Preparation, release and physicochemical characterisation of ethyl butyrate and hexanal inclusion complexes with ß- and γ-cyclodextrin.

Complexes of ethyl butyrate and hexanal encapsulated by ß-cyclodextrin (ß-CD) and γ-cyclodextrin (γ-CD) were prepared by coprecipitation, and gas chromatography was used to quantity the flavour compounds in the complexes. The ethyl butyrate-γ-CD complex had the highest inclusion ratio (12.20%) followed by the ethyl butyrate-ß-CD, hexanal-ß-CD and hexanal-γ-CD complexes (11.29, 4.41 and 3.33%, respectively). Release experiments were performed under different relative humidities (RH 93, 75 and 52%) and temperatures (4 and 25 °C). The flavour release behaviours of the complexes were described by the Avrami equation. The rate of flavour release was enhanced with both increasing temperature and RH, although the effect of RH was stronger. Physicochemical characterisation using FT-IR, XRD, DSC and SEM analyses demonstrated that crystalline complexes were formed. Both ß-CD and γ-CD were able to encapsulate ethyl butyrate and hexanal, and lower RH and temperature were more suitable for the storage of these complexes.

Characterization of polar organosulfates in secondary organic aerosol from the green leaf volatile 3-Z-hexenal.

Evidence is provided that the green leaf volatile 3-Z-hexenal serves as a precursor for biogenic secondary organic aerosol through the formation of polar organosulfates (OSs) with molecular weight (MW) 226. The MW 226 C6-OSs were chemically elucidated, along with structurally similar MW 212 C5-OSs, whose biogenic precursor is likely related to 3-Z-hexenal but still remains unknown. The MW 226 and 212 OSs have a substantial abundance in ambient fine aerosol from K-puszta, Hungary, which is comparable to that of the isoprene-related MW 216 OSs, known to be formed through sulfation of C5-epoxydiols, second-generation gas-phase photooxidation products of isoprene. Using detailed interpretation of negative-ion electrospray ionization mass spectral data, the MW 226 compounds are assigned to isomeric sulfate esters of 3,4-dihydroxyhex-5-enoic acid with the sulfate group located at the C-3 or C-4 position. Two MW 212 compounds present in ambient fine aerosol are attributed to isomeric sulfate esters of 2,3-dihydroxypent-4-enoic acid, of which two are sulfated at C-3 and one is sulfated at C-2. The formation of the MW 226 OSs is tentatively explained through photooxidation of 3-Z-hexenal in the gas phase, resulting in an alkoxy radical, followed by a rearrangement and subsequent sulfation of the epoxy group in the particle phase.

Differential metabolisms of green leaf volatiles in injured and intact parts of a wounded leaf meet distinct ecophysiological requirements.

Almost all terrestrial plants produce green leaf volatiles (GLVs), consisting of six-carbon (C6) aldehydes, alcohols and their esters, after mechanical wounding. C6 aldehydes deter enemies, but C6 alcohols and esters are rather inert. In this study, we address why the ability to produce various GLVs in wounded plant tissues has been conserved in the plant kingdom. The major product in completely disrupted Arabidopsis leaf tissues was (Z)-3-hexenal, while (Z)-3-hexenol and (Z)-3-hexenyl acetate were the main products formed in the intact parts of partially wounded leaves. (13)C-labeled C6 aldehydes placed on the disrupted part of a wounded leaf diffused into neighboring intact tissues and were reduced to C6 alcohols. The reduction of the aldehydes to alcohols was catalyzed by an NADPH-dependent reductase. When NADPH was supplemented to disrupted tissues, C6 aldehydes were reduced to C6 alcohols, indicating that C6 aldehydes accumulated because of insufficient NADPH. When the leaves were exposed to higher doses of C6 aldehydes, however, a substantial fraction of C6 aldehydes persisted in the leaves and damaged them, indicating potential toxicity of C6 aldehydes to the leaf cells. Thus, the production of C6 aldehydes and their differential metabolisms in wounded leaves has dual benefits. In disrupted tissues, C6 aldehydes and their α,ß-unsaturated aldehyde derivatives accumulate to deter invaders. In intact cells, the aldehydes are reduced to minimize self-toxicity and allow healthy cells to survive. The metabolism of GLVs is thus efficiently designed to meet ecophysiological requirements of the microenvironments within a wounded leaf.

Effects of crop development on the emission of volatiles in leaves of Lycopersicon esculentum and its inhibitory activity to Botrytis cinerea and Fusarium oxysporum.

