Evaluation of the adsorption affinity of proteins to calcium hydroxyapatites by desorption and pre-adsorption methods

The adsorption affinity of bovine serum albumin (BSA) and lysozyme (LSZ) to calcium hydroxyapatite (CaHAP) was evaluated by desorption and two step adsorption methods. These experiments were carried out at 15°C in a 1×10⁻⁴ mol dm⁻³ KCl solution of pH 6.0. BSA molecules were scarcely desorbed, exhibiting an irreversible adsorption of BSA, though LSZ slightly desorbed. This result supports our previous findings that LSZ adsorbs weakly onto phosphate ions exposed on ac or bc faces of CaHAP while BSA adsorbs strongly onto positively charged sites on ac or bc faces of CaHAP. The amount of adsorbed LSZ was markedly increased by the pre-adsorption of BSA, where LSZ was adsorbed onto BSA-covered CaHAP. On the other hand, the amount of adsorbed BSA was not changed by the pre-adsorption of LSZ. In both pre-adsorption systems it was confirmed by an HPLC method that no protein molecule pre-adsorbed was desorbed after the post-adsorption procedure. Therefore, it was interpreted that the enhancement of adsorption of positively charged LSZ is induced by an electrostatic attractive force through pre-adsorption of negatively charged BSA molecules with a high coverage. However, since the coverage of LSZ onto CaHAP is considerably low, no stimulation of BSA adsorption occurred on the LSZ-covered surface. The formation of double protein adsorbed layers consisting of pre- and post-adsorbed proteins was proposed.     

Unambiguous Determination of the Absolute Configurations of Acetylene Alcohols by Combination of the Sonogashira Reaction and the CD Exciton Chirality Method – Exciton Coupling between Phenylacetylene and Benzoate Chromophores

The CD exciton chirality method was applied to various phenylacetylene alcohols to determine their absolute configurations; the long axis polarized –^* transition (_max=252nm) of the 4-methoxyphenylacetylene chromophore couples with the transition (_max=257nm) of the 4-methoxybenzoate group to generate intense exciton split CD Cotton effects, from the signs of which the absolute configurations of phenylacetylene alcohols were unambiguously determined. As an extension of the results, a new methodology for determining the absolute configurations of acetylene alcohols having the HCCCH(OH)-moiety by combination of the Sonogashira reaction and the CD exciton chirality method has been developed and applied. Since the –^* transition of acetylene triple bond is located below 180nm, it is difficult to observe ideal bisignate CD Cotton effects due to the exciton coupling between acetylene and benzoate chromophores. To observe the ideal exciton split Cotton effects necessary for the unambiguous determination of absolute configuration, the terminal acetylene group was converted, by the Sonogashira reaction, to the 4-methoxyphenylacetylene moiety, which exhibits an intense –^* absorption band polarized along the long axis of the chromophore at 252nm. As a partner of exciton coupling, 4-methoxybenzoate showing a –^* band at 257nm was introduced into the alcohol moiety, and the benzoates formed showed intense bisignate CD Cotton effects, from the signs of which the absolute configurations of original acetylene alcohols could be determined in an unambiguous manner.     

Extraction of sodium and potassium perchlorates with benzo-18-crown-6 into various organic solvents. Quantitative elucidation of anion effects on the extraction-ability and -selectivity for Na(+) and K(+)

