Development and validation of a sensitive method for simultaneous determination of rosuvastatin and N‐desmethyl rosuvastatin in human plasma using liquid chromatography/negative electrospray ionization/tandem mass spectrometry

A sensitive and specific liquid chromatography tandem mass spectrometric method was developed and validated for the simultaneous determination of rosuvastatin (ROS) and N‐desmethyl rosuvastatin (NOR‐ROS) in human plasma using deuterium‐labeled internal standards. The plasma samples were prepared using liquid–liquid extraction with diethyl ether. Chromatographic separation was accomplished on an Xterra MS C₁₈ column. The mobile phase consisted of a gradient mixture of 15 µmol/L ammonium acetate in water and in methanol, maintained at a flow rate of 0.4 mL/min. Mass spectrometric detection was carried out in negative electrospray ionization mode and monitored by quantification and qualification transitions for each analyte. Using 300 μL plasma samples, the lower limits of quantification of ROS and NOR‐ROS were 0.05 and 0.02 µg/L respectively. The linearity of ROS and NOR‐ROS ranged from 0.05 to 42 and 0.02 to 14 µg/L respectively. The relative standard deviations of ROS and NOR‐ROS were <13 and 9%, respectively, while the deviations from expected values were within ?4.7–9.8 and ?5.2–4.6%, respectively. The present method offered high sensitivity and was successfully applied to a 24 h pharmacokinetic study of ROS and NOR‐ROS in healthy subjects receiving a single dose of 10 mg ROS. Copyright © 2013 John Wiley & Sons, Ltd.     

DFT analysis of the active site in catalytic metabolic redox reactions of mononuclear molybdenum enzymes

Abstract

Model complexes [Mo^VIO₂(S₂C₂Me₂)SMe]^? (A, derived from the X-ray crystal structure of native sulfite oxidase (SO)) and [Mo^VIO₂(mnt)₂]^2? (B, coordination mode similar to the active site of selenate reductase (SeR)) were computed at the COSMO-B3LYP/SDDp//B3LYP/Lanl2DZ(p) energy level of Density Functional Theory in order to study their behavior in oxidation of selenite (Se^IV) and sulfite (S^IV) to selenate (Se^VI) and sulfate (S^VI), respectively. For the oxidation of sulfite, computational model A, which resembles the SO active site, is clearly the best choice (lowest barrier, minor exothermicity). For the reduction of selenate, a smaller activation is computed for model A; however, the reaction is less exothermic with model B, which resembles the SeR active site.     

Synthesis and structural characterization of nitrite-coordinating CoII and CoIII complexes as models for the reaction center of Co-substituted nitrite reductase

Co^II and Co^III complexes containing nitrite and tridentate aromatic amine compounds [bis(6-methyl-2-pyridylmethyl)amine (Me₂bpa) and bis(2-pyridylmethyl)amine (bpa)] have been prepared as models of the catalytic center in Co-substituted nitrite reductase: [Co^II(Me₂bpa)(NO₂)Cl]₂ · acetone (2), Co^II(Me₂bpa)(NO₂)₂ (3), Co^II(bpa)(NO₂)Cl (4), Co^II(bpa)(NO₂)₂ (5), Co^III(Me₂bpa)(NO₂)(CO₃) (6), and Co^III(bpa)(NO₂)₃ (7). The X-ray crystal structure analyses of these Co^II and Co^III complexes indicated that the geometries of the cobalt centers are distorted octahedral and the Me₂bpa and bpa with three nitrogen donors exhibit mer- (2, 3, and 7) and fac-form (4 and 6). The coordination mode of nitrite depends on the cobalt oxidation state, to Co^II through the oxygen (nitrito coordination, O- and O,O-coordination) and to Co^III through nitrogen (nitro coordination, N-coordination mode). These findings are consistent with the results of their IR spectra, except that another oxygen of the O-coordinated nitrito group in 3 might interact weakly with Co^II according to its IR spectrum. Reductions of the nitrite in 2, 3, 4, and 5 to nitrogen monoxide were not accelerated in the presence of proton, perhaps due to the nitrito coordination in these Co^II complexes.     

羰基还原酶辅酶结合域位点突变对其不对称催化性能的影响

基于同源模型的比较和分析,发现羰基还原酶SCR1辅酶结合域P124和W125位点对辅酶NADPH的结合形成了一定的空间位阻效应.通过对该位点进行小侧基氨基酸的取代突变,该酶的底物专一性和立体选择性均发生了不同程度的改变,表明该位点是酶与辅酶有效结合的关键位点,而且它与辅酶结合的空间效应进一步影响了底物结合域活性中心对不同构型的底物及其对映体产物的亲和作用.在底物专一性方面,野生型酶对2-羟基苯乙酮和2-溴苯乙酮及其衍生物等底物表现出较高的催化活性,而突变株W125A,W125G,P124A/W125A和P124G/W125G对苯乙酮及其部分衍生物和2-辛酮等底物的催化活性均有所提高.对于酶的立体选择性,部分突变株发生了转化产物对映体构型反转的现象,突变株P124A/W125A和P124G/W125G催化还原2-羟基苯乙酮和4-氯乙酰乙酸乙酯均生成了(R)-型产物.     

