- DNA-catalyzed glycosylation using aryl glycoside donors
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We report the identification by in vitro selection of Zn2+/Mn2+-dependent deoxyribozymes that glycosylate the 3′-OH of a DNA oligonucleotide. Both β and α anomers of aryl glycosides can be used as the glycosyl donors. Individual deox
- Hesser, Anthony R.,Brandsen, Benjamin M.,Walsh, Shannon M.,Wang, Puzhou,Silverman, Scott K.
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Read Online
- Preparation method of substituted benzyl or substituted phenyl beta-D-hexuronic acid glucoside
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The invention relates to the technical field of pharmaceutical and chemical industries, and discloses a preparation method of substituted benzyl or substituted phenyl beta-D-hexuronic acid glucoside.The preparation method comprises the steps of taking hexuronic acid as a raw material, and performing acetylation, selective acyl removal, methyl esterification, bromization, aethrization and alkalinealcoholysis to form substituted benzyl or substituted phenyl beta-hexuronic acid glucoside with a structural formula as follows as shown in the description, wherein n is equal to 0-1; and R is o-, m-or para-hydrogen, nitryl, methoxy or halogen. The method is mild in reaction condition, simple in step and suitable for large-scale preparation, and a reaction reagent is easy to obtain.
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Paragraph 0062; 0085; 0086; 0087
(2018/10/19)
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- Characterization of the zebrafish Ugt repertoire reveals a new class of drug-metabolizing UDP glucuronosyltransferases
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The zebrafish genome contains a gene superfamily of 40 Ugt genes that can be divided into Ugt1, Ugt2, and Ugt5 families. Because the encoded zebrafish UDP glucuronosyltransferase (UGT) proteins do not display orthologous relationships to any of the mammalian and avian UGT enzymes based on molecular phylogeny, it is difficult to predict their substrate specificity. Here, we mapped their tissue-specific expression patterns. We showed that the zebrafish UGT enzymes can be glycosylated. We determined their substrate specificity and catalytic activity toward diverse aglycone substrates. Specifically, we measured mRNA levels of each of the 40 zebrafish Ugt genes in 11 adult tissues and found that they are expressed in a tissue-specific manner. Moreover, functional analyses with the donor of UDP glucuronic acid (UDPGA) for each of the 40 zebrafish UGT proteins revealed their substrate specificity toward 10 important aglycones. In particular, UGT1A1, UGT1A7, and UGT1B1 displayed good glucuronidation activities toward most phenolic aglycones (4-methylumbelliferone, 4-nitrophenol, 1-naphthol, bisphenol A, and mycophenolic acid) and the two carboxylic acids (bilirubin and diclofenac). Importantly, some members of the UGT5, a novel UGT family identified recently, are capable of glucuronidating multiple aglycones with the donor cofactor of UDPGA. In particular, UGT5A5, UGT5B2, and UGT5E1 glucuronidate phenols and steroids with high specificity toward steroid hormones of estradiol and testosterone and estrogenic alkylphenols 4-tert-octylphenol. These results shed new insights into the mechanisms by which fish species defend themselves against vast numbers of xenobiotics via glucuronidation conjugations and may facilitate the establishment of zebrafish as a model vertebrate in toxicological, developmental, and pathologic studies. Copyright
- Wang, Yuanming,Huang, Haiyan,Wu, Qiang
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- Accurate prediction of glucuronidation of structurally diverse phenolics by human UGT1A9 using combined experimental and in silico approaches
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Purpose: Catalytic selectivity of human UGT1A9, an important membrane-bound enzyme catalyzing glucuronidation of xenobiotics, was determined experimentally using 145 phenolics and analyzed by 3D-QSAR methods. Methods: Catalytic efficiency of UGT1A9 was determined by kinetic profiling. Quantitative structure activity relationships were analyzed using CoMFA and CoMSIA techniques. Molecular alignment of substrate structures was made by superimposing the glucuronidation site and its adjacent aromatic ring to achieve maximal steric overlap. For a substrate with multiple active glucuronidation sites, each site was considered a separate substrate. Results: 3D-QSAR analyses produced statistically reliable models with good predictive power (CoMFA: q 2=0.548, r2=0.949, r pred 2 =0.775; CoMSIA: q2=0.579, r2=0.876, rpred2 =0.700). Contour coefficient maps were applied to elucidate structural features among substrates that are responsible for selectivity differences. Contour coefficient maps were overlaid in the catalytic pocket of a homology model of UGT1A9, enabling identification of the UGT1A9 catalytic pocket with a high degree of confidence. Conclusion: CoMFA/CoMSIA models can predict substrate selectivity and in vitro clearance of UGT1A9. Our findings also provide a possible molecular basis for understanding UGT1A9 functions and substrate selectivity.
