.

Semisynthetic derivatives of the fungal metabolite eupenifeldin via targeting the tropolone hydroxy groups.

Eupenifeldin (1) is a fungal secondary metabolite possessing bis-tropolone moieties that demonstrates nanomolar cytotoxic activity against a number of cancer cell types. As a potential anticancer lead, this meroterpenoid was used to access 29 semisynthetic analogues via functionalization of the reactive hydroxy groups of the bis-tropolones. A series of ester (2-6), carbonate (7-8), sulfonate (9-16), carbamate (17-20), and ether (21-30) analogues of 1 were generated via 22 reactions. Most of these compounds were disubstituted, produced via functionalization of both of the tropolonic hydroxy moieties, although three mono-functionalized analogues (6, 8, and 24) and one tri-functionalized analogue (3) were also obtained. The cytotoxic activities of 1-30 were evaluated against human melanoma and ovarian cancer cell lines (i.e., MDA-MB-435 and OVCAR3, respectively). Ester and carbonate analogues of 1 (i.e., 2-8) maintained cytotoxicity at the nanomolar level, and the greatest improvement in aqueous solubility came from the monosuccinate analogue (6), which was acylated on the secondary hydroxy at the 11 position.

8-acetoxy-trisulfopyrene as the first activatable fluorogenic probe for add-and-read assessment of Organic anion-transporting polypeptides, OATP1B1, OATP1B3, and OATP2B1.

Organic anion-transporting polypeptides, OATP1B1, OATP1B3, and OATP2B1 are multispecific membrane proteins mediating the hepatocellular uptake of structurally diverse endo- and exogenous compounds, including various kinds of drugs. Co-administration of OATP1B/2B1 substrates may lead to altered pharmacokinetics or even toxicity. Therefore, the study of the interaction with these OATPs is essential in drug development and is recommended by international regulatory agencies, the FDA, EMA, and PMDA. In general, radiolabeled indicators are used to measure drug interactions of OATPs, and, lately, fluorescent probes are also gaining wider application in OATP tests. However, all of the currently available methods (either radioactive or fluorescence-based) comprise multiple steps, including the removal of the indicator in the end of the experiment. Hence, they are not ideally suited for high-throughput screening. In the current study, in order to find an indicator allowing real-time assessment of hepatic OATP function, we searched for an activatable fluorogenic OATP substrate. Here, we show that 8-acetoxypyrene-1,3,6-trisulfonate (Ace), a fluorogenic derivative of the hepatic OATP substrate pyranine (8-hydroxypyrene-1,3,6-trisulfonate) enters the cells via OATP1B1/3 or OATP2B1 function. In living cells, Ace is then converted into highly fluorescent pyranine, allowing "no-wash" measurement of OATP function and drug interactions. Furthermore, we demonstrate that Ace can be used in an indirect assay termed as competitive counterflow suitable to distinguish between transported substrates and inhibitors of OATP1B1. The fluorescence-based methods described here are unique and open the way toward high-throughput screening of interactions between new molecular entities and OATPs.

Spatiotemporal Control of Supramolecular Polymerization and Gelation of Metal-Organic Polyhedra.

In coordination-based supramolecular materials such as metallogels, simultaneous temporal and spatial control of their assembly remains challenging. Here, we demonstrate that the combination of light with acids as stimuli allows for the spatiotemporal control over the architectures, mechanical properties, and shape of porous soft materials based on metal-organic polyhedra (MOPs). First, we show that the formation of a colloidal gel network from a preformed kinetically trapped MOP solution can be triggered upon addition of trifluoroacetic acid (TFA) and that acid concentration determines the reaction kinetics. As determined by time-resolved dynamic light scattering, UV-vis absorption, and 1H NMR spectroscopies and rheology measurements, the consequences of the increase in acid concentration are (i) an increase in the cross-linking between MOPs; (ii) a growth in the size of the colloidal particles forming the gel network; (iii) an increase in the density of the colloidal network; and (iv) a decrease in the ductility and stiffness of the resulting gel. We then demonstrate that irradiation of a dispersed photoacid generator, pyranine, allows the spatiotemporal control of the gel formation by locally triggering the self-assembly process. Using this methodology, we show that the gel can be patterned into a desired shape. Such precise positioning of the assembled structures, combined with the stable and permanent porosity of MOPs, could allow their integration into devices for applications such as sensing, separation, catalysis, or drug release.