Fabrication of Redox‐Mediator Supported Potentiometric Nitrate Biosensor with Nitrate Reductase

Fabrication of a more superior nitrate potentiometric biosensor than previously achieved with NaR and NADH has been accomplished by co‐entrapment of redox mediators and NaR into polypyrrole (PPy) film during galvanostatic polymerization of pyrrole. The replacement of NADH with redox mediators such as thionin acetate (ThAc), safranin (Saf), and azure A (AzA) gave more sensitive potentiometric responses, better minimum detectable concentration, linear concentration range and response time for nitrate than possible with NADH. The co‐entrapment of ThAc, Saf, AzA and methyl viologen (MV) with NaR into PPy films also improved the Nernstian behavior of the electrode process beyond the capability of the PPy‐NaR‐NADH biosensor. Substantial reduction in volume and quantity of cofactor/mediator and, hence cost, was achieved by the replacement of NADH with a redox mediator. Only 50 μM of AzA was required to form a PPy‐NaR‐AzA biosensor which gave the most sensitive potentiometric response for nitrate, achieving a minimum detectable concentration of 10 μM, a linear concentration range of 50–5000 μM and a response time of 2–4 s.     

Electrocatalytic Nitrite Reduction to Nitrogen Oxide by a Synthetic Analogue of the Active Site of Cu‐Containing Nitrite Reductase Incorporated in Nafion Film

The electrocatalytic reduction of nitrite to NO by [CuMe₂bpa(H₂O)(ClO₄)]⁺ ( 1 ), which is a model for the active site of copper‐containing nitrite reductase, incorporated in Nafion film was investigated. The Cu complex in the Nafion matrix exhibits an intense band at 267 nm and a broad band around 680 nm, assigned to d–d and ligand field transitions, respectively. The 77‐K EPR spectrum of 1 in the Nafion matrix reveals the typical axial signals (g_//=2.28, g =2.08, A_//=13.3 mT) of a tetragonal Cu²⁺ chromophore. The redox potential, which is related to the Cu⁺/Cu²⁺ couple, was ?146 mV (ΔE=72 mV) at pH 5.5. The redox reaction of 1 in Nafion was not dependent on pH and was a diffusion‐controlled process. The electronic structure and redox properties of 1 in the negatively charged polymer matrix were almost the same as those in aqueous solution. In the presence of nitrite, an increase in the cathodic current was observed in the cyclic voltammogram of 1 in the Nafion matrix. The current increase was dependent on the nitrite concentration and pH in solution. Upon reaching ?400 mV, a linear generation of NO was observed for the 1 /Nafion film coated electrode. The relationship between the rate of NO generation and the nitrite concentration in solution was analyzed with the Michaelis–Menten equation, where V_max=45.1 nM s^?1 and K_m=15.8 mM at pH 5.5. The Cu complex serves the function of both the catalyst and electron transport in the Nafion matrix. The sensitivity of the electrode was estimated to be 3.23 μA mM^?1 in the range of 0.1–0.4 mM nitrite.     

Molecular docking studies of selected tricyclic and quinone derivatives on trypanothione reductase of Leishmania infantum

Visceral leishmaniasis, most lethal form of Leishmaniasis, is caused by Leishmania infantum in the Old world. Current therapeutics for the disease is associated with a risk of high toxicity and development of drug resistant strains. Thiol‐redox metabolism involving trypanothione and trypanothione reductase, key for survival of Leishmania, is a validated target for rational drug design. Recently published structure of trypanothione reductase (TryR) from L. infantum, in oxidized and reduced form along with Sb(III), provides vital clues on active site of the enzyme. In continuation with our attempts to identify potent inhibitors of TryR, we have modeled binding modes of selected tricyclic compounds and quinone derivatives, using AutoDock4. Here, we report a unique binding mode for quinone derivatives and 9‐aminoacridine derivatives, at the FAD binding domain. A conserved hydrogen bonding pattern was observed in all these compounds with residues Thr335, Lys60, His461. With the fact that these residues aid in the orientation of FAD towards the active site forming the core of the FAD binding domain, designing selective and potent compounds that could replace FAD in vivo during the synthesis of Trypanothione reductase can be deployed as an effective strategy in designing new drugs towards Leishmaniasis. We also report the binding of Phenothiazine and 9‐aminoacridine derivatives at the Z site of the protein. The biological significance and possible mode of inhibition by quinone derivatives, which binds to FAD binding domain, along with other compounds are discussed. © 2010 Wiley Periodicals, Inc. J Comput Chem, 2010     

Efficient Synthesis of a Water-Soluble Glucoamide Inhibitor Against Human Aldose Reductase by Click Chemistry

While D-glucose is the natural substrate of aldose reductase (AR) in the polyol pathway, the K _m value of D-glucose against AR is large. A glucoamide 1 was designed as a tool to investigate whether AR has a strong affinity for the open form of D-glucose. Glucoamide 1 was synthesized in high yield by modification of the reaction condition for click chemistry. It was found that our modified condition was applicable for highly polar alkynes and gave coupling products in excellent yield (90% to 100%). Although weak inhibitory activity against AR was observed, kinetic studies showed that AR does not accept glucoamide 1 in its active site.