On-chip type cation-exchange chromatography with ferrocene-labeled anti-hemoglobin antibody and electrochemical detector for determination of hemoglobin A1c level

An on-chip type cation-exchange chromatography system with electrochemical detection of HbA_1c, which is one of the most important diabetes marker protein, was developed using ferrocene-conjugated anti-human hemoglobin (Hb) monoclonal antibody (FcAb). The FcAb was used as an electrochemical probe for the detection of each Hb. The system contains syringe pump, on-chip type cation-exchange column consisted of PDMS and cation-exchange resin beads, and a three-electrode flow-cell system. The separation conditions of HbA_1c in blood calibrator samples from other Hbs, e.g. HbA₀, HbA_1a or HbA_1b, were optimized using the on-chip type system. The electrochemical oxidation current from FcAb reacting with each Hb was measured at 350 mV (vs. Ag/AgCl). Hbs including HbA_1a and HbA_1b, HbA_1c and HbA₀ fractions were eluted in this order. A linear relationship between HbA_1c levels and electrochemical oxidation currents was obtained in the range from 4.0% to 12.6% HbA_1c. All procedure including antigen-antibody reaction was completed in 15 min. Furthermore, a good correlation was obtained between KO500 method (HPLC) and our proposed method. These results indicate that the on-chip type system with electrochemical detection can be applied to a novel POCT device for rapid and precise detection of HbA_1c.     

Modelling Drugs and Receptors Using Potentials: Examples in the GPCRs' Domain

Abstract

Shape complementarity, electrostatic and hydrophobic matching, were used to model drugs and receptors. From known experimental data on α_1A/α_2A?adrenergic ligands and α_1A/α_2A?alrenoceptors, a model for the ligand binding sites, based on the structure of bacteriorhodopsin as a template, was proposed and built. Agonists and antagonists have overlapping but different binding sites. Emphasis was given on the role of the disulphide bridge and on the role of the sodium site. The model was extended to other G-protein coupled receptors.     

Design and Synthesis of Arylpiperazine Serotonergic/Dopaminergic Ligands with Neuroprotective Properties

Long-chain arylpiperazine scaffold is a versatile template to design central nervous system (CNS) drugs that target serotonin and dopamine receptors. Here we describe the synthesis and biological evaluation of ten new arylpiperazine derivatives designed to obtain an affinity profile at serotonin 5-HT_1A, 5-HT_2A, 5-HT₇ receptor, and dopamine D₂ receptor of prospective drugs to treat the core symptoms of autism spectrum disorder (ASD) or psychosis. Besides the structural features required for affinity at the target receptors, the new compounds incorporated structural fragments with antioxidant properties to counteract oxidative stress connected with ASD and psychosis. All the new compounds showed CNS MultiParameter Optimization score predictive of desirable ADMET properties and cross the blood–brain barrier. We identified compound 12a that combines an affinity profile compatible with antipsychotic activity (5-HT_1A K_i = 41.5 nM, 5-HT_2A K_i = 315 nM, 5-HT₇ K_i = 42.5 nM, D₂ K_i = 300 nM), and compound 9b that has an affinity profile consistent with studies in the context of ASD (5-HT_1A K_i = 23.9 nM, 5-HT_2A K_i = 39.4 nM, 5-HT₇ K_i = 45.0 nM). Both compounds also had antioxidant properties. All compounds showed low in vitro metabolic stability, the only exception being compound 9b, which might be suitable for studies in vivo.     

Facilitated electron transfer of hemoglobin embedded in nanosized Fe3O4 matrix based on paraffin impregnated graphite electrode and electrochemical catalysis for trichloroacetic acid

Nanosized Fe₃O₄, coated with acrylic acid copolymer approximately 5 nm in size, was synthesized and used for fabrication of hemoglobin (Hb) immobilized electrochemical sensor. Facilitated electron transfer (eT) of Hb embedded in the nanosized Fe₃O₄ solid matrix on paraffin impregnated graphite electrode (PIGE) was observed corresponding to Hb heme Fe(III)/Fe(II) conversion. A pair of stable and well-defined redox peaks at approximately −0.293 V vs. saturated calomel electrode was obtained at the Hb-Fe₃O₄ modified PIGE. It was found that nanosized Fe₃O₄ solid matrix could offer a friendly platform to assemble Hb molecules and greatly enhance the eT in the absence of conventional mediators. The standard rate constant, k_s, was determined as 4.2 s⁻¹. Spectroscopic studies revealed that Hb retained the native-like structure in the nano-Fe₃O₄ solid matrix. Embedded Hb showed a great catalytic activity toward trichloroacetic acid (TCA) with 900 mV decrease in reduction overpotential. This was used for voltammetric determination of TCA with detection limit of approximately 0.116 mmol l⁻¹.     

