Tautomerism of xanthine and alloxanthine: A model for substrate recognition by xanthine oxidase

Summary Tautomerism of neutral xanthine and alloxanthine has been examined both in the gas phase and in aqueous solution. The tautomeric preference in the gas phase has been studied by means of semiempirical and ab initio quantum-mechanical computations with inclusion of correlation effects at the Møller-Plesset level, and from density-functional calculations. The influence of solvent on the relative stability between tautomers has been estimated from self-consistent reaction field calculations performed with different models. The results provide a detailed picture of tautomerism for these biologically relevant purine bases. The functional implications in the recognition by xanthine oxidase are analyzed from inspection of the interaction patterns of the most stable tautomeric forms. A model for the recognition of these purine derivatives in the enzyme binding site is discussed.     

X-ray spectroscopy of heterocyclic biochemicals: xanthine, hypoxanthine, and caffeine

The electronic structures of the purine derivatives xanthine, hypoxanthine and caffeine have been investigated in the gas phase using C, N, and O 1s X-ray photoemission (XPS) and near edge X-ray absorption fine structure (NEXAFS) spectroscopy. The results have been interpreted by means of ab initio calculations using the third-order algebraic-diagrammatic construction (ADC(3)) method for the one-particle Green's function and the second-order ADC method (ADC(2)) for the polarization propagator. The carbon, nitrogen and oxygen K-edge NEXAFS spectra of xanthine and caffeine are very similar, since the molecules differ only by substitution of three hydrogen atoms by methyl groups. For hypoxanthine, the electronic structure and spectra differ considerably from xanthine as the purine ring is more highly conjugated, and there is one less oxo group. Effects due to oxo-hydroxy tautomerism were not observed. However, the two oxo tautomeric forms of hypoxanthine oxo-N(9)-H and oxo-N(7)-H are populated in the gas phase, and the C 1s spectra can be simulated only by taking account of these two tautomers, with appropriate Boltzmann population ratios which we have also calculated. For xanthine and caffeine, single tautomeric forms were observed.     

Gas phase prototropic equilibrium studies of 2,4-dihydroxyquinoline by cndo/2 calculations, mass spectrometry and derivatization

The tautomerism of 2,4-dihydroxyquinoline in the gas phase is discussed in terms of its electron impact fragmentation pattern and of quantum chemical calculations by the LCAO-MO method in the CNDO/2 approximation. Dihydroxyquinoline methylated at position 4 can only exist in two tautomeric forms. Thus, comparison of mass spectrometric data of this compound with dihydroxyquinoline provided additional evidence for the gas phase equilibrium of the latter. The equilibria of these compounds in the gas phase were found to be displaced towards the hydroxyl tautomers.     

The tautomerism of 1,2,3-triazole, 3(5)-methylpyrazole and their cations

The annular tautomerism of 1,2,3-triazole and 3(5)-methylpyrazole is discussed by means of a combination of theoretical calculations and experimental (ICR) gas-phase basicities and acidities. In the gas phase 1,2,3-triazole exists as the 2H-tautomer, whereas both tautomers of 3(5)-methylpyrazole are of similar energy. The solvent effects on these prototropic equilibria are discussed taking into account solvent properties as polarity/polarizability, acidity, and basicity. In nonhydrogen bonding solvents, the difference in dipole moments between both tautomers plays a role that has usually been underestimated.     

别嘌呤醇对黄嘌呤氧化酶的抑制作用研究

用聚苯胺黄嘌呤氧化酶电极（生物传感器）研究了别嘌呤醇对黄嘌呤对氧化酶的抑制作用，实验结果表明：别嘌呤醇对黄嘌呤氧化酶有明显的抑制作用，但这种抑制作用是可逆的，抑制剂在存在使固定酶的表观米氏常数增大但并没有影响催化反应的最大速率，所以别嘌呤是黄嘌呤氧化酶的一种可逆竞争抑制剂，抑制剂对固定酶的最适ｐＨ值没有影响，而对反应活化能略能影响。     

Amperometric Study of Au‐Colloid Function on Xanthine Biosensor Based on Xanthine Oxidase Immobilized in Polypyrrole Layer

