Detection of Intracellular Free Na+ Concentration of Synaptosomes by a Fluorescent Indicator, Na+-Binding Benzofuran Isophthalate: The Effect of Veratridine, Ouabain, and α-Latrotoxin

Abstract: A novel fluorescent Na⁺ indicator, Na⁺-binding benzofuran isophthalate (SBFI), was used to follow changes in the intracellular free Na⁺ concentration ([Na⁺]₁) of synaptosomes. The dye, when loaded into synapto- somes in the form of its acetoxymethyl ester, was responsive to changes of [Na⁺]₁. Calibration was made using the 340/380 nm excitation ratio when the cytoplasmic Na⁺ concentration was equilibrated with different concentrations of extracellular Na⁺ in the presence of 2 μ M gramicidin D. The basal value of [Na⁺]₁ in synaptosomes in the presence of 140 m M extracellular Na⁺ was found to be 10.9 ± 1.8 m M. Veratridine, which opens potential-dependent Na⁺ channels, caused a sudden increase in [Na⁺]₁ in a concentration-dependent manner (1 -20 μ M ), whereas the effect of ouabain (20 and 50 μ M ), the inhibitor of the plasma membrane Na⁺,K⁺-ATPase, was more gradual. The rise in the fluorescence intensity upon addition of veratridine was prevented completely by 2 μ M tetrodotoxin. α-Latrotoxin, the black widow spider toxin, caused an increase in the fluorescence intensity, which became evident 1 min after the addition of the toxin. The rate of increase was proportional to the concentration of the toxin (0.19–1.5 n M ). This report confirms our earlier finding demonstrating a Na⁺-dependent component in the action of α-Iatrotoxin, and shows that changes in [Na⁺]₁ in synaptosomes can be followed by SBFI.     

Green synthesis of enantiopure (S)‐1‐(benzofuran‐2‐yl)ethanol by whole‐cell biocatalyst

Optically active aromatic alcohols are valuable chiral building blocks of many natural products and chiral drugs. Lactobacillus paracasei BD87E6, which was isolated from a cereal‐based fermented beverage, was shown as a biocatalyst for the bioreduction of 1‐(benzofuran‐2‐yl) ethanone to (S)‐1‐(benzofuran‐2‐yl) ethanol with highly stereoselectivity. The bioreduction conditions were optimized using L. paracasei BD87E6 to obtain high enantiomeric excess (ee) and conversion. After optimization of the bioreduction conditions, it was shown that the bioreduction of 1‐(benzofuran‐2‐yl)ethanone was performed in mild reaction conditions. The asymmetric bioreduction of the 1‐(benzofuran‐2‐yl)ethanone had reached 92% yield with ee of higher than 99.9% at 6.73 g of substrate. Our study gave the first example for enantiopure production of (S)‐1‐(benzofuran‐2‐yl)ethanol by a biological green method. This process is also scalable and has potential in application. In this study, a basic and novel whole‐cell mediated biocatalytic method was performed for the enantiopure production of (S)‐1‐(benzofuran‐2‐yl)ethanol in the aqueous medium, which empowered the synthesis of a precious chiral intermediary process to be converted into a sophisticated molecule for drug production.     

Molecular dynamics simulation and binding free energy studies of novel leads belonging to the benzofuran class inhibitors of Mycobacterium tuberculosis Polyketide Synthase 13

In this work, the binding mechanism of new Polyketide Synthase 13 (Pks13) inhibitors has been studied through molecular dynamics simulation and free energy calculations. The drug Tam1 and its analogs, belonging to the benzofuran class, were submitted to 100 ns simulations, and according to the results obtained for root mean square deviation, all the simulations converged from approximately 30 ns. For the analysis of backbone flotation, the root mean square fluctuations were plotted for the Cα atoms; analysis revealed that the greatest fluctuation occurred in the residues that are part of the protein lid domain. The binding free energy value (ΔG_bind) obtained for the Tam16 lead molecule was of ?51.43 kcal/mol. When comparing this result with the ΔG_bind values for the remaining analogs, the drug Tam16 was found to be the highest ranked: this result is in agreement with the experimental results obtained by Aggarwal and collaborators, where it was verified that the IC₅₀ for Tam16 is the smallest necessary to inhibit the Pks13 (IC₅₀ = 0.19 μM). The energy decomposition analysis suggested that the residues which most interact with inhibitors are: Ser1636, Tyr1637, Asn1640, Ala1667, Phe1670, and Tyr1674, from which the greatest energy contribution to Phe1670 was particularly notable. For the lead molecule Tam16, a hydrogen bond with the hydroxyl of the phenol not observed in the other analogs induced a more stable molecular structure. Aggarwal and colleagues reported this hydrogen bonding as being responsible for the stability of the molecule, optimizing its physic-chemical, toxicological, and pharmacokinetic properties.     

Schistosomicidal Activity of Dihydrobenzofuran Neolignans

We have evaluated the antischistosomal activity of synthetic dihydrobenzofuran neolignans (DBNs) derived from (±)‐trans‐dehydrodicoumaric acid dimethyl ester ( 1 ) and (±)‐trans‐dehydrodiferulic acid dimethyl ester ( 2 ) against adult Schistosoma mansoni worms in vitro. Compound 4 ((±)‐trans‐4‐O‐acetyldehydrodiferulic acid dimethyl ester) displayed the most promising activity; at 200 μm , it kills 100 ± 0% of worms after 24 h, which resembles the result achieved with praziquantel (positive control) at 1.56 μm . The hydrogenation of the double bond between C7′ and C8′, the introduction of an additional methyl group at C3′, and a double bond between C7 and C8 decreased the schistosomicidal activity of DBNs. On the other hand, the presence of the acetoxy group at C4 played an interesting role in this activity. These results demonstrated the interesting schistosomicidal potential of DBNs, which could be further exploited.     

