BUNSEKI KAGAKU Abstracts

Analytical reagents have a function to recognize the state of an experimental system such as a species and its concentration by visualization. The sensitivity and selectivity are strongly requested in the development of a novel indicator. To fulfill these purposes, twisted intramolecular charge transfer (TICT) mechanisms have been employed for the development of novel fluorescent chemosensors in our study. In this paper, we report on the TICT mechanism of N-phenyl-9-anthacenecarboxamide (9-AA) as a fundamental structure and 9-AA derivatives to detect alkaline earth metal ions and a surfactant.

Macrocyclic ligands, calixarene derivatives have been preparaed as solvent extraction reagents. Based on the obtained data, four types of ion-exchange resins and an ion-selective electrode containing calix[4]arene derivatives have been prepared for applications to separation analysis. For resins containing calix[4]arene tetraacetic acid, the metal adsorption behavior was investigated by batchwise and column chromatographic methods. The resins containing calix[4]arene tetraacetic acid exhibited high lead selectivity. Methylene-crosslinked resin exhibited high lead capacity. Column chromatographic separation of lead and zinc ions by using the present resins was successfully achieved. An ion-selective electrode containing calix[4]arene tetraketone and tetra(2-pyridine) derivatives exhibited high silver selectivity. Calix[4]arene derivatives with high separation efficiency as solvent extraction reagents also exhibited a high efficiency when used as ion-exchange resins and ion-selective electrodes.

A variety of analytical applications were developed using tris(β-diketonato)lanthanides. In addition to high coordination numbers and fast kinetics, their lanthanide centers have high Lewis acidities to form ternary complexes between mother lanthanide complexes and external substrates. Such ternary complexation offered anion selective electrode determination, naked-eye anion detection, near-infrared luminescence sensing of amino acids and separation of lanthanide compounds. These examples provided effective separation and analytical methods for current bioscience and medical applications based on common tris(β-diketonato)lanthanides.

¹¹B NMR measurements showed that boric acid reacts with the polyol compounds, chromotropic acid, Tiron and 2,3-dihydroxynaphthalene-6-sulfonate, to form complexes with 1 : 1 and 1 : 2 stoichiometries. The equilibria of the complexation depend on the pH in the solution. The 1 : 2 complex is formed in an acidic or a neutral solution, while the 1 : 1 complex occurs in a higher pH range. Kinetic studies revealed that the reaction of 1 : 1 complex formation is much faster than that of 1 : 2 complex formation. A proton catalyzes both the reactions of 1 : 2 complex formation and its decomposition. On the basis of equilibrium and kinetic information, a novel method of on-line absorptiometric determination for trace amounts of boron was developed using a chromotropic acid presorbed anion-exchange column. On-line reaction and separation were achieved by controlling the pH conditions in solutions to accelerate 1 : 2 complex formation in the concentration process at pH 3 and to stabilize the complex in the separation process at pH 8. The 1 : 2 complex was satisfactorily separated from excess free ligand and matrix components by changing the concentration of NaClO₄ in the eluent ; its peak height on a chromatogram monitored at 350 nm was used for boron determination. The sensitivity could be enhanced by increasing the sample amount introduced, and the detection limit (3σ) was 0.05 μg dm⁻³ of boron when 13.4 cm³ of the sample was used. The method has been successfully applied to the determination of trace boron in river water, ion-exchanged water and steels. Based on the electrochemical activities of respective species in the boron-chromotropic acid and boron-Tiron systems, methods for monitoring the boron concentration in RO desalination processes were also proposed.

Protein kinases (PKs) play crucial roles in intracellular signal transduction and they are one of the representative targets of drug development because their hyper-activation is related to many diseases including cancer. Kinomics is a technique to evaluate the activities of whole cellular PKs and will be important for drug development and diagnosis for personalized medicine. Peptide microarray is one of the representative methods to evaluate kinomics. We have proposed several techniques to improve the detection of peptide microarrays and have achieved quantitative evaluation of cellular PKs based on both fluorescence and SPR detection. We have also succeeded in screening of highly reactive substrate peptides of target PKs by using our microarrays.

