Enantioseparation by HPLC using phenylcarbonate, benzoylformate, p-toluenesulfonylcarbamate, and benzoylcarbamates of cellulose and amylose as chiral stationary phases

Phenylcarbonate, benzoylformate, and p-toluenesulfonylcarbamate of cellulose and five new benzoylcarbamate derivatives of both cellulose and amylose were synthesized and their chiral recognition abilities were evaluated as chiral stationary phases (CSPs) for high-performance liquid chromatography (HPLC). Cellulose benzoylcarbamate has a higher chiral recognition ability compared to phenylcarbonate, p-toluenesulfonylcarbamate, and benzoylformate of cellulose. The benzoylcarbamate derivatives exhibited a characteristic chiral recognition for the racemates, which bear a hydrogen atom capable of hydrogen bonding to the carbonyl group of the benzoylcarbamates. The structures of the benzoylcarbamates were investigated by CD spectroscopy.     

Purification,Cloning and Functional Expression of hydroxyphenylpyruvate Reductase Involved in Rosmarinic Acid Biosynthesis in Cell Cultures of Coleus Blumei

Hydroxyphenylpyruvate reductase (HPPR) is an enzyme involved in the biosynthesis of rosmarinic acid in Lamiaceae reducing hydroxyphenylpyruvates in dependence of NAD(P)H to the corresponding hydroxyphenyllactates. The HPPR protein was purified from suspension cells of Coleus blumei accumulating high levels of rosmarinic acid by ammonium sulfate precipitation, anion exchange chromatography, hydroxylapatite chromatography, chromatography on 2',5'-ADP-Sepharose 4B and SDS-polyacrylamide gel electrophoresis. The protein was tryptically digested and the peptides sequenced. Sequence information was used to isolate a full-length cDNA-clone for HPPR (EMBL accession number AJ507733) by RT-PCR, screening of a C. blumei cDNA-library and 5'-RACE-PCR. The open reading frame of the HPPR-cDNA consists of 939 nucleotides encoding a protein of 313 amino acid residues. The sequence showed that HPPR belongs to the family of D-isomer-specific 2-hydroxyacid dehydrogenases. The HPPR-cDNA was heterologously expressed in Escherichia coli and the protein was shown to catalyse the NAD(P)H-dependent reduction of 4-hydroxyphenylpyruvate to 4-hydroxyphenyllactate and 3,4-dihydroxyphenylpyruvate to 3,4-dihydroxyphenyllactate.     

Enantioseparation on 4-halogen-substituted phenylcarbamates of amylose as chiral stationary phases for high-performance liquid chromatography

Four 4-halogen-substituted phenylcarbamate derivatives of amylose were prepared and their chiral recognition abilities as chiral stationary phases (CSPs) for high-performance liquid chromatography (HPLC) were evaluated and compared with those of the corresponding cellulose derivatives. The amylose derivatives with fluoro, chloro, bromo, or iodo group at the four-position on the phenyl group were found to show higher chiral resolving ability than the corresponding cellulose derivatives. Among four amylose derivatives 4-fluoro- and 4-chlorophenylcarbamates showed an excellent chiral recognition ability. Especially, amylose tris(4-chlorophenylcarbamate) resolved (±)-1,2,2,2-tetraphenylethanol with a very high α value (α = 8.29). In order to obtain useful information concerning the chiral recognition mechanism of this resolution, we also performed enantioseparation of a variety of analogous racemic alcohols, and found that both the hydroxy and bulky triphenylmethyl groups of the racemate are essential for the effective chiral recognition. Chirality 9:63–68, 1997. © 1997 Wiley-Liss, Inc.     

Growth effects and metabolism of arginine in stationary and rapidly-dividing sugarcane cell suspensions

The effect of 300 μ M arginine on growth of sugarcane cell suspensions was studied. Cells transferred to defined media in the stationary growth stage showed a greater requirement for exogenous arginine than cells similarly transferred in the rapidly dividing stage. Cell arginine levels, rates of arginine synthesis, and enzymes of arginine synthesis all decreased in cells entering the stationary stage. It is concluded that stationary stage cells are deficient in their ability to synthesize arginine and are therefore dependent upon an exogenous supply to resume growth in fresh media.     

Catabolism of flavonol glucosides in plant cell suspension cultures

Catabolism of flavonol glucosides was investigated in plant cell suspension cultures using kaempferol 3-O-β-d-glucoside and kaempferol 7-O-β-d-glucoside labelled with ¹⁴C either in the glucose or in the flavonol moiety. Catabolic rates of glucosides were compared with those of free glucose and kaempferol. All substrates were degraded efficiently by cell cultures of mungbean, soybean, garbanzo bean and parsley. Based on ¹⁴CO₂-formation, glucose from position 3 of kaempferol is 3–5 times more rapidly metabolized than that from position 7. The flavonol nucleus from both isomers is, however, oxidized to the same extent with a considerable portion of the flavonol being incorporated into insoluble polymeric cell material.     