Determination of the absolute configuration of marine oxylipin topsentolide A1 by the synthesis of the enantiomer of the natural product

Two possible stereoisomers of topsentolide A₁, a cytotoxic oxylipin against human solid tumor cell lines, were prepared in order to determine the stereochemistry of natural product. That is, the enantiomer of topsentolide A₁, (8S,11S,12R)-isomer, and its diastereomer was efficiently synthesized in a stereoselective manner. The stereochemistry of topsentolide A₁ was determined to be 8R,11R,12S by comparing NMR spectra and specific rotations of the synthetic isomers and the natural product.     

Synthesis of an o-Nitrobenzyl Attached A1 Adenosine Receptor Antagonist,a Prodrug Approach

Abstract

The o-nitrobenzyl group, possessing distinct advantage of being photolabile under mild conditions, was successfully connected to 8-(5,6-epoxynorbornan-2-yl)-1,3-dipropylxanthine (5), a high specific A₁ adenosine receptor antagonist. The resulting compound 4 would have potential use as a prodrug.     

Current Knowledge on Mammalian Phospholipase A1, Brief History,Structures, Biochemical and Pathophysiological Roles

Phospholipase A₁ (PLA₁) is an enzyme that cleaves an ester bond at the sn-1 position of glycerophospholipids, producing a free fatty acid and a lysophospholipid. PLA₁ activities have been detected both extracellularly and intracellularly, which are well conserved in higher eukaryotes, including fish and mammals. All extracellular PLA₁s belong to the lipase family. In addition to PLA₁ activity, most mammalian extracellular PLA₁s exhibit lipase activity to hydrolyze triacylglycerol, cleaving the fatty acid and contributing to its absorption into the intestinal tract and tissues. Some extracellular PLA₁s exhibit PLA₁ activities specific to phosphatidic acid (PA) or phosphatidylserine (PS) and serve to produce lysophospholipid mediators such as lysophosphatidic acid (LPA) and lysophosphatidylserine (LysoPS). A high level of PLA₁ activity has been detected in the cytosol fractions, where PA-PLA₁/DDHD1/iPLA₁ was responsible for the activity. Many homologs of PA-PLA₁ and PLA₂ have been shown to exhibit PLA₁ activity. Although much has been learned about the pathophysiological roles of PLA₁ molecules through studies of knockout mice and human genetic diseases, many questions regarding their biochemical properties, including their genuine in vivo substrate, remain elusive.     

Brain Iron Deficiency Changes the Stoichiometry of Adenosine Receptor Subtypes in Cortico-Striatal Terminals: Implications for Restless Legs Syndrome

Brain iron deficiency (BID) constitutes a primary pathophysiological mechanism in restless legs syndrome (RLS). BID in rodents has been widely used as an animal model of RLS, since it recapitulates key neurochemical changes reported in RLS patients and shows an RLS-like behavioral phenotype. Previous studies with the BID-rodent model of RLS demonstrated increased sensitivity of cortical pyramidal cells to release glutamate from their striatal nerve terminals driving striatal circuits, a correlative finding of the cortical motor hyperexcitability of RLS patients. It was also found that BID in rodents leads to changes in the adenosinergic system, a downregulation of the inhibitory adenosine A₁ receptors (A₁Rs) and upregulation of the excitatory adenosine A_2A receptors (A_2ARs). It was then hypothesized, but not proven, that the BID-induced increased sensitivity of cortico-striatal glutamatergic terminals could be induced by a change in A₁R/A_2AR stoichiometry in favor of A_2ARs. Here, we used a newly developed FACS-based synaptometric analysis to compare the relative abundance on A₁Rs and A_2ARs in cortico-striatal and thalamo-striatal glutamatergic terminals (labeled with vesicular glutamate transporters VGLUT1 and VGLUT2, respectively) of control and BID rats. It could be demonstrated that BID (determined by measuring transferrin receptor density in the brain) is associated with a selective decrease in the A₁R/A_2AR ratio in VGLUT1 positive-striatal terminals.     

