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Modification of nucleic acids by reactive groups for bioconjugations and cross-linking with lysine-containing peptides and proteins
Ivancová, Ivana ; Hocek, Michal (advisor) ; Míšek, Jiří (referee) ; Urban, Milan (referee)
In the first part of this thesis, I developed reactive DNA probe for selective cross-linking with lysine residues of DNA-binding proteins. I synthesized 2'-deoxycytidine 5'-O-mono- and triphosphate bearing squaramate moiety tethered to the position 5 via propargylamine linker. The monophosphate was used as a model compound to test the reactivity of this mixed squaramate in cross-linking reactions with lysine and short lysine containing peptides. Squaramate modified 2'-deoxycytidine 5'-O-triphosphate was found to be suitable substrate for KOD XL polymerase in both PEX and PCR synthesis of modified DNA. Squaramate modified DNA forms stable diamide linkage with primary amines. I tested the reactivity of this DNA probe in bioconjugation reactions with sulfo-Cy5-amine and lysine containing peptides. Afterwards, squaramate-linked DNA was successfully cross-linked with lysine rich histone proteins. This reactive squaramate modified nucleotide showed potential for following bioconjugation reactions of nucleic acids with amines or lysine containing peptides and proteins without the need of external reagent. Based on positive results of experiments with squaramate modified DNA, in the second part of the thesis I developed and synthesized squaramate modified ribonucleotide to study cross- linking with RNA...
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Modifications of DNA by reactive groups for bioconjugations and cross-links with arginine-containing peptides and proteins
Leone, Denise Liu ; Hocek, Michal (advisor) ; Urban, Milan (referee) ; Vrábel, Milan (referee)
This PhD thesis describes the development and the synthesis of DNA-reactive probes bearing 1,3- diketone or phenylglyoxal moieties which can be used for cross-linking with arginine-containing peptides or proteins. The general strategy was based on the functionalization of the position 5 of pyrimidines via aqueous Sonogashira coupling or click reaction (CuAAC) with reactive building blocks to synthesize modified 2'-deoxyribonucleoside monophosphates and triphosphates (dNMPs/dNTPs). The following step was the enzymatic incorporation of such functionalized dNTPs into DNA via primer extension (PEX). In parallel, the monophosphate derivatives were used as model compounds for reactions with arginine and arginine-containing peptides to test the reactivity of the probes. For the reactive candidates, model reactions on the modified-DNA with arginine or Arg-containing peptides were performed and the DNA-probes were used in cross- linking reactions with Arg-rich DNA-binding proteins. The development of such probes was not straightforward but required the investigation of diverse candidates. More importantly the balance between reactivity and stability of the reactive group was the focus point and the challenge in the design of the reactive building blocks. The first candidate consisted of thiazolidine masked...
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Tandem Anionic Sigmatropic Rearrangement/Radical Reactions and Their Application Toward the Total Synthesis of Natural Products
Šimek, Michal ; Jahn, Ullrich (advisor) ; Urban, Milan (referee) ; Švenda, Jakub (referee)
The thesis describes the development of unprecedented tandem reactions merging anionic oxy-Cope rearrangement with single-electron transfer oxidation of thus generated metal enolates by ferrocenium hexafluorophosphate. The formed α-carbonyl radicals were utilized in oxygenation reactions by coupling them with persistent radical TEMPO, furnishing rearranged α-aminoxy carbonyl compounds. Suitable reaction conditions and factors influencing the rearrangement aptitudes were determined. The obtained polyfunctional products proved versatile in diversifications by polar reactions producing diverse scaffolds. Furthermore, rearranged α-aminoxy carbonyls with a double bond located in the δ-position of the carbonyl group are applicable in all-carbon 5-endo-trig radical cyclizations governed by the persistent radical effect. Despite the low kinetic rate, this rare cyclization mode furnished a number of substituted cyclopentanes. The reaction scope, including competitive cyclization modes, was studied. The developed methodology was utilized as a key reaction step to synthesize appropriately substituted cyclopentane core in a divergent approach to meroterpenoid fungal metabolites isolated from Ganoderma applanatum. The accomplished synthesis of applanatumols V and W and their epimers enabled the correction of...
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Design, Synthesis, and Application of Novel Bifunctional (Thio)urea Organocatalysts Derived from Saccharides & Catalytic Asymmetric C−H Arylation of (η6-Arene)Chromium Complexes
Gergelitsová, Ivana ; Veselý, Jan (advisor) ; Sedlák, Miloš (referee) ; Urban, Milan (referee)
The first part of the thesis is focused on the design, synthesis, evaluation, and application of novel bifunctional (thio)urea organocatalysts derived from saccharides. Combination of H-bonding donor (thio)urea moiety with Lewis base active site in a single chiral scaffold (e.g., 1,2-trans-cyclohexyl, cinchona alkaloids, 1,1'-binaphthyl) is a popular motif in catalyst design. Only limited attention has been paid to the synthesis of bifunctional (thio)ureas using saccharides as a chiral scaffold. Saccharides bring the advantage of the availability of various diastereomeric forms. Moreover, they offer modification of steric, electronic, and solubility properties via O-substitution. Three types of novel organocatalysts were designed: C2-symmetrical thiourea/tertiary amines entirely derived from 2-amino-2-deoxy saccharides, thiourea/primary amines based on pentopyranose and a cyclohexane skeleton, and (thio)urea/tertiary phosphines containing both saccharide and α-amino acid unit. Both functional groups of organocatalysts of the first type are located on the saccharide unit, and it is the only element which fully determines stereoselectivity. This is an exceptional approach as the majority of saccharide-based organocatalysts use saccharide as a bulky electron- withdrawing substituent. However, the...
