- 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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- 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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- 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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- 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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- 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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- 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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