Hierarchical Zeolites Prepared Using a Surfactant-Mediated Strategy: ZSM-5 vs. Y as Catalysts for Friedel-Crafts Acylation Reaction.

Hierarchical ZSM5 and Y zeolites were prepared through a surfactant-mediated strategy with NH4OH changing the duration of the treatment and the amount of CTAB surfactant and taking as reference multiples of the critical micellar concentration (CMC). The materials were characterized using powder X-ray diffraction, N2 adsorption isotherms at -196 °C, and SEM and TEM microscopy. The catalytic performance was evaluated in Friedel-Crafts acylation of furan with acetic anhydride at 80 °C. The alkaline surfactant-mediated treatment had different effects on the two zeolites. For ZSM5, the CTAB molecular aggregates can hardly diffuse inside the medium-size pores, leading mainly to intercrystalline mesoporosity and increased external surface area, with no positive catalytic impact. On the other hand, for large-pore Y zeolite, the CTAB molecular aggregates can easily diffuse and promote the rearrangement of crystal units around micelles, causing the enlargement of the pores, i.e., intracrystalline porosity. The optimized Y-based sample, treated for 12 h with a CTAB amount 32 times the CMC, shows an increase in product yield and rate constant that was not observed when a higher amount of surfactant was added. The reuse of spent catalysts upon thermal treatment at 400 °C shows a regeneration efficiency around 90%, showing good potentialities for the modified catalysts.

Examination of the Butler Equation for the Surface Tension of Liquid Mixtures.

The classical Butler equation used to describe surface tension and the surface composition of liquid mixtures is revisited. A straightforward derivation is presented, separating basic chemical thermodynamics and assumptions proper to Butler's model. This model is shown to conceal an approximation not recognized by other researchers. The shortcoming identified consists of not allowing surface standard values to vary with surface tension by virtue of the changing composition. A more rigorous equation is derived and shown to yield the Butler equation in case of incompressible surface phases. It is concluded that the Butler equation slightly overestimates ideal surface tensions. Butler's surface-phase concentrations of the surface-active component are also slightly overestimated in the surface-active component dilute range, being just underestimated at higher concentrations. Despite this, Butler's model stands as a very good standard due to its versatility.

Unveiling fungal detoxification pathways of the cruciferous phytoalexin rapalexin A: Sequential L-cysteine conjugation, acetylation and oxidative cyclization mediated by Colletotrichum spp.

The metabolism of the phytoalexin rapalexin A, a unique indole isothiocyanate (ITC) produced by crucifers (family Brassicaceae), was investigated. Three phytopathogenic fungal species were examined: Colletotrichum dematium (Pers.:Fr.) Grove, a broad host range pathogen, C. higginsianum Sacc., a host-selective pathogen of crucifers and C. lentis Damm, a host-selective pathogen of lentils (Lens culinaris Medik.). The metabolism of rapalexin A by C. dematium and C. higginsianum was similar, taking place via one common intermediate and two divergent pathways, but C. lentis was unable to transform rapalexin A. Both C. higginsianum and C. dematium transformed rapalexin A to two previously undescribed metabolites, the structures of which were confirmed by chemical synthesis: N-acetyl-S-(8-methoxy-4H-thiazolo[5,4-b]indol-2-yl)-L-cysteine and 4-hydroxy-3-(4-methoxy-1H-indol-3-yl)-2-thioxothiazolidine-4-carboxylic acid. That is, both fungal pathogens metabolized and detoxified rapalexin A by addition of the thiol group of L-Cys residue to the isothiocyanate carbon of rapalexin A, a transformation usually catalyzed by glutathione transferases. Coincidentally, this metabolic pathway is employed by mammals and insects to detoxify isothiocyanates and other xenobiotics. Hence, C. higginsianum could be a useful model fungus to uncover genes involved in the detoxification pathways of ITCs and related xenobiotics. Our overall results suggest that increasing rapalexin A production in specific crucifers could increase crop resistance to certain fungal pathogens.

Quercetin dual interaction at the membrane level.

Quercetin impact on lipid bilayers suggests a dual action mechanism at cell membranes. This widespread polyphenol displays high partition with low interference in the more fluid membrane domains, more vulnerable to oxidative attack, but strong perturbation of cholesterol/sphingolipid enriched domains, where signalling platforms are expected to assemble.