Four kinds of xanthine oxidase (XOD) based amperometric biosensors were fabricated and their analytical performances were compared. Polypyrrole (PPY)/XOD biosensor was constructed by electrochemical oxidation of pyrrole in the solution containing xanthine oxidase and pyrrole in this paper. Colloidal Au was then immobilized on the biosensor. On the other hand, electron mediator, Prussian Blue (PB), was deposited on the electrode before the immobilization of PPY/XOD to enhance electron‐transfer rate and current response. The results showed that PPY/XOD, PPY/XOD/Au‐colloid, PB/PPY/XOD and PB/PPY/XOD/Au‐colloid biosensors exhibit good response to xanthine in 1×10^?6 M and 2×10^?5 M and Michaelis‐Menten constants (K_m) of these biosensors were 242.2, 113.4, 144.5, 43.2 μmol?L^?1, respectively. The dependence of current responses with applied voltages was discussed, and different mechanisms of these biosensors were discussed. It has been found that colloidal Au can enhance the current response at the same concentration of xanthine solution and decrease the energy‐barrier of electron‐transfer reaction on the electrode.     

伞形酮对黄嘌呤氧化酶的抑制作用研究

The investigation on inhibition of xanthine oxidase by umbelliferone was carried out using a polyaniline-xanthine oxidase electrode. The experimental results indicated significant inhibition caused by umbelliferone. The apparent Michaelis-Menten constant (K'm) and the maximum rate (im) of the immobilized xanthine oxidase were both affected by umbelliferone. This indicated that umbelliferone acts as a mixed type of inhibitor of xanthine oxidase. The optimal pH of the immobilized enzyme was hardly affected by umbelliferone; The response current increases with increasing potential in the potential range 0.55~0.68V.     

Electron ionization mass spectra and tautomerism of substituted 2‐phenacylquinolines

Tautomerism has been studied conventionally in solutions or in the solid state. However, the importance of mass spectrometry in the gas phase was realized relatively late. 2‐Phenacylquinolines are known to undergo ketimine‐enaminone tautomerism. The ratio of tautomers is dependent on the nature of the phenyl ring substituent and the Hammett substituent constants σ. Theoretical calculations indicate the presence of ketimine and enaminone tautomers in the gas phase. The electron ionization mass spectra of eight 2‐phenacylquinolines (ketimine form) were recorded at 70 eV in order to determine the fragmentation routes and to screen for the presence of their enaminone tautomers, (Z)‐2‐benzoylmethylene‐1,2‐dihydroquinolines, in the gas phase. The relative abundances or total ion currents of some ions correlated with the Hammett substituent constants and Hammett‐Brown constants. The product ions [M–CO]^{+ .} and [M–HCO]⁺ were observed. A reaction mechanism is suggested for the formation of these ions, requiring skeletal rearrangements. The results furnish information relating to tautomerism in the gas phase. Copyright © 2009 John Wiley & Sons, Ltd.     

Xanthine and hypoxanthine sensors based on xanthine oxidase immobilized in poly(mercapto-p-benzoquinone) film

The construction and performance of an enzyme electrode as an amperometric sensor of xanthine and hypoxanthine is described. Xanthine oxidase has been immobilized in a conductive redox polymer, poly(mercapto-p-benzoquinone), by means of an electropolymerization of mercaptohydroquinone in the presence of the enzyme. An Au-electroplated glassy carbon electrode coated with the resulting polymer film functioned well as a direct response type of sensor, where the polymer chain served as a conductive molecular chain between the active sites in the enzyme and the substrate electrode. Response characteristics as well as kinetic parameters have been evaluated.     

Unique tautomeric properties of isoguanine

The tautomeric properties of isoguanine (also named 2-oxoadenine or 2-hydroxyadenine) have been studied in the gas phase, in different pure solvents, and in the DNA environment using state of the art theoretical methods. Our results show that isoguanine constitutes an unique example of how tautomerism can be modulated by the environment. Compared to the tautomeric preference in the gas phase, both polar solvents and the DNA microenvironment dramatically change the intrinsic tautomeric properties of isoguanine. Tautomers which are important in physiological conditions are less than 1/10(5) of the total population of isoguanine in the gas phase. The impact of the present findings in the understanding of spontaneous mutations and in the design of new nucleobases with multiple recognition properties is discussed.     

