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The development of an HPLC method for monitoring the preparation of oligodeoxynucleotide probes
Šemlej, Tomáš ; Kučera, Radim (advisor) ; Pilařová, Pavla (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Chemistry and Pharmaceutical Analysis Candidate: Mgr. Tomáš Šemlej Supervisor: doc. PharmDr. Radim Kučera, Ph.D. Title of rigorosum thesis: The development of an HPLC method for monitoring the preparation of oligodeoxynucleotide probes An artificially prepared oligodeoxynucleotide sequence with the attached label is referred to as a molecular probe. Molecular probes are mainly used to quantify multiplication in RT-PCR and identify mutations in PCR analyses. There are typically two different types of probes - mono-labeled molecular probes and double-labeled molecular probes. The mono-labeled molecular probes are constructed from fluorophore- labeled oligodeoxynucleotide sequence or quencher-labeled oligodeoxynucleotide sequence. Whereas double-labeled molecular probes consist of an oligodeoxynucleotide sequence with a fluorophore attached at one end together with a quencher attached at the other end of the chain. In this work, we first paid attention to the separation of double- labeled oligodeoxynucleotides from mono-labeled oligodeoxynucleotides. Several types of stationary phases were tested and based on that the column Clarity® Oligo-RP™ was chosen to separate a mixture of labeled oligodeoxynucleotides. This...
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Development of catalytic systems suitable for the enantioselective synthesis of heterocycles
Dočekal, Vojtěch ; Veselý, Jan (advisor) ; Cibulka, Radek (referee) ; Pospíšil, Jiří (referee)
The development of novel methodologies and synthetic strategies for the preparation of complex 3D heterocyclic compounds with the well-defined spatial arrangement of functional groups represents one of the most studied research problems in current synthetic chemistry. Nowadays, organocatalysis (beside transition-metal catalysis and biocatalysis) became the most popular tool, because of usage small chiral organic compound for asymmetric induction and acceleration of reaction. In this thesis, the development of novel organocatalytic methodologies for the preparation of various heterocyclic compounds is described. The utility of novel methodologies is demonstrated by easy access to chiral building block molecules or products with valuable physical or biological properties. In the first part, a novel [4+2] cycloaddition reaction of sulfur-containing heterocyclic electron-deficient alkenes with allenic compounds catalyzed by chiral tertiary amine is described. Organocatalytic cycloaddition reaction of 3-benzylidene benzo[b]thiophenones with allenoates was investigated and optimized. Dihydro-2H-pyranes were obtained in high isolated yields (up to 92%) and optical purities (up to 99% ee). The robustness and synthetic utility of the developed methodology was demonstrated by a simple work-up of...
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Hydrosolubilization of BODIPY for optical labelling of biomolecules
Bartoň, Jan ; Kotek, Jan (advisor) ; Vrábel, Milan (referee)
1 Abstract This work aims at showing synthesis and potential use of water-soluble fluorescent probes based on BODIPY. The preparation of probes containing bioorthogonal mono- and heterobifunctional functional groups was demonstrated. Ground work was done at the optimisation of reliable, scalable and fast sulfonation of BODIPY in 2,6-positions. A protocol for handling sulfonated BODIPY has been established; especially for the exchange of counterions. In counterion se- lection, their relation to synthetic pathway and biocompatibility were taken into consideration. The second part of the work shows series of water-soluble fluorescent probes, into which can be easily introduced bioactive or bioorthogonal functional groups. This can be used for click chemistry in connection with turn off/on probes or fluorescent sensing of molecules or ions. All this can be done in aqueous solution without organic solvents, which is relevant for biochemical, analytical and imaging applications. Keywords BODIPY, bifunctional, water-soluble, fluorescent probe, solubilization, biocompa- tible probes, bioorthogonal reaction, BODIPY sulfonation
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Bifunctional BODIPY for optical labelling of biomolecules
Bartoň, Jan ; Kotek, Jan (advisor) ; Henke, Petr (referee)
The water-soluble derivate of BODIPY was prepared, which will be further modified in order to prepare bioorthogonal bifunctional BODIPY. Target application of this derivate is fluorescent probe for labelling of biomolecules. Main goals of this thesis were optimalization of synthesis of BODIPY core and sulfonation to positions 2,6. It was found out that sodium salt of sulfonated BODIPY shows good solublility in water and methanol, but it is poorly soluble in less polar solvents. Based on these findings DIPEA salt of sulfonated BODIPY was prepared, which shows outstanding range of solubility from water to dichlormethane. These findings will be used for synthesis of bifunctional water- soluble BODIPY. Further, the potential of Pd-BODIPY complex for detection of CO was reasserted which will be further investigated. Keywords BODIPY, bifunctional, water-soluble, fluorescent probe, fluorescence, protein labelling, solubilization
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