Ecological Roles of Tryptanthrin, Indirubin and N-Formylanthranilic Acid in Isatis indigotica: Phytoalexins or Phytoanticipins?

Leaves of the plant species Isatis indigotica Fortune ex Lindl. (Chinese woad) produce the metabolites tryptanthrin, indirubin and N-formylanthranilic acid upon spraying with an aqueous solution of copper chloride but not after spraying with water. The antifungal activities of these metabolites against the phytopathogens Alternaria brassicicola, Leptosphaeria maculans and Sclerotinia sclerotiorum established that tryptanthrin is a much stronger growth inhibitor of L. maculans than the phytoalexin camalexin. The biosynthetic precursors of tryptanthrin and N-formylanthranilic acid are proposed based on the deuterium incorporations of isotopically labeled compounds. The overall results suggest that tryptanthrin is a phytoalexin and indirubin and N-formylanthranilic acid are phytoanticipins in the plant species I. indigotica and that chemical diversity and biodiversity are intimately connected.

Methoxycamalexins and related compounds: Syntheses, antifungal activity and inhibition of brassinin oxidase.

The phytoalexin camalexin is a competitive inhibitor of brassinin oxidase, an enzyme that detoxifies the phytoalexin brassinin and is produced by an economically important plant pathogen. For this reason, the camalexin scaffold has guided the design of inhibitors of brassinin detoxification. To further understand the structure-activity relationships of camalexin related compounds, the syntheses of monomethoxy and dimethoxycamalexins were undertaken. Four monomethoxy camalexins together with 4,6-dimethoxy and 5,7-dimethoxy camalexins were prepared from the corresponding methoxyindoles using the Ayer's method. The dimethoxy derivatives were prepared from the corresponding dimethoxyindole-3-thiocarboxamides using the Hantzsch reaction; however, this method did not work for the syntheses of 4,6-dimethoxy and 5,7-dimethoxycamalexins due to the lower reactivities of the corresponding indole-3-thiocarboxamides. The antifungal activity and brassinin oxidase inhibitory activity of all methoxycamalexins and ten camalexin related compounds were investigated. Among the 20 compounds evaluated, monomethoxycamalexins were stronger antifungals than the dimethoxy derivatives. However, remarkably, 5,6-dimethoxycamalexin, 6,7-dimethoxycamalexin and 5-methoxycamalexin displayed the strongest inhibitory activity against brassinin oxidase, while 4,5-dimethoxycamalexin displayed no inhibitory effect. Altogether the structure-activity relationships of camalexin related compounds suggest that the targets for fungal growth inhibition and brassinin oxidase inhibition are unrelated and emphasize that brassinin oxidase inhibitors do not need to be antifungal.

Interrogation of biosynthetic pathways of the cruciferous phytoalexins nasturlexins with isotopically labelled compounds.

The discovery of the first non-indolyl cruciferous phytoalexins nasturlexins A and B together with cyclonasturlexin and brassinin, all chemical defenses of watercress plants (Nasturtium officinale R. Br.), revealed the co-occurrence of two parallel defense pathways, the tryptophan (Trp) pathway and the phenylalanine (Phe) pathway in crucifers. Similar to watercress, winter cress (Barbarea vulgaris R. Br.) and upland cress [B. verna (P. Mill.) Aschers] produce Phe derived phytoalexins, the nasturlexins C and D together with their counterpart sulfoxides. A detailed chemical understanding of the biosynthetic pathways of these phytoalexins facilitates their metabolic engineering. To this end, the biosynthetic pathways of cyclonasturlexin, nasturlexins A-D and corresponding sulfoxides in cress plants were investigated using isotopically labelled compounds. Except for the carbon atom of the thiomethyl groups of nasturlexins, the origin of all carbon atoms and nitrogen of nasturlexins was established to be homophenylalanine. A detailed map of the biosynthetic intermediates between phenylethyl isothiocyanates and nasturlexins A-D and sulfoxides in upland cress, winter cress and watercress is proposed. An application beyond these findings could lead to "designer crops" containing a wider range of chemical defenses that could make such crops more resistant to pests and diseases, a greatly advantageous trait.

Differential targeting of membrane lipid domains by caffeic acid and its ester derivatives.