Determination of thiamine in blood serum and urine by high-performance liquid chromatography with direct injection and post-column derivatization

Thiamine (vitamin B₁) was determined in human serum and urine by HPLC with fluorimetric detection of its oxidation product, thiochrome. The samples were injected directly into the chromatographic system without previous treatment or dilution. A column filled with an ultra-high molecular weight surface-modified polyethylene (PE) was able to separate matrix components from analyte and also to allow a good chromatographic resolution of thiamine. The interaction of thiamine, thiocrome and both matrices (serum and urine) with PE was studied off- and on-line to determine the optimal procedure for vitamin B₁ determination. When carried off-line, matrix adsorption yield was 49 mg serum proteins/g polymer and components of 1000 μl urine/g polymer. In an on-line arrangement, the yield dropped to 10 mg/g and 150 μl/g, respectively. The matrix/analyte separation was carried out in an on-line procedure on a 50×4.6-mm, 25-μm PE column, using a water-sodium phosphate-methanol gradient elution. Part of the matrix was eluted within the first 2 min and thiamine after 3.8 min. The rest of the matrix retained on the column was eluted after thiamine at the last step of the gradient elution. Analysis time was 12 min. The within-day and day-to-day precision gave C.V. varying from 3.6% to 14.5% and recoveries from spiked samples were in the range of 84.8-98.8%.     

Exploring the molecular basis of selectivity in A1 adenosine receptors agonists: a case study

Adenosine is a naturally occurring purine nucleoside that has a wide variety of well-documented regulatory functions and physiological roles. Selective activation of the adenosine A₁ receptor has drawn attention in drug discovery for the therapeutic effects on neural and cardiovascular disorders. We have developed a model of the human A₁ adenosine receptor using bovine rhodopsin as a template. A flexible docking approach has been subsequently carried out for evaluating the molecular interactions of twenty-one selective A₁ agonists with the receptor model. The results of these studies are consistent with mutational and biochemical data. In particular, they highlight a wide hydrogen-bonding network between the nucleoside portion of the ligands and the A₁ receptor as well as key amino acids for hydrophobic interactions with the different N⁶-groups of the agonists. The models presented here provide a detailed molecular map for the selective stimulation of the adenosine A₁ receptor subtype and a steady basis for the rational design of new A₁ selective ligands.     

Synthesis, Structure, and 31P NMR of Sulfur/Oxygen-Bridged Incomplete Cubane-Type Molybdenum and Tungsten Clusters with Triphenylphosphine Ligands

Sulfur/oxygen-bridged incomplete cubane-type triphenylphosphine molybdenum and tungsten-clusters [Mo₃S₄Cl₄(H₂O)₂(PPh₃)₃]·3THF (1A), [Mo₃S₄Cl₄(H₂O)₂(PPh₃)₃]·2THF (2A), [Mo₃OS₃Cl₄(H₂O)₂(PPh₃)₃]·2THF (1B), and [W₃S₄Cl₄(H₂O)₂(PPh₃)₃]·2THF (1C) were prepared from the corresponding aqua clusters and PPh₃ in THF/MeOH. On recrystallization from THF, procedures with and without addition of hexane to the solution gave 1A and 2A, respectively, while the procedures gave no effect on the formation of 1B and 1C. Crystallographic results obtained are as follows: 1A: monoclinic, P2₁/n, a=17.141(4) Å, b=22.579(5) Å, c=19.069(4) Å, =96.18(2)°, V=7337(3) ų, Z=4, R(R _w)=0.078(0.102); 1C: monoclinic, P2 ₁/c, a=12.635(1) Å, b=20.216(4) Å, c=27.815(3) Å, =96.16(1)°, V=7062(2) ų, Z=4, R(R _w)=0.071(0.083). If the phenyl groups are ignored, the molecule [Mo₃S₄Cl₄(H₂O)₂(PPh₃)₃] in 2A has idealized CS symmetry with the mirror plane perpendicular to the plane determined by the metal atoms, while the molecule in 1A does not have the symmetry. The tungsten compound 1C is isomorphous with the molybdenum compound 2A. ³¹P NMR spectra of 1A, 2A, and 1C were obtained and compared with similar clusters with dmpe (1,2-bis(dimethylphosphino)ethane) ligands.     

Synthesis of Benzo[b]thiophene Carboxamides Connected to 4‐Arylpiperazines through a Benzylic Spacer: Potential Ligands with 5‐HT1A Binding Affinity

New benzothiophene arylpiperazine derivatives 8 (a–f) were synthesized as potential serotoninergic agents with 5‐HT_1A receptor affinity. Preparation of the derivatives was performed by treating N‐[2‐(chloromethyl)phenyl]‐4,7‐dimethoxybenzo[b]thiophene‐2‐carboxamide (7) with a series of substituted 4‐arylpiperazines.     

A Novel and Convenient Synthesis of Coenzyme Q1

Abstract

A convenient and efficient synthetic route to Coenzyme Q₁ (6) starting from 3,4,5‐trimethoxytoluene (1) is described. The key features of this synthesis include the Diels–Alder reaction of 2,3‐dimethoxy‐l,4‐benzoquinone (3) with cyclopentadiene and the introduction of a C₅ side chain to 4,5‐dimethoxy‐2‐methyltricyclo[6.2.1.0^2,7]undeca‐4,9‐diene‐3,6‐dione (4) under mild conditions, (6) was obtained in overall 60% yield.     