Metal chelators were covalently attached to the end of synthetic oligodeoxyribonucleotide (ODN) to prepare several ODN conjugates. The sequences of a pair of the conjugates were designed so as to form a ternary duplex or triplex with the targets, where their auxiliary units face each other, providing a microenvironment to accommodate a metal ion. The thermal stability of the ternary complexes is significantly stabilized by the addition of appropriate metal ions. That is, the two conjugates formed a dimer with an appropriate metal ion on the target DNA to form a stable complex. The triplex DNA structures containing convergently bound idaODN (the conjugate with iminodiacetic acid) and gluODN (the conjugate with glutamic acid) conjugates were stabilized by lanthanide ions and Cu²⁺, respectively. The cooperativity (ω, the ratio of the equilibrium constants of the second binding to the first one) was estimated to be ca. 165 for the later conjugate. The techniques were applied to colorimetric SNP (single nucleotide polymorphism) analysis using a luminescent lanthanide (Ln³⁺ : Tb³⁺ or Eu³⁺) as metal ions. Among the library of the conjugates with complexane-type chelators and heterocyclic aromatic ligands, only the combination of EDTA (ethylenediaminetetraacetic acid) conjugates (edtaODN) and phen (1,10-phenanthroline) conjugates (phenODN) provided significant emissions with quantum yields of 12 and 5.3% for Tb³⁺ and Eu³⁺, respectively, in the presence of the target. Their luminescence lifetimes were 1.26 and 1.34 ms for Tb³⁺ and Eu³⁺ ; they are long enough to be subjected to time-resolved luminescence measurements. Biallelic polymorphism in the TPMT (thiopurine S-methyltransferase) gene, wt/wt (G/G), mut/mut (C/C), and wt/mut (G/C), were distinguished as emissions in green, red, and yellow, respectively ; the colors were identified even by the naked eye.

Porphyrin-manganese(III) complexes were used as Cl⁻-selective ionophores for sol-gel derived membranes encapsulating and binding covalently them. The ion-sensing membrane encapsulating the ionophore showed hardly any EMF response to Cl⁻-activity changes, probably because the ionophore was not uniformly dispersed in the sol-gel-derived membranes. On the other hand, the ion-sensing membrane binding covalently the ionophore, in which the ionophore was dispersed in the membranes uniformly, showed near-Nernstian responses to Cl⁻-activity changes. The sol-gel-derived membranes were relatively Cl⁻-selective over interfering hydrophobic anions, as compared with the case of the membranes containing only a cationic additive. The Cl⁻-selective electrode is, therefore, promising for applications to clinical analysis in the future.

For selective dopamine (DA) recognition in an aqueous solution, a novel azoprobe possessing crown ether and boronic acid {2-[3,4-(18-crown-6)phenylazo]-5-hydroxy-phenylboronic acid} was designed. This probe was water soluble and changed its color from yellow to red to orange with increasing pH. From UV-vis spectra in the presence of DA or analogous compounds, based on 1 : 1 complex formation of the probe with a guest, the highest binding constant of (1.11 ± 0.021) × 10² M⁻¹ was noted for DA. At pH 8, the solution color changed from red to orange upon the addition of DA. This color change was ascribed to bronate ester formation followed by hydrolysis or by the B–N interaction between boronate ester and the azo moiety. This peculiar behavior was found to be strongly affected by the cis-diol moiety of guest molecules.

Three types of cyclic bisbipyridinium host compounds (Cpp, Cpm, Cmm), each being structural isomers containing two bipyridinium units bridged by a p- or m-xylene unit at N-position of the bipyridine, and two acyclic hosts of their analogues (ACp, ACm) have been prepared to investigate their molecular-recognition abilities for indole and naphthalene carboxylic acid derivatives by charge-transfer absorption in acetonitrile. When the solution contained the bisbipyridinium host and the guest, charge-transfer absorption was observed in the visible region with three different modes, in which the absorption maximum was observed at around 450 nm, 530 nm, and no maximum appeared. Such development of the charge transfer absorption is associated with the combination of a bisbipyridinium host and a guest. By using dual-wavelength analysis at 450 and 530 nm, it was found that ACp and ACm recognized 2-naphthalene carboxylic acid derivatives rather than the isomer with 1-substituted ones. Since the ester derivatives of the naphthalein carboxylic acid guest exhibited no response based on charge transfer, the carboxylic group would play an important role for the recognition system.

