Christopher K. Jankowski

Electrospray-mass spectrometric studies of selectivity of alkali metal cations extraction by calix(4)arene crowns

Electrospray Ionization Mass Spectrometry (ESI/MS) has quickly become a versatile method of qualitative analysis of a wide variety of host-guest complexes formed in solution. However, considerable controversy exists on how ESI spectra quantitatively reflect and compare to the results obtained on these complexes. The extraction of alkali cations from acidic aqueous solutions by nine various calix(4)arenes-crown-6 diluted in NPOE, was studied by using ESI/MS. The stoichiometry of cesium complex and Cs + /Na + selectivity were evaluated.

Acetylcholinesterase-inhibiting Alkaloids from Zephyranthes concolor

The bulbs and aerial parts of Zephyranthes concolor (Lindl.) Benth. & Hook. f. (Amaryllidaceae), an endemic species to Mexico, were found to contain the alkaloids chlidanthine, galanthamine, galanthamine N-oxide, lycorine, galwesine, and epinorgalanthamine. Since currently only partial and low resolution 1H-NMR data for chlidanthine acetate are available, and none for chlidanthine, its 1D and 2D high resolution 1H- and 13C-NMR spectra were recorded. Unambiguous assignations were achieved with HMBC, and HSQC experiments, and its structure was corroborated by X-ray diffraction. Minimum energy conformation for structures of chlidanthine, and its positional isomer galanthamine, were calculated by molecular modelling. Galanthamine is a well known acetylcholinesterase inhibitor; therefore, the isolated alkaloids were tested for this activity. Chlidanthine and galanthamine N-oxide inhibited electric eel acetylcholinesterase (2.4 and 2.6 × 10−5 M, respectively), indicating they are about five times less potent than galanthamine, while galwesine was inactive at 10−3 M. Inhibitory activity of HIV-1 replication, and cytotoxicity of the isolated alkaloids were evaluated in human MT-4 cells; however, the alkaloids showed poor activity as compared with standard anti-HIV drugs, but most of them were not cytotoxic.

Recognition and oxidative metabolism of cyclodipeptides by hepatic cytochrome P450.

Possible recognition of peptide derivatives by hepatic cytochrome P450 3A has been suggested by binding and metabolism of numerous pseudopeptidic compounds such as ergot derivatives and cyclosporin. Natural linear or cyclic dipeptides containing hydrophobic amino acids produced by microorganisms and present in mammals are able to interact with the P450 active site through either iron-amine interactions (Type II) or hydrophobic Type I interactions. P450 3A from dexamethasone-treated rats or yeast-expressed P450 human 3A4 are the most potent in such interactions, which are particularly strong with peptides containing a histidyl residue. Some cyclodipeptides are rapidly transformed by rat cytochrome P450 3A to mono- or dihydroxylated metabolites, with turnovers around 3 nmoles min(-1) P450(-1). Linear peptides are poorly transformed in these conditions. This metabolism of cyclodipeptides occurs in 8 species including man. Such interactions and metabolism have only minor consequences in terms of P450 3A binding and metabolism of classical P450 3A substrates. These data reinforce the concept that, in addition to their effect on the regulation of P450 neosynthesis, naturally occurring endogenous peptides are also substrates of P450 3A. The physiological activities of these peptides may be modulated by their metabolism.

Essential Oils in Mexican Bays (Litsea spp., Lauraceae): Taxonomic Assortment and Ethnobotanical Implications1