Discovery of pyrazole N-aryl sulfonate: A novel and highly potent cyclooxygenase-2 (COX-2) selective inhibitors.

Based on a new pyrazole sulfonate synthetic method, a novel class of molecules with a basic structure of pyrazole N-aryl sulfonate have been designed and synthesized. The interest in conducting intensive research stems from quite evident anti-inflammatory effects exhibited by the compounds in preliminary animal experiments. A series of compounds were synthesized by different substitutions of the R1, R2, and R3 groups. Within the series, 4-iodophenyl 5-methyl-3-(p-tolyl)-1H-pyrazole-1-sulfonate and phenyl 5-methyl-3-(4-(trifluoromethyl) phenyl)-1H-pyrazole-1-sulfonate exhibited excellent anti-inflammatory activity (% inhibition of auricular edemas = 27.0 and 35.9, respectively); the in vivo analgesic activity of phenyl 5-methyl-3-(p-tolyl)-1H-pyrazole-1-sulfonate and 2-chlorophenyl 5-methyl-3-(p-tolyl)-1H-pyrazole-1-sulfonate was confirmed to be effective (inhibition ratio of writhing = 50.7% and 48.5% separately), and compounds phenyl 5-methyl-3-(p-tolyl)-1H-pyrazole-1-sulfonate , 4-iodophenyl 5-methyl-3-(p-tolyl)-1H-pyrazole-1-sulfonate and 2-chlorophenyl 5-methyl-3-(p-tolyl)-1H-pyrazole-1-sulfonate were identified as selective COX-2 inhibitors (SI = 455, 10,497 and >189 severally). In Acute Oral Toxicity assays conducted in vivo, the lethal dose 50 (LD50) of 4-iodophenyl 5-methyl-3-(p-tolyl)-1H-pyrazole-1-sulfonate and 2-chlorophenyl 5-methyl-3-(p-tolyl)-1H-pyrazole-1-sulfonate to mice was >2000 mg/kg BW.

Influence of molecular rebinding on the reaction rate of complex formation.

We simulated Brownian diffusion and reaction-diffusion processes to study the influence of molecular rebinding on the reaction rates of bimolecular reactions. We found that the number of rebinding events, Nreb, is proportional to the target's size and inversely proportional to the diffusion coefficient D and simulation time-step Δt. We found the proportionality constant close to π-1/2. We confirmed that Nreb is defined as a ratio of the activation-limited rate constant ka to the diffusion-limited rate constant, kD. We provide the formula describing the reactivity coefficient κ, modelling the transient-native complex transition for the activation-controlled reaction rates. We show that κ is proportional to (D/Δt)1/2. Finally, we apply our rebinding-including reaction rate model to the real reactions of photoacid dissociation and protein association. Based on literature data for both types of reactions, we found the Δt time-scale. We show that for the photodissociation of a proton, the Δt is equal to 171 ± 18 fs and the average number of rebinding events is approximately equal to 40. For proteins, Δt is of the order of 100 ps with around 20 rebinding events. In both cases the timescale is similar to the timescale of fluctuation of the solvent molecules surrounding the reactants; vibrations and bending in the case of photoacid dissociation and diffusional motion for proteins.

Pd(NHC)(cinnamyl)Cl-catalyzed Suzuki cross-coupling reaction of aryl sulfonates with arylboronic acids.

A series of N-heterocyclic carbene (NHC)-palladium catalysts have been synthesized and applied to catalyze the Suzuki coupling reaction efficiently between aryl sulfonates and arylboronic acids with the potassium phosphate heptahydrate as a base. The desired yields are obtained even with less reactive aryl tosylates or aryl mesylates as substrates. This method was applied successfully to the synthesis of the (R)-2-(t-butoxycarbonylamino)-3-(biphenyl-4-yl)-propan-1-ol which is the key intermediate of sacubitril, a component of the newly approved antihypertensive drug "Entresto."