哒嗪酮类α1-肾上腺素受体拮抗剂的合成和生物活性研究

将苯(氧)乙胺和苯氧烷胺类α₁-肾上腺素受体拮抗剂中的苯(氧)乙胺、苯氧烷胺片段引入哒嗪酮类化合物中, 设计、合成了30个新的含哒嗪酮环的α₁-肾上腺素受体拮抗剂. 所有新化合物的结构均经¹H NMR, IR, HRMS确证. 生物活性测试表明28个目标物对α₁-肾上腺素受体有较好的拮抗作用(pA₂＞6.00), 化合物6o, 6p, 6q, 6v, 6x, 6y, 10c, 10d的pA₂值＞7.00.     

Hydrogen Peroxide Sensor Based on Hemoglobin Immobilized on Glassy Carbon Electrode with SiO2 Nanoparticles/Chitosan Film as Immobilization Matrix

Abstract

A novel amperometric sensor of hydrogen peroxide was constructed. Hemoglobin (Hb) was successfully immobilized on nanometer‐sized SiO₂, which was supported by chitosan. Chitosan was acted as dispersant. The determination of hydrogen peroxide was performed in the presence of an electron mediator hydroquinone. Hb immobilized on the SiO₂/chitosan composite film displayed excellent electrocatalytical activity to the reduction of H₂O₂. The linear range of detection towards H₂O₂ was from 6.25×10^?7 to 1.63×10^?4mol/L with a detection limit of 1.8×10^?7mol/L (S/N=3). The apparent Michaelis‐Menten constant (K ^app _M) was found to be 0.75mmol/L.     

Lonijaposides, novel cerebrosides from Lonicera japonica

Six novel cerebrosides, lonijaposides A₁-A₄, B₁ and B₂ (1-6) have been isolated from the flowers of Lonicera japonica. Their structures were established on the basis of 1D, 2D (DEPT, HMQC, HMBC and COSY) NMR, ESI-QTOF-MS/MS and chemical evidence.     

Structural studies of cerium tantalates

Structural data obtained from neutron diffraction studies of some cerium tantalate phases are presented, including the first report of the high temperature structure of a CeTaO₄ phase, Ce_0.85TaO_3.84 deduced from in situ data recorded at 900°C in vacuum. It was found that this material transformed from the low temperature LaTaO₄ type phase to the orthorhombic A2₁am structure reported here, with a unit cell of a=5.64062(2) Å, b=14.81609(6) Å and c=3.93482(1) Å. This data agrees well with the previously proposed structural transformations.     

Electrocatalytic activity of hemoglobin in sodium alginate/SiO<Subscript>2</Subscript> nanoparticle/ionic liquid BMIMPF<Subscript>6</Subscript> composite film

A novel biocompatible composite film containing sodium alginate (SA), room temperature ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate (BMIMPF₆), SiO₂ nanoparticle, and hemoglobin (Hb) was fabricated and covered on the surface of a traditional carbon paste elecrode (CPE). The immobilized Hb on the electrode surface showed good direct electrochemical behaviors, and a pair of quasi-reversible redox peaks of Hb was obtained, which indicated that the direct electron transfer of Hb with the electrode surface had been achieved. The SA/nano-SiO₂/BMIMPF₆/Hb/CPE showed dramatically electrocatalytic activity to the reduction of trichloroacetic acid, hydrogen peroxide (H₂O₂), and oxygen (O₂). The kinetic parameters for the electrocatalytic reactions were evaluated. The composite film showed the potential to the biosensor and biocatalysis.     

Hemoglobin Modified Carbon Paste Electrode: Direct Electrochemistry and Electrocatalysis

Abstract

A new hemoglobin (Hb) modified carbon paste (CP) electrode was fabricated by simply mixing the hemoglobin with carbon powder and paraffin homogeneously. To prevent the leakage of Hb from the electrode surface, a Nafion film was further applied on the surface of Hb-carbon composite paste electrode. Direct electrochemistry of hemoglobin in the paste electrode was easily achieved, and a pair of well-defined quasi-reversible redox peak of heme Fe(III)/Fe(II) couple appeared with the formal potential (E^0′) as ?0.335 V (vs. Saturated calomel electrode; CE) in pH 7.0 phosphate buffer solution (PBS). The fabricated Hb modified electrode showed good electrocatalytic ability to the reduction of trichloroacetic acid (TCA) and H₂O₂.