Molecular insights into substrate recognition and catalysis by phthalate dioxygenase from Comamonas testosteroni

Phthalate, a plasticizer, endocrine disruptor, and potential carcinogen, is degraded by a variety of bacteria. This degradation is initiated by phthalate dioxygenase (PDO), a Rieske oxygenase (RO) that catalyzes the dihydroxylation of phthalate to a dihydrodiol. PDO has long served as a model for understanding ROs despite a lack of structural data. Here we purified PDO_KF1 from Comamonas testosteroni KF1 and found that it had an apparent k_cat/K_m for phthalate of 0.58 ± 0.09 μM⁻¹s⁻¹, over 25-fold greater than for terephthalate. The crystal structure of the enzyme at 2.1 Å resolution revealed that it is a hexamer comprising two stacked α₃ trimers, a configuration not previously observed in RO crystal structures. We show that within each trimer, the protomers adopt a head-to-tail configuration typical of ROs. The stacking of the trimers is stabilized by two extended helices, which make the catalytic domain of PDO_KF1 larger than that of other characterized ROs. Complexes of PDO_KF1 with phthalate and terephthalate revealed that Arg207 and Arg244, two residues on one face of the active site, position these substrates for regiospecific hydroxylation. Consistent with their roles as determinants of substrate specificity, substitution of either residue with alanine yielded variants that did not detectably turnover phthalate. Together, these results provide critical insights into a pollutant-degrading enzyme that has served as a paradigm for ROs and facilitate the engineering of this enzyme for bioremediation and biocatalytic applications.     

A rearranged eudesmane and further verbesindiol derivatives from Verbesina eggersii

A reinvestigation of Verbesina eggersii gave in addition to compounds isolated previously two further verbesindiol derivatives, a rearranged eudesmane and a benzofuran related to tremetone.     

Neolignans from Aniba terminalis

A benzene extract of the trunk wood of Aniba terminalis (Lauraceae) contained besides benzyl benzoate, benzyl salicylate, d,1-camphor and sitosterol, (2S,3S,3aR)- and (2R,3S,3aS)-3a-allyl-5-methoxy-3-methyl-2-piperonyl-2,3,3a,6-tetrahydro-6-oxobenzofurans, which may be responsible, through sequential rearrangements of the Cope, retro-Claisen and Claisen types, and finally dehydrogenation, for the formation of the co-occurring (2S,3S,5S)- and (2R,3S,5R)-5-allyl-5-methoxy-3-methyl-2-piperonyl-2,3,5,6-tetrahydro-6-oxobenzofurans, the (2S,3S)-6-O-allyl-5-methoxy-3-methyl-2-piperonyl-2,3-dihydrobenzofuran, the (2S,3S)- and (2R,3S)-7-allyl-6-hydroxy-5-methoxy-3-methyl-2-piperonyl-2,3-dihydrobenzofuran and the 7-allyl-6-hydroxy-5-methoxy-3-methyl-2-piperonylbenzofuran.     

Highly selective and sensitive ‘on–off’ fluorescent chemosensor for Fe3+ ions crafted by benzofuran moiety in both experimental and theoretical methods

A benzofuran glycinamide-based chemosensor, 3-(2-([4-fluorobenzyl]amino)acetamido)benzofuran-2-carboxamide ( BGA ) was developed and synthesized for the selective and sensitive detection of Fe³⁺ ions. The photophysical properties of the probe BGA were studied using UV–visible light absorption and fluorescence spectrophotometers. The chemosensor BGA showed a marked ‘on–off’ fluorescence response towards Fe³⁺ ions in the presence of other metal ions in DMSO/H₂O solution (9/1, v/v). The very low limits of detection (LOD) were calculated to be 10 nM and 43 nM using UV–visible light absorption and fluorescence spectrophotometers, respectively. Job's plot analysis revealed the formation of a BGA -Fe³⁺ complex with a 1:1 binding stoichiometry ratio using UV–visible light spectroscopy. The sensing mechanism was also demonstrated using density functional theory calculation.     

Effect of chemical structure of benzofuran derivatives and reaction conditions on enantioselective properties of Aureobasidium pullulans microorganism contained in Boni Protect antifungal agent

Bioorganic asymmetric reduction of carbonyl compounds is one of the most important fundamental and practical reactions for producing chiral alcohols. The stereoselective bioreduction of prochiral ketones of benzofuran derivatives in the presence of yeast-like fungus Aureobasidium pullulans contained in the antifungal Boni Protect agent was studied. Biotransformations were carried out under moderate conditions in an aqueous and two-phase system and without multiplication of the bioreagent. Despite similar chemical structure, each of the used ketone has been reduced with varying efficiency and selectivity. One of the reasons for these results is the presence of a whole set of oxidoreductases in A. pullulans cells that are sensitive to the smallest changes in the structure of prochiral substrate. The unsymmetrical methyl ketones were biotransformed with the highest selectivity. Aureobasidium pullulans microorganism is less effective in the reduction of unsymmetrical halomethyl ketones. The presence of a heteroatom in the alkyl group significantly decreases the selectivity of the process. Finally, as a result of the preferred hydride ion transfer from the dihydropyridine ring of the cofactor to the carbonyl double bond on the re side, secondary alcohols of the S and R configuration were obtained with moderate to high enantioselectivity (55-99%).