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Role of brassinosteroids in plant response to drought stress
Trubanová, Nina ; Rothová, Olga (advisor) ; Urban, Milan (referee)
Brassinosteroids are steroid phytohormones with a wide range of effects. They improve survivance of plants exposed to drought stress, increase their resistence and yield therethrough. They regulate the response of plants directly (via synthesis of metabolits) and/or indirectly (via the induction of antioxidant compounds and enzymes), often in interaction with other phytohormones. Experiments studying influence of brassinosteroids on response of plants stressed with water deficit differ in several parameters. Their correct interpretation is complicated conseguently.
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Application of fluoroalkyl hypervalent iodine reagents in C-H functionalization of small molecules and aromatic amino acid residues
Rahimidashaghoul, Kheironnesae ; Beier, Petr (advisor) ; Kvíčala, Jaroslav (referee) ; Urban, Milan (referee)
The chemistry of fluorolkyl hypervalent iodine reagents has witnessed a great boast in recent years. These compounds are highly attractive as drug candidates, advanced materials and agrochemicals as described in detail in the Introduction. Despite this fact, applications of these reagents in biological studies are rather rare and under developed. The goal of this thesis is therefore the development of mild and metal-free methods in order to fill this gap. Two ways of application of fluoroalkyl hypervalent iodine reagents in labeling of biologically relevant compounds was explored. First, the applicability of previously reported parent Togni CF3 and their analogous tetrafluoroethyl reagents in radical fluoroalkylation of electron-rich substrates such as indole and pyrrole derivatives using sodium ascorbate as reductant was described. This afforded trifluoromethyl or 1,1,2,2-tetrafluoroethyl containing products in moderate to high yields. Next, same reagents were applied for labeling of several peptides and proteins bearing aromatic amino acids in their structure. This way, peptides and proteins containing electron-rich aromatics such as Trp, Phe, Tyr and His were reacted with fluoroalkyl groups with high selectivity toward Trp. In the second part of the work, a different approach of radical...
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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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New redox labels for DNA
Simonova, Anna ; Hocek, Michal (advisor) ; Urban, Milan (referee) ; Vyskočil, Vlastimil (referee)
The aim of my thesis was the synthesis of the modified 2'-deoxyribonucleoside triphosphates (dNTPs) bearing electrochemically oxidizable labels and their incorporation into DNA for the application in bioanalysis. In the first part of my thesis, I developed the synthesis of modified dNTPs bearing 2,3- dihydrobenzofuran (DHB) or 2-methoxyphenol (MOP) labels at 5-position of 2'- deoxycytidine 5'-O-triphosphate and at the 7-position of 7-deaza-2'-deoxyadenosine 5'-O- triphosphate by Suzuki-Miyaura cross-coupling reactions. Then modified dNTPs were used as substrates for DNA polymerases in enzymatic synthesis of modified DNA by PCR and primer extension. Electrochemical properties of the DHB and MOP-labeled nucleosides, dNTPs and DNA were studied by using of a square-wave voltammetry (SWV) at the pyrolytic graphite electrode (PGE) giving signals of MOP oxidation around 0.5 V and DHB oxidation around 0.85 V. The use of DHB group in combination with other electrochemical active labels was limited by close position of its oxidation peak to the signals of oxidation of natural nucleobases, whereas MOP moiety was successfully used for redox coding of nucleobases in combination with aminophenyl or benzofurazane label giving two independently readable redox signals in each case. In the second part of this...
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The physiological and proteomic characterisation of winter oilseed rape upon abiotic stress
Urban, Milan ; Vítámvás, Pavel (advisor) ; Hnilička, František (referee) ; Valentová, Olga (referee)
- Ph.D. thesis - Milan Urban, 2017 In some years, the agricultural production of oilseed rape, an important crop in the Czech Republic, is - besides biotic stress - facing the problem of damage caused by frost or drought. Together with special attention paid to proteins revealing responses between crop genotypes with differential abiotic stress tolerance levels we reviewed possible applications of proteomic results in crop breeding programs aimed at an improvement of crop stress tolerance (paper 1). For first original result, cold temperature was imposed upon non-vernalized plants in the stage of leaf rosette. The article (paper 2) shows a significant correlation between frost tolerance (FT), dehydrin (DHN) accumulation, and photosynthetic acclimation in five cultivars (cvs). Newly, the specific DHN D97 was shown to accumulate and other DHNs were shown to have qualitative differences in accumulation. These results imply that proper FT assessment is based on rapid photosynthetic acclimation together with higher accumulation of protective compounds. Drought stress (paper 3) was imposed in the water- demanding stem prolongation phase before flowering, because late-spring drought before and during flowering decreases the yield and seed quality significantly. This paper newly describes two water-uptake...
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