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Design and Synthesis of cell nucleus-targeted compounds for use in bioorthogonal reactions
Hrušák, Jáchym ; Vrábel, Milan (advisor) ; Míšek, Jiří (referee)
This bachelor thesis focuses on the design and synthesis of 1,2,4,5-tetrazine and trans- cyclooctene derivatives containing functional moieties which are able to target cellular nucleus and participate in bioorthogonal reactions. Based on literature research, the targeting moieties and the structure of the tetrazine and trans-cyclooctene derivatives were designed with the aim to achieve selective nuclear targeting and to ensure sufficient reactivity of the resulting conjugates under biological conditions. Selected candidates were then synthesized and their properties together with cellular reactivity were investigated in model experiments.
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In Vitro Selection of Aptamers for Methionine Sulfoxide
Jureček, Matěj ; Míšek, Jiří (advisor) ; Bařinka, Cyril (referee)
Oxidation of methionine to methionine sulfoxide in proteins is considered one of important post-translational modifications of proteins. This modification can activate and also inhibit functions of many proteins and it is a part of regulation mechanisms of various (patho)physiological processes. For further research of the effects of methionine oxidation in proteins it would be very helpful to find its bioindicator. So far however, there has not been found any such antibody, nor any of its alternatives. This thesis was concerned with the search of ssDNA aptamer specific for methionine sulfoxide by the method of in vitro selection (SELEX). Several conditions for in vitro selection of methionine sulfoxide were tested in this diploma thesis. None of them led to the enrichment of the starting oligonucleotide pool and no selective aptamer for methionine sulfoxide has been found. Such results don't necessarily point to the impossibility of finding such aptamer, but the conventional methods used in this thesis weren't suitable for this task. In a control in vitro selection there has been found an enriched ssDNA pool for sulforhodamine B as a ligand. Sequencing of clones of this enriched pool has shown oligonucleotides with G-rich sequences, which is typical for already published aptamers for sulforhodamine B.
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Functional screening of de novo proteins
Melikov, Aleksandr ; Hlouchová, Klára (advisor) ; Míšek, Jiří (referee)
Synthetic biology relies upon working with two main types of biological macromolecules - nucleic acids and proteins. Natural proteins represent only a small percentage of the whole amino-acid sequence space. Most of it may conceal an enormous potential (unexplored by nature as well as scientific endeavor), which has started to be carefully explored only in the recent decades. Characterization of non-native proteins includes several key aspects: structure and its stability, function, patterns of interaction with other molecules (of different chemical nature) and in vivo tolerance. This work focuses on the functional testing of de novo polypeptide molecules, either appearing as novelties of genome non-coding regions or as products of artificial bioengineering design. Key words: de novo proteins, function screening, protein libraries, protein design, sequence space
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Dynamers based on the reversible formation of hemiacetals
Nosek, Vladimír ; Drahoňovský, Dušan (advisor) ; Míšek, Jiří (referee)
This work deals with the design and synthesis of building blocks, usable for creating dynamic polymers based on the reversible formation of hemiacetals bond. Next part is focused on the study of the formation of hemiacetal between polyfunctional alcohols and trifluoromethylketones via NMR spectroscopy. Key words: constitutional dynamic chemistry, hemiacetals, trifluoromethylketones, diols and polyols
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Preparation of a library of methionine sulfoxide reductase for applications in synthesis of chiral sulfoxides
Havelka, Václav ; Míšek, Jiří (advisor) ; Hlouchová, Klára (referee)
Chiral sulfoxides are important compounds in the pharmaceutical and chemical industries, however, their enantioselective synthesis providing only one desired enantiomer is not fully mastered. Some natural enzymes can be used for the biocatalytic preparation of chiral sulfoxides. One of such enzymes is methionine sulphoxide reductase. Methionine sulphoxide reductase is an enzyme limiting the effects of reactive oxygen radicals in the organism resulting from oxygen metabolism. Its function is the reduction of methionine sulfoxide in proteins to methionine. There are two types of methionine sulphoxide reductase, methionine sulphoxide reductase A reducing only (S)-methionine sulphoxide and methionine sulphoxide reductase B reducing (R)-methionine sulphoxide. Methionesulfoxide reductase B is suitable for the preparation of (S)-sulfoxides, however its catalytic activity is not sufficient for practical use. Using the recombinant DNA and mutagenic PCR techniques, a methionine sulphoxide reductase B mutant library was prepared, and the extent and nature of mutation introduced was determined. This library will serve as a starting point for the controlled evolution of the enzyme to obtain clones with increased activity and reduced substrate specificity.
