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Study of DNA-compatible Reactions and Their Utilisation for DNA Encoded Libraries
Havelka, Václav ; Míšek, Jiří (advisor) ; Kožíšek, Milan (referee)
DNA-encoded peptide libraries are the basis for in vitro selection methods that use various biological systems (phage display; yeast display; mRNA display). Despite the great success of these selection methods, their obvious disadvantage is the limited number of building blocks, which consist of only twenty proteinogenic amino acids. The involvement of other non- proteinogenic amino acids and other building blocks could significantly expand the range of possible applications of these selection methods. For example, the introduction of chemical modifications in amino acid side chains in such libraries would allow the effective study of post-translational modifications (phosphorylation, acylation, glycosylation, methylation, etc.) in living organisms. The aim of this work was to develop a method for preparation of a fully synthetic DNA encoded library of peptides. The basic steps for the preparation were the chemical synthesis of the peptide and associated enzymatic synthesis of encoding DNA. Compatibility of chemical reactions with DNA is essential for the synthesis of DNA-encoded peptide libraries. Because the final acidic deprotection of the side chains in the peptide is not compatible with DNA, two approaches have been tested to overcome this problem. The first was an attempt to develop finer...
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Phenotyping of proteolytic activities enabled by fluorogenic libraries
Pospíšil, Šimon ; Míšek, Jiří (advisor) ; Hocek, Michal (referee)
This work deals with the preparation of combinatorial libraries of peptides serving as platforms for proteolytic phenotyping. The primary objective was to prepare a solid phase fluorogenic peptide library and screen proteases by fluorescence. Further, the possibility of preparing solid phase DNA-encoded libraries was studied. Due to the non-reactivity of the specific proteases with the solid phase peptides, the solid phase was completely abandoned and DNA-encoded peptide library was prepared in the solution. Using this model of DNA-encoded dipeptide with terminal biotin, the new principle of testing proteolytic activities of proteases was verified. A combinatorial library of DNA-encoded hexapeptides was also prepared. Despite the low yield of the library, the possibility of DNA encoding, the amplifiability of the prepared molecules and the possibility of biotin-based separation were verified. The integrity of the hexapeptide sequence and the protease testing is the subject of further study.
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The synthesis of ortho-condensed polyaromatic compounds and their physico-chemical properties
Mildner, Daniel ; Starý, Ivo (advisor) ; Míšek, Jiří (referee)
This Bachelor Thesis deals with the preparation of laterally/axially extended polyaromatic compounds with helically shaped structure ("long/wide" helicenes) with major focus on optimizing key steps in the synthesis of tetrabenzo[9]helicene and its derivatives. In Theoretical Background the author summarises the physico- chemical properties of helicenes and methods of their preparation describing approaches that can be used for the preparation of extended helicenes. In chapter Results and Discussion the synthesis of tetrabenzo[9]helicene and its derivatives is presented including their preparation in a nonracemic form, resolution of racemates into enantiomers and study on their properties. The Experimental Part provides the reader with practical information on experimental procedures and characterization of the prepared compounds.
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Organocatalytic preparation of spirocycles derived from heterocycles
Koberová, Tereza ; Veselý, Jan (advisor) ; Míšek, Jiří (referee)
This bachelor work is focused on enantioselective preparation of heterocyclic spirocompounds using organocatalytic approach. First part deals with enantioselective synthesis of spirocycles by reaction of α,β-unsaturated aldehydes with heterocyclic nucleophiles under chiral secondary amin catalysis. This tandem Michael/Michael/aldol reaction is based on iminium and enamine activation of subtrate. Second part of the work is focused on the following oxidation of prepared spirocyclic aldehydes to the coresponding carboxylic acid.
