In order to evaluate the chemiluminescence (CL) reagents for selective detection of reactive oxygen species (ROS), we comprehensively measured the CL responses of 20 CL reagents (three luminol derivatives, two imidazopyrazinone derivatives, eight lophine derivatives, six acridinium ester derivatives and lucigenin) against six types of ROS (superoxide anion: O(2)(*-), hydroxyl radical: *OH, hydrogen peroxide: H(2)O(2), hypochlorite anion: ClO(-), singlet oxygen: (1)O(2), and nitric oxide: NO). As a result of the screening, it was found that nine CL reagents selectively detected O(2)(*-) while one CL reagent selectively detected *OH. However, no CL reagent had selectivity on the detection of H(2)O(2), ClO(-), (1)O(2) and NO. Our screening results could help to select the most suitable CL reagent for selective determination of different ROS. As an application study, 4-methoxyphenyl-10-methylacridinium-9-carboxylate (MMAC), one of the acridinium ester derivatives, showed high selectivity on the detection of O(2)(*-), and thus was applied to the assay of superoxide dismutase (SOD) activity. The dynamic range and detection limit of the developed CL assay were 0.1-10 and 0.06 U mL(-1), respectively. Significant correlation (r=0.997) was observed between the results by the CL assay using MMAC and the spectrophotometric assay using 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt.

Fluorometric analysis is one of the most sensitive methods for detecting organic and/or inorganic compounds and therefore it has been widely used in many scientific fields with improving analytical instruments such as high-performance liquid chromatography (HPLC), capillary electrophoresis (CE), fluorescence confocal microscopy, etc. For these purposes, a great deal of effort has gone into the development of fluorescent reagents using various fluorophores, which can be utilized for derivatizing biologically active small molecules, labeling macromolecules such as proteins and DNAs, and probing ions such as biologically relevant metal cations and inorganic or organic anions. [1][2][3][4] In the development of such fluorescent reagents, it should first be ensured that the fluorophore used has a high fluorescence quantum yield because of the reliably high sensitivity in the detection. Fluorescein is one of the most widely used fluorophore for biological experiments, since it has a high fluorescence quantum yield of 0.90 or grater in aqueous solution and its excitation and emission wavelengths are in the visible region. Recently, Nagano and coworkers have succeeded in developing fluorescent reagents for the specific detection of nitric oxide (NO), [5][6][7][8] singlet oxygen ( 1 O 2 ), 9,10) and others 11,12) using fluorescein as the fluorophore. 4,4-Difluoro-4-bora-3a,4a-diazas-indacenes (BODIPYs) are also well-known fluorophores that have very sharp and narrow fluorescence bandwidths in addition to their high fluorescence quantum yield in aqueous solution. 4,13,14) Therefore they have been used as mother fluorophores for many analytical purposes. [15][16][17][18] However, the difference between the excitation and emission wavelengths of these derivatives are very small, less than approximately 30 nm for fluoresceins and 15 nm for BODIPYs, and it is necessary to correct their spectra from interferences such as Rayleigh or Raman scattering light.On the other hand, coumarin (2H-benzopyran-2-one), is also another interesting fluorophore, since its fluorescence changes drastically with substituents and their introduced positions. [19][20][21] In previous papers, we reported the fluorescence characteristics of methoxycoumarins and discussed the structural features of strongly fluorescing methoxycoumarins from the viewpoint of intramolecular charge transfer (ICT) between push-and pull-substituents in the ground and the excited states.22,23) Based on the above findings, we succeeded in developing novel fluorescent reagents with both high sensitivity and functions such as self-catalytic reactivity. [24][25][26] In the course of those studies, we suspected that most coumarin derivatives showed large Stokes shifts in their fluorescence spectra as compared with other fluorophores. These large Stokes shifts appear to be advantageous for escaping autofluorescence from biological molecules and reducing the self-absorption of chromophores. Further, if the emission bands of coumarins could be shifted to longer wavelengths with larg...

The fluorescence characteristics of various methoxycoumarin fluorophores for the development of fluorescence reagents were examined in relation to their structures. The fluorescence emission mechanisms were also considered from the viewpoint of the intramolecular charge-transfer (CT) between methoxyl substituents and the coumarin ring. The arrangement of 6-methoxyl and 3-acetyl group pairs on the coumarin ring significantly contributed to the fluorescence enhancement through the intramolecular CT. We found that the structural features of methoxycoumarins required for intense fluorescence are to hold both diether bonds at the 6-and 7-positions and an electron-withdrawing group at the 3-position, as shown in 3-acetyl-6,7-dimethoxycoumarin with 0.52 in quantum yield.

π-Conjugated helicenes containing heteroatoms have attracted significant attention due to their diverse chemical and electronic structures, as well as tunable physical properties. It was rationally anticipated that the self-assembly of coumarin-fused helicenes would be controlled by the effects of a substituent on the internal edge of the helix. Here, this work reports the efficient syntheses of coumarin-fused helicenes 1 a,b (R=Ph, Me), and the enantioselective synthesis of 1 a (R=Ph) by chiral Au -catalyzed hydroarylation. The helical structure of 1 was unambiguously determined by X-ray crystallography. Of particular note, the enantiomerically pure crystal of 1 a adopted a one-dimensional columnar structure based on π-π stacking interactions, as expected. Furthermore, a significant difference between the fluorescence quantum yields of the enantiomerically pure form and racemate of 1 a was observed.

Keywords3-Bromoacetyl-6,7-methylenedioxycoumarin, fatty acid, fluorescence labeling reagent, high-performance liquid chromatographyCoumarin derivatives have been known to be useful labeling reagents owing to their strong fluoroscence in solution, suitable molecular size, easy synthesis and good storage. Of these coumarins, 4-bromomethyl-7-acetoxy-1, 4-bromomethyl-6,7-dimethoxy-(BrDMC)2, and 4-bromomethyl-6,7-methylenedioxy-coumarins (BrMDC)3 were listed as highly sensitive reagents for carboxylic acids, which were mostly comparable in sensitivities or quantum yields to other reagents, e.g., 1-bromoacetylpyrene4, 3-bromomethyl-6,7-dimethoxy-lmethyl-2(1 H)-quinoxaline5, 9-anthryldiazomethane6, p-(9-anthroyloxy)phenacyl bromide', 9,10-diaminophenanthrene8, 9-aminophenanthrene9, and monodansyl cadaverine,lo Thus, 3-bromoacetyl-6,7-methylenedioxycoumarin (BrAMDC, Fig. 1) was sythesized in a search for the most excellent reagent regarding both reactivity and sensitivity among coumarins for carboxylic acids analysis. Free fatty acids in human blood plasma were readily labeled with BrAMDC under quite mild conditions. Experimental ApparatusProton nuclear magnetic resonance (1H-NMR) spectra were obtained with a JEOL JNM-GSX 500FT-NMR spectrometer employing tetramethylsilane as an internal standard. Absorption and fluorescence spectra were measured with a Hitachi 150-20 spectrophotometer and a Hitachi F-4000 fluorescence spectrophotometer, respectively. The fluorescence quantum yields were determined according to the method of Paker and Rees11, quinine sulfate in 0.5 M H2SO4 was used as a standard. Mass spectra were taken with a JEOL JMS-DX303 spectrometer. A Hitachi 655A high-performance liquid chromatograph was used, equipped with a Hitachi F-1050 fluorescence spectrophotometer (excitation wavelength 388 nm; emission wavelength 475 nm) and a Hitachi L-6200 solvent gradient device. A Unisil Pack 5C18-250A column (250X4.6 mm i.d.; particle size, 5 µm;