离心超滤质谱法筛选中药复方二妙丸中黄嘌呤氧化酶抑制剂

采用离心超滤质谱分析技术(UF-UPLC/Q-TOF/MS), 结合体外酶活性实验方法, 对中药复方二妙丸提取物中的黄嘌呤氧化酶抑制剂进行了筛选. 体外酶活性实验测得二妙丸水提液对黄嘌呤氧化酶的半数抑制浓度(IC₅₀)为(0.218±0.0034) mg/mL, 表明二妙丸具有较强的黄嘌呤氧化酶抑制活性. 进一步采用离心超滤质谱技术对二妙丸水提物中潜在的黄嘌呤氧化酶抑制剂进行筛选, 从中筛选并鉴定了9种具有潜在黄嘌呤氧化酶抑制活性的化合物, 为开发黄嘌呤氧化酶抑制剂及阐明二妙丸治疗痛风和高尿酸血症的作用机制提供了一定的依据.     

Mass spectrometry of beta-ketoesters. Some evidence of their tautomerism

Mass spectrometric evidence of tautomerism is reported for beta-ketoesters. The analysis of the corresponding mass spectra has allowed specific assignment of fragment ions to tautomers. The predictive value of this methodology is supported by the influence of substitution pattern of these compounds on these equilibria. Experimental data are strongly supported by Austin Model 1 semiempirical calculations indicating that mass spectrometry could be resourced as a tool for the investigation of tautomerism of neutral species in the gas phase.     

Theoretical study of three predominant tautomers of 2-oxo-6-methylpurine and their two transition state structures

The three predominantly stable tautomers of 2-oxo-6-methylpurine were studied in gas phase and aqueous solution by means of quantum mechanical calculations. Two transition state structures connecting these three tautomeric forms on the free energy surface were determined. The activation free energy for the intramolecular proton transfer in gas phase was calculated to be considerably smaller than the bond energy of either N-H or O-H: 59.01 and 30.37 kcal/mol for N9→N3 and N1→O2, respectively, obtained at the QCISD(T)/6-31G+(d)//MP2(full)/6-31G(d) level of theory.     

用聚苯胺膜从牛乳中直接分离黄嘌呤氧化酶

酶电极;新鲜牛乳;固定分离;用聚苯胺膜从牛乳中直接分离黄嘌呤氧化酶     

Electrochemical Investigation on Kinetics of Xanthine Metabolism and Inhibition Effect of Febuxostat on Xanthine Oxidase Activity

Xanthine is a significant biomolecule and its concentration level in urine and blood plasma is an indicator of specified pathological states. Here, a new sensing platform was designed, which showed excellent analytical performance for xanthine. Importantly, it is the first time to investigate the kinetics of xanthine metabolic reaction by electrochemical method. The results demonstrated that the conversion of xanthine to uric acid completely conformed to the Michaelis-Menten kinetics. Furthermore, we also studied the inhibitory effect of febuxostat on xanthine oxidase activity detailed. As expected, the work may offer potential value for researchers in the treatment of hyperuricemia and gout.     

黄嘌呤及其互变异构体的密度泛函理论研究

嘌呤碱及其衍生物在生物系统中起重要作用。对人具有兴奋和利尿作用的茶碱和咖啡碱就是黄嘌呤的甲基衍生物。黄嘌呤存在多种互变异构体，从理论计算的角度研究这些互变异构体的几何结构、电子结构及相对稳定性，进一步研究溶剂对其结构和性质的影响是有意义旧。密度泛函理论方法既考虑了电子相关，又较其它CI（组态相互作用）或MPn（n级微扰）方法节省机时。其计算结果较好，被广泛应用于研究各种化合物。本文采用密度泛函B3LYP／6—311G方法对14种黄嘌呤可能的互变异构体（见图1），分别在气相和水相中进行几何构型全自由度优化和能量计算，讨论了异构体的相对稳定性，水的溶剂化作用对异构体的能量、几何构型、电荷分布和偶极矩的影响，探讨了溶剂极性对异构体的能量和偶极矩的影响。     

