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C-H activations of deazapurine heterocycles
Klečka, Martin ; Hocek, Michal (advisor) ; Hlaváč, Jan (referee) ; Rádl, Stanislav (referee)
Direct C-H borylations of 7-deazapurines (7H-pyrrolo[2,3-d]pyrimidine) were developed at position 8 using B2pin2 and Ir catalysis. The obtained boronates were efficiently applied in the Suzuki cross-couplings with aryl halides and other functional group transformations to give diverse 6-substituted 8-aryl-7-deazapurine derivatives. Furthermore, I was also interested in the synthesis of biologically relevant 8-aryl-7- deazaadenines and -7-deazahypoxanthines. As the direct C-H borylation of 7- deazaadenines was unsuccessful and the borylation/Suzuki reaction of 6-chloro-7- deazapurine gave only low yield (20%) of the desired 8-aryl derivative, I focused on the one-pot borylation/arylation of SEM-protected 6-methylsulfanyl- or 6-methoxy-7- deazapurines. The one-pot borylation/Suzuki coupling reactions were followed either by demethylation and deprotection to yield deazahypoxanthine base, or by oxidation of sulfide to sulfone, amination and deprotection to give deazaadenines. In addition, the boronate intermediates were successfully converted to 8-halo- or 8-trifluoromethyl-7- deazapurine derivatives. While the 7-deazahypoxantine analogues were almost entirely inactive, most of the 8-subtituted 6-methoxy-7-deazapurine and 7-deazaadenines bases showed significant cytostatic activities. Also a general...
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Modified ribonucleotides as building blocks for enzymatic construction of functionalized RNA or as antiviral compounds
Milisavljević, Nemanja ; Hocek, Michal (advisor) ; Baszczyňski, Ondřej (referee) ; Krečmerová, Marcela (referee)
The aim of this thesis was to study the steric influence of the base-modified nucleoside triphosphates (NTPs) on the enzymatic incorporation into RNA, as well as to study their inhibitory effect on different viral RNA polymerases in vitro. Their parent nucleosides and prodrug derivatives were also prepared and their antiviral activity evaluated. In the first part of the thesis, NTPs bearing groups varying in size from small methyl and ethynyl substituents via medium-size phenyl and benzofuryl groups, up to large dibenzofuran ring were prepared. Aromatic substituents were installed via Suzuki coupling on iodinated triphosphates or, in the case of modified guanosines, by the phosphorylation of modified nucleosides. Methyl and ethynyl NTPs were prepared via Pd-catalyzed coupling with AlMe3 and Sonogashira coupling, respectively, followed by the phosphorylation of modified nucleoside. To examine their incorporation into RNA by T7 RNA polymerase, templates coding for 35mer RNA containing one, three or seven modifications were designed. Modified pyrimidine triphosphates worked well for all the sequences, while the biggest dibenzofuryl group was not accepted in the difficult sequence with seven modifications. In the case of AR TPs dibenzofuryl modification did not incorporate at all, while other...
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Novel modified nucleosides with antiviral or cytostatic activity
Tokarenko, Anna ; Hocek, Michal (advisor) ; Rádl, Stanislav (referee) ; Dvořák, Dalimil (referee)
A general and modular synthetic approach to 4-substituted phenyl, 2-substituted pyridin- 5-yl and 5-substituted pyridin-2-yl 2′-C-methyl-C-ribonucleosides as potential anti-HCV agents was developed. Addition of halo(het)aryllithium reagents to benzylated 2-C-methyl-D- ribonolactone gave the corresponding hemiketals, which were subsequently converted to the β-anomeric benzyl-protected bromo(het)aryl-C-nucleosides via either direct reduction (in the case of phenyl derivative) or acetylation followed by reduction of the resulting hemiketal acetates (in the case of pyridyl derivatives). The key halogenated (het)aryl-C-nucleoside intermediates were further transformed by Pd-catalyzed cross-coupling, hydroxylation and amination reactions affording series of protected C-nucleosides with small hydrophilic and hydrophobic substituents. The final protecting group removal was rather problematic, and different debenzylation methods, such as hydrogenation on Pd/C or treatment with BCl3, had to be optimized for each derivative to minimize the formation of side-products. The final C- nucleosides were also converted into their 5′-O-triphosphates, and biological activity screenings revealed that none of the free C-nucleosides possesses any antiviral activity in the HCV replicon assay, and none of their NTPs...
