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Analysis and isolation of intermediates involved in the biosynthetic pathway of alkylproline derivatives
Cudlman, Lukáš ; Bosáková, Zuzana (advisor) ; Michalíková, Klára (referee)
(EN) This work aims at preparation, analysis and isolation of intermediates of biosynthetic pathways of 4-alkyl-L-proline derivatives for their structural elucidation. Compounds with incorporated 4-alkyl-L-proline derivatives include clinically used lincosamide antibiotic, lincomycin A, antitumor pyrrolobenzodiazepines and bacterial hormone hormaomycin. Detailed knowledge of biosynthetic pathways of these biological active substances can be used to prepare new, more efficient derivatives. The first part of this work focuses on yellow-coloured dicarboxylic intermediates 1 and 2 of the biosynthetic pathway of 4-propyl-L-proline - the precursor of lincomycin A. In the presence of the methylation agent, S-adenosyl-L-methionine, and LmbW C- -methyltransferase, 1 was partially converted into intermediate 2. Using ultra-high performance liquid chromatography, both intermediates were identified from absorption and mass spectrometry spectra. A semi-preparative chromatographic method for isolation of both intermediates was developed. Surprisingly, a significantly lower stability of 2 compared to intermediate 1 was observed in an in vitro enzymatic reaction mixture. The second part of the work focuses on 4-ethylidene-L-proline - the precursor of tomaymycin belonging to pyrrolobenzodiazepines. After...
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Analysis and isolation of intermediates involved in the biosynthetic pathway of alkylproline derivatives
Cudlman, Lukáš ; Bosáková, Zuzana (advisor) ; Michalíková, Klára (referee)
(EN) This work aims at preparation, analysis and isolation of intermediates of biosynthetic pathways of 4-alkyl-L-proline derivatives for their structural elucidation. Compounds with incorporated 4-alkyl-L-proline derivatives include clinically used lincosamide antibiotic, lincomycin A, antitumor pyrrolobenzodiazepines and bacterial hormone hormaomycin. Detailed knowledge of biosynthetic pathways of these biological active substances can be used to prepare new, more efficient derivatives. The first part of this work focuses on yellow-coloured dicarboxylic intermediates 1 and 2 of the biosynthetic pathway of 4-propyl-L-proline - the precursor of lincomycin A. In the presence of the methylation agent, S-adenosyl-L-methionine, and LmbW C- -methyltransferase, 1 was partially converted into intermediate 2. Using ultra-high performance liquid chromatography, both intermediates were identified from absorption and mass spectrometry spectra. A semi-preparative chromatographic method for isolation of both intermediates was developed. Surprisingly, a significantly lower stability of 2 compared to intermediate 1 was observed in an in vitro enzymatic reaction mixture. The second part of the work focuses on 4-ethylidene-L-proline - the precursor of tomaymycin belonging to pyrrolobenzodiazepines. After...
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Development of microemulsion electrokinetic chromatographic method for the analysis of fat-soluble dyes illegally used in foodstuffs
Bradová, Jana ; Michalíková, Klára (advisor) ; Pospíšilová, Marie (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Jana Bradová Supervisor: Mgr. Klára Petrů Ph.D. Consultant: Doc. RNDr. Miroslav Polášek, CSc. Title of diploma thesis: Development of microemulsion electrokinetic chromatography method for the analysis of illegal fat-soluble foodstuff dyes A microemulsion electrokinetic chromatography (MEEKC) method was developed and proposed for the determination of fat-soluble dyes (Sudan I, Sudan II, Sudan III, Sudan Red 7B, Sudan Orange G, and Methyl Red) illegally used in foodstuffs. The effect of surfactant, co-surfactant, organic modifier and oil as well as the capillary length were examined in order to optimize the separation. Final background electrolyte (solution of the microemulsion) for MEEKC was composed of 30mM phosphate buffer (pH 7.5), 1.2 % (w/v) sodium dodecyl sulfate, 1.2% (v/v) of n-hexane, 15% (v/v) of butan-1-ol, and 20% (v/v) of acetonitrile. A baseline separation of these six dyes was achieved within 11 min by using fused-silica capillary with 75 µm i.d. and effective length 36.5 cm. The applied voltage was 20 kV and temperature 25řC was maintained. The VIS detection wavelengths were 500 and 400 nm. The repeatability of the migration times and peak areas were characterized by RSD...
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Separation and assay of indomethacin and its degradation products by micellar electrokinetic chromatography
Mitlenerová, Dagmar ; Michalíková, Klára (advisor) ; Honegr, Jan (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Dagmar Mitlenerová Supervisor: PharmDr. Klára Petrů, Ph.D. Consultant: doc. RNDr. Miroslav Polášek, CSc. Title of Diploma Thesis: Micellar electrokinetic chromatography for the separation and assay of indomethacin and its degradations products A new method of micellar electrokinetic chromatography (MEKC) for analysis of indomethacin and its degradation products, 4-chlorbenzoic acid (4CHBA) and 5-methoxy- 2methy-3indolylacetic acid (MMIAA), was developed and optimized. The separation was carried out in uncoated fused silica capillary (with i.d. 75 µm, total length 33 cm and effective length 24,5 cm) with applied voltage 15 kV and temperature of capillary 25ř C. Wave length for UV detection was 224 nm and 234 nm. The optimized background electrolyte contained: 10mM phosphate buffer, 60mM SDS (pH 7,0) and 10% (v/v) of methanol. Internal standard was 1-naftylacetic acid. Large volume sample stacking method was used for sensitivity enhancement. The mixture of standards was injected hydrodynamically at 100 mBar for 10 s (24% of capillary total length). Voltage -7,5 kV was applied for stacking of the analytes. Polarity was switched to +15 kV (time of switching 1,5 min), when 95% of original...
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Communication Taboo in the Family
Michalíková, Klára ; Rampouchová, Jaroslava (advisor) ; Krahulcová, Beáta (referee)
In the preface of this work I talking about quality of our life. Work on the assumption life should be lived as good as possible. We can't do this without understanding who we are? What we are feeling? What we need? What our neighbour need? What they are feeling? It's obvious, our family formed us in the most sensitive period of our life. We are without experiences, vulnerable. We will formed our children in the same way. I show the taboo from the many views and positions. Taboo is unremitting and still changing component of our lifes. The separation taboo from natural keeping secrets - privacy is very important. I used taboo as an affect negatively instrument in family communication. Taboo can interrupt the family communication.
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