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Modified Substrates in β-N-Acetylhexosaminidase-Catalyzed Synthesis
Bojarová, Pavla ; Křen, Vladimír (advisor) ; Moravcová, Jitka (referee) ; Walterová, Daniela (referee)
4 Conclrrsion 4 CoNcrusroN This Ph. D. thesis is a systematic study of the substrate specificity and the synthetic potential of B-N-acetylhexosaminidases (EC 3.2.1.52) with structurally modified substrates. It comprises four publications in intemational journals, one review and 17 oral and poster contributions. The following parts of the substrate molecule were modified: 2-acetamido moiety, the C-6 hydroxyl (oxidations, introduction of a cyano group) and the aglycon part (glycosyl azides - C-N bond hydrolysis). Thirteen modified substrates were synthesized, seven of them were described for the first time. They were tested for hydrolysis and transglycosylation by over thirty fungal $-N-acetylhexosaminidases (culture collections at Charles University and at the Institute of Microbiology, Academy of Sciences of the Czech Republic) and the results were discussed in relation to the conclusions of molecular modeling (B-N-acetylhexosaminidase from Aspergillus oryzae CCF 1066). Eight oligosaccharidic structures (six of them novel) were prepared by semi- preparative transglycosylation reactions (tens of miligrams), isolated (mostly 16_377a yields, even 787o yie\ď) and fully characterized. Noteworthy properties like immunoactivity (binding to natural killer cell activation receptors) and inhibitory potential were...
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New Microbial Glycosidases
Charvátová, Andrea ; Křen, Vladimír (advisor) ; Králová, Blanka (referee) ; Walterová, Daniela (referee)
Sunrvranv Glycosidases from fungi are useful in the preparation of various glycosides mainly by transglycosylation or reversed glycosylation. The lack ofany requirement for protection- deprotection sequences, mild conditions and easier synthesis of thermodynamically not preferred glycosidic bonds are the main advantages of glycosidase-catalysed synthesis of glycosides. In ttris thesis we concentratedon exoglycosidases,mainly B.N.aceýlhexosaminidases, cr-galactosidases and cr-L-rhamnosidases. For the enzyme preparation taxonomically characterised fungal strains from public collections were used. A library comprising more than 200 various glycosidases was developed by modification ofcultivation conditions and by the use ofspecific inducers. The enrymes were used for screening ofsubstrate specificity and stability in organic solvents and subsequently for synthesis and modification of various substrates. Both B-N-acetylgalactosaminidase and B-N-acetylglucosaminidase activities in the series of B-N-acetylhexosaminidases were determined. Saccharides with strong immunomodulation activity $-o-GalpNAc-(i-+4)-o-GlcpNAc and B-o-GalpNAc-(1-+6)-o-GlcpNAc were synthesisedby transglycosylation using B-N-acetylhexosaminidase from Penicillium oxalicum CCF 2430' enryme having the highest B.N-acetylgalactosaminidase activiý....
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Development of new glycosylation methods for the synthesis of nucleosides
Downey, Alan Michael ; Hocek, Michal (advisor) ; Křen, Vladimír (referee) ; Kočovský, Pavel (referee)
As they make up DNA and RNA, nucleosides are considered the key to life. Synthetic nucleosides also constitute many drugs that treat viral infections and cancer. As a result, more efficient methods to access these crucial molecules would have implications that extend beyond a synthetic chemist's benchtop and into medicinal chemistry and medical research. One of the most challenging steps in the synthesis of nucleosides is the glycosylation step between the acceptor heterocycle (nucleobase) and the saccharide-based donor. Often to obtain satisfactory yield of this step with good regio- and stereochemical control the extensive use of protecting groups must be employed to squelch reactivity at unwanted reactive groups. Consequently, this process of protection−glycosylation−deprotection is laborious, inefficient, and often requires the use of toxic reagents. It would be, therefore, highly welcomed if new methodology to effect this glycosylation step was designed that reduces or removes the need to use protecting groups, but would still provide nucleosides in good yield, regio- and stereoselectively. Herein, this thesis presents my efforts into achieving this end. By employing modified Mitsunobu conditions, I determined that it is possible to directly glycosylate a nucleobase with D-ribose to afford...