Essential Oils in Mexican Bays (Litseaspp., Lauraceae): Taxonomic Assortment and Ethnobotanical Implications. The seven species of Litsea found in Mexico, all of them popularly known as “laurel,” were surveyed by gas chromatography-mass spectrometry for their foliar essential oils composition and related ethnobotanical applications. Litsea glaucescens is in high demand as a condiment, and is sold in rural and urban markets all over Mexico. However, four other species are also locally used for food seasoning. Litsea guatemalensis is the species most used in traditional medicine, especially to treat fever, chills, infectious diseases of the digestive system, and arthritis. No reports of culinary, medicinal, or other applications were located for L. muelleri, and L. pringlei. This is the first report on the essential oils for L. neesiana, L. muelleri, L. parvifolia, L. pringlei, and L. schaffneri. The terpenoids commonly found in all the Litsea species studied were 1,8-cineole, linalool, α-pinene, β-pinene, m-cymene, terpinen-4-ol, α-terpineol, caryophyllene, and caryophyllene oxide. Nevertheless, each species can be distinguished by its characteristic assortment of terpenoids. According to hierarchical cluster analysis, three groups of species were recognized: (1) 1,8-cineole group (C-10 terpenes), consisting of L. glaucescens, L. schaffnerii, L. pringlei, and L. muelleri; (2) limonene-rich group (C-10 oxygenated terpenes), including L. guatemalensis, and L. neesiana, and (3) oxygenated sesquiterpenes-rich group (C-15 oxygenated terpenes), comprising L. parvifolia. The chemical profiles of L. glaucescens and L. guatemalensis suggest a correlation with the culinary and medicinal uses of these species due to the known properties of their main constituents.ResumenAceites esenciales de laureles mexicanos(Litseaspp., Lauraceae): Distribución taxonómica e implicaciones etnobotánicas. La composición de los aceites esenciales foliares de las siete especies de Litsea encontradas en México, todas ellas conocidas popularmente como “laurel,” fue analizada por cromatografía de gases acoplada a espectrometría de masas y relacionada con sus usos tradicionales. Litsea glaucescens es altamente demandada como condimento y se vende en los mercados rurales y urbanos a lo largo de todo el país. Sin embargo, otras cuatro especies, también se utilizan localmente como especias. Litsea guatemalensis es la especie más usada en la medicina tradicional para tratar fiebre, resfriados, infecciones del aparato digestivo y artritis. No se registró ningún uso para L. muelleri y L. pringlei. Se reporta por primera vez la composición de los aceites esenciales de L. neesiana, L. muelleri, L. parvifolia, L. pringlei y L. schaffneri. Los terpenoides comunes en todas las especies estudiadas de Litsea fueron: 1,8-cineol, linalool, α-pineno, β-pineno, m-cimeno, terpinen-4-ol, α-terpineol, cariofileno y óxido de cariofileno, sin embargo, cada una de las siete especies puede distinguirse por un perfil característico de terpenoides. Con base en sus compuestos mayoritarios se distinguieron tres grupos: (1) rico en 1,8-cineol (monoterpenos C-10) constituido por L. glaucescens, L. schaffnerii, L. pringlei y L. muelleri; (2) rico en limoneno (monoterpenos oxigenados C-10) incluye a L. guatemalensis y L. neesiana; y (3) rico en sesquiterpenos oxygenados (C-15) que incluye a L. parvifolia, la cual posee semejanza química con otras especies asiáticas. Nuestros resultados sugieren que los perfiles químicos de L. glaucescens y L. guatemalensis están relacionados con sus aplicaciones culinarias y medicinales en razón a las propiedades conocidas de sus componentes mayoritarios.

Comparison of the Stability of Calix[4]arene‐crown‐6‐cation Binary Complexes Under Electrospray Mass Spectrometry

Electrospray/mass spectrometry ESI/MS analyses were performed to study the stability of calix[4]‐arene‐crown‐6/alkali cation complexes in the gas phase, and in acetonitrile/water mixtures. This approach allowed a comparison with previous investigations by molecular‐dynamic simulations,which demonstrated a complementarity between calculation and experiment. Experimental results obtained from ESI/MS confirm that the stability of calixarene/cation complexes depends upon the medium used. Indeed, the calixarene in solution presents a strong affinity for cesium, whereas in the gas phase, it has a stronger affinity for sodium. Similarly, the stability of [calixarene + Na]+‐type complexes in the solvent phase is increased by the presence of water in the dilution system (up to 40% in acetonitrile), whereas other alkaline complexes are destabilized by water in any proportion. Finally, calixarenes that bear benzo groups on their crowns have an affinity for sodium, which is weak in solution, but considerably stronger in the gas phase. ‡Some of the results of this paper were presented as a CEA Scientific Report R‐5835 (1998), pp. 94–98.

The Problems Associated with Enzyme Purification

This chapter aims to highlight the difficulties encountered during the purification of native cellular and membrane-bound enzymes from whole cell extracts. There are many reasons for wanting to purify enzymes, such as to fully characterize them or to mass produce them for commercial purposes. Regardless of the reason for wanting to purify an enzyme, the general extraction and purification procedures are essentially the same. However, depending on the properties of the enzyme, certain modifications of the methods must be considered regarding specific problems that can be encountered throughout the process, such as enzyme insolubility and loss of enzyme activity. Although several classic and more modern methods are available to solve these kinds of challenges, the enzyme purification step remains a major challenge for any method of extraction used.

