69-89-6Relevant articles and documents
Label-Free Surface Enhanced Raman Scattering Approach for High-Throughput Screening of Biocatalysts
Westley, Chloe,Xu, Yun,Carnell, Andrew J.,Turner, Nicholas J.,Goodacre, Royston
, p. 5898 - 5903 (2016)
Biocatalyst discovery and directed evolution are central to many pharmaceutical research programs, yet the lack of robust high-throughput screening methods for large libraries of enzyme variants generated (typically 106-108) has hampered progress and slowed enzyme optimization. We have developed a label-free generally applicable approach based on Raman spectroscopy which results in significant reductions in acquisition times (>30-fold). Surface enhanced Raman scattering (SERS) is employed to monitor the enzyme-catalyzed conversion by xanthine oxidase of hypoxanthine to xanthine to uric acid. This approach measures the substrates and products directly and does not require chromogenic substrates or lengthy chromatography, was successfully benchmarked against HPLC, and shows high levels of accuracy and reproducibility. Furthermore, we demonstrate that this SERS approach has utility in monitoring enzyme inhibition illustrating additional medical significance to this high-throughput screening method.
Identification of function and mechanistic insights of guanine deaminase from Nitrosomonas europaea: Role of the C-terminal loop in catalysis
Bitra, Aruna,Hussain, Bhukya,Tanwar, Ajay Singh,Anand, Ruchi
, p. 3512 - 3522 (2013)
NE0047 from Nitrosomonas europaea has been annotated as a zinc-dependent deaminase; however, the substrate specificity is unknown because of the low level of structural similarity and sequence identity compared to other family members. In this study, the function of NE0047 was established as a guanine deaminase (catalytic efficiency of 1.2 × 105 M-1 s-1), exhibiting secondary activity towards ammeline. The structure of NE0047 in the presence of the substrate analogue 8-azaguanine was also determined to a resolution of 1.9 A?. NE0047 crystallized as a homodimer in an asymmetric unit. It was found that the extreme nine-amino acid C-terminal loop forms an active site flap; in one monomer, the flap is in the closed conformation and in the other in the open conformation with this loop region exposed to the solvent. Calorimetric data obtained using the full-length version of the enzyme fit to a sequential binding model, thus supporting a cooperative mode of ligand occupancy. In contrast, the mutant form of the enzyme (ΔC) with the deletion of the extreme nine amino acids follows an independent model of ligand occupancy. In addition, the ΔC mutant also does not exhibit any enzyme activity. Therefore, we propose that the progress of the reaction is communicated via changes in the conformation of the C-terminal flap and the closed form of the enzyme is the catalytically active form, while the open form allows for product release. The catalytic mechanism of deamination was also investigated, and we found that the mutagenesis of the highly conserved active site residues Glu79 and Glu143 resulted in a complete loss of activity and concluded that they facilitate the reaction by serving as proton shuttles.
The Influencing of Preanodized Inlaying Ultrathin Carbon Paste Electrode on the Oxidation for the Xanthine and Hypoxanthine by the Hydrogen Bond
Qiao, Yue-Chun,Li, Jing,Li, Yao,Li, Quan-Min
, p. 1011 - 1019 (2015)
In this paper, a pre-anodized inlaying ultrathin carbon paste electrode (PAIUCPE) with 316L as a matrix was constructed by a simple and fast electrochemical pretreatment. Using xanthine (Xa) and hypoxanthine (HXa) as the target compounds, the pH effects compositions of buffer solution, the accumulation times, hydrogen bond catalysis, degree of auxiliary electrode reaction on the size of peak currents (Ip) of Xa and HXa was discussed in detail. Also, it was proposed that Xa and HXa were respectively absorbed at the surface of PAIUCPE through hydrogen bonding. The influencing mechanisms of the PAIUCEP on electrochemical oxidation of Xa and HXa were explained in detail. Moreover, the linear relationships for the Xa and HXa were obtained in the range of 6×10-8-3×10-5 mol/L and 2×10-7-7×10-5 mol/L, respectively. The detection limits for the Xa and HXa were 1.2×10-8 mol/L and 5.7×10-8 mol/L, respectively. Moreover, this proposed method could be applied to determine the Xa and HXa in human urine simultaneously with satisfactory results.
