Chiral separation of phospine‐containing α‐amino acid derivatives using two complementary cellulosic stationary phases in supercritical fluid chromatography

Enantiomeric separations of six amino‐acid derivatives have been studied using packed‐column supercritical fluid chromatography with two polysaccharide‐based enantioselective stationary phases: cellulose tris(3,5‐dimethylphenylcarbamate) and cellulose tris(3‐chloro‐4‐methylphenylcarbamate) (Lux Cellulose‐1 and ‐2). The effect of analyte structure on retention and separation was studied. Varied mobile phase compositions were investigated: alcohol modifier percentage was increased from 3 to 40% but smaller amounts were most effective in separating these compounds. Besides, ethanol was preferred to methanol or isopropanol as it proved to be a good compromise to achieve sufficient resolution in a reasonable analysis time. Moreover, a carbon dioxide‐ethanol mixture allows performing analyses in safe and green conditions. The effect of temperature at constant mobile phase composition was explored between 10 and 40°C. In most cases, increasing the temperature improved the chiral separation, up to an optimum temperature. The results are discussed in line with the structure variation of the racemic derivatives analyzed and the two columns are compared. The two columns were shown to provide complementary selectivities for the investigated solutes: whereas Lux 1 provided separation for five of the six racemates, Lux 2 could resolve the last racemic mixture. Finally, optimized conditions of separation are defined. Chirality, 2010. © 2009 Wiley‐Liss, Inc.     

Effects of solvent on chiral and enantiomeric separation of Koga bases using derivatized amylose chiral stationary phase

The separation of R,R‐, S,S‐, and meso‐Koga bases on derivatized amylose chiral stationary phases (CSP) has been studied using different alcohol and alcohol‐hexane mixtures as eluant. Straight‐chain and branched alcohols with carbon numbers from one to four were investigated. The carbon number and geometry of the alcohol impacts the separation of Koga bases. The optimal separations were obtained using a mixture of methanol with linear or branched alcohol. Also, the elution order of meso‐ and R,R‐Koga base was switched as content of branched alcohol increases in cosolvent. The study of acidic and basic additive effects demonstrated that maintaining analytes in the free base state is crucial in order to achieve retention and separation. TEA alone or TEA and TFA mixture were used in the studies. Chirality 2010. © 2009 Wiley‐Liss, Inc.     

A calorimetric and spectroscopic comparison of the effects of ergosterol and cholesterol on the thermotropic phase behavior and organization of dipalmitoylphosphatidylcholine bilayer membranes

We performed comparative DSC and FTIR spectroscopic measurements of the effects of cholesterol (Chol) and ergosterol (Erg) on the thermotropic phase behavior and organization of DPPC bilayers. Ergosterol is the major sterol in the biological membranes of yeasts, fungi and many protozoa. It differs from Chol in having two additional double bonds, one in the steroid nucleus at C7-8 and another in the alkyl chain at C22-23. Erg also has an additional methyl group in the alkyl chain at C24. Our DSC studies indicate that the incorporation of Erg is more effective than Chol is in reducing the enthalpy of the pretransition. At lower concentrations Erg is also more effective than Chol in reducing the enthalpies of both the sharp and broad components of main phase transition. However, at sterol concentrations from 30 to 50 mol%, Erg is generally less effective at reducing the enthalpy of the broad components and does not completely abolish the cooperative hydrocarbon chain-melting phase transition at 50 mol%, as does Chol. Nevertheless, in this higher ergosterol concentration range, there is no evidence of the formation of ergosterol crystallites. Our FTIR spectroscopic studies demonstrate that Erg incorporation produces a similar ordering of liquid-crystalline DPPC bilayers as does Chol, but an increased degree of hydrogen bonding of the fatty acyl carbonyl groups in the glycerol backbone region of the DPPC bilayer. These and other results indicate that Erg is less miscible in DPPC bilayers at higher concentrations than is Chol. Finally, we provide a tentative molecular explanation for the comparative experimental and computation results obtained for Erg and Chol in phospholipid bilayers, emphasizing the dynamic conformational differences between these two sterols.     