Application of gas chromatography–tandem mass spectrometry to the analysis of inhibition of dimerisation of tributylphosphate under radiolysis: Identification of isomeric tributylphosphate-alkylbenzene inhibitor coupling products

Tributylphosphate (TBP), solvent used as extractant for reprocessing spent nuclear fuel, can dimerise under radiolysis. This occurs by radical radical recombination, leading to 10 isomeric dimers (TBP-TBP). These species are complexation agents and are responsible of fission product retention in the organic phase that increases the solvent degradation. In order to limit their formation two free radical inhibitors (In), isopropyl and 1,4-diisopropylbenzenes, were used. These additives reduce by about 50% the concentration of TBP-TBP dimers but this reduction is not strictly followed by TBP regeneration as mixed coupling products from TBP and inhibitor are detected. By using GC-MS-MS and selectively deuterated compounds, the identification of these different isomers (TBP-In) has been realised. From these identifications and from the analysis of the proportion of the different isomers, the major primary TBP radical generated under radiolysis was determined.

A new approach for the purification and characterisation of PA49.5, the main prebiotic of Lactococcus lactis subsp. cremoris

The main autolysin PA49.5, an enzyme that hydrolyzes or destroys the components of a biological endogenous cell or a tissue, was purified 3045 times from the homogenate of a whole cell extract of Lactococcus lactis subsp. cremoris ATCC 9596 (Mc5), with a recovery yield of 52%. The purification of the protein was carried out through a micro-purification technique using SDS-BigCHAP polyacrylamide gel electrophoresis and concentrated with a Microcon-10 filtration system. SDS-polyacrylamide gel electrophoresis of the purified enzyme confirmed the presence of only one band having a molecular weight of 49.5 kDa. In view of its insolubility, PA49.5 contained in the cell extract precipitate was solubilized in the presence of 0.1% (w/v) of BigCHAP, a non-ionic detergent. Higher concentrations of this detergent completely inhibited the activity of solubilized PA49.5 or prevented its solubilization. The optimal pH and temperature for PA49.5 enzymatic activity are 7.5 and 45 degrees C respectively. In addition 0.1% or less of PA49.5 significantly increased Mc5 lysis. We observed 55% more lysis with 0.25 mug of purified PA49.5 compared to the control. Gas chromatography analysis of the components of the crude cell extract, of the precipitate and of the supernatant indicates the presence of at least 6 fatty acids. The long-chained fatty acids (e.g. C18:0 and C18:3) detected represent 81.65% of the precipitate from which PA49.5 was purified. Of these two acids, the C18:0 (stearic acid) alone represents 47.40% of the precipitate. Mc5 releases proteins at the beginning (major peak) and at the end (moderate peak) of the exponential stage of growth. Analysis by denaturing polyacrylamide gel electrophoresis with Mc5 cell walls incorporated as the autolysins substrate identified a band corresponding to PA49.5 in the second peak of protein secretion.

XAS examination of glutathione–cobalt complexes in solution

In the present work, we have investigated the coordination modes of cobalt with glutathione (γ-l-glutamyl-l-cysteinyl-glycine, GSH). A systematic study of cobalt-GSH complexes at basic and neutral pH has been undertaken with a multi-spectroscopic approach combined with quantum chemistry calculations. XAS (x-ray absorption spectroscopy) has been performed at the cobalt K edge in order to shed light into the cation coordination sphere and formal oxidation states. XANES (x-ray absorption near edge structure) enabled to show that in basic and neutral media, cobalt oxidation state is equal to +III and +II respectively. EXAFS (extended x-ray absorption fine structure) provided indications on the donor atoms involved in the coordination with cobalt as well as the bond lengths. DFT (density functional theory)-based calculations and NMR experiments have been performed to assess the most stable structure of the cobalt-GSH complex in basic conditions.

On a novel dihalocyclopropane– dihalomethylvinyl rearrangement: Additional mechanistic evidence

Some additional evidences on the mechanism of new rearrangment of dihalocyclopropane–dihalomethylvinyl reaction assisted by Hiyama reagent Cr