Volatiles emitted from the leaves of Lycopersicon esculentum at the two-, ten-leaf and anthesis periods were collected by a gas absorbing method and analyzed by gas chromatography (GC)-mass spectrometry. In total, 33 compounds of volatiles emitted from three developmental stage plants were separated and identified, and quantitatively analyzed by the internal standard addition method. All of the samples of volatile were found to be rich in monoterpenes and sesquiterpenes. beta-phellandrene and caryophyllene predominated in the volatiles of the leaves of plants at the two- and ten-leaf stages. Furthermore, (E)-2-hexenal were the dominant components in the volatiles emitted from anthesis plants. The results of volatiles analyzed show that the compositions varied depending on the developmental stages. The volatiles emitted from crushed tomato leaves of plants at the anthesis stage had the most strongly inhibitory activity against the spore germination and hyphal growth of Botrytis cinerea and Fusarium oxysporum, followed by ten- and two-leaf plants. However, the activity of volatiles, emitted from the leaves of plants at the two-leaf stage, in inhibiting F. oxysporum was greater than B. cinerea.

Ozonolysis of geraniol-trans, 6-methyl-5-hepten-2-one, and 6-hydroxy-4-methyl-4-hexenal: kinetics and mechanisms.

A combined density functional theory and transition-state theory study of the mechanisms and reaction coefficients of gas-phase ozonolysis of geraniol-trans, 6-methyl-5-hepten-2-one, and 6-hydroxy-4-methyl-4-hexenal is presented. The geometries, energies, and harmonic vibrational frequencies of each stationary point were determined by B3LYP/6-31(d,p), MPW1K/cc-pVDZ, and BH&HLYP/cc-pVDZ methods. According to the calculations, the ozone 6-methyl-5-hepten-2-one reaction is faster than the ozone 6-hydroxy-4-methyl-4-hexenal reaction, but both are slower than the ozone geraniol-trans reaction. By using the BH&HLYP/cc-pVDZ data, a global rate coefficient of 5.9 x 10(-16) cm(3) molecule(-1) s(-1) was calculated, corresponding to the sum of geraniol-trans, 6-methyl-5-hepten-2-one, and 6-hydroxy-4-methyl-4-hexenal reactions with the ozone. These results are in good agreement with the experimental studies.

Mung bean lipoxygenase in the production of a C6-aldehyde. Natural green-note flavor generation via biotransformation.

Mung bean was investigated as a novel source of lipoxygenase in the natural production of the green-note aroma compound hexanal. Lipoxygenase extracted from mung bean catalyzed the oxidative reaction of linoleic acid, after which the intermediate hydroperoxide compound was split via green bell pepper hydroperoxide lyase to produce hexanal. In comparison to soybean lipoxygenase, mung bean lipoxygenase was found to be a good substitute as it produced 15.4 mM (76% yield) hexanal while soybean gave 60% yield. The mung bean pH profile comprised a wide peak (optimum pH 6.5) representing lipoxygenase-2 and lipoxygenase-3 isozymes, whereas two narrower peaks representing lipoxygenase-1 and lipoxygenase-2/3 isozymes were observed for soybean (optimum pH 10). Extraction at pH 4.5 was preferred, at which specific lipoxygenase activity was also the highest.

Stereoselective hexobarbital 3'-hydroxylation by CYP2C19 expressed in yeast cells and the roles of amino acid residues at positions 300 and 476.

We examined the enzymatic function of recombinant CYP2C19 in enantiomeric hexobarbital (HB) 3'-hydroxylation, and searched the roles of amino acid residues, such as Phe-100, Phe-114, Asp-293, Glu-300, and Phe-476 of CYP2C19 in the stereoselective HB 3'-hydroxylation, using a yeast cell expression system and site-directed mutagenesis method. CYP2C19 wild-type exerted substrate enantioselectivity of (R)-HB>>(S)-HB and metabolite diastereoselectivity of 3'(R)<3'(S) in 3'-hydroxylation of HB enantiomers. The substitution of Asp-293 by alanine failed to yield an observable peak at 450 nm in its reduced carbon monoxide-difference spectrum. CYP2C19-E300A and CYP2C19-E300V with alanine and valine, respectively, in place of Glu-300 exerted total HB 3'-hydroxylation activities of 45 and 108%, respectively, that of the wild-type. Interestingly, these two mutants showed substrate enantioselectivity of (R)-HB<(S)-HB, which is opposite to that of the wild-type, while metabolite diasteroselectivity remained unchanged. The replacement of Phe-476 by alanine increased total HB 3'-hydroxylation activity to approximately 3-fold that of the wild-type. Particularly, 3'(S)-OH-(S)-HB-forming activity elevated to 7-fold that of the wild-type, resulting in the reversal of the substrate enantioselectivity. In contrast, the substitution of phenylalanine at positions 100 and 114 by alanine did not produce a remarkable change in the total activity or the substrate enantioselectivity. These results indicate that Glu-300 and Phe-476 are important in stereoselective oxidation of HB enantiomers by CYP2C19.