Binding of human serum albumin to silica particles by means of polymers: a liquid chromatographic study of the selectivity of resulting chiral stationary phases

Chiral stationary phases obtained by immobilization of human serum albumin (HSA) on various polymer-coated silicas were tested to resolve DL-tryptophan, DL-NBP, RS-oxazepam and RS-warfarin racemic mixtures. HSA immobilized on anion exchangers [quaternized poly(vinylimidazole)-coated silica] was highly selective. Stable and selective chiral stationary phases were also prepared by covalent binding of HSA to silica particles via reactive-polymers. Poly(acryloyl chloride), poly(methacryloyl chloride) and poly(vinyl chloroformate) derivatives were compared. Parameters that govern the selectivity of resulting chiral supports were evaluated, especially the orientation of HSA after immobilization, the mobility of polymer chains and the number of covalent linkages between the protein and the polymer.     

INDUCED DIMORPHIC LIFE CYCLE OF A COCCOLITHOPHORID,CALYPTROSPHAERA SPHAEROIDEA (PRYMNESIOPHYCEAE,HAPTOPHYTA)1

The holococcolith Calyptrosphaera sphaeroidea Schiller was collected at Miyake‐jima Island, Japan and unialgal cultures established. Alternation of the holococcolith and heterococcolith phases was induced using new culture media (MNK, TR, and LO). Cells synchronized in the holococcolith phase were transferred into TR medium to induce a life cycle change. The heterococcolith phase, which has never been reported before, appeared after more than 40 days. The heterococcoliths were very small elliptical discs, about 0.5 μm wide and 1 μm long. Typical diploid‐type organic scales on the cell surface were observed. This phase was very stable in culture and was tolerant of unfavorable conditions. To reverse the life phase, cells in the heterococcolith phase were transferred into cold LO medium and exposed to low temperature (4°C) and low light (2 μmol photons·m^?2·s^?1) for 30 min before culturing at normal conditions (22.5°C and 20 μmol photons· m^?2·s^?1). The swimming behavior of the holococcolith cells seemed to be an indicator of the life cycle phase transition. This article reports for the first time a set of conditions that could control the transition of a coccolithophorid from one life phase to the other. Selected vitamins and trace metals induced the heterococcolith phase, whereas a slightly higher concentration of components in the basic medium along with concomitant stresses of light and temperature induced the holococcolith phase. Based on the results, we propose a hypothesis that the alternation of coccolithophorid life phases is regulated by changes between pelagic and coastal environments coupled with changes in seasonal conditions.     

Chirality of amino acids of microorganisms used in food biotechnology

Bacteria of the genera Acetobacter, Bifidobacterium, Brevibacterium, Lactobacillus, Micrococcus, Propionibacterium, and Streptococcus, which are used as so-called starter cultures for the large-scale production of fermented foods and beverages in food biotechnology, have been investigated for the chirality of their amino acids (AA) by gas chromatography (GC). Bacteria were grown in complex media, centrifuged, and washed with 0.85% aqueous NaCl. Aliquots were totally hydrolyzed (6 M HCl, 110°C, 18 h), or extracted with 70% aqueous ethanol in order to isolated free AA. The AA were adsorbed on Dowex WX 8 cation-exchanger, eluted with 4 M ammonia and converted into their N(O)-trifluoroacetyl(TFA) 2-propyl esters or TFA methyl esters. The AA derivatives were investigated by capillary GC using the chiral stationary phases Chirasil-L -Val, Chirasil-D -Val, and Lipodex E. Besides L -AA, in all bacteria D -amino acids (D -AA) were detected; those in the highest relative amounts were D -Ala and D -Asp (occurring in all bacteria) and, in several cases, D -Glu. Lower, but significant amounts of other D -AA such as D -Ser, D -Pro, D -Val, D -Thr, D -Ile, D -Leu, D -Met, D -Phe, D -Tyr, D -Orn, and D -Lys were also detected in certain bacteria. These findings explain the origin of D -AA found in all fermented foods and drinks produced with the aid of bacterial starter cultures. © 1993 Wiley-Liss, Inc.     