To investigate quantitatively the anion effect on the extraction-ability and -selectivity of benzo-18-crown-6 (B18C6) for alkali metal ions, the constants for overall extraction into various diluents having low dielectric constants (K(ex)) and aqueous ion-pair formation (K(MLA)) of B18C6-sodium and potassium perchlorate 1:1:1 complexes (MLA) were determined at 25 degrees C. The K(ex) value was analyzed by the four fundamental equilibrium constants. The K(MLA) values were determined by applying our established method to this perchlorate extraction system. The K(M(B18C6)A) value of the perchlorate is much larger for K(+) than for Na(+), and is much smaller than that of the picrate. The K(M(B18C6)A) value makes a minor contribution to the magnitude of K(ex) for the perchlorate system, but a major contribution to that for the picrate one. The distribution behavior of the B18C6 1:1:1 complexes with the alkali metal perchlorates follows the regular solution theory. For the diluent with a high dipole moment, however, the 1:1:1 complexes somewhat undergo the dipole-dipole interaction. B18C6 always shows very high extraction selectivity for KClO(4) over NaClO(4), which is determined mostly by the much greater log/(log K(MLA)) value for K(+) than for Na(+). The extraction-ability and -selectivity of B18C6 for Na(+) and K(+) ions with a perchlorate ion were compared with those with a picrate ion in terms of the fundamental equilibrium constants. The K(+) extraction-selectivity of B18C6 over Na(+) for the perchlorate system is superior to that for the picrate one, which is caused largely by the greater log/(log K(K(B18C6)A))-log/(log K(Na(B18C6)A)) value for the perchlorate than for the picrate. The perchlorate system is recommended for extraction separation of K(+) from Na(+).     

Determination of selenium in human serum by liquid chromatography/electron capture atmospheric pressure chemical ionization mass spectrometry after acid digestion and derivatization using 2,3-diaminonaphthalene

Analysis of selenium in biological samples is very important and numerous analytical methods for the element have been developed. One of the most convenient and widely used methods for routine determination of serum selenium is a fluorometric method using 2,3-diaminonaphthalene (DAN); however, this method lacks specificity. We observed that 4,5-benzopiazselenol (BPS), a selenium derivative of DAN, is ionized with electron capture in an atmospheric pressure chemical ionization (APCI) interface, and subsequently established a method for determining total human serum selenium by means of liquid chromatography/atmospheric pressure chemical ionization mass spectrometry. All pretreatment procedures were carried out in a single test tube to minimize selenium loss. The recovery of organic or inorganic selenium spiked to human serum was 97-103%.The detection limit of BPS was equivalent to 0.2 ng of selenium and the lower quantitative limit of serum selenium was 10 ng mL(-1). The coefficient of variation of standard concentrations in control serum samples was 4.5%. The purity of the observed peak obtained from serum samples was confirmed using the ion cluster technique.     

A high‐performance liquid chromatography assay with a triazole‐bonded column for evaluation of d‐amino acid oxidase activity

Elution profiles of kynurenic acid (KYNA) and 7‐chlorokynurenic acid (Cl‐KYNA) were examined by high‐performance liquid chromatography (HPLC) using a triazole‐bonded stationary phase column (Cosmosil® HILIC) under isocratic elution of a mobile phase consisting of CH₃CN–aqueous 10 mm ammonium formate between pH 3.0 and 6.0. The capacity factors of KYNA and Cl‐KYNA varied with both the CH₃CN content and the pH of the mobile phase. The elution order of KYNA and Cl‐KYNA was reversed between the CH₃CN‐ and H₂O‐rich mobile phases, suggesting that hydrophilic interactions and anion‐exchange interactions caused retention of KYNA and Cl‐KYNA in the CH₃CN‐ and H₂O‐rich mobile phases, respectively. The present HPLC method using a triazole‐bonded column and fluorescence detection (excitation 250 nm, emission 398 nm) was applied to monitor in vitro production of KYNA from d ‐kynurenine (d ‐KYN) by d ‐amino acid oxidase (DAO) using Cl‐KYNA as an internal standard. A single KYNA peak was clearly observed after enzymatic reaction of d ‐KYN with DAO. Production of KYNA from d ‐KYN was suppressed by the addition of commercial DAO inhibitors. The present HPLC method can be used to evaluate DAO activity and DAO inhibitory effects in candidate drugs for the treatment of schizophrenia. Copyright © 2015 John Wiley & Sons, Ltd.     

