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Use of chiral stationary phases based on teicoplanin and teicoplanin aglycone for enantioseparation of FMOC-derivatized amino acids.
Repko, Pavel ; Bosáková, Zuzana (advisor) ; Tesařová, Eva (referee)
In this work, an enantioselective HPLC method with UV and fluorimetric detection was developed and subsequently optimized for chiral separation of four aminoacids (D/L-alanine, D/L-valine, D/L-leucine, D/L-isoleucine) in native and particularly in derivatized form with an emphasis on enantioseparation of D-analogues. Retention and enantioseparation behavior of studied analytes was investigated on three chiral stationary phases based on teicoplanin (Chirobiotic T, Chirobiotic T2) and teicoplanin aglycone (Chirobiotic TAG). At the Chirobiotic T column, enantioseparations of underivatized aminoacids were performed with UV detection at 205 nm in the mobile phases methanol/water with different volume ratios. Baseline separation of L- and D-forms was achieved, however, the sensitivity of detection was very low. In order to increase detection sensitivity, derivatization of aminoacids was performed using 9-fluorenylmethyl chloroformate (FMOC-Cl) and the derivatization procedure was monitored on Chirobiotic T column with fluorimetric detection (λEx = 254 nm, λEm = 314 nm) in a buffered mobile phase methanol/0.5% TEAA buffer, pH 6.0 40/60 (v/v). In terms of derivatization, volume ratio D/L-aminoacid/derivatization agent 1/1 with ten times higher concentration of derivatization agent was found to be the most suitable....
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Chiral HPLC separation of newly synthesized amino acids
Kučerová, Gabriela ; Kalíková, Květa (advisor) ; Riesová, Martina (referee)
The aim of this work was to develop and to optimize HPLC method for enantioseparation of newly synthesized derivatives of amino acids. The set of these analytes contained four N- blocked derivatives of D,L-Phenylalanine, three N-unblocked derivatives of D,L- Phenylalanine, , one methylated derivative of D,L-Tyrosine and D,L-Tyrosine. Two separation modes i.e. reversed phase and polar-organic modes and two columns i.e. Chirobiotic® T and Chirobiotic® T2 were used. Chiral stationary phases of these columns were composed of macrocyclic antibiotic teicoplanin coated on silica gel support. As mobile phases in revesed phase mode, methanol and acetate buffer were used with Chirobiotic® T column. The most suitable concentration were 20 mM and the most suitable pH value were 4.00. Under the above mentioned conditions eight analytes of ten were separated and the optimal conditions were found. Polar-organic mode and Chirobiotic® T column were suitable only for enantioseparation of three N-blocked derivatives of D,L- Phenylalanine. Mobile phases were composed of methanol with small additions of triethylamine and acetic acid. Chirobiotic® T2 column in reversed phase mode was not suitable for tested set of analytes. Only partial separation of D,L-Tyrosine derivative and one N-unblocked D,L-Phenylalanine...
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The effect of vanZTei and vanZg expression on resistance to glycopeptide antibiotics in Staphylococcus aureus
Zieglerová, Leona ; Balíková Novotná, Gabriela (advisor) ; Lichá, Irena (referee)
A membrane protein VanZTei which is encoded by the gene vanZ from the vanA glycopeptide resistance gene cluster is a part of the large family of VanZ proteins. VanZTei confers resistance to teicoplanin in Enterococcus faecalis without the presence of other proteins encoded by the cluster. The aim of my work was to compare the ability of two orthologous proteins VanZTei and VanZg (from the genome of Enterococcus faecium) to confer resistance to glycopeptides in Staphylococcus aureus RN4220 and Enterococcus faecium. We have shown that VanZg increases resistance to teicoplanin (Tei) 8 to 16 times the and also to dalbavancin (Dalb) 8 times. VanZTei also confers resistance to Tei and Dalb, but the increase is only twofold. Conversely VanZTei confers resistance to newly synthetized glycopeptides more effectively than VanZg (fourfold increase of resistance confered by VanZTei and two to fourfold increase of resistance confered by VanZg). It suggests that both proteins have different specificity to antibiotics. In despite the mutants of S. aureus RN4220 VanZTei pRMC2 with increased resistance to teicoplanin (MICTei> 8 µg/ml) in which the resistance is dependent on vanZTei expression were selected. These resistant mutants do not carry mutation in a gene vanZTei or in its ribosomal binding site. Neither of the...
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Chiral HPLC separation of newly synthesized amino acids
Kučerová, Gabriela ; Kalíková, Květa (advisor) ; Riesová, Martina (referee)
The aim of this work was to develop and to optimize HPLC method for enantioseparation of newly synthesized derivatives of amino acids. The set of these analytes contained four N- blocked derivatives of D,L-Phenylalanine, three N-unblocked derivatives of D,L- Phenylalanine, , one methylated derivative of D,L-Tyrosine and D,L-Tyrosine. Two separation modes i.e. reversed phase and polar-organic modes and two columns i.e. Chirobiotic® T and Chirobiotic® T2 were used. Chiral stationary phases of these columns were composed of macrocyclic antibiotic teicoplanin coated on silica gel support. As mobile phases in revesed phase mode, methanol and acetate buffer were used with Chirobiotic® T column. The most suitable concentration were 20 mM and the most suitable pH value were 4.00. Under the above mentioned conditions eight analytes of ten were separated and the optimal conditions were found. Polar-organic mode and Chirobiotic® T column were suitable only for enantioseparation of three N-blocked derivatives of D,L- Phenylalanine. Mobile phases were composed of methanol with small additions of triethylamine and acetic acid. Chirobiotic® T2 column in reversed phase mode was not suitable for tested set of analytes. Only partial separation of D,L-Tyrosine derivative and one N-unblocked D,L-Phenylalanine...
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Use of chiral stationary phases based on teicoplanin and teicoplanin aglycone for enantioseparation of FMOC-derivatized amino acids.
Repko, Pavel ; Bosáková, Zuzana (advisor) ; Tesařová, Eva (referee)
In this work, an enantioselective HPLC method with UV and fluorimetric detection was developed and subsequently optimized for chiral separation of four aminoacids (D/L-alanine, D/L-valine, D/L-leucine, D/L-isoleucine) in native and particularly in derivatized form with an emphasis on enantioseparation of D-analogues. Retention and enantioseparation behavior of studied analytes was investigated on three chiral stationary phases based on teicoplanin (Chirobiotic T, Chirobiotic T2) and teicoplanin aglycone (Chirobiotic TAG). At the Chirobiotic T column, enantioseparations of underivatized aminoacids were performed with UV detection at 205 nm in the mobile phases methanol/water with different volume ratios. Baseline separation of L- and D-forms was achieved, however, the sensitivity of detection was very low. In order to increase detection sensitivity, derivatization of aminoacids was performed using 9-fluorenylmethyl chloroformate (FMOC-Cl) and the derivatization procedure was monitored on Chirobiotic T column with fluorimetric detection (λEx = 254 nm, λEm = 314 nm) in a buffered mobile phase methanol/0.5% TEAA buffer, pH 6.0 40/60 (v/v). In terms of derivatization, volume ratio D/L-aminoacid/derivatization agent 1/1 with ten times higher concentration of derivatization agent was found to be the most suitable....
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