- Wu, Baojian,Wang, Xiaoqiang,Zhang, Shuxing,Hu, Ming
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experimental part
p. 1544 - 1561
(2012/07/27)
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- Phenylalanine 93 of the human UGT1A10 plays a major role in the interactions of the enzyme with estrogens
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Little is currently known about the substrate binding site of the human UDP-glucuronosyltransferases (UGTs) and the structural elements that affect their complex substrate selectivity. In order to further understand and extend our earlier findings with phenylalanines 90 and 93 of UGT1A10, we have replaced each of them with Gly, Ala, Val, Leu, Ile or Tyr, and tested the activity of the resulting 12 mutants toward eight different substrates. Apart from scopoletin glucuronidation, the F90 mutants other than F90L were nearly inactive, while the F93 mutants' activity was strongly substrate dependent. Hence, F93L displayed high entacapone and 1-naphthol glucuronidation rates, whereas F93G, which was nearly inactive in entacapone glucuronidation, was highly active toward estradiol, estriol and even ethinylestradiol, a synthetic estrogen that is a poor substrate for the wild-type UGT1A10. Kinetic analyses of 4-nitrophenol, estradiol and ethinylestradiol glucuronidation by the mutants that catalyzed the respective reactions at considerable rates, revealed increased Km values for 4-nitrophenol and estradiol in all the mutants, whilst the K m values of F93G and F93A for ethinylestradiol were lower than in control UGT1A10. Based on the activity results and a new molecular model of UGT1A10, it is suggested that both F90 and F93 are located in a surface helix at the far end of the substrate binding site. Nevertheless, only F93 directly affects the selectivity of UGT1A10 toward large and rigid estrogens, particularly those with substitutions at the D ring. The effects of F93 mutations on the glucuronidation of smaller or less rigid substrates are indirect, however.
- H?glund, Camilla,Sneitz, Nina,Radominska-Pandya, Anna,Laakonen, Liisa,Finel, Moshe
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experimental part
p. 1465 - 1473
(2011/11/29)
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- Interaction analyses of amyloid β peptide (1-40) with glycosaminoglycan model polymers
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We synthesized a novel glycopolymer library with 6-sulfo-GlcNAc and glucuronicacid(GlcA) based on the structure of glycosaminoglycans. The molecular weights of the polymers were controlled via living radical polymerization. The interactions of Aβ(1-40)ith glycopolymers were analyzed by inhibition activity of protein aggregation using ThT fluorescence assay, atomic force microscopy observation, and CD spectra. The inhibition activity of Aβ was much affected by the sugar structure and molecular weight of the polymer. The glycopolymers carrying 6-sulfo-GlcNAc showed inhibition activity toward Aβ aggregate, and those with 6-suflo-GlcNAc and GlcA showed the strong inhibition activity. The glycopolymer libraries yielded valuable information about Aβ aggregate with glycosaminoglycans.
- Miura, Yoshiko,Mizuno, Hikaru
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supporting information; experimental part
p. 1004 - 1009
(2010/12/19)
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- Development of a rapid and detailed structural identification system with an on-line immobilized enzyme reactor integrated into LC-NMR
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Immobilized enzyme reactors (IMERs) integrated into an LC-NMR system were developed for rapid and detailed structural identification of enzymatic reaction products. An on-column enzymatic reaction was achieved for immobilized cytochrome-c and dog microsomes. After the reaction, these products were analyzed by LC-NMR without any work-up processes. The immobilized cytochrome-c column integrated into LC-NMR was used to characterize the reaction product formed on N-demethylation by measurement of 1H-NMR. In the case of reaction taking place on the microsome column, a glucuronidation product was identified by 1H-NMR and 1H-1H correlated spectroscopy. The chemical structures of the enzymatic reaction products could be elucidated by IMER-LC-NMR without the need for authentic samples or isolation processes.