2-(5-Bromo-2-pyridylazo)-5-diethylaminophenol (5-Br-PADAP) nanoparticle-coated membranes were prepared by simple filtration of an aqueous dispersion of 5-Br-PADAP nanoparticles through a cellulose ester membrane filter (0.45 μm pore). By letting the dispersion stand for 3 min at room temperature for sufficient particle growth, more than 99.92% of the 5-Br-PADAP nanoparticles were coated uniformly and firmly on the membrane filter without any additives. A 5-Br-PADAP thin layer was located within 1 μm from the top surface of the filter. The membranes were cut into 6 pieces, and then test strips were immersed in 20 mL of a sample solution containing Cd^II and pH buffer (immersion test). The test strip changed color from yellow to reddish-orange based on complex formation with Cd^II. Stirring increased the reaction rate ; the color formation at 100 ppb was completed in ca. 30 min under stirring in contrast to more than 90 min without stirring. A very low extraction percentage (0.77%) of 700 ppb Cd^II to the 5-Br-PADAP thin layer was obtained by ICP-MS analysis. A detection curve was obtained using ΔE*(a*,b*) of a colorimeter. The equation is expressed as ΔE*(a*,b*) = 73.677 (1–e^{−0.0036283 C}) and the 3σ detection limit is 7.14 ppb based on 7 blank measurements. To improve the reaction rate and extraction percentage of an immersion test, a drop test was attempted by dropping 50 μL of a sample solution on the test strips equipped with No.1 filter paper as a water absorber. The extraction percentage by the drop test of 1 ppm Cd^II was 30.2% for ca. 15 min.

The separation of metal ions was examined using a poly{4-[2-(vinyloxy)-ethoxyl]-benzoic acid} [Poly(VEBA)s] as a pH-sensitive polymer. It showed phase separation at around pH 6.5. As a result, several specific characteristics of scavenging metals were confirmed. Among them, a specific result for rare earth elements was obtained. A separation and recovery system of rare earth elements was developed by using pH change and filtration. The recovery for 16 kinds of rare earth elements was approximately 100%. It was possible to separate the metal from Poly(VEBA)s by remelting Poly(VEBA)s with sodium hydroxide solution. Poly(VEBA)s could be recycled by recovery of polymer. This system has high safety to the environment and organism because of an all-water system without-using any organic solvent.

Telomerase is an enzyme that elongates repeated six bases of TTAGGG of the telomere DNA, and is known to be expressed in tumor cells, but not in normal cells. For this reason, this enzyme is expected to serve as a new tumor marker. The telomerase activity has been detected by the telomerase repeat amplification protocol (TRAP), which involves a tedious polymerase chain reaction (PCR) and gel electrophoresis. We have been developing an electrochemical telomerase assay with ferrocenylnaphthalene diimide (FND) as an electrochemical indicator. Where telomerase activity is present in the sample, the telomerase substrate (TS)-primer immobilized on the electrode is elongated to form a telomeric repeat sequence ; the resulting products form a tetraplex DNA. FND binds to the tetraplex DNA thus formed to give rise to an electrochemical signal whose magnitude reflects the telomerase activity. We tested saliva, oral epithelial cells, and solid tumor for telomerase activity for diagnosis of tongue cancer using disposable electrode chips coupled with a FND derivative. The samples were lysed, and supernatants (2000 and 200 ng of protein) were used in TRAP and the FND-based electrochemical assay with Osteryoung square wave voltammetry (SWV). In both assays, all of the samples from a solid tumor turned out to be telomerase-positive. However, the telomerase activity was detected at a higher rate, even in saliva and oral epithelial cells in the latter assay than in the former. In conclusion, the FND-based electrochemical telomerase assay was more sensitive than TRAP in terms of the detection of cancerous samples taken from tongue cancer patients. This electrochemical method will enable early diagnosis of tongue cancer in clinical dentistry.