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Enzymatic and Metabolic Transformation of Silybin and its Congeners
Purchartová, Kateřina ; Křen, Vladimír (advisor) ; Macek, Tomáš (referee) ; Vítek, Libor (referee)
Natural flavonoids and flavonolignans feature beneficial properties for living organisms such as antioxidant and hepatoprotective effects, anticancer, chemoprotective, dermatoprotective and hypocholesterolemic activities. Their metabolism in mammals is complex, the exact structure of their metabolites still remains partly unclear and the standards are usually not commercially available. Hence, this project focused on the preparation of potential and defined biotransformation Phase II sulfated metabolites of silymarin flavonolignans: silybin, 2,3-dehydrosilybin, isosilybin, silychristin, silydianin and flavonoids quercetin, taxifolin, rutin and isoquercitrin. Pure sulfated derivatives were prepared using aryl sulfotransferase from Desulfitobacterium hafniense and aryl sulfotransferase from rat liver. Using heterologously expressed PAPS (3'-phosphoadenosine-5'-phosophosulfate) - independent arylsulfotransferase from Desulfitobacterium hafniense and cheap p-nitrophenyl sulfate as sulfate donor, sulfated flavonolignans and flavonoids were obtained in high yields. Silymarin flavonolignans afforded exclusively monosulfates at the position C-20 (C-19 in the case of silychristin), except 2,3-dehydrosilybin that yielded also the 7,20-O-disulfated derivative. Isoquercitrin and rutin were selectively sulfated...
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Mammalian Serine Racemase as a Pharmaceutical Terget
Vaníčková, Jana ; Konvalinka, Jan (advisor) ; Kotora, Martin (referee) ; Křen, Vladimír (referee)
PH.D. DISSERTATION Mammalian Serine Racemase as a Pharmaceutical Target Jana Jirásková Supervisor: Jan Konvalinka Department of Biochemistry Faculty of Science Charles University Institute of Organic Chemistry and Biochemistry Gilead Sciences & IOCB Research Centre Academy of Sciences of the Czech Republic Praha 2010 Introduction Serine racemase is a pyridoxal-5'-phosphate (PLP) -dependent enzyme that is responsible for D- serine production. D-serine is a neurotransmitter that acts, together with L-glutamate, as agonist of ionotropic N-methyl-D-aspartate (NMDA) receptors, which are important for neuronal tissue signalization. Recent serine racemase knock-out mouse studies revealed that SR produces approximately 90% of brain D-serine. SR was first isolated from a pool of rat brains about a decade ago. Its orthologs are present in mammals as well as plants and yeast. Mammalian SRs share high sequence identity, about 90%. Mouse and human SRs are similar enzymatically, suggesting that mouse SR and mouse model are suitable to shed light on human SR. SR forms homodimers in solution and has a molecular weight of approximately 37 kDa per monomer. In addition to pyridoxal-5'-phosphate, the enzyme requires divalent cations such as Ca2+ or Mg2+ , nucleotides such as ATP, and reducing agents for full activity. The...
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Characterization of recombinant human serine racemase
Hoffman, Hillary Elizabeth ; Konvalinka, Jan (advisor) ; Křen, Vladimír (referee) ; Čeřovská, Noemi (referee)
6 Brief Abstract The pyridoxal-5'-phosphate-dependent enzyme serine racemase (SR) is responsible for the biosynthesis of D-serine in the mammalian central nervous system. D-serine acts as a neurotransmitter and coagonist, together with L-glutamate, of ionotropic N-methyl-D-aspartate receptors (NMDARs). Excitotoxic D-serine levels have been implicated in neuropathologies including Alzheimer's disease and amyotrophic lateral sclerosis. SR inhibitors offer a novel and potentially highly specific approach for attenuation of NMDAR-mediated glutamate excitotoxicity and for further study of the pathway. Many of the SR inhibitors described to date are small, naturally occurring compounds, and novel structures capable of influencing SR's activity are highly sought after. Moreover, structural information about this enigmatic enzyme is lacking, and suitable animal models need to be identified for inhibitor studies. This thesis presents the first published biochemical comparison of mouse and human SR orthologs, validating, at least in part, the use of mouse models in SR research. Additionally, hydroxamic acids are introduced as a novel class of SR inhibitors. While the experimentally determined structure of a mammalian SR remains elusive, random and site-directed mutagenesis experiments in combination with multiple...
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Electrochemistry of Flavonolignans and their Interactions with DNA and Proteins
Pyszková, M. ; Zatloukalová, M. ; Biedermann, David ; Křen, Vladimír ; Ulrichová, J. ; Ramešová, Šárka ; Sokolová, Romana ; Vacek, J.
Electrochemical oxidation of flavonolignans silybin, silydianin, silychristin and their 2,3-dehydroderivatives, was studied using ex situ and in situ cyclic and square wave voltammetry at pyrolytic graphite electrode. The pilot results presented here could be used for further investigation of mechanism of oxidation and reactivity of flavonolignans to DNA and proteins.
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