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Investigation of the compounds influencing the melting temperature of oligonucleotide probes
Kostelanský, Filip ; Zimčík, Petr (advisor) ; Hocek, Michal (referee) ; Krátký, Martin (referee)
Charles University, Faculty of Pharmacy in Hradec Kralove Department: Department of Pharmaceutical Chemistry and Pharmaceutical Analysis Author: Mgr. Filip Kostelanský Supervisor: prof. PharmDr. Petr Zimčík, Ph.D. Doctoral Thesis: Investigation of the compounds influencing the melting temperature of oligonucleotide probes Real-time PCR is widely used method in various research fields like biomedicine, microbiology, veterinary medicine, etc. Quantification of gene expression, allelic discrimination and alteration detection are new and interesting application possibilities. Mismatch discrimination is not optimal when longer probes are used. Melting temperature difference between fully complementary duplex and mismatched duplex is negligible in the case of longer probes. The short oligodeoxynucleotide (ODN) probes, on the other hand, are suitable for good discrimination of single nucleotide variants. However, these probes suffer from low melting temperatures. Adding minor groove binders (MGB), or other substances, which can strongly interact with DNA such as intercalating dyes or polyamines, can increase the melting temperature of short probes duplexes. In this work, several series of acridine-4-carboxamide intercalators were synthesized. MGB (Hoechst 33258) and polyamine (spermine) were modified and...
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Synthesis and evaluation of probes for fluorescent microscopy based on cyanopyridines and merocyanine scaffolds and lanthanide complexes
Bajnok, Juraj ; Kučerová, Marta (advisor) ; Zimčík, Petr (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Chemistry and Pharmaceutical Analysis Student: Juraj Bajnok Supervisor: PharmDr. Marta Kučerová, Ph.D. Consultant: Assist. prof. Stane Pajk, M. Pharm., Ph.D. Title of diploma thesis: Synthesis and evaluation of probes for fluorescent microscopy based on cyanopyridines and merocyanine scaffolds and lanthanide complexes Fluorescence as part of luminescence is a process, which describes how electrons of suitable materials are excited by the energy absorption of a photon, followed by the immediate emission of a photon with less energy compared to the absorbed photon. Suitable compounds must contain a system of conjugated double bonds. In this thesis, we present the synthesis and evaluation of fluorescence on derivates of cyanopyridines, merocyanine-type fluorescent probes, and lanthanide complexes of terbium, dysprosium, and europium. Lanthanide ions have low molar extinction coefficients, therefore they cannot be excited directly, without a proper ligand. We explored the influence of pH on the emission of synthesized derivates of cyanopyridines. We synthesized two merocyanine dyes with new alkyl moieties, which should change their emission spectra. Then, the merocyanine dyes could serve as new fluorescent probes for...
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Bioorthogonal reactions on DNA for regulation of transcription
Chakrapani, Aswathi ; Hocek, Michal (advisor) ; Zimčík, Petr (referee) ; Vrábel, Milan (referee)
This PhD thesis describes the design and synthesis of photocaged or glucosylated derivatives of epigenetic 5-(hydroxymethyl)pyrimidine-modified nucleotides and DNA using chemical and enzymatic methods and the studies on their regulation of gene expression in bacterial (Escherichia coli RNA polymerase) in vitro transcription level. In the first part of the thesis, the design and syntheses of 5-(nitrobenzyloxymethyl)-2'-deoxyuridine (dUNB) and -cytidine (dCNB) phosphoramidites are described. These photocaged nucleoside phosphoramidite building blocks were used in the automated solid-phase synthesis of oligonucleotides (ONs) modified at specific positions. The ONs were used as forward primers in a polymerase chain reaction (PCR) to construct DNA templates modified at specific sites of the promoter region. The specific site photocaged DNA was then irradiated with light to result in the corresponding specific site 5-(hydroxymethyl)-modified DNA. Bacterial in vitro transcription studies of both the specific site photocaged and uncaged DNA were carried out. The incorporation of the photocaged epigenetic pyrimidine nucleotides at the -35 region of the promoter region of the template DNA inhibited transcription partially while the presence of the same outside the -35 region did not have any significant...