Nickel(II) and copper(II) complexes with a hexadentate ligand, derived from a condensation reaction between 1-phenyl-1,3,5-hexanetrione and diamine, were characterized by electrochemical measurements to elucidate the usefulness as an electrode material for a plasticized PVC membrane electrode. Among the complexes tested, a nickel(II) mononuclear complex with 5,5-(1,2-ethanediyldinitrilo)bis(1-phenyl-1,3-hexanedione) has functionality as a perchlorate ionophore and lipophilic salt. A PVC membrane electrode that consists of the nickel(II) complex (30 mg), o-NPOE (125 mg) and poly(vinyl chloride) PVC (50 mg) shows good results with a Nernstian response, 53 mV/decade, in accordance with a wide ClO₄⁻ activity change in the activity range, 1 × 10⁻⁷–8 × 10⁻² M and ClO₄⁻ selectivity with respect to Cl⁻, log k ^pot _ClO4,Cl is –5.35. In addition, to evaluate the application of this electrode, a calibration curve of ClO₄⁻ was obtained by adding ClO₄⁻ in commercial milk. It was found that the lower detection limit of the concentration of ClO₄⁻ is ca. 1 ppm.

The adsorption-desorption behaviors of hydrophobic anion on the solid phase coated with redox-active metal complexes (ML₂^+/0 ; M, Co^III/II ; L⁻, pyridylazo compounds) were investigated. The combination of a cobalt complex of poly vinyl phenol introduced with a 2-pyridylazo group and a solid-phase extraction cartridge packed with styrene-divinyl benzene copolymer sorbent showed the highest adsorption capacity for methyl orange as a hydrophobic anion. The percentage adsorption of methyl orange (1.0 μmol) on this solid phase was more than 99%, even in the presence of a 100-fold stoichiometric excess of coexisting anions (ClO₄⁻, Cl⁻). Methyl orange molecules were desorbed by reducing the cobalt complexes in the solid phase with a 1.0 × 10⁻² M ascorbic acid solution.

The Aliquat 336 (material of ISE) dissolved in 1,2-dichloroethan solvent was used for the solvent extraction of a anion species of triethylentetramine hexa acetic acid (TTHA, H₆X), diethylenetetraminepentaaceticacid (DTPAH₅X) and cyclohexanediaminetetraacetic acid (CyDTA). The extraction behavior was examined. The extraction efficiency (%E) was indicated in the enlarge order as CyDTA > EDTA > DTPA > TTHA in the pH range from 4.0 to 12.0. The extraction constants (K_n^ex) were calculated by using consecutive dissociation constant (k_m), overall acid formation constants of polyaminocarboxylic acid. (β_n). The extraction coefficients of all partitions of the extraction constants of anion species of TTHA are indicated as follows : K₂^ex = 3.6 × 10⁻⁵, K₃^ex = 5.49 × 10⁻⁶, K₄^ex = 9.08 × 10⁻⁶, K₅^ex = 1.15 × 10⁻⁵, K₆^ex = 1.81 × 10⁻⁵. All partitions of the extraction constants of the anion species of DTPA were also obtained as follows : K₂^ex = 1.25 × 10⁻³, K₃^ex = 9.26 × 10⁻⁵, K₄^ex = 3.11 × 10⁻⁴, K₅^ex = 5.22 × 10⁻⁴. All partitions of the extraction constants of CyDTA are also indicated as follows : K₂^ex = 0.043, K₃^ex = 0.014, K₄^ex = 0.02. The steric rearrangement by bonding between the aminocarboxylic acid anion in ligands and the triocthylmethylammonium cation species is important in solvent extraction. It was also found that the steric existence of cycloring in CyDTA affected the extraction.