Real-Time Monitoring of Human Guanine Deaminase Activity by an Emissive Guanine Analog
Bucardo, Marcela S.,Wu, You,Ludford, Paul T.,Li, Yao,Fin, Andrea,Tor, Yitzhak
, p. 1208 - 1214 (2021/07/26)
Guanine deaminase (GDA) deaminates guanine to xanthine. Despite its significance, the study of human GDA remains limited compared to other metabolic deaminases. As a result, its substrate and inhibitor repertoire are limited, and effective real-time activity, inhibitory, and discovery assays are missing. Herein, we explore two emissive heterocyclic cores, based on thieno[3,4-d]pyrimidine (thN) and isothiazole[4,3-d]pyrimidine (tzN), as surrogate GDA substrates. We demonstrate that, unlike the thieno analog, thGN, the isothiazolo guanine surrogate, tzGN, does undergo effective enzymatic deamination by GDA and yields the spectroscopically distinct xanthine analog, tzXN. Further, we showcase the potential of this fluorescent nucleobase surrogate to provide a visible spectral window for a real-time study of GDA and its inhibition.
Inhibition of xanthine oxidase by theaflavin: Possible mechanism for anti-hyperuricaemia effect in mice
Chen, Jianmin,Jin, Nan,Li, Qinglian,Ran, Mengnan,Ruan, Zhipeng,Ye, Yaling
, p. 11 - 18 (2020/07/03)
Xanthine oxidase (XO) catalyzes the oxidation of hypoxanthine to xanthine and then to uric acid. Excessive production of uric acid leads to hyperuricaemia. Due to the serious side effects of allopurinol, it is an urgent need to explore new XO inhibitors. Herein, the effects of theaflavin (TF1) on XO and anti-hyperuricaemia effect in hyperuricemic mice were investigated. Kinetic analysis indicate that TF1 is a reversible competitive inhibitor and has a significant inhibitory effect on XO with an IC50 value of 63.17 ± 0.13 μmol/L. Analysis of fluorescence spectra suggests that TF1 causes the obvious fluorescence quenching of XO, which is mainly driven by hydrophobic interactions and hydrogen bonds. Docking studies demonstrate that TF1 interacts with dozens of amino acid residues surrounded in the active cavity of XO, including Glu-879, Pro-1012, Thr-1010, Val-1011, Lys-771, Glu-802, Pro-1076, Leu-873, Leu-1014, Asn-768, Leu-648 and Phe-649. The inhibitory mechanism may be the insertion of TF1 into the active site of XO, which hinders the substrate xanthine to enter into the site. Furthermore, the results from animal experiments demonstrate that TF1 is effective in reducing serum uric acid in mice. These findings suggest that TF1 may be a potential drug candidate for the treatment of hyperuricaemia.
SUBSTITUTED XANTHINE DERIVATIVES
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Page/Page column 41, (2020/07/07)
The present invention relates to compounds of formula (I) a process for their manufacture, pharmaceutical compositions containing them and their use in therapy, particularly in the treatment of conditions having an association with TRPC5 containing ion channels. R1, R2, R3, R4 and R5 have meanings given in the description.
Preparation method of xanthine
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Paragraph 0052-0061, (2019/02/10)
The invention discloses a preparation method of xanthine. The preparation method comprises the following steps that 1, guanine performs diazo-reaction with sodium nitrite in a dilute acid or diazotized recovered mother solution; 2, after the diazo-reaction is completed, solid-liquid separation is performed, the step 3 is conducted on the obtained solid, and optionally the obtained liquid serves asthe diazotized recovered mother solution is reused in the step 1; 3, the solid obtained in the step 2 is subjected to hydrolysis reaction, and after reaction is completed, the xanthine is obtained posttreatment. The guanine is adopted as an initial raw material, the new preparation method is adopted, prepared diazonium salt is extracted from a system, the side reaction is inhibited, meanwhile reuse of the diazo-reaction mother solution is achieved, and the yield of three wastes is greatly reduced; in addition, a new refining method is adopted, so that the molar yield and product purity are far higher than literature values.