- Kashima, Yousuke,Okabayashi, Yoshito
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experimental part
p. 423 - 425
(2011/02/26)
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- On-line drug metabolism in capillary electrophoresis. 1. Glucuronidation using rat liver microsomes
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A rat liver microsome pseudostationary phase has been used for the on-line capillary electrophoresis monitoring of glucuronidation. Uridine diphosphate glucuronosyltransferase (EC 2.4.1.17) containing microsomes was isolated from rat liver and directly injected onto neutrally coated capillary containing polymeric replaceable gels followed by injection of the substrate mixture. On-line glucuronidation was observed within 15 min without any sample preparation. The factors affecting the separation of glucuronides and parent compounds were investigated by varying the applied electric fields and the size (length and internal diameter) of capillary. The Michaelis-Menten parameters (Km and Vmax) for the glucuronidation of 4-methyl-7-hydroxy coumarin and 4-nitrophenol were determined using the CE method and by off-line microsomal incubation. No significant differences were observed for Km and Vmax values for 4-methyl-7- hydroxycoumarin and 4-nitrophenol between on-line and off-line glucuronidation of these two compounds. This method was also used to determine the inhibition constant (IC50 value) for the competitive inhibition of morphine glucuronidation by codeine, IC50 (on-line) = 170 vs 580 μM (off-line). The results demonstrate that this method can be used to screen for the glucuronidation of test compounds and should reduce the time required for this screening process.
- Kim, Hee Seung,Wainer, Irving W.
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p. 7071 - 7077
(2007/10/03)
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- Purification and characterization of a novel α-glucuronidase from Aspergillus niger specific for O-α-D-glucosyluronic acid α-D-glucosiduronic acid
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A new α-glucuronidase that specifically hydrolyzed O-α-D-glucosyluronic acid α-D-glucosiduronic acid (trehalose dicarboxylate, TreDC) was purified from a commercial enzyme preparation from Aspergillus niger, and its properties were examined. The enzyme did not degrade O-α-D-glucosyluronic acid α-D-glucoside, O-α-D-glucosyluronic acid β-D-glucosiduronic acid, O-α-D-glucosyluronic acid-(1 → 2)-β-D-fructosiduronic acid, p-nitrophenyl-O-α-D-glucosiduronic acid, methyl-O-α-D-glucosiduronic acid, or 6-O-α-(4-O-α-D- glucosyluronic acid)-D-glucosyl-β-cyclodextrine. Furthermore, it showed no activity on α-glucuronyl linkages of 4-O-methyl-D-glucosyluronic acid-α-(1 → 2)-xylooligosaccharides, derived from xylan, a supposed substrate of α-glucuronidases. The molecular mass of the enzyme was estimated to be 120 kDa by gel filtration and 58 kDa by SDS-PAGE suggesting, the enzyme is composed of two identical subunits. It was most active at pH 3.0-3.5 and at 40°C. It was stable in pH 2.0-4.5 and below 30°C. It hydrolyzed O-α-D-glucosyluronic acid α-D-glucosiduronic acid to produce α- and β-anomers of D-glucuronic acid in an equimolar ratio. This result suggests that inversion of the anomeric configuration of the substrate is involved in the hydrolysis mechanism.
- Kiryu, Takaaki,Nakano, Hirofumi,Kiso, Taro,Murakami, Hiromi
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p. 522 - 529
(2008/02/03)
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- Enzymic synthesis of β-glucuronides of estradiol, ethynylestradiol and other phenolic substrates on a preparative scale employing UDP-glucuronyl transferase
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By incubation of bovine liver UDP-glucuronyl transferase with various phenolic aglycone substrates, preparation of the corresponding β-glucuronides could be achieved in milligram quantities. Concomitantly, simple access was generated to estrogen steroid glucuronides of great biological importance. In terms of experimental feasibility, direct application of the commercially available crude microsomal product was compared to and proven to be competitive with the usually preferred utilization of an immobilized enzyme preparation.
- Werschkun, Barbara,Wendt, Andreas,Thiem, Joachim
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p. 3021 - 3023
(2007/10/03)
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