Quick identification of xanthine oxidase inhibitor and antioxidant from Erycibe obtusifolia by a drug discovery platform composed of multiple mass spectrometric platforms and thin‐layer chromatography bioautography

As a final step of the purine metabolism process, xanthine oxidase catalyzes the oxidation of hypoxanthine and xanthine into uric acid. Our research has demonstrated that Erycibe obtusifolia has xanthine oxidase inhibitory properties. The purpose of this paper is to describe a new strategy based on a combination of multiple mass spectrometric platforms and thin‐layer chromatography bioautography for effectively screening the xanthine oxidase inhibitory and antioxidant properties of E. obtusifolia. This strategy was accomplished through the following steps. (i) Separate the extract of E. obtusifolia into fractions by an autopurification system controlled by liquid chromatography with mass spectrometry. (ii) Determine the active fractions of E. obtusifolia by thin‐layer chromatography bioautography. (iii) Identify the structure of the main active compounds with the information provided by direct analysis in real time mass spectrometry. (iv) Calculate the IC₅₀ value of each compound against xanthine oxidase using high‐performance liquid chromatography. Using the caulis of E. obtusifolia as the experimental material, seven target peaks were screened out as xanthine oxidase inhibitors or antioxidants. Our screening strategy allows for rapid analysis of small molecules with almost no sample preparation and can be completed within a week, making it a useful assay to identify unstable compounds and provide the empirical foundation for E. obtusifolia as a natural remedy for gout and oxidative‐stress‐related diseases.     

THE ROLE OF O2- IN THE CHEMILUMINESCENCE OF LUMINOL*

Abstract— The chemiluminescence of luminol in buffered aqueous solutions is inhibited by superoxide dismutase. This occurs whether the luminescence is induced by ferricyanide, persulfate, hypochlorite, or by the action of xanthine oxidase on xanthine. Since superoxide dismutase inhibits reactions which involve O₂^-, we conclude that this radical is a constant factor in the chemiluminescence of luminol in aqueous solutions. The kinetics of light production are discussed in terms of hypothetical mechanisms that fit the available data. The strong luminescence of luminol in aprotic solvents or in aqueous systems containing relatively high concentrations of H₂O₂ could not be explored in this way, because superoxide dismutase is inactive under such conditions.     

2-羟基咪唑在气相和水中的异构平衡和质子迁移的从头算计算和Monte Carlo模拟

采用从头算方法在MP2/6-31＋G^*水平上研究了2-羟基咪唑分子在孤立分子和一水合物的异构体的相对稳定性和可能的质子迁移反应, 分析了一个水分子的参与对2-羟基咪唑分子异构体的相对稳定性和质子迁移速率的影响, 采用Monte Carlo模拟方法研究了反应体系在水溶液中反应的溶剂化效应. 结果表明: 2-羟基咪唑分子的孤立分子和一水合物的最稳定异构体相同, 都为酮式. 直接质子迁移反应在水溶液中活化能垒有轻微增加, 但产物能量得到降低; 水助催化质子迁移反应在水溶液中的活化能垒和产物能量都得到明显降低. 综合气相和水相的计算结果, 2-羟基咪唑水助催化的质子迁移反应较易进行, 且在水溶液中进行容易, 可以很容易被实验观察到.     

Tautomerism in Schiff bases. The cases of 2-hydroxy-1-naphthaldehyde and 1-hydroxy-2-naphthaldehyde investigated in solution and the solid state

Schiff bases derived from hydroxyl naphthaldehydes and o-substituted anilines have been prepared and their tautomerism assessed by spectroscopic, crystallographic, and computational methods. Tautomeric equilibria have also been studied and reveal in most cases a slight preference of imine tautomers in solution; a fact supported by DFT calculations in the gas phase as well as incorporating solvent effects through the SMD model. To simulate the effect exerted by the crystal lattice on tautomer stability, we have developed a computational protocol in the case of 1-tert-butyl-2-(2-hydroxy-1-naphthylmethylene)aminobenzene whose data have been obtained experimentally at 120 K. Although a rapid imine-enamine interconversion may be occurring in the solid state, the imine tautomer becomes the most stable form and the energy difference should be related to the difference in the packing of the molecules.