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Laccase & galactose oxidase: way to glycomimetics

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Information about Laccase & galactose oxidase: way to glycomimetics
Science-Technology

Published on October 24, 2008

Author: karel.krenek

Source: authorstream.com

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Laccase and galactose oxidase:an elegant way to glycomimetics : Laccase and galactose oxidase:an elegant way to glycomimetics Pavla Bojarová, Karel Křenek Laboratory of Biotransformation www.biotrans.cas.cz Institute of Microbiology Academy of Sciences of the Czech Republic Laboratory of Biotransformation : Laboratory of Biotransformation Glycomimetics and glycoconjugates for immunoaplications Oxidases for natural product and carbohydrate modification Biocatalysts for biotransformations of nitriles and amides Flavonoids and antioxidants silybin & quercetin derivatives Library of Glycosidases Enzyme and Substrate Engineering Laboratory of Biotransformation : Laboratory of Biotransformation Glycomimetics and glycoconjugates for immunoapplications Laboratory of Biotransformation : Laboratory of Biotransformation Oxidases for natural product and carbohydrate modification Laboratory of Biotransformation : Laboratory of Biotransformation Biocatalysts for biotransformations of nitriles and amides Laboratory of Biotransformation : Laboratory of Biotransformation Flavonoids and antioxidants silybin & quercetin derivatives Laboratory of Biotransformation : Laboratory of Biotransformation Library of Glycosidases Enzyme and Substrate Engineering Laboratory of Biotransformation : Laboratory of Biotransformation Institute of Microbiology Academy of Sciences of Czech Republic www.biotrans.cas.cz Center of Biocatalysis and Biotransformation www.cbb.cas.cz Cascade Chemo-EnzymaticProcesses : Cascade Chemo-EnzymaticProcesses Multistep synthetic pathway combining: chemical modifications enzymatic modifications chemo-enzymatic modifications Immunological application Laccase and galactose oxidase:an elegant way to glycomimetics Natural Killer Cells : Natural killer cells (NK) recognize tumour cells and upon activation their destruction mechanism kills them. Activation receptor of the NK-cells (NKR-P1, CD69) is a C-type lectin and interacts with carbohydrates (aminosugars). Higher affinity towards compounds carrying groups with negative charge (carboxylate, sulfate, etc.). Natural Killer Cells Target carbohydrate structures : Target carbohydrate structures gluco galacto Oxidation of pNP-GalNAc : Oxidation of pNP-GalNAc Selective oxidation of pNP-GalNAc by galactose oxidase. Oxidation of pNP-GalNAc : Oxidation of pNP-GalNAc Galactose oxidase needs oxygen supply TUBE REACTOR Galactose oxidase: tube reactor : Galactose oxidase: tube reactor stirred reaction vessel oxygen bottle (1.5 bar oxygen pressure) peristaltic pump Commercially available alternative: bioreactor for lab scale cell cultivation silicon tubing (Tygon ®) Galactose oxidase: tube reactor : reaction vessel oxygen bottle peristaltic pump manometer magnetic stirrer oxygen supply tube reactor with oxygen supply quantitative yield low enzyme consumption Galactose oxidase: tube reactor Oxidation of pNP-GlcNAc : Oxidation of pNP-GlcNAc Selective oxidation of pNP-GlcNAc by laccase & TEMPO Cooperation with prof. Sergio Riva, Italy TEMPO = 2,2,6,6-Tetramethylpiperidine-1-oxyl Oxidation of pNP-GlcNAc : Oxidation of pNP-GlcNAc DMP - 1,1,1-Triacetoxy-1,1-dihydro-1,2-benziodoxol-3(1H)-one Selective oxidation of pNP-GlcNAc according to Dess-Martin Oxidized substrates forβ-N-acetylhexosaminidases : Oxidized substrates forβ-N-acetylhexosaminidases β-N-Acetylhexosaminidases : β-N-Acetylhexosaminidases glycosidases (EC 3.2.1.52; CAZy GH 20) cleavage / synthesis of glycosidic bonds stereoselective, broad substrate specificity availability (CCF), robustness extracellular enzymes Activity of hexosaminidases : Activity of hexosaminidases To what extent are oxidized substrates cleaved? Acremonium, Aspergillus, Fusarium, Penicillium, Talaromyces % cleavage by hexosaminidase from Talaromyces flavus related to standard (pNP-GlcNAc or pNP-GalNAc) Molecular modeling: galacto : Molecular modeling: galacto ∆G = -241 kJ/mol ∆G = -138 kJ/mol Transglycosylation : Transglycosylation GalNAc C6-aldehyde 37 % Transglycosylation : Transglycosylation GalNAc C6-aldehyde 35 % Transglycosylation : Transglycosylation GlcNAc C6-aldehyde 23 % β-N-Acetylhexosaminidase (Talaromyces flavus) NaClO2, H2O, 0C, 1h Transglycosylation : Transglycosylation 25 % GlcNAc C6-uronate Ligands for NK-cells receptors : Ligands for NK-cells receptors -log IC50 (inhibition binding assay), CD69 (human) Cascade Chemo-EnzymaticProcesses : Cascade Chemo-EnzymaticProcesses Multistep synthetic pathway combining: chemical modifications enzymatic modifications chemo-enzymatic modifications Immunological application Acknowledgement : Laboratory of Biotransformation P. Bojarová V. Křen R. Gažák Institute of Microbiology, Prague K. Bezouška, et al. P. Sedmera, et al. V. Havlíček, et. al. Czech and international collaboration S. Riva, et al., CNR Milan, I L. Elling, et al., RWTH Aachen, D R. Ettrich & N. Kulik, Nové Hrady, CZ Acknowledgement Selected Publications : Selected Publications A. Pišvejcová, C. Rossi, L. Hušáková, V. Křen, S. Riva, D. Monti: -1,4-Galactosyltransferase-catalyzed glycosylation of sugar derivatives: new acquisitions J. Mol. Catal: B-Enzymatic. 39, 98-104 (2006). P. Simerská, M. Kuzma, D. Monti, S. Riva, M. Macková, V. Křen: Unique transglycosylation potential of extracellular -D-galactosidase from Talaromyces flavus. J. Mol. Catal./B Enzymatic. 39, 128-134 (2006). P. Bojarová, L. Petrásková, E. Ferrandi, D. Monti, H. Pelantová, M. Kuzma, P. Simerská, V. Křen: Glycosyl Azides – An Alternative Way to Disaccharides. Adv. Synth. Catal. 349, 1514–1520 (2007). P. Bojarová, K. Křenek, M. Kuzma, L. Petrásková, K. Bezouška, D.-J. Namdjou, L. Elling, V. Křen: N-Acetylhexosamine triad in one molecule: Chemoenzymatic introduction of 2-acetamido-2-deoxy-β-D-galactopyranosyluronic acid residue into a complex oligosaccharide. J. Mol. Catal./B Enzymatic. 50, 69-73 (2008).  R. Gažák, P. Sedmera, M. Marzorati, S. Riva, V. Křen: Laccase-mediated dimerization of the flavonolignan silybin. J. Mol. Catal./B Enzymatic 50, 87-92 (2008). B. Sauerzapfe, K. Křenek, J. Schmiedel, W. W. Wakarchuk, H. Pelantová, V. Křen, L. Elling: Chemo-enzymatic synthesis of poly-N-Acetyllactosamine (poly-LacNAc) structures and their characterization for galectin-mediated binding of ECM glycoproteins to biomaterial surfaces. Glycoconjugate J. in press 2008. www.biotrans.cas.cz/publications.html Natural Killer Cells : Natural Killer Cells Natural Killer Cells : Natural killer cells (NK) recognize tumour cells and upon activation their destruction mechanism kills them. Activation receptor of the NK-cells (NKR-P1, CD69) is a C-type lectin and interacts with carbohydrates (aminosugars). Natural Killer Cells Natural Killer Cells : Natural Killer Cells Malignant tumour cells are either not recognised by the NK cells (masking) or the NK cells lack sufficient activation signal resulting in uncontrolled proliferation of the tumour. External activation of the NK cells by glycomimetics can trigger their killing activity targeted to the resistant tumours leading eventually to their complete destruction. NKR-P1 Structural Preferences : NKR-P1 Structural Preferences Affinity of monosaccharides ManNAc > GalNAc > GlcNAc >> Glc > Man Type of O-glycosidic linkage b anomer is the best Length of oligosaccharidic chain 4 carbohydrate units is the optimum length Influence of aromatic system (enhances the binding) Multivalency effect (strongly enhances the binding) CD69 Structural Preferences : CD69 Structural Preferences Different order of monosaccharide affinity: GalNAc ~ GlcNAc >> … Increased preferences of branched oligosaccharidic structures to linear ones. Higher affinity towards compounds carrying groups with negative charge (carboxylate, sulfate, etc.).

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