Phenolic acids have been associated to a wide range of important health benefits underlain by a common molecular mechanism of action. Considering that significant membrane permeation is prevented by their hydrophilic character, we hypothesize that their main effects result from the interplay with cell membrane surface. This hypothesis was tested using the paradigmatic caffeic acid (CA) and two of its ester derivatives, rosmarinic (RA) and chlorogenic (CGA) acids, for which we predict, based on molecular dynamics simulations, a shallow location in phospholipid bilayers dependent on the protonation-state. Using complementary experimental approaches, an interaction with the membrane was definitely revealed for the three compounds, with RA exhibiting the highest lipid bilayer partition, and the redox signals of membrane-bound RA and CA being clearly detected. Cholesterol decreased the compounds bilayer partition, but not their ability to lower membrane dipole potential. In more complex membrane models containing also sphingomyelin, with liquid disordered (ld)/ liquid ordered (lo) phases coexistence, mimicking domains in the external leaflet of human plasma membrane, all compounds were able to affect nanodomains lateral organization. RA, and to a lesser extent CGA, decreased the size of lo domains. The most significant effect of CA was the possible formation of a rigid gel-like phase, enriched in sphingomyelin. In addition, all phenolic acids decreased the order of lo domains. In sum, phenolic acid effects on the membrane are enhanced in cholesterol-rich lo phases, which predominate in the outer leaflet of human cell membranes and are involved in many key cellular processes.

Synthesis of stable isotope-labeled nasturlexins and potential precursors to probe biosynthetic pathways of cruciferous phytoalexins.

The syntheses of perdeuterated phytoalexins nasturlexins A and C, and putative biosynthetic precursors, including phenylethyl isothiocyanates and phenylethyl dithiocarbamates, using commercially available [2,3,4,5,6-D5 ]phenylalanine, [2,3,4,5,6-D5 ]nitrobenzene, and [2,3,4,5,6-D5 ]benzaldehyde are described. In addition, application of an efficient deuterium-hydrogen exchange transformation to nonlabeled starting materials allowed access to new deuterated compounds, including 3-hydroxyphenylethyl glucosinolate.

Inhibitors of the Detoxifying Enzyme of the Phytoalexin Brassinin Based on Quinoline and Isoquinoline Scaffolds.

The detoxification of the phytoalexin brassinin to indole-3-carboxaldehyde and S-methyl dithiocarbamate is catalyzed by brassinin oxidase (BOLm), an inducible fungal enzyme produced by the plant pathogen Leptosphaeria maculans. Twenty-six substituted quinolines and isoquinolines are synthesized and evaluated for antifungal activity against L. maculans and inhibition of BOLm. Eleven compounds that inhibit BOLm activity are reported, of which 3-ethyl-6-phenylquinoline displays the highest inhibitory effect. In general, substituted 3-phenylquinolines show significantly higher inhibitory activities than the corresponding 2-phenylquinolines. Overall, these results indicate that the quinoline scaffold is a good lead to design paldoxins (phytoalexin detoxification inhibitors) that inhibit the detoxification of brassinin by L. maculans.

Defense and signalling metabolites of the crucifer Erucastrum canariense: Synchronized abiotic induction of phytoalexins and galacto-oxylipins.

Erucastrum canariense Webb & Berthel. (Brassicaceae) is a wild crucifer that grows in rocky soils, in salt and water stressed habitats, namely in the Canary Islands and similar environments. Abiotic stress induced by copper chloride triggered formation of a phytoalexin and galacto-oxylipins in E. canariense, whereas wounding induced galacto-oxylipins but not phytoalexins. Analysis of the metabolite profiles of leaves of E. canariense followed by isolation and structure determination afforded the phytoalexin erucalexin, the phytoanticipin indolyl-3-acetonitrile, the galacto-oxylipins arabidopsides A, C, and D, and the oxylipin 12-oxophytodienoic acid. In addition, arabidopsides A and D were also identified in extracts of leaves of Nasturtium officinale R. Br.

Biotransformation of rutabaga phytoalexins by the fungus Alternaria brassicicola: Unveiling the first hybrid metabolite derived from a phytoalexin and a fungal polyketide.