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Synthesis and delivery of novel fluorescently-labelled nucleotides and their nucleic acids for bio-analytical applications
Güixens Gallardo, Pedro ; Hocek, Michal (advisor) ; Zimčík, Petr (referee) ; Klán, Petr (referee)
1 Abstract The goals of the thesis were to synthesise novel fluorescently labelled nucleotides and the corresponding nucleic acids for bio-analytical applications as well as their delivery into cells. The thesis also aimed at the development of an effective method to inhibit non-templated incorporation of nucleotides. The problematic non-templated enzymatic incorporation of nucleotides is addressed by using several commercially available 5'-modified-oligonucleotides. The oligonucleotides (ONs) that we tested bore ortho twisted intercalating nucleic acid (oTINA), a trityl group, or biotin at the 5'-end. The modified ONs were used as templates in the enzymatic primer extension (PEX) experiments in the presence of either modified nucleotides or only natural deoxynucleoside triphosphates (dNTPs). The oTINA templates underwent PEX reaction using natural dNTPs and different DNA polymerases of the A or B family. In parallel, two types of fluorescent nucleoside derivatives were independently designed and synthesised. Firstly, we envisaged new fluorescent nucleotide tags containing the hexamethylated BODIPY moiety as a bright fluorescent label. Conversely, we focused on the improvement of fluorescent nucleotide probes sensitive to the viscosity or polarity. The fluorescently labelled methylated BODIPY nucleotides...
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Enzymatic synthesis of DNA modified in the minor groove
Matyašovský, Ján ; Hocek, Michal (advisor) ; Hlaváč, Jan (referee) ; Urban, Milan (referee)
In the first part of the thesis, a series of six modified 2'-deoxyadenosine triphosphates, bearing small functional groups (chloro, amino, methyl, vinyl, ethynyl and phenyl) at position 2 of adenine, was designed and synthesised. They were then tested as substrates for DNA polymerases in enzymatic synthesis of minor-groove modified DNA. The 2-phenyl modified dATP was the only triphosphate unable to be incorporated, meaning that the phenyl group is already too big for minor-groove incorporations. All of the other tested nucleotides were good substrates for tested DNA polymerases [KOD XL, Vent(exo-) and Bst LF] affording minor- groove modified DNA bearing one or four modifications. The vinyl- and ethynyl-modified DNAs were then used for post-synthetic modification of DNA minor groove with fluorescent labels utilising click reactions. Ethynyl group reacted in copper-catalysed alkyne-azide cycloaddition (CuAAC), whereas the vinyl group participated in thiol-ene reaction. This procedure allowed for the attachment of big functional groups otherwise unable to be installed into the DNA minor groove using direct enzymatic incorporation. The second part of the thesis was devoted to the study of 2-alkylamino-2'- deoxyadenosine triphosphates and their use in enzymatic synthesis of base-modified ONs and DNA....
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Synthesis and studies of modified DNA: (i) development of DNA targeting molecular scissors and (ii) competitive enzymatic incorporation of base-modified nucleotides
Panattoni, Alessandro ; Hocek, Michal (advisor) ; Urban, Milan (referee) ; Fojta, Miroslav (referee)
In the first part of this work, a series of site-specific artificial metallonucleases (AMNs) was developed conjugating clamped-phenanthroline (Clip-Phen) copper complexes to triplex- forming oligonucleotides (TFOs). Several synthetic routes were explored for the synthesis of the TFO-AMNs hybrids, all sharing a copper-catalyzed alkyne-azide cycloaddition (CuAAC) reaction as the key step. As a consequence, building blocks for enzymatic or chemical synthesis of oligonucleotides (ONs) containing clickable groups, or already conjugated to the Clip-Phen ligand via CuAAC, were prepared. Two new alkynyl-linked nucleoside-5'-O-triphosphates (dNTPs) were designed and developed in order to obtain an efficient polymerase incorporation of clickable alkynyl-tethers into ONs and, at the same time, enhance the efficiency of CuAAC reactions on modified DNA. The relative 3'-O- phosphormaidites were also prepared in order to insert the same alkynyl-linkers into ONs via solid-phase synthesis. The AMN was linked at the 5'- or 3'-ends or in the middle of the TFO stretch, using diverse likers. The hybridization of all the synthesized TFOs with a target DNA duplex was studied. Finally, an extensive study of cleavage efficiency and specificity of the TFO-AMN conjugates towards the target DNA was performed, exploring the...
