Voltammetric investigation of avidin-biotin complex formation using an electroactive bisbiotinyl compound

Formation of avidin-biotin complex was investigated using bisbiotinyl thionine (BBT) by means of voltammetric techniques. Thionine is an electroactive compound and has two amino groups that are necessary for the reaction with a biotinylation reagent. The biotinylation of thionine produces a new reagent with two biotin moieties at each end of thionine. Three BBTs of different lengths of the spacer that connects the biotin moiety to the thionine moiety were prepared. The avidin-biotin binding assay was achieved by measuring the electrode response of the thionine moiety in BBT. The binding affinity and the conformation of complex, which depended on the length of spacer, are discussed. BBT in which the spacer is shortest (BBT-S, distance between carbonyl group of the two biotin moieties: 11 Å) binds with only one avidin molecule. BBT with medium length of spacer (BBT-M, 28.8 Å) forms the complex with two avidin molecules. BBT with the longest spacer (BBT-L, 46.6 Å) allows binding with two avidin molecules as well as intramolecular binding within one avidin molecule. The affinity constants of BBT-S, BBT-M and BBT-L for avidin were estimated to be 7.0 × 10¹² M⁻¹, 3.2 × 10¹² M⁻¹ and 4.0 × 10¹² M⁻¹, respectively.     

4,7‐Diiodo‐2,1,3‐benzothiadiazole and 7,7′‐diiodo‐4,4′‐bi(2,1,3‐benzothiadiazole)

In the crystal structures of the title compounds, C₆H₂I₂N₂S, (I), and C₁₂H₄I₂N₄S₂, (II), respectively, a large number of short inter‐heteroatom contacts, such as S?N, I?I and N?I, are observed. In (II), which is non‐centrosymmetric, two halves of the mol­ecule are related by a crystallographic twofold axis.     

Potentiometric flow-injection determination of boron by using a flow-through tetrafluoroborate ion-selective poly(vinyl chloride) membrane electrode

A highly sensitive tetrafluoroborate ion-selective poly(vinyl chloride) membrane electrode was constructed and applied to a detector in the flow-injection determination of boron. Boron at the 10 ng ml^?1 level can be determined by converting it to tetrafluoroborate ion in a flow of hydrofluoric acid. A sampling rate of 30 h^?1 was achieved with a relative standard deviation of less than 2%.     

Construction of an asymmetric quaternary carbon via an asymmetric aza-Claisen rearrangement and its application in the total synthesis of (+)-α-cuparenone

Excess lithium hexamethyldisilazide (LHMDS) with LiCl prompted the asymmetric aza-Claisen rearrangement of carboxamide and retarded the decomposition of its amide enolate. The addition of these two reagents was a key step that led to the total synthesis of (+)-α-cuparenone with a stereogenic quaternary center.     

Electrochemical Sensing of Ovalbumin Based on the Interaction between Lysozyme Origin/Tyrosine‐rich Peptides Modified on Magnetic Beads and Oligothreonine/Ovalbumin‐origin Peptide

We developed a highly sensitive electrochemical system for the sensing of ovalbumin (OVA). Lysozyme origin/tyrosine‐rich peptides (RNRCKGTDVQAWY₄C) were immobilized on magnetic beads, and the competitive reaction between OVA and oligothreonine/OVA origin peptide probe (T₈VLLPDEVSG) could then be measured. In a previous study, the detection of OVA at the 10^?13 M level was achieved using RNRCKGTDVQAWY₄C‐modified beads via a cross‐linker. To improve the sensitivity to OVA, this system uses T₈VLLPDEVSG peptide probe to measure the interaction to RNRCKGTDVQAWY₄C immobilized on magnetic beads. The peak of Y₄C actually was an electron‐transfer peptide, which represented the oxidation of a phenolic hydroxyl group. First, we confirmed that the oxidation response of Y₄C was increased based on an improvement in the electron transfer accessibility by oligothreonine. Next, T₈VLLPDEVSG peptide probe was used for the electrochemical sensing of OVA in solutions that contained consistent amounts of RNRCKGTDVQAWY₄C on magnetic beads. As a result, the peak current decreased as the concentration of OVA increased. The sensitivity to OVA was improved compared with the use of only RNRCKGTDVQAWY₄C on magnetic beads. The OVA detection level was 10^?14 M, which approximates the results from antibody‐antigen reactions. Consequently, the proposed system is a powerful new concept in protein sensing.     

Synthesis of the ABC ring system of Jiadifenin via Pd-catalyzed cyclizations

An efficient route toward the central ABC system of jiadifenin has been developed using two key Pd-catalyzed cyclizations. A protic solvent-activated Mizoroki-Heck reaction was used to construct the C(9) quaternary carbon and the A ring. A cascading Tsuji-Trost cyclization/lactonization sequence was employed to establish the BC ring system and the C(5,6) stereochemistry.     

Adsorptive accumulation voltammetry of copper(II) using complex formation reaction with salicylideneamino-2-thiophenol

Summary The cathodic stripping voltammetry of copper(II) was investigated with a method, based on the adsorptive accumulation of the Cu(II)-salicylideneamino-2-thiophenol (SATP) complex on a hanging mercury drop electrode. The copper(II)-SATP complex could be accumulated on the electrode at –0.20 V in 0.01 mol/l nitric acid. The reduction peak of the copper complex was observed by scanning the potential in a negative direction in the differential pulse mode. The calibration curve for copper was linear over the range 5×10^–9–1×10^–7 mol/l. This method was applied to determine copper(II) in GSJ (Geological Survey of Japan) standard rock reference materials.     

Micro flow injection analysis combined with a separation technique for the urinary glucose assay.

A urinary glucose assay has been investigated, employing a micro flow injection analysis (microFIA) combined with a separation technique of glucose from the analyte. The adsorption part using activated alumina for the glucose in the analyte can be successively integrated onto a microFI chip. The selective adsorption-desorption of glucose in the artificial urine can progress on the adsorption part. Along with this selective preconcentration of glucose, the typical FI peak of glucose can be obtained just by feeding the sample and deionized water as an elutant sandwiched with the reagent on the carrier stream. The glucose concentration in artificial urine can be quantitatively determined with the present microFIA system, while the interference of other components coexisting in urine occurs in the case of the conventional FIA system without any separation part. The described method serves as a template for improving the selectivity for the analyte in the multi-component system.     

Voltammetric homogeneous binding assay of biotin without a separation step using iminobiotin labeled with an electroactive compound.

Avidin, which is one type of glycoprotein, has a strong affinity with biotin (Ka = 10(15) M(-1)). Iminobiotin also forms a complex with avidin (Ka = 10(8) M(-1) at pH 9.5). The avidin-iminobiotin complex changes to the avidin-biotin complex in the presence of biotin because of the difference of the binding constant to avidin. In this study, the interaction between avidin and iminobiotin labeled with an electroactive compound was investigated by voltammetry. After avidin and the labeled iminobiotin (LI) were incubated in 0.1 M phosphate buffer (pH 7.0), the peak currents of LI were measured in various concentrations of biotin. The peak currents increased with increasing the concentration of biotin. Thus, this observation indicates the formation of avidin-biotin complex. On the other hand, the formation of avidin-iminobiotin complex depended on the pH of the solution. LI combines with the avidin at pH 5.6-8.9 and dissociates at pH 4.6.