Fluorescence Enhancement by Calixarene Supramolecular Aggregate.

We herein constructed supramolecular assemblies from guanidinocalixarenes and sulfonatocalixarenes by exploiting multiple salt bridge interactions. They encapsulate six different kinds of fluorescent dyes (both cationic and anionic), leading to a fluorescence enhancement that could not be achieved by either single calixarene. As such, this study advances the research on high-performance fluorophores.

Biological effects of the free and embedded metribuzin and tribenuron-methyl herbicides on various cultivated weed species.

The present study addresses the herbicidal activity and biological effects of the metribuzin (MET) and tribenuron-methyl (TBM) herbicides used to control various weed species (Amaranthus retroflexus, Sinapis arvensis, and Leucanthemum maximum). The effects of the free herbicides and the herbicides embedded in granules of degradable polymer poly-3-hydroxybutyrate [P(3HB)] blended with birch wood flour were compared. Metribuzin, regardless of the form, caused 100% mortality of the three weeds by day 21. The herbicidal activity of tribenuron-methyl was lower than that of metribuzin, but the embedded TBM was superior to the free herbicide in the length and strength of its action on the weeds. Both metribuzin forms dramatically decreased the main parameters of fluorescence: maximum quantum yield of photosystem-II [Y(II)max], maximum quantum yield of non-photochemical quenching [Y(NPQ)max], and maximum rate of non-cyclic electron transport [ETRmax] and concentrations of chlorophyll a and b. The effect of the embedded TBM on the photosynthetic activity of the weeds was lower in the first two weeks of the growth of herbicide-treated plants but lasted longer than the effect of the free TBM and increased over time. Embedding of metribuzin in the matrix of degradable blend did not decrease its herbicidal activity.

Divergent Total Synthesis of Chaetoglines C to F.

The first total syntheses of chaetoglines C-F via a bioinspired and divergent synthetic strategy are reported. Chaetolines C and D were obtained from the condensation of hemiacetal and tryptophan methyl ester building blocks followed by functional group transformations. The synthesis of chaetogline E employed the diastereoselective Pictet-Spengler reaction, and the tetrahydro-carboline skeleton was further utilized as a precursor for an oxidative aromatization reaction to introduce the ß-carboline moiety of chaetogline F.

Pyranine-Modified Amphiphilic Polymer Conetworks as Fluorescent Ratiometric pH Sensors.

The fluorescent dye 8-hydroxypyrene-1,3,6-trisulfonate (pyranine) combines high photostability with ratiometric pH detection in the physiological range, making it a prime candidate for optical sensors in biomedical applications, such as pH-based chronic wound monitoring. However, pyranine's high water solubility and the difficulty of covalent attachment pose severe limitations in terms of leaching from sensor matrices. Herein, pyranine-modified nanophase-separated amphiphilic polymer conetworks (APCNs) are reported as fluorescent ratiometric pH sensors. The thin, freestanding APCN membranes composed of one hydrophilic and one hydrophobic polymer provide an optically transparent, flexible, and stable ideal matrix that enables contact between dye and aqueous environment. An active ester-based conjugation approach results in a highly homogeneous and stable pyranine modification of the APCN's hydrophilic phase. This concept effectively solves the leaching challenge for pyranine without compromising its functionality, which is demonstrated by ratiometric pH detection in the range of pH 5-9.

Aryl sulfonate based anticancer alkylating agents.

This research work revolves around synthesis of antineoplastic alkylating sulfonate esters with dual alkylating sites for crosslinking of the DNA strands. These molecules were evaluated as potential antineoplastic cross linking alkylating agents by reaction with the nucleoside of Guanine DNA nucleobase at both ends of the synthesized molecule. Synthesis of the alkylating molecules and the crosslinking with the guanosine nucleoside was monitored by MALDITOF mass spectroscopy. The synthesized molecule's crosslinking or adduct forming rate with the nucleoside was compared with that of 1,4 butane disulfonate (busulfan), in form of time taken for the appearance of [M+H]+. It was found that aryl sulfonate leaving group was causing higher rate of nucleophilic attack by the Lewis basic site of the nucleobase. Furthermore, the rate was also found to be a function of electron withdrawing or donating nature of the substituent on the aryl ring. Compound with strong electron withdrawing substituent on the para position of the ring reacted fastest. Hence, new alkylating agents were synthesized with optimized or desired reactivity.