The biotransformations of the rutabaga phytoalexins rutalexin, brassicanate A, isalexin and rapalexin A by the plant pathogenic fungus Alternaria brassicicola are reported. While the biotransformations of rutalexin, brassicanate A, and isalexin are fast, rapalexin A is resistant to fungal transformation. Unexpectedly, biotransformation of rutalexin yields a hybrid metabolite named rutapyrone, derived from rutalexin metabolism and phomapyrone G, a fungal metabolite produced by A. brassicicola. These fungal transformations are detoxification reactions likely carried out by different enzymes. The discovery of rapalexin A resistance to detoxification suggests that this phytoalexin in combination with additional phytoalexins could protect crucifers against this pathogen. Phytoalexins resistant to degradation by A. brassicicola are expected to provide the producing plants with higher disease resistance levels.

The biosynthesis of brassicicolin A in the phytopathogen Alternaria brassicicola.

Alternaria brassicicola (Schwein.) Wiltshire is a phytopathogenic fungus that together with A. brassicae causes Alternaria black spot disease in Brassica species. Brassicicolin A is the major host-selective phytotoxin produced in cultures of A. brassicicola. Biosynthetic studies to establish the metabolic precursors of brassicicolin A were carried out with isotopically labeled compounds. Incorporation of D-[13C6]glucose, L-[15N]valine, or L-[2H8]valine into brassicicolin A was established using 1H, 13C, 15N NMR and INADEQUATE spectroscopy and HPLC-ESI-MS spectrometry. Based on analyses of the spectroscopic data, the labeling patterns of brassicicolin A isolated from cultures incubated with the labeled precursors are found to be consistent with both the glycolytic and the valine pathways. That is, the carbons of mannitol and acetyl units and the isocyanide carbon atoms are derived from D-[13C6]glucose whereas the hydroxyisopentanoyl and isocyanoisopentanoyl units are derived from L-valine, including the nitrogen atoms of both isocyanide groups.

Revealing microheterogeneities and second order phase transitions in aqueous mixtures of 1-propoxypropan-2-ol at 298 K.

Second order phase transitions corresponding to discontinuities in the plots of Kirkwood-Buff integrals as a function of composition were observed in aqueous mixtures of the amphiphilic molecule, 1-propoxypropan-2-ol, revealing the formation of hydrophobic aggregates and generating microheterogeneities over a limited range of compositions. Electrospray mass spectra, surface tension measurements and solvatochromic parameters confirmed the onsets of different aggregation patterns over the entire composition range, and allowed us to scrutinize the prevailing types of aggregate species. This is seemingly the first time that such discontinuities are clearly assumed as second order phase transitions in a system macroscopically homogeneous and corroborated by other independent tools.

Expanding the nasturlexin family: Nasturlexins C and D and their sulfoxides are phytoalexins of the crucifers Barbarea vulgaris and B. verna.

The metabolites produced in leaves of the crucifers winter cress (Barbarea vulgaris) and upland cress (Barbarea verna) abiotically elicited were investigated and their chemical structures were elucidated by analyses of spectroscopic data and confirmed by syntheses. Nasturlexins C and D and their sulfoxides are cruciferous phytoalexins displaying antifungal activity against the crucifer pathogens Alternaria brassicicola, Leptosphaeria maculans and Sclerotinia sclerotiorum. The biosynthesis of these metabolites is proposed based on pathways of cruciferous indolyl phytoalexins. This work indicates that B. vulgaris and B. verna have great potential as sources of defense pathways transferable to agriculturally important crops within the Brassica species.

Metabolite diversity in the plant pathogen Alternaria brassicicola: factors affecting production of brassicicolin A, depudecin, phomapyrone A and other metabolites.