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Synthesis of sulphanylaminoderivatives of cyclodextrines and computational simulations of their complexes formation
Provazníková, Adéla ; Jindřich, Jindřich (advisor) ; Míšek, Jiří (referee)
This master thesis deals with synthesis of 6I -N-monosubstituted β-CD bearing a linear spacer of various lenghts containing disulfidic bond and terminated by amine. Whole series was succesfully prepared by nucleophilic substitution of toluenesulfonyl group on tosyl β-CD by a spacer's amine group. In case of the shortest spacer (cystamine) 35 % yield was achieved. Synthesis of β-CD derivatives using longer and more lipophilic spacer derived from di-, tri- and tetraethylenglycol yielded 66 to 85 %. The oligoethylene spacers were prepared by standard chemical modifications. A separation method using n-butanol elution mixtures was optimized for isolation of β-CD derivatives. To better understand the β-CD derivative behavior in solution, molecular dynamics (MD) computations were used. The synthetized species were intended to be used for fluorescent sensor construction. For the same reason the inclusion of small organic molecules in β-CD cavity was studied with molecular dynamic simulations. MD method for computation of relative binding energy was optimized and gave values of right sign and order of magnitude.
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Synthesis of novel helquats and their properties
Joshi, Vishwas ; Teplý, Filip (advisor) ; Šindelář, Vladimír (referee) ; Míšek, Jiří (referee)
The subject of this thesis comprises synthesis of novel helquats, exploration of their different properties, such as nonlinear optical properties, target-specific fluorescence light-up properties, molecular recognition properties (including chiral recognition of dsDNA using enantiopure helquat dyes) and their applicability based on these properties. Four novel helquats containing activated methyl groups were synthesized and one of the synthesized helquats has been successfully resolved in gram scale into two separate enantiomers for further applications. Synthesis of functionalized helquat derivatives employing Knoevenagel condensation reaction between helquats having activated methyl groups and various substituted aromatic and heteroaromatic aldehydes has been developed. This methodology opened an easy access to a structurally diverse library of more than 500 compounds via chromatography-free purifications in moderate to high yield. Members of this compound library have been explored in various directions: as novel nonlinear optical chromophores, target-specific fluorescence light-up probes for recognition of targets like heparin and dsDNA. Few helquat derivatives have shown fluorescence light-up in presence of AT-rich DNA sequences. This feature has been further studied in detail using various...
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Synthesis of cyclodextrin derivatives using methoxymethyl protecting groups
Kasal, Petr ; Jindřich, Jindřich (advisor) ; Míšek, Jiří (referee)
This work deals with the use of methoxymethyl protecting group in chemistry of cyclodextrins, where this group has not been used very often so far. The work deals with development of optimal methods for introduction and for subsequent removal of the group and shows its usefulness in the preparation of new 6I -O-monosubstituted cyclodextrin derivatives, which are hard to get by standard methods. Key words: cyclodextrin derivatives, methoxymethyl group, protecting group
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Characterization of organogold compounds
Shcherbachenko, Elena ; Roithová, Jana (advisor) ; Míšek, Jiří (referee)
Geminally diaurated complexes are important intermediates involved in some of the gold catalyzed reactions and therefore they have become a subject of the current research. The aim of this thesis was to assess the influence of electronic structure of an aryl ligand on the stability of gold complexes with three-center-two-electron interaction Au2C. A series of geminally diaurated complexes was obtained by a reaction of gold complex [(IPr)Au(NCMe)]BF4 (IPr = 1,3-bis(2,6-di-iso-propylphenyl)imidazol-2-ylidene) with arylboronic acids ArB(OH)2 bearing different substituents at the aryl group (Ar = para- O2NC6H4, para-NCC6H4, para-ClC6H4, C6H5, para-H3CC6H4, para-H3COC6H4, meta- O2NC6H4, meta-NCC6H4, meta-H3CC6H4, ortho-O2NC6H4, ortho-NCC6H4, ortho-H3CC6H4). Mass spectrometry (MS) was used as the main experimental method in this study. Appearance energies (AE) for the fragmentation of geminally diaurated complexes were estimated by the methods MS/MS. The obtained energies were plotted against the corresponding Hammett σ-constants to assess the correlation between the electronic structure of a given aryl ligand and the stability of the Au2C bond. It was found that structures of gold complexes derived from the cyanophenylboronic acids do not correspond to the geminally diaurated complexes but to the...
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