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Study of azaphthalocyanines as fluorescence quenchers in oligodeoxynucleotide probes
Demuth, Jiří ; Nováková, Veronika (advisor) ; Hocek, Michal (referee) ; Vávrová, Kateřina (referee)
Charles University, Faculty of Pharmacy in Hradec Kralove Department: Department of Pharmaceutical Chemistry and Pharmaceutical Analysis Author: Mgr. Jiří Demuth Supervisor: doc. PharmDr. Veronika Nováková, Ph.D. Consultant: prof. PharmDr. Petr Zimčík, Ph.D. Doctoral Thesis: Study of azaphthalocyanines suitable for quenching of fluorescence in oligodeoxynucleotide probes Phthalocyanines (Pcs) and azaphthalocyanines (AzaPcs) are planar macrocyclic compounds. These synthetic compounds are derived from naturally occurring dyes - porphyrins. Pcs and AzaPcs have very interesting photophysical properties due to their extensive 18 -electron conjugated system. They are used in electrotechnics, material industry or in medicinal applications. Octaalkylamino substituted tetrapyrazinoporphyrazines (TPyzPzs), which belong to AzaPc family, can quench fluorescence of other molecules due to the ultrafast intramolecular charge transfer. This thesis studies this quenching ability of TPyzPzs with the aim to develop efficient fluorescence probes. Such probes could find application in a variety of experiments across scientific disciplines i.e. - biochemistry, forensic or genetic analysis. Synthetic part of dissertation thesis is a continuation of previous investigation of TPyzPzs in our research group. It describes...
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Synthesis of 9-deazaguanine derivatives as potential inhibitors of tRNA-guanine transglycosylase (TGT)
Chylíková, Barbora ; Hocek, Michal (advisor) ; Smrček, Stanislav (referee)
Shigellosis is an acute diarrheal disease caused by enterobacteria of the genus Shigella. Worldwide, these bacteria are responsible for about 1 million deaths out of 165 million cases of the disease yearly, of which children under the age of 5 from developing countries are the most vulnerable group. The enzyme tRNA-guanine transglycolase (TGT for short) has been identified as essential for the effective pathogenesis of Shigella bacteria and therefore becomes a potential target for the selective treatment of shigellosis. This bachelor thesis describes the optimization of the preparation of a key intermediate for the synthesis of potential TGT inhibitors based on 8-aryl-9-deazaguanine structure. The synthesis starts from commercially available 2,6-dichloro-9-deazapurine. The main steps of the synthesis are the hydrolysis at position 6, the nucleophilic aromatic substitution of the chlorine group for the amino group in position 2, further the iodation and the introduction of the 3-thienyl group in position 8 by Suzuki coupling.
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Synthesis of fluorinated nucleosides
Nguyen, Van Hai ; Hocek, Michal (advisor) ; Baszczyňski, Ondřej (referee)
The key intermediate 6-amino-7-iodo-7-deazapurine 3'-deoxy-3'-fluororibonucleoside was synthesized using multistep sequence of several reactions, which started from the commercially available D-xylose and 6-chloro-7-deazapurine. The synthetic strategy was based on fluorination of sugar and glycosylation with corresponding nucleobase afterwards. The fluorination of 5-protected-1,2-isopropylidine xylose with different protecting groups at position 5 always led to elimination. It was later discovered that isopropylidine forces the conformation, which is unfavorable for substitution. During the extensive optimization it was also found out that DAST appears to be an optimal fluorinating agent. Fluorination was performed on 2,3-unprotected xylose, which was subsequently used for glycosylation. After several unsuccessful attempts on "protection group free" glycosylation, Vorbrüggen glycosylation was successful and gave desired 3'-fluoro nucleoside in good yield. However, benzoyl group had to be introduced into position 2'. The protected nucleoside was then aminated and simultaneously deproctected with solution of aqueous NH3 and 1,4-dioxane. The obtained key intermediate was used for synthesis of a small series of desired 6-amino-7-hetaryl nucleoside using Pd-catalyzed Suzuki reaction under aqueous...
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