A systematic investigation of the metabolites of Alternaria brassicicola produced under various culture conditions is reported. The phytotoxin brassicicolin A is produced in significantly larger amounts in potato dextrose broth than in minimal medium cultures. In general an increase in the incubation temperature of cultures 23-30 C increases the production of brassicicolin A but decreases depudecin production. Reducing or eliminating nitrate from culture media or adding ammonium chloride increases the production of brassicicolin A at 30 C, depudecin at 23 C and α-acetylorcinol at either temperature, suggesting that nitrogen represses their biosynthesis. Siderophores are detected in cultures of A. brassicicola containing low and high ferric ion concentrations. The metabolites α-acetylorcinol and tyrosol are isolated for the first time from cultures of A. brassicicola, and α-acetylorcinol is synthesized in four steps and 36% overall yield. Only brassicicolin A and no other isolated metabolites, including depudecin and phomapyrone A, display phytotoxicity on leaves of Brassica species (up to 5.0 mM). Epigenetic modifiers, 5-azacitidin (5-AZA), suberoylanilide hydroxamic acid (SAHA) and suberoyl bis-hydroxamic acid (SBHA) do not affect the metabolite profiles of liquid cultures of this fungal pathogen.

Plant chemical defenses: are all constitutive antimicrobial metabolites phytoanticipins?

A critical perspective on phytoanticipins, constitutive plant secondary metabolites with defensive roles against microbes is presented. This mini-review focuses on the chemical groups and structural types of defensive plant metabolites thus far not reviewed from the phytoanticipin perspective: i) fatty acid derivatives and polyketides, ii) terpenoids, iii) shikimates, phenylpropanoids and derivatives, and iv) benzylisoquinoline and pyrrolizidine alkaloids. The more traditional groups of phytoanticipins are briefly summarized, with particular focus on the latest results: i) benzoxazinoids, ii) cyanogenic glycosides, iii) glucosinolates and their metabolic products, and iv) saponins. Current evidence suggests that a better understanding of the functions of plant metabolites will drive their application to protect crops against microbial diseases.

Non-indolyl cruciferous phytoalexins: Nasturlexins and tridentatols, a striking convergent evolution of defenses in terrestrial plants and marine animals?

Highly specialized chemical defense pathways are a particularly noteworthy metabolic characteristic of sessile organisms, whether terrestrial or marine, providing protection against pests and diseases. For this reason, knowledge of the metabolites involved in these processes is crucial to producing ecologically fit crops. Toward this end, the elicited chemical defenses of the crucifer watercress (Nasturtium officinale R. Br.), i.e. phytoalexins, were investigated and are reported. Almost three decades after publication of cruciferous phytoalexins derived from (S)-Trp, phytoalexins derived from other aromatic amino acids were isolated; their chemical structures were determined by analyses of their spectroscopic data and confirmed by synthesis. Nasturlexin A, nasturlexin B, and tridentatol C are hitherto unknown phenyl containing cruciferous phytoalexins produced by watercress under abiotic stress; tridentatol C is also produced by a marine animal (Tridentata marginata), where it functions in chemical defense against predators. The biosynthesis of these metabolites in both a terrestrial plant and a marine animal suggests a convergent evolution of unique metabolic pathways recruited for defense.

Activity coefficients in the surface phase of liquid mixtures.

A novel equation for evaluating surface activity coefficients is obtained from a recent thermodynamic formalism describing the surface phase of liquid mixtures. The input quantities are the surface tension, bulk activity coefficients and pure constituent thermophysical properties. It is demonstrated thermodynamically that the order of magnitude of each component surface and bulk activity coefficients must be the same. This order is intrinsically associated with the sign of excess surface tension. Reliable activity coefficients of ethanol and water in the surface phase of their mixtures are computed and reported for the first time, by using literature data for the required input quantities. It is shown that the so-called transferring method for estimating surface activity coefficients is severely flawed, because it leads to contradictory values of predicted excess surface tensions depending on which component this prediction is based.

New thermodynamics for evaluating the surface-phase enrichment in the lower surface tension component.

Regarding the surface phase of liquid mixtures as a thermodynamic phase, ideal surface phases are designed so that at fixed bulk-phase composition, real and ideal surface phases have the same chemical composition and identical limiting slopes for the dependence of surface tension on mole fraction. Standard chemical potentials are introduced for surface phase components, and quasi-exact expressions are worked out to compute ideal surface tensions and surface-phase compositions of real liquid mixtures. Guidelines for choosing molecular models to estimate the molar surface area of pure constituents are given. Ideal and excess surface tensions are calculated by using literature data for aqueous ethanol solutions at 298 K. These results show treatment based on Butler's equations grossly overestimate predicted surface tensions, thus leading to lower ethanol content in the surface phase. These inaccuracies are ascribed to the use of molar surface areas in model equations that are too small.