Investigation of the enantioselectivity and enantiomeric elution order of some piperidine-2,6-dione drugs on chiralcel OD column

The enantioselectivity and enantiomeric separation of five racemic piperidine-2,6-dione compounds, on the cellulose tris(3,5-dimethylphenyl carbamate) chiral stationary phase Chiralcel OD-CSP were investigated under the same chromatographic conditions. This class of drugs includes glutethimide, aminoglutethimide, cyclohexylaminoglutethimide, pyridoglutethimide, and phenglutarimide. The results revealed that chiral recognition and the binding sites of these drugs on the Chiralcel OD column are similar, regardless of the absolute configuration of the individual enantiomers. A possible chiral recognition mechanism(s) for this class of drugs and the CSP is presented. © 1994 Wiley-Liss, Inc.     

Selection and characterization of an L-thiazolidine-4-carboxylic acid resistant callus culture of Vigna radiata (L.) Wilczek var. radiata

Callus cultures were established from seedling root tips of mungbean (Vigna radiata (L.) Wilczek var. radiata) cv. K 851. The growing calli were exposed to increasing concentrations of thioproline — an analog of proline, in the medium. A concentration of 3.0 mM thioproline completely inhibited the growth of the cells. However, after 25 days incubation 5 cell clones were obtained which could grow on this concentration of thioproline. Out of them one vigorously growing cell clone was further characterized. This selected clone contained higher endogenous levels of free proline (5 fold) and K⁺ (1.5 fold) and exhibited elevated tolerance, not only to thioproline but also to exogenously applied NaCl in the growth medium, as compared to the normal sensitive callus cells. Higher endogenous levels of free proline and K⁺ appear to impart dual resistance to thioproline and NaCl to the selected cell strain.     

Foaming and cell flotation in suspended plant cell cultures and the effect of chemical antifoams

Foam development and stability in Atropa belladonna suspensions were investigated as a function of culture conditions. Foaming was due mainly to properties of the cell-free broth and was correlated with protein content; effects due to presence of cells increased towards the end of batch culture. Highest foam levels were measured 11 days after inoculation. Air flow rate was of major importance in determining foam volume; foam volume and stability were also strongly dependent on pH. Foam flotation of plant cells was very effective. After 30 min foaming, ca. 55% of cells were found in the foam; this increased to ca. 75% after 90 min. Polypropylene glycol 1025 and 2025, Pluronic PE 6100, and Antifoam-C emulsion were tested as chemical antifoams. Polypropylene glycol 1025 and Antifoam C at concentrations up to 600 ppm had no adverse effect on growth in shake flasks; Pluronic PE 6100 has an inhibitory effect at all levels tested. Concentrations of polypropylene glycol 2025 and Pluronic PE 6100 as low as 20 ppm reduced foam volumes by a factor of ca. 10. Addition of antifoam reduced k(L)a values in bubble-column and stirred-tank bioreactors. After operation of a stirred reactor for 2 days using Antifoam C for foam control, cell production was limited by oxygen due to the effect of antifoam on mass transfer. Theoretical analysis showed that maximum cell concentrations and biomass levels decline with increasing reactors working volume due to greater consumption of antifoam to prevent foam overflow. The results indicate that when chemical foam control is used in plant cell cultures, head-space volume and tolerable foam levels must be considered to optimize biomass production. (c) 1994 John Wiley & Sons, Inc.     

Hormonal effects on diosgenin biosynthesis and growth in Dioscorea deltoidea tissue cultures

Analysis of Dioscorea deltoidea tissue cultures grown in the presence of 2,4-D, indole-3-butyric acid, isopentenyladenine, benzyladenine and GA singly and in combination showed that the medium with 2,4-D most consistently favored diosgenin production. GA and high benzyladenine concentrations were toxic.     

Elicitation of different Panax ginseng transformed root phenotypes for an improved ginsenoside production

We tested the effect of the presence in the culture medium of chitosan, vanadyl sulfate or methyl jasmonate on growth and ginsenoside production of three stable hairy root lines of Panax ginseng C.A. Meyer showing different morphological phenotypes C-M, HR-M and T-M. The response depended upon line phenotype, specificity of the elicitor and the stage of growth at which the lines were treated. The highest ginsenoside yield was found when methyl jasmonate was added during the progressive deceleration growth phase (on day 25 of culture). In this case, the ginsenoside content reached at the end of the culture (day 28) by root lines C-M, HR-M and T-M was, respectively, 2, 1.8 and 4 times higher than the highest content achieved, also at the end of the culture, by the corresponding untreated root lines. Under the same conditions, the ginsenoside content in the presence of vanadyl sulfate also increased considerably, while with chitosan it clearly decreased. The ginsenoside pattern in response to the presence of the elicitors is also considered.