One-Step Synthesis of 2-Fluoroadenine Using Hydrogen Fluoride Pyridine in a Continuous Flow Operation
Salehi Marzijarani, Nastaran,Snead, David R.,McMullen, Jonathan P.,Lévesque, Fran?ois,Weisel, Mark,Varsolona, Richard J.,Lam, Yu-Hong,Liu, Zhijian,Naber, John R.
supporting information, p. 1522 - 1528 (2019/07/10)
We report the development of a one-pot synthesis of 2-fluoroadenine from an inexpensive 2,6-diaminopurine starting material using diazonium chemistry in a continuous fashion. Given the sensitivity of this transformation to temperature, we conducted critical experiments to study the exothermicity of the reaction and the heat removal, which were critical for the development of the process. Our goal was to improve the yield and purity of this pharmaceutical intermediate (2-fluoroadenine) and develop a more robust process.
Hydroxyl radical induced oxidation of theophylline in water: A kinetic and mechanistic study
Sunil Paul,Aravind,Pramod,Saha,Aravindakumar
, p. 5611 - 5620 (2014/07/22)
Oxidative destruction and mineralization of emerging organic pollutants by hydroxyl radicals (OH) is a well established area of research. The possibility of generating hazardous by-products in the case of OH reaction demands extensive investigations on the degradation mechanism. A combination of pulse radiolysis and steady state photolysis (H2O2/UV photolysis) followed by high resolution mass spectrometric (HRMS) analysis have been employed to explicate the kinetic and mechanistic features of the destruction of theophylline, a model pharmaceutical compound and an identified pollutant, by OH in the present study. The oxidative destruction of this molecule, for intermediate product studies, was initially achieved by H2O 2/UV photolysis. The transient absorption spectrum corresponding to the reaction of OH with theophylline at pH 6, primarily caused by the generation of (T8-OH), was characterised by an absorption band at 330 nm (k2 = (8.22 ± 0.03) × 109 dm3 mol-1 s-1). A significantly different spectrum (λmax: 340 nm) was observed at highly alkaline pH (10.2) due to the deprotonation of this radical (pKa ～ 10.0). Specific one electron oxidants such as sulphate radical anions (SO4-) and azide radicals (N 3) produce the deprotonated form (T(-H)) of the radical cation (T+) of theophylline (pKa 3.1) with k2 values of (7.51 ± 0.04) × 109 dm3 mol-1 s-1 and (7.61 ± 0.02) × 109 dm3 mol-1 s-1 respectively. Conversely, oxide radicals (O -) react with theophylline via a hydrogen abstraction protocol with a rather slow k2 value of (1.95 ± 0.02) × 109 dm3 mol-1 s-1. The transient spectral studies were complemented by the end product profile acquired by HRMS analysis. Various transformation products of theophylline induced by OH were identified by this technique which include derivatives of uric acids (i, iv & v) and xanthines (ii, iii & vi). Further breakdown of the early formed product due to OH attack leads to ring opened compounds (ix-xiv). The kinetic and mechanistic data furnished in the present study serve as a basic frame work for the construction of OH induced water treatment systems as well as to understand the biological implications of compounds of this kind. the Partner Organisations 2014.
Characterization of inosine-uridine nucleoside hydrolase (RihC) from Escherichia coli
Arivett, Brock,Farone, Mary,Masiragani, Ranjith,Burden, Andrew,Judge, Shelby,Osinloye, Adedoyin,Minici, Claudia,Degano, Massimo,Robinson, Matthew,Kline, Paul
, p. 656 - 662 (2014/03/21)
A non-specific nucleoside hydrolase from Escherichia coli (RihC) has been cloned, overexpressed, and purified to greater than 95% homogeneity. Size exclusion chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis show that the protein exists as a homodimer. The enzyme showed significant activity against the standard ribonucleosides with uridine, xanthosine, and inosine having the greatest activity. The Michaelis constants were relatively constant for uridine, cytidine, inosine, adenosine, xanthosine, and ribothymidine at approximately 480 μM. No activity was exhibited against 2′-OH and 3′-OH deoxynucleosides. Nucleosides in which additional groups have been added to the exocyclic N6 amino group also exhibited no activity. Nucleosides lacking the 5′-OH group or with the 2′-OH group in the arabino configuration exhibited greatly reduced activity. Purine nucleosides and pyrimidine nucleosides in which the N7 or N3 nitrogens respectively were replaced with carbon also had no activity.
