Keiyu Ueno

A highly water-soluble disulfonated tetrazolium salt as a chromogenic indicator for NADH as well as cell viability

A highly water soluble disulfonated tetrazolium salt, 4-[3-(2-methoxy-4-nitrophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate sodium salt, was synthesized. The compound is reduced by NADH in good yields at neutral pHs in the presence of 1-methoxy PMS to produce the corresponding formazan dye that absorbs at 460 nm. The formazan is soluble to water at concentrations higher than 0.1 M. The tetrazolium salt thus proved to be useful as a sensitive chromogenic indicator for NADH. It is also applicable to cell proliferation assays as a cell viability indicator.

Novel disulfonated tetrazolium salt that can be reduced to a water-soluble formazan and its application to the assay of lactate dehydrogenase

A new tetrazolium salt, 4-[3-(4-iodophenyl)-2-(2,4-dinitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate, sodium salt, that produces a highly water-soluble formazan dye upon reduction by reduced nicotinamide adenine dinucleotide (NADH) was synthesized. The reduction of the compound by NADH at a neutral pH is fast owing to its small reduction potential. The applicability of the compound to the assay of lactate dehydrogenase is described in comparison with a prevalent tetrazolium salt.

2-(5-hydrazinocarbonyl-2-oxazolyl)-5,6-dimethoxybenzothiazole as a precolumn fluorescence derivatization reagent for carboxylic acids in high-performance liquid chromatography and its application to the assay of fatty acids in human serum.

Abstract A unique acid hydrazide, 2-(5-hydrazinocarbonyl-2-oxazolyl)-5,6-dimethoxybenzothiazole, that is characterized by the benzothiazole structure conjugated to an oxazoline moiety was synthesized, and its applicability as a precolumn derivation reagent for carboxylic acids in HPLC was examined in view of sensitivity and separability. The sensitivity of the hydrazide for carboxylic acids was determined using lauric acid to be 0.1 pmol (per 10-μ1 injection volume) at a signal-to-noise ratio of 3. The reagent allowed rapid assays of carboxylic acids within 20 min with satisfactory separability. The method was applied to the determination of fatty acids in human sera from healthy volunteers as well as from patients with diabetes or thyroid dysfunction.

Benzothiazole derivatives as substrates for alkaline phosphatase assay with fluorescence and chemiluminescence detection

Unique fluorogenic and chemiluminescence substrates for alkaline phosphatase (ALP), 2-(N-phenylphthalimidyl)benzothiazolyl-5-phosphate and 2-(2′,3′-dihydro-1′,4′-dionephthalazyl)benzothiazolyl-5-phosphate, respectively, that contain a highly fluorescent benzothiazole moiety as the fluorophore were synthesized and their applicabilities to the assay of ALP were examined. The chemiluminogenic substrate exhibited a linear chemiluminescence response to the amount of ALP in the range 0–20 pmol.

2-(5-hydrazinocarbonyl-2-thienyl)-5,6-methylenedioxybenzofuran and 2-(5-hydrazinocarbonyl-2-furyl)-5,6-methylenedioxybenzofuran as novel fluorescence derivatisation reagents for carboxylic acids in liquid chromatography

Abstract The new acid hydrazides, 2-(5-hydrazinocarbonyl-2-thienyl)-5,6-methylenedioxybenzofuran (TMBH) and 2-(5-hydrazin-ocarbonyl-2-furyl)-5, 6-methylenedioxybenzofuran (FMBH), were synthesized as precolumn fluorescence derivatisation reagents for carboxylic acids in liquid chromatography. These compounds readily react with carboxylic acids in the presence of a coupling reagent under mild conditions in aqueous solution to give fluorescent derivatives. The detection limits of lauric acid were both 0.1 pmol per 10 μl injection volume at a signal-to-noise ratio of 3. The methods in which these compounds are used were applied to the assay of a standard mixture of prostaglandins (25 μM) and prostaglandins in a human seminal fluid.

Synthesis of a new tetrazolium salt giving a water-soluble formazan and its application in the determination of lactate dehydrogenase activity

Abstract A pyridine analog of 3-(4-,5dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide was synthesized. The new reagent, 2-phenyl-3-(4,5-dimethyl-2-thiazolyl)-5-(4-pyridyl)tetrazolium bromide, gave a water-soluble formazan dye on enzymatic reduction and was applied in the spectrophotometric determination of lactate dehydrogenase (0–84 mU ml −1 ) in blood serum. The sensitivity (ϵ = 1.83 × 10 4 l mol cm −1 at 539 nm) was about 70% of that obtainable with nitrotetrazolium blue.