Tetrakis(2-hydroxyphenyl)ethene and derivatives. A structurally preorganized tetradentate ligand system for polymetallic coordination chemistry and catalysis

A topologically unique, conformationally constrained tetradentate ligand system for polymetallic coordination chemistry has been developed: tetrakis(2-hydroxyphenyl)ethene (1a) and substituted derivatives. The design exploits the planarity of the tetraphenylethylene core to impart rigidity to the roughly square oxygen binding array, while maintaining a degree of conformational mobility associated with rotation about the aryl-ethylene carbon-carbon bonds. Tetrakis(2-hydroxyphenyl)ethene derivatives are designed to promote multiple metal bridging over chelating coordination modes. The ligand is synthesized from anisole or 4-tert-butylanisole in four steps via the 2,2'-dimethoxybenzophenone hydrazones 4a,b. The sterically hindered ortho-substituted tetraphenylethylene core is produced in high yield by acid-catalyzed decomposition of the corresponding diaryl diazomethane prepared in situ by oxidation of the hydrazone using nickel peroxide. Deprotection of the methyl ethers using boron tribromide gives tetrakis(2-hydroxyphenyl)ethene (1a), characterized by X-ray crystallography, and tetrakis(5-tert-butyl-2-hydroxyphenyl)ethene (1b). Sterically isolating substituents in the 3-position can be installed via Claisen rearrangement/hydrogenation, providing tetrakis(3-n-propyl-2-hydroxyphenyl)ethene (6) efficiently. To illustrate potential applications of this unprecedented ligand class, two coordination complexes are reported, including tetrakis(2-diethylaluminoxyphenyl)ethene (8), a structurally robust eight-membered-ring aluminum/oxygen crown complex characterized both in solution and in the solid state.     

New Chiral Kemp's Acid Diamides for Chiral Amine Recognition by 1 H NMR

Two Kemp's acid diamides were synthesized and applied to chiral amine recognition using ¹H NMR analysis. One derivative based on 1-(1-naphthyl)ethylamine had good chiral recognition of six amines and was useful to determine the optical purity for three amines, i.e., methylbenzylamine, 1-(1-naphthyl)ethylamine and 1- henylpropylamine,however, the cyclohexylethylamine derivative showed little discrimination for the amines studied. Together with the results for alkylamines, it was shown that aromatic structure was important for aromatic shielding anisotropy and –– interactions between host and guest. The structure of the 1-(1-naphthyl)ethylamine derivative in solution was also considered based on ¹H NMR data and computer simulation.     

The effect of fluorination: a crystallographic and computational study of mesogenic alkyl 4-[2-(perfluorooctyl)ethoxy]benzoates

The series of alkyl 4-[2-(perfluorooctyl)ethoxy]benzoates (F8-n) shows a systematic change of crystal structures depending on the length of the alkyl chain: separate packing of perfluorooctyl (Rf) and alkyl (Rh) chains from each other for shorter (n=2) and longer (n=11) members, alternate packing of Rf and Rh chains for middle (n=6,7) members, and an intermediate type of packing for n=4. Semiempirical MO calculations show slightly repulsive interactions between the Rf chains, and attractive ones between Rf and Rh chains and between Rh and the core of a molecular pair. It is concluded that fluorination determines the molecular shape of the crystal structures by making the chain rigid. It is confirmed that the interactions between Rf chains are small compared with those between other moieties and that they are forced to aggregate owing to the exclusion from other moieties. Thus, the effect is dependent on the geometries and intermolecular interactions of the other moieties.     

Sample enrichment by using monolithic precolumns in microcolumn liquid chromatography

An on-line sample enrichment system was designed using monolithic precolumns in microcolumn LC. The monolithic ODS capillary columns were prepared via in situ sol-gel processes. The enrichment efficiency of the monolithic columns was tested by using phthalates as the analytes. The relative standard deviations (n = 6) for the retention time, peak area and peak height were between 0.4 and 1.2%, 0.9 and 5.5% and 0.4 and 3.9%, respectively. The system was linear (R2 > 0.99) within the working sample concentration and sample volume ranges. Comparing to 0.2 microl injection with a typical sample injector, the theoretical plate number of a same separation column was increased by 3-6-fold when the precolumn unit was used for sample injection. The recoveries of the analytes were between 88 and 120%, and the sample volume that could be injected into the system was increased up to 5000-fold. The limits of detection were improved by more than 2000-fold and were between 0.21 and 0.87 ng ml(-1) even with a UV absorbance detector. This system was applied to the determination of phthalates contained in laboratory distilled water and tap water samples.