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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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The synthesis of extended helical aromatics
Trávníková, Veronika ; Starý, Ivo (advisor) ; Míšek, Jiří (referee)
Helicenes are inherently chiral polycyclic aromatic molecules. Laterally extended helicenes have their outer rim extended by one or more aromatic rings, which gives them unique optical and electron properties and makes them hot candidates for applications in asymmetric catalysis or molecular electronics, for instance. However, the larger the outer rim is, the more difficult the synthesis of wide helicenes becomes. Frequently bad solubility of laterally extended helicenes, as well as their intermediates, requires comprehensive optimisation of the reaction conditions, which are commonly used in helicene synthesis. In my Bachelor Thesis, I focus on synthesis of a laterally extended helicene with two hexabenzocoronene moieties incorporated in its structure. One of the essential intermediates, bromoiodohexaphenylbenzene derivative, was prepared by Diels-Alder reaction. Optimised conditions of this reaction enabled the synthesis of crucial intermediates necessary for further steps in the target helicene synthesis. Key words: helicene, hexabenzocoronene, hexaphenylbenzene, Diels-Alder, polyaromatics
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Synthesis of 1,2-disubstituted cyclopentadienes and their application in syntheses of metallocenes
Vician, Marek ; Kotora, Martin (advisor) ; Míšek, Jiří (referee)
In this master thesis 1,2-disubstituted cyclopentadienes were prepared by method composed of three transition metal catalysed reactions. Then, the cyclopentadienes were converted into cyclopentadienyl manganese, rhenium and iron complexes. A one-pot synthesis of the cyclopentadienyl metal complexes was developed without isolation of the intermediate cyclopentadienes. The second goal of this thesis was preparation of vinyl-substituted cyclopentadienes using ring closing enyne metathesis. The last part of thesis dealt with synthesis of a chiral cyclopentadiene and preparation of the corresponding metal complexes. The important part of this work was preparation of starting materials using palladium catalysed cross-coupling reactions. Key words: cyclopentadienes, carbometallation, cross-coupling, metathesis, metallocenes.
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Synthesis of polycyclic compounds containing quaternary carbon centers
Vašíček, Tomáš ; Matoušová, Eliška (advisor) ; Míšek, Jiří (referee)
In this diploma Thesis, a method for the preparation of polycarbocyclic compounds containing all-carbon quaternary centers embedded in triquinane skeleton was prepared. The key reaction sequence leading towards the preparation of these carbocycles was Pd-catalyzed tandem cyclization/Suzuki cross-coupling reaction followed by halocarbocyclization. In the first part of synthetic project, a model oxygen-containing polycyclic derivative was prepared. In the second part, 3 novel polycarbocyclic compounds containing all-carbon quaternary centers were successfully prepared. Synthetic procedure for the starting material required for the key reaction sequence was developed. This approach represents another pathway for the synthesis of polycarbocyclic natural products. Keywords: synthesis, polycyclic compounds, quaternary centers, catalysis
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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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Preparation of benzoylated derivatives of cyclodextrins
Jůza, Radomír ; Jindřich, Jindřich (advisor) ; Míšek, Jiří (referee)
This master thesis deals with the preparation of the cyclodextrin (CD) derivatives that are suitable for complexation of the electron-rich aromatic compounds such as helicenes. The basic skeleton of native CD was perbenzoylated in all positions 2, 3 and 6 in the first place. Then the nucleophilic attack of carboxylates of benzoic acids with electron- withdrawing groups (NO2) or electron-donating group (NH2) to per(6-deoxy-6-iodo)-CD derivatives were used to prepare the perbenzoylated CD derivatives in the postition 6. Binding constant was determined for the prepared benzoylated derivatives of CD and the racemate of the hexahelicene using the NMR titration. Perbenzoylated derivatives of β-CD in all position 2,3 and 6 with one azido group in position 6 were prepared as well. These derivatives could be covalently bonded as chiral selectors in the stationary phase for the HPLC separation of aromatic enantiomers. Keywords: cylodextrins, persubstitution, benzoyl, 4-nitrobenzoyl, 3,5-dinitrobenzoyl, helicenes, NMR titration, binding constants
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