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Aminoazaphtalocyanine peripheral substitution effect on J-dimer formation
Machan, Radek ; Zimčík, Petr (advisor) ; Zitko, Jan (referee)
Charles University, Faculty of Pharmacy in Hradec Kralove Department: Department of Pharmaceutical Chemistry and Pharmaceutical Analysis Author: Mgr. Radek Machan Supervisor: prof. PharmDr. Petr Zimčík, Ph.D. Rigorosum thesis: Aminoazaphtalocyanine peripheral substitution effect on J-dimer formation Azaphthalocyanines (AzaPcs) are classified as subgroup of phthalocyanine family, which are planar synthetic macromolecules derived from naturally occurring porphyrins. AzaPcs are aza-analogues of phthalocyanines, where benzenes are isosterically replaced by nitrogen heterocycles (pyrazine, pyridine or pyridazine). Due to the extensive system of conjugated double bonds, AzaPcs exhibit interesting photophysical properties, for which they are commonly used as industrial dyes, photosensitisers in photodynamic therapy, fluorescent sensors, fluorescence quenchers etc. Octakis(dialkylamino) substituted tetrapyrazinoporphyrazines (TPyzPzs), which belong to the AzaPcs family, can be used as effective fluorescence quenchers due to their ability to relax from excited state by intramolecular charge transfer (ICT) that leads to zero intrinsic fluorescence. This thesis deals with the preparation and subsequent study of the properties of symmetric TPyzPzs. Above all, the ability of TPyzPzs to form aggregates of the...
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Synthesis and evaluation of probes for fluorescent microscopy based on cyanopyridines and merocyanine scaffolds and lanthanide complexes
Bajnok, Juraj ; Kučerová, Marta (advisor) ; Zimčík, Petr (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Chemistry and Pharmaceutical Analysis Student: Juraj Bajnok Supervisor: PharmDr. Marta Kučerová, Ph.D. Consultant: Assist. prof. Stane Pajk, M. Pharm., Ph.D. Title of diploma thesis: Synthesis and evaluation of probes for fluorescent microscopy based on cyanopyridines and merocyanine scaffolds and lanthanide complexes Fluorescence as part of luminescence is a process, which describes how electrons of suitable materials are excited by the energy absorption of a photon, followed by the immediate emission of a photon with less energy compared to the absorbed photon. Suitable compounds must contain a system of conjugated double bonds. In this thesis, we present the synthesis and evaluation of fluorescence on derivates of cyanopyridines, merocyanine-type fluorescent probes, and lanthanide complexes of terbium, dysprosium, and europium. Lanthanide ions have low molar extinction coefficients, therefore they cannot be excited directly, without a proper ligand. We explored the influence of pH on the emission of synthesized derivates of cyanopyridines. We synthesized two merocyanine dyes with new alkyl moieties, which should change their emission spectra. Then, the merocyanine dyes could serve as new fluorescent probes for...
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Modulation of pKa of the recognition moiety of azaphthalocyanine sensors II.
Karlíková, Martina ; Zimčík, Petr (advisor) ; Zitko, Jan (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Chemistry and Pharmaceutical Analysis Candidate: Martina Karlíková Supervisor: prof. PharmDr. Petr Zimčík, Ph.D. Title of diploma thesis: Modulation of pKa of the recognition moiety of azaphthalocyanine sensors II. Our research group recently developed the sensors based on azaphthalocyanine (AzaPc) core containing one or two phenolic groups as recognition moiety and because of them, the AzaPcs can be utilized as fluorescent sensors. The fluorescent state of the molecule depends on the pH of the environment and the pKa of the recognition moiety. The latter can be modified by altering the substituents on phenol. The formation of AzaPcs was initiated by the synthesis of their precursors. In most of the cases, the starting material was 4-hydroxyacetophenon that was modified by electrophilic substitution. The products were oxidized to corresponding vicinal ketoaldehydes using selenium dioxide and immediately condensed with diaminomaleonitrile to substituted pyrazine-2,3-dicarbonitriles. Synthesis were completed by the cyclotetramerization reaction of this dicarbonitrile with 5,6-bis(tert-butylsulfanyl)pyrazine-2,3-dicarbonitrile using magnesium butoxide as initiator. Six different congeners were obtained by this...
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Synthesis of substituted acridines
Voštová, Kateřina ; Zimčík, Petr (advisor) ; Miletín, Miroslav (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department Department of Pharmaceutical Chemistry and Pharmaceutical Analysis Candidate Kateřina Voštová Supervisor prof. PharmDr. Petr Zimčík, Ph.D. Consultant Mgr. Filip Kostelanský Title of Thesis Synthesis of substituted acridines Real-time PCR is a widely used method in many laboratories for diagnostic purposes. This method combines PCR with the use of fluorescent signaling agents, probes, to monitor the number of amplicons during each PCR cycle. There are several types of probes used for DNA analysis. Short probes do not form sufficiently stable duplexes with the target DNA sequence to be able to detect single base mismatching. This work provides an overview of the oligodeoxynucleotides (ODN) probes in use and in what way is their thermostability is affected. In the practical part, two structurally distinct acridine derivatives were prepared because they may increase duplex thermostability due to their ability to intercalate between DNA bases. By increasing the thermostability, the melting temperature of the duplex between probe and the target sequence is also increased. For this reason, acridine derivatives form promising compounds suitable for short ODN probes that are characterized by low thermal stability. The described reactions...