Lipid composition of Sporendonema epizoum

Triglycerides, free fatty acids, free and esterified ergosterol, Q₉, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, lysophosphatidylethanolamine, and three different acylglycoses were identified in the soluble lipids of Sporendonema epizoum mycelium. The same compounds as well as a sterol glycoside were also found in conidia. The mycelium is richer than the conidia in phospholipids, Q₉ and free and esterified ergosterol but contains less glycolipids. The most abundant fatty acid in all non-polar fractions is C_18:2. The prevalent fatty acid of the phospholipids is C_18:1, except for conidial phosphatidylethanolamine and mycelial lysophosphatidylethanolamine.     

Fluorenone 1,4-dihydropyridine derivatives with cardiodepressant activity: Enantiomeric separation by chiral HPLC and conformational aspects

The direct HPLC enantiomeric separation of five fluorenone-1,4-dihydropyridine-3,5-dicarboxylic diesters has been achieved using a Chiralpak AD stationary phase obtaining simultaneously good enantioselectivities, resolution factors, and elution times. CD spectra of the individual enantiomers for two compounds were measured. Thermodynamic parameters associated with the adsorption equilibria of the enantiomers with the chiral stationary phase were obtained from HPLC runs at various temperatures. The conformational preferences of the synperiplanar fluorenone group and of the cis/cis ester groups were obtained by ¹H NMR spectra, including NOE experiments. © 1996 Wiley-Liss, Inc.     

Activity of yeast multidrug resistance pumps during growth is controlled by carbon source and the composition of growth-depleted medium: DiS-C3(3) fluorescence assay

Like other tested wild-type strains (DTXII and IL-125-2B), exponential glucose- and/or fructose-grown cells of Saccharomyces cerevisiae BY4742 exhibit the previously described high activity of Pdr5p and Snq2p pumps (measured as export of the potentiometric fluorescent probe diS-C₃(3)). Upon saccharide depletion from the medium the pump activity in these cells, which differ from other strains in having a lower membrane potential, sharply drops to a very low level similar to that found in cells grown on ethanol or glycerol. This negligible pump activity in respiring cells thus appears to have a universal character.

Addition of glucose or fructose to respiring BY4742 cells grown to low culture densities restores multidrug resistance pump activity due partly to pump synthesis in pre-existing cells and partly to the high pump activity of newly grown cells; no such pump activity boost occurs when the sugar is added to high-density cultures of ethanol-grown or post-diauxic glucose-grown cells, even if these cultures are diluted to low density by their original growth-depleted medium. A strong sugar-induced increase in pump activity is found solely if respiring cells from high-density cultures are resuspended in fresh YPD or YPE medium before sugar addition. Its absence in respiring cells suspended in growth-depleted medium reflects an as yet unidentified effect of the composition of the growth-exhausted medium (depletion of some components and/or accumulation of extracellular metabolites during yeast growth) on sugar-induced pump activity rise.     

Chromatographic enantioseparation by cycloalkylcarbamate derivatives of cellulose and amylose

Cyclopentyl and (+/-)-exo-2-norbornylcarbamates of cellulose and amylose were prepared and their chiral recognition abilities as chiral stationary phases for high-performance liquid chromatography (HPLC) were evaluated. Among these carbamates, cellulose tris(cyclopentylcarbamate) and amylose tris((+/-)-exo-2-norbornylcarbamate) showed particularly high chiral recognition, which is comparable to that of several well-known phenylcarbamate derivatives. The chiral recognition mechanism of cellulose tris(cyclohexylcarbamate), which was previously found to be an effective chiral stationary phase for HPLC, was investigated using NMR spectroscopy. The derivative dissolved in chloroform exhibited the chiral discrimination of several enantiomers in NMR as well as in HPLC. For example, the 1,1'-bi-2-naphthol enantiomers were distinctly discriminated in the (1)H, (13)C, and 2D-NOESY spectra.