Photoreceptor for curling behavior in Peranema trichophorum and evolution of eukaryotic rhodopsins.

When it is gliding, the unicellular euglenoid Peranema trichophorum uses activation of the photoreceptor rhodopsin to control the probability of its curling behavior. From the curled state, the cell takes off in a new direction. In a similar manner, archaea such as Halobacterium use light activation of bacterio- and sensory rhodopsins to control the probability of reversal of the rotation direction of flagella. Each reversal causes the cell to change its direction. In neither case does the cell track light, as known for the rhodopsin-dependent eukaryotic phototaxis of fungi, green algae, cryptomonads, dinoflagellates, and animal larvae. Rhodopsin was identified in Peranema by its native action spectrum (peak at 2.43 eV or 510 nm) and by the shifted spectrum (peak at 3.73 eV or 332 nm) upon replacement of the native chromophore with the retinal analog n-hexenal. The in vivo physiological activity of n-hexenal incorporated to become a chromophore also demonstrates that charge redistribution of a short asymmetric chromophore is sufficient for receptor activation and that the following isomerization step is probably not required when the rest of the native chromophore is missing. This property seems universal among the Euglenozoa, Plant, and Fungus kingdom rhodopsins. The rhodopsins of animals have yet to be studied in this respect. The photoresponse appears to be mediated by Ca2+ influx.

Attraction of male European tarnished plant bug, Lygus rugulipennis to components of the female sex pheromone in the field.

Previous work showed that females of the European tarnished plant bug, Lygus rugulipennis Poppius (Heteroptera: Miridae), produced three chemicals, hexyl butyrate, (E)-2-hexenyl butyrate, and (E)-4-oxo-2-hexenal, and that these were suspected to be components of the female sex pheromone. In field experiments, traps baited with blends of these chemicals dispensed from polyethylene vials and sachets failed to catch significant numbers of males. Here, we report more recent field experiments in which the chemicals were released from glass microcapillary tubes. A blend of hexyl butyrate and (E)-4-oxo-2-hexenal was significantly attractive to male L. rugulipennis. In addition, whereas the mixture of all three components attracted fewer L. rugulipennis males, this tertiary blend captured significantly greater numbers of males of the congeneric species Lygus pratensis than the binary mixture. The possible reasons for the success of the microcapillaries compared with other dispensers are discussed.

Enantioselective synthesis of (+)-cryptophycin 52 (LY355703), a potent antimitotic antitumor agent.

A highly enantioselective and convergent synthesis of cryptophycin 52 (2), an exceedingly potent cytotoxic agent, is described. Cryptophycin 52, a synthetic variant of the cryptophycin family, is currently undergoing clinical trials. The synthesis is convergent and involves assembly of three fragments, phenyl hexenal 3, d-tyrosine phosphonate 4, and protected beta-amino acid derivative 5. The synthesis of fragment 3 involves an efficient and stereocontrolled construction of both stereogenic centers at C-3 and C-4 by cleavage of a substituted tetrahydrofuran ring via an acyloxycarbenium ion intermediate. Both of these stereogenic centers were derived from optically active 4-phenylbutyrolactone, synthesized enantioselectively by Corey-Bakshi-Shibata reduction.

Enantiomer separation by capillary electrochromatography on a cyclodextrin-modified monolith.

A chiral monolithic stationary phase was prepared by packing a capillary with bare porous silica and sintering the silica bed at high temperature. The resulting silica monolith was polymer-coated with Chirasil-Dex, a permethylated beta-cyclodextrin covalently linked via an octamethylene spacer to dimethylpolysiloxane. Subsequently, Chirasil-Dex was thermally immobilized on the silica support and a chiral monolith of very high stability (30 kV, more than 400 bar pressure) was obtained. The enantiomer separation of various chiral compounds by monolithic (rod) capillary electrochromatography (rod-CEC) was feasible. This method was compared with capillary liquid chromatography (LC) in a single-column mode using unified equipment. About two to three times higher efficiency was found in the rod-CEC mode as compared to rod-LC. The influence of pressure-driven flow support on efficiency, resolution, elution time and baseline stability was investigated. The amount and nature of organic modifier strongly influences efficiency and resolution.

Effects of volatile aldehydes from Aspergillus-resistant varieties of corn on Aspergillus parasiticus growth and aflatoxin biosynthesis.