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Synthesis of aerothionin analogs as potential antimycobacterial agents
Šimovičová, Martina ; Zitko, Jan (advisor) ; Zimčík, Petr (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Chemistry and Pharmaceutical Analysis Author: Martina Šimovičová Supervisors: Assoc. Prof. PharmDr. Jan Zitko, Ph.D.; Adjunct Prof. Paula Kiuru, Ph.D. Title of diploma thesis: Synthesis of aerothionin analogs as potential antimycobacterial agents Key words: antimycobacterial; tuberculosis; synthesis; aerothionin; bromotyrosines Drugs currently used for the treatment of tuberculosis are the result of studies carried out 50 or 60 years ago. With the constantly growing bacterial resistance to these pharmaceuticals grows also the importance of research for new antimycobacterially active compounds. The marine environment undoubtedly holds an enormous potential for discovering new leads for the development of antitubercular agents. One of these leads is a spirocyclic compound called aerothionin (1), which was found to be active against multidrug-resistant strains of Mycobacterium tuberculosis, as well as three non-tuberculosis mycobacteria (Figure 1). In addition, several spirocyclic structures (not only from marine origin) were discovered to affect on the M. tuberculosis in recent years, making this structure segment attractive for antitubercular research. Figure 1: Aerothionin (1) and general structure of the...
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Synthesis of novel 5,6-disubstituted derivatives of uracil as potential drugs
Vu, Lien Phuong ; Kučerová, Marta (advisor) ; Zimčík, Petr (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Chemistry and Pharmaceutical Analysis Candidate: Vu Lien Phuong Supervisors: PharmDr. Marta Kučerová, Ph.D. Tanja Bruun, M.Sc. (Pharm.) Prof. Jari Yli-Kauhaluoma Title of diploma thesis: Synthesis of novel 5,6-disubstituted derivatives of uracil as potential drugs In this thesis, uracil was used as the core structure given its many biological activities that were reported such as antitumor, antiviral, antibiotic, hypoglycemic, diuretic and many others. The work was focused on the preparation of new 5,6- disubstituted uracil derivatives as potential biologically active agents. 2,4,6-Trichloropyrimidine was used for the preparation of 6-chlorouracil that was condensed with phenols or anilines to give the respective 6-phenoxyuracils and 6- phenylaminouracils. These intermediates were then modified in position 5 to give the final products. For this very challenging last step, various alkylating and acylating agents were used, e.g. Vilsmeier reagent, alkylchlorides, chloroacetyl chloride, ethyl chlorooxoacetate and ethyl bromoacetate. In the end, ethyl bromoacetate gave the most promising results affording four novel 5,6-disubstituted uracil derivatives. During the experimental work it was found that pH of water used...
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Study of barrier lipids in the skin and skin models
Sochorová, Michaela ; Vávrová, Kateřina (advisor) ; Zimčík, Petr (referee) ; Salavec, Miloslav (referee)
1 Abstract Charles University, Faculty of Pharmacy in Hradec Králové Department of Organic and Bioorganic Chemistry Candidate: Mgr. Michaela Sochorová Supervisor: prof. PharmDr. Kateřina Vávrová, Ph.D. Title of doctoral thesis: Study of barrier lipids in the skin and skin models The barrier lipids are found in the intercellular spaces of the uppermost layer of epidermis - stratum corneum (SC). These lipids comprise an approximately equimolar mixture of ceramides (Cer), fatty acids (FA) and cholesterol (Chol). The composition and organization of the lipid mixture is unique and provides a barrier, which protects organism against harmful effects from the surroundings and, at the same time, it protects body from excessive water loss and contributes to homeostasis maintenance. Skin barrier disruption, including altered barrier lipid composition and organization, was described in many skin diseases, for example atopic dermatitis, psoriasis or skin cancer. Study and therapy of these diseases are limited by their complex pathophysiology and a lack of biological material (human skin). Therefore, skin models are appropriate and useful tools for study of skin diseases. The skin models differ in their simplicity/complexity and their preparation therefore they can provide different information. Model lipid membranes are...
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