Palladium Catalyzed Arylation and Benzylation of Nitroarenes Using Aryl Sulfonates and Benzyl Acetates.

Pd-catalyzed arylation or benzylation of nitroazoles using aryl sulfonates or benzyl acetates is described. Electronically varied aryl tosylates and mesylates, as well as benzyl acetates, afford the arylated and benzylated products. Arylation of nitrobenzene is also reported. The relative rate for the arylation of halides is greater than that of tosylates using the reported reaction parameters. These studies enhance the scope of electrophiles for nitroarene arylations and benzylations, which was hitherto limited to the use of halide electrophiles.

A sialosyl sulfonate as a potent inhibitor of influenza virus replication.

A new direction for influenza virus sialidase inhibitor development was identified using a sulfonate congener of 2-deoxy-2-ß-H N-acetylneuraminic acid. Sialosyl sulfonates can be synthesised efficiently in four steps from N-acetylneuraminic acid via a microwave assisted decarboxylation. The presence of the sulfonate group significantly increases inhibition of influenza virus sialidase and viral infection when compared to the carboxylate congener, and also to the benchmark sialidase inhibitor 2,3-dehydro-2-deoxy-N-acetylneuraminic acid, Neu5Ac2en.

Herbicidal activity of slow-release herbicide formulations in wheat stands infested by weeds.

The present study reports the herbicidal activity of metribuzin and tribenuron-methyl embedded in the degradable matrix of natural poly-3-hydroxybutyrate [P(3HB)/MET and P(3HB)/TBM]. The developed formulations were constructed as films and microgranules, which were tested against the weeds such as white sweet clover Melilotus albus and lamb's quarters Chenopodium album in the presence of soft spring wheat (Triticum aestivum, cv. Altaiskaya 70) as the subject crop for investigation. The activity was measured in laboratory scale experiments by determining the density and weight of the vegetative organs of weeds. The study was also aimed at testing the effect of the experimental formulation on the growth of wheat crop as dependent on the method of herbicide delivery. The experimental MET and TBM formulations showed pronounced herbicidal activity against the weed species used in the study. The effectiveness of the experimental formulations in inhibiting weed growth was comparable to and, sometimes, higher than that of the commercial formulations (positive control). The amount of the biomass of the wheat treated with the experimental herbicide formulations was significantly greater than that of the wheat treated with commercial formulations.

Reversible Stabilization of Vesicles: Redox-Responsive Polymer Nanocontainers for Intracellular Delivery.

We present the self-assembly of redox-responsive polymer nanocontainers comprising a cyclodextrin vesicle core and a thin reductively cleavable polymer shell anchored via host-guest recognition on the vesicle surface. The nanocontainers are of uniform size, show high stability, and selectively respond to a mild reductive trigger as revealed by dynamic light scattering, transmission electron microscopy, atomic force microscopy, a quantitative thiol assay, and fluorescence spectroscopy. Live cell imaging experiments demonstrate a specific redox-responsive release and cytoplasmic delivery of encapsulated hydrophilic payloads, such as the pH-probe pyranine, and the fungal toxin phalloidin. Our results show the high potential of these stimulus-responsive nanocontainers for cell biological applications requiring a controlled delivery.

Weak C-H acids as protonophores can carry hydrogen ions through lipid membranes and mitochondria: a case of o-carborane.

ortho-Carborane (1,2-C2B10H12) was found to be a carrier of protons in both mitochondrial and artificial lipid membranes, suggesting that this dicarborane can reversibly release hydrogen ions and diffuse through the membranes in neutral and anionic forms. Similar to conventional uncouplers (e.g. 2,4-dinitrophenol), o-carborane stimulated mitochondrial respiration and decreased the membrane potential at concentrations of tens of micromoles. Protonophoric activity of o-carborane was observed both by a fluorometric assay using pyranine-loaded liposomes and electrical current measurements across planar lipid bilayers. Substantial contribution of the proton flux to the o-carborane-mediated current was proved by a shift of the zero current voltage upon imposing a pH gradient across the membrane. Meta-carborane (1,7-C2B10H12) lacked the protonophoric activity in line with its reduced C-H acidity. The results suggest that weak C-H acids can exhibit protonophoric activity in the biological environment. The finding of a new class of protonophoric compounds is of substantial interest due to promising anti-obesity and anti-diabetic properties of uncouplers.

