Fluorimetric determination of α-keto acids with 4,5-dimethoxy-1,2-diaminobenzene and its application to high-performance liquid chromatography

A highly sensitive fluorimetric method for the determination of α-keto acids of biological importance is described. The α-keto acids react in dilute hydrochloric acid with 4,5-dimethoxy-1,2-diaminobenzene to give a compound which fluoresces in neutral solution. The method is selective for α-keto acids and the limits of detection are 30–750 pmol ml^?1 of test solution. The fluorescent compounds in a reaction mixture of ten α-keto acids are separated within 18 min by high-performance liquid chromatography on a reversed-phase column with isocratic elution. The limits of detection for the acids are in the range 9–780 fmol in a 10-μl injection volume.     

Phosphorimetric assay for β-glucuronidase in biological materials

A highly sensitive phosphorimetric method for the assay of β-glucuronidase in biological samples is described. p-Nitrophenol, formed enzymatically from p-nitrophenyl β-D-glucuronide, is extracted with ether and determined phosphorimetrically in a mixture of ether and ethanolic potassium hydroxide. The method is rapid, precise, and very sensitive, requiring as little as 0.5–5 μl of human serum or urine, or 0.3–3.0 μg of protein of rat tissue. The limit of detection for the p-nitrophenol formed is 20 pmol.     

The exo‐Outstretching Effect Governing the Exclusive Stereoselectivity of the Intramolecular [2+2] Photocycloaddition of Vinylarenes

Intramolecular [2+2] photocycloaddition of a dimeric vinylpyridine 1 through a singlet‐excited species efficiently formed the corresponding syn‐ and anti‐pyridinophanes 2 and 3 . The syn‐isomers were elucidated spectroscopically and by X‐ray crystallography as exo,syn‐configured. The high selectivity under formation of exo,syn‐ 2 was thoroughly investigated. Consequently, an exo‐outstretching effect, which is observed around the periphery of a face‐to‐face‐oriented system between two aromatic nuclei as a transition state, on cyclobutane ring formation was discovered for the first time.     

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

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.     

Enantioselective synthesis of near enantiopure compound by asymmetric autocatalysis triggered by asymmetric photolysis with circularly polarized light

Right- and left-handed circularly polarized light (CPL) has been proposed as one of the origins of homochirality of biomolecules. However, the enantiomeric excess induced by CPL has been only very low (<2% ee). We found the unprecedented example of asymmetric autocatalysis triggered directly by a chiral physical factor, that is, right- and left-handed CPL, leading to a near enantiopure compound. Asymmetric photolysis of racemic pyrimidyl alkanol by r-CPL irradiation followed by asymmetric autocatalysis affords (R)-pyrimidyl alkanol with >99.5% ee. On the other hand, irradiation with l-CPL affords (S)-pyrimidyl alkanol with >99.5% ee. Thus, chiral physical power, such as CPL, in conjunction with asymmetric autocatalysis, provides a highly enantioenriched compound.     

Sensitive flow-injection determination of l-lactate in human blood with immobilized enzyme columns and fluorimetric detection

Enzyme columns prepared by packing l-lactate oxidase and horseradish peroxidase immobilized chemically on controlled-pore glass beads are connected in series. Hydrogen peroxide formed in the enzymatic conversion of l-lactate in the first column is mixed with 3-(p-hydroxyphenyl)propionic acid before passage through the peroxidase column and fluorimetric measurement. Linear calibration was obtained for 0.5–500 pmol of L-lactate in 20 μl of 1000-fold diluted, deproteinated whole blood. A rapid sampling rate (60 h^?1) was possible.     

Fluorimetric assay for argininosuccinate lyase in human plasma and erythrocytes with a benzoin reagent

A fluorimetric method for the assay of argininosuccinate lyase in human plasma (15–150 units) and erythrocytes (40–1200 units) is established. The arginine formed enzymatically is quantified by means of its fluorescent reaction with benzoin. The method is rapid, simple and sensitive enough to allow the enzyme activity to be determined in as little as 100 μl of plasma or 12.5 μl of packed erythrocytes.