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Chiral separation of biologically active compounds by chromatography
Landl, David ; Kalíková, Květa (advisor) ; Kubíčková, Anna (referee)
- 4 - Abstract in English This diploma thesis is focused on the evaluation and comparison of the enantioselective potential of two columns CHIRAL ART Amylose-SA and CDShell-RSP using a set of 29 chiral drugs in high performance liquid chromatography. The separations of enantiomers were performed in three modes: reversed-phase, normal-phase, and polar- organic mode. The CHIRAL ART Amylose-SA column was tested in normal-phase mode, the CDShell-RSP column in reversed-phase and polar-organic modes. The CHIRAL ART Amylose-SA column contains amylose tris(3,5- dimethylphenylcarbamate) immobilized on 3 µm porous silica gel particles. The CDShell-RSP column contains a chiral selector hydroxypropyl-β-cyclodextrin, which is covalently bonded on 2.7 µm superficially porous particles. In the normal-phase mode, mobile phases composed of hexane and propane-2-ol were used. Furthermore, the effect of various additives (triethylamine, diethylamine, trifluoroacetic acid, and the mixture of diethylamine and trifluoroacetic acid) in the mobile phase on the enantioseparation of chiral drugs was tested. The most universal additive was a mixture of diethylamine and trifluoroacetic acid. A total of 22 chiral drugs were enantioseparated on the CHIRAL ART Amylose-SA column, 10 of them were baseline separated. Mobile phases for...
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Preparation of cytochrome b5 mutants containing photoreactive amino acids, and their crosslinking with the interaction partners
Landl, David ; Ječmen, Tomáš (advisor) ; Kukačka, Zdeněk (referee)
Cytochrome b5 is an electron transport protein of a clinically prominent mixed-function oxygenase (MFO) system. This system participates in the first stage of xenobiotic biotransformation. The hydrofility of xenobiotics is increased by introduction of an oxygen atom into their structure. The MFO system also activates or deactivates certain drugs and carcinogens. Cytochrome b5 affects reactions catalyzed by the terminal oxygenases of the system - cytochromes P450. Electrons are donated to cytochrome b5 by redox partners NADH:cytochrome b5 reductase and NADPH:cytochrome P450 reductase. The aim of this work was to demonstrate capability of photo-crosslinking approach to fixate transient interactions within MFO system. Covalent complexes obtained by this technique could be further analyzed by mass spectrometry, which can provide structural information about the binding sites of the proteins. We prepared a mutant cytochrome b5 comprising photo-reactive methionine analogue in the position 41 of the sequence. We expressed the protein employing E. coli B834 (DE3) auxotrophic strain in 300 ml of the limit medium supplemented with L-2- amino-5,5-azi-hexanoic acid (photo-methionine). The rate of the unnatural amino acid incorporation was determined by mass spectrometry and it was about 40 % after 16 hours of...
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Chiral separation of biologically active compounds by chromatography
Landl, David ; Kalíková, Květa (advisor) ; Kubíčková, Anna (referee)
- 4 - Abstract in English This diploma thesis is focused on the evaluation and comparison of the enantioselective potential of two columns CHIRAL ART Amylose-SA and CDShell-RSP using a set of 29 chiral drugs in high performance liquid chromatography. The separations of enantiomers were performed in three modes: reversed-phase, normal-phase, and polar- organic mode. The CHIRAL ART Amylose-SA column was tested in normal-phase mode, the CDShell-RSP column in reversed-phase and polar-organic modes. The CHIRAL ART Amylose-SA column contains amylose tris(3,5- dimethylphenylcarbamate) immobilized on 3 µm porous silica gel particles. The CDShell-RSP column contains a chiral selector hydroxypropyl-β-cyclodextrin, which is covalently bonded on 2.7 µm superficially porous particles. In the normal-phase mode, mobile phases composed of hexane and propane-2-ol were used. Furthermore, the effect of various additives (triethylamine, diethylamine, trifluoroacetic acid, and the mixture of diethylamine and trifluoroacetic acid) in the mobile phase on the enantioseparation of chiral drugs was tested. The most universal additive was a mixture of diethylamine and trifluoroacetic acid. A total of 22 chiral drugs were enantioseparated on the CHIRAL ART Amylose-SA column, 10 of them were baseline separated. Mobile phases for...
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Preparation of cytochrome b5 mutants containing photoreactive amino acids, and their crosslinking with the interaction partners
Landl, David ; Ječmen, Tomáš (advisor) ; Kukačka, Zdeněk (referee)
Cytochrome b5 is an electron transport protein of a clinically prominent mixed-function oxygenase (MFO) system. This system participates in the first stage of xenobiotic biotransformation. The hydrofility of xenobiotics is increased by introduction of an oxygen atom into their structure. The MFO system also activates or deactivates certain drugs and carcinogens. Cytochrome b5 affects reactions catalyzed by the terminal oxygenases of the system - cytochromes P450. Electrons are donated to cytochrome b5 by redox partners NADH:cytochrome b5 reductase and NADPH:cytochrome P450 reductase. The aim of this work was to demonstrate capability of photo-crosslinking approach to fixate transient interactions within MFO system. Covalent complexes obtained by this technique could be further analyzed by mass spectrometry, which can provide structural information about the binding sites of the proteins. We prepared a mutant cytochrome b5 comprising photo-reactive methionine analogue in the position 41 of the sequence. We expressed the protein employing E. coli B834 (DE3) auxotrophic strain in 300 ml of the limit medium supplemented with L-2- amino-5,5-azi-hexanoic acid (photo-methionine). The rate of the unnatural amino acid incorporation was determined by mass spectrometry and it was about 40 % after 16 hours of...
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