Photoinduced electron transfer reactions of pyranine with benzoquinone and titanium dioxide.

The fluorescence quenching of pyranine by benzoquinone in acetonitrile medium was studied using steady-state and time-resolved fluorescence techniques. The quenching process was characterized by a Stern-Volmer plot, which displayed a linear aspect. From the linear plot, the bimolecular quenching rate constant was obtained. The obtained rate constants are within diffused controlled limits. The results show that benzoquinone can efficiently quench the fluorescence of pyranine with dynamic quenching rate constants in the order of 1010 M-1 s-1 , suggesting that the pyranine can act as a good electron donor for photoinduced electron transfer in artificial photosynthesis and organic solar cells. In addition, the electron injection dynamics of a pyranine/titanium dioxide semiconductor film was also investigated and electron injection from the excited state pyranine into the conduction band of titanium dioxide is suggested. These preliminary results hold promise for the possibility of using pyranine in dye-sensitized solar cells. Copyright © 2016 John Wiley & Sons, Ltd.

Extended N-Arylsulfonylindoles as 5-HT6 Receptor Antagonists: Design, Synthesis & Biological Evaluation.

Based on a known pharmacophore model for 5-HT6 receptor antagonists, a series of novel extended derivatives of the N-arylsulfonyindole scaffold were designed and identified as a new class of 5-HT6 receptor modulators. Eight of the compounds exhibited moderate to high binding affinities and displayed antagonist profile in 5-HT6 receptor functional assays. Compounds 2-(4-(2-methoxyphenyl)piperazin-1-yl)-1-(1-tosyl-1H-indol-3-yl)ethanol (4b), 1-(1-(4-iodophenylsulfonyl)-1H-indol-3-yl)-2-(4-(2-methoxyphenyl)piperazin-1-yl)ethanol (4g) and 2-(4-(2-methoxyphenyl)piperazin-1-yl)-1-(1-(naphthalen-1-ylsulfonyl)-1H-indol-3-yl)ethanol (4j) showed the best binding affinity (4b pKi = 7.87; 4g pKi = 7.73; 4j pKi = 7.83). Additionally, compound 4j was identified as a highly potent antagonist (IC50 = 32 nM) in calcium mobilisation functional assay.

Steroidal Surfactants: Detection of Premicellar Aggregation, Secondary Aggregation Changes in Micelles, and Hosting of a Highly Charged Negative Substance.

CHAPSO and CHAPS are zwitterionic surfactants derived from bile salts which are usually employed in protein purification and for the preparation of liposomes and bicelles. Despite their spread use, there are significant discrepancies on the critical concentrations that determine their aggregation behavior. In this work, we study the interaction between these surfactants with the negative fluorescent dye pyranine (HPTS) by absorbance, fluorescence, and infrared spectrometry to establish their concentration-dependent aggregation. For the studied surfactants, we detect three critical concentrations showing their concentration-dependent presence as a monomeric form, premicellar aggregates, micelles, and a second type of micelle in aqueous medium. The nature of the interaction of HPTS with the surfactants was studied using analogues of their tails and the negative bile salt taurocholate (TC) as reference for the sterol ring. The results indicate that the chemical groups involved are the hydroxyl groups of the polar face of the sterol ring and the sulfonate groups of the dye. This interaction causes not only the incorporation of the negative dye in CHAPSO and CHAPS micelles but also its association with their premicellar aggregates. Surprisingly, this hosting behavior for a negative charged molecule was also detected for the negative bile salt TC, bypassing, in this way, the electrostatic repulsion between the guest and the host.