The fungi Aspergillus flavus and Aspergillus parasiticus produce a potent class of hepatocarcinogens known as aflatoxins. Corn-derived volatile compounds have been previously found to affect growth and aflatoxin production in A. flavus. In this study, the effects on A. parasiticus of three corn-derived volatile compounds, n-decyl aldehyde, hexanal and octanal, were measured. These three compounds were previously found to be variably expressed in five Aspergillus-resistant maize strains and three susceptible strains. In this study, A. parasiticus radial growth was restricted least by n-decyl aldehyde and most by octanal. Treatments of 100 microl of both hexanal and octanal were found to completely inhibit radial growth of the fungus using an agar plate assay method. While the volatile compound n-decyl aldehyde had less of an effect on radial growth than the other volatiles, the n-decyl aldehyde treated colonies had a predominance of uniquely aerial hyphae. These colony structures were found to have more complex hyphae and significantly fewer conidiophores than the control and other aldehyde treatments. Furthermore, aflatoxin production by the fungus was reduced by n-decyl aldehyde and hexanal, but was stimulated by octanal. The results presented here indicate that all three volatile compounds reduce radial growth but only n-decyl aldehyde significantly inhibits aflatoxin biosynthesis in A. parasiticus.

Molecular modelling of mammalian CYP2B isoforms and their interaction with substrates, inhibitors and redox partners.

1. The construction of three-dimensional models of CYP2B isozymes from rat (CYP2B1), rabbit (CYP2B4) and man (CYP2B6), based on a multiple sequence alignment with CYP102, a unique eukaryotic-like bacterial P450 (in terms of possessing an NADPH-dependent FAD- and FMN-containing oxidoreductase redox partner) of known crystal structure, is reported. 2. The enzyme models described are shown to be consistent with experimental evidence from site-directed mutagenesis studies, antibody recognition sites and amino acid residues identified as being associated with redox partner interactions, together with the location of a key serine residue (Ser-128) likely to be involved in protein kinaseA-mediated phosphorylation. 3. A substantial number of known substrates and inhibitors of CYP2B isozymes are shown to fit the putative active sites of the enzyme models in agreement with their reported position of metabolism or mode of inhibition respectively. In particular, there is complementarity between the characteristic non-planar geometries of CYP2B substrates and key groups in the enzymes' active sites. 4. Molecular modelling of CYP2B isozymes appears to rationalize a number of the reported findings from quantitative structure-activity relationship investigations on series of CYP2B substrates and inhibitors.

Análisis cuantitativo de la 3-hidroxi, 3-etil-, 3-fenil propionamida en plasma por cromatografía de líquidos de alta resolución / Quantitatyve analysis of 3-hydroxi, 3-ethyl, 3-phenyl propionomide in plasma by high resolution liquid cromatography

Se describe un método sensible y específico de cromatografía de alta resolución (HPCL) para el análisis en plasma humano de 3-hidroxi, 3-etil, 3-fenil propionami-da (HEPP), un nuevo anticonvulsivante. El estándar interno (2-hidroxi, 2-etil, 2-fenil acetamida HEPA) y la HEPP se extrajeron en acetonitrilo de plasma amortiguado, la extracción fue cercana al 100 por ciento respecto a la cantidad extraída de cada fármaco de solución salina amortiguada. El método consiste en HPCL en fase reversa (Lichro Spher 100 RP-18 en Lichro Cart 125-4), la fase móvil está compuesta de metanol-acetonitrilo-amortiguador de fosfatos (35/15/50 por vol) y detección ultravioleta a 200 nm. Las curvas de calibración fueron lineales y repetibles (coeficiente de correlación > 0.999). Las determinaciones de HEPP en plasma humano o en solución salina fueron lineales en el intervalo 0 - 10 µg/ml y el coeficiente de variación fue menor que 10 por ciento. HEPP es muy estable a temperatura ambiente y a 4 ºC, y puede ser cuantificada en presencia de otros antiepilépticos tales como: difenilhidantoina, clonazepam. carbamezepina y hexobarbital

Application of Empore C-8 extraction disks for screening urine in systematic toxicological analysis.

Solid-phase extraction (SPE) by means of disposable columns has become a widely accepted technique for sample pretreatment in toxicology, both for directed analyses and for screening analyses. However, the sample capacity in SPE is usually limited to a few millilitres. Therefore, we have investigated to what extent these problems can be overcome by using Empore extraction disks, consisting of chemically modified C-8 reversed-phase silica, embedded in an inert polytetrafluoroethylene (PTFE) matrix. Human urine was selected as the matrix and dexetimide and mepyramine were initially used as test drugs because these drugs were available in tritiated form. Additional drugs investigated included codeine, hexobarbital, imipramine, methamphetamine, and nitrazepam. In these investigations, the sample capacity for untreated urine was at least 25 mL, and analyte quantities up to 250 micrograms could be retained by these filters. Washing with water/methanol mixtures was successful in removing substantial amounts of endogenous interferences, and methanol proved to be an acceptable eluent. Thus, these disks seem to have interesting potential for toxicological analysis in that sample concentration and cleanup can be achieved at the same time.