Tsunehiko Higuchi

Fluorescent Indicators for Imaging Nitric Oxide Production

The membrane-permeating indicator DAF-FM DA is transformed by intracellular esterases into the highly water-soluble dye DAF-FM, which traps NO produced by NO synthase (NOS) to yield a highly fluorescent triazole compound in cells (see schematic diagram). Monitoring with a fluorescence microscope should allow direct identification of intracellular production and location of NO.

Novel Fluorescent Probes for Singlet Oxygen.

The first fluorescent chemical traps for (1)O(2) have been developed. DPAXs react specifically with (1)O(2) to yield the corresponding endoperoxides, DPAX-EPs (see scheme; X = H, Cl, F). DPAXs scarcely fluoresce, while DPAX-EPs are strongly fluorescent. Since the fluorescence of these probes is unaffected by H(2)O(2), superoxide, and nitric oxide, they are useful for the selective detection of (1)O(2) in biological systems.

Imaging of caspase-3 activation in HeLa cells stimulated with etoposide using a novel fluorescent probe.

Microscopic visualization of intracellular enzyme activity can provide information about the physiological role of the enzyme. Caspases are cysteine proteases that have critical roles in the execution of apoptosis. General fluorometric substrates of caspase‐3, such as DEVD‐MCA, are unsuitable for imaging because they are excited at short wavelength, so we designed and synthesized novel fluorescent probes that are excited at suitable wavelengths for detecting caspase‐3 activity in living cells. Using one of these probes, we succeeded in microscopic visualization of caspase‐3‐like activity within HeLa cells treated with etoposide. The caspase‐3‐like activity was increased in the cytosol at first, then expanded to the whole cell.

Highly efficient epoxidation of olefins with pyridine n-oxides catalyzed by ruthenium porphyrins

Abstract Oxygen transfer reactions from 2,6-disubstituted pyridine N-oxides to olefins were efficiently catalyzed by ruthenium porphyrins under mild conditions, and various olefins were converted into their epoxides in high yield.

Identification of a novel sugar, 4-amino-4,6-dideoxy-2-O-methylmannose in the lipopolysaccharide of Vibrio cholerae O1 serotype Ogawa

A novel sugar in the lipopolysaccharide of Vibrio cholerae O1 serotype Ogawa has been identified. The sugar was liberated from the lipopolysaccharide when hydrolyzed in 10 M HCl at 90 degrees C for 15 min. The sugar was purified and identified as 4-amino-4,6-dideoxy-2-O-methylmannose (2-O-methylperosamine). Since it was found only in the lipopolysaccharide of Vibrio cholerae O1 serotype Ogawa, it seems that the sugar is one of the specific constituents determining Ogawa serotype specificity.

Fluorescent indicators for nitric oxide based on rhodamine chromophore

Abstract We have developed a novel fluorescent indicator for NO, DAR-M, which features long-wavelength excitation, high photostability and no pH dependency over a wide pH range.

Four recent studies in cytochrome P450 modelings: A stable iron porphyrin coordinated by a thiolate ligand; a robust ruthenium porphyrin-pyridine N-oxide derivatives system; polypeptide-bound iron porphyrin; application to drug metabolism studies

Abstract (1) A distinctive structural feature of P450 is the unusual thiolate coordination to heme. We have succeeded in the preparation of the first synthetic thiolato-iron porphyrin ( SR complex) which retains its structure during catalytic oxidation. Experiments using SR complex have revealed that the thiolate ligand greatly accelerates the rate of the Oue5f8O bond cleavage and its heterolysis even in highly hydrophobic media. (2) Heteroaromatic N -oxides were found to be excellent oxidants in the presence of ruthenium porphyrin. 2,6-disubstituted pyridine N -oxides plus a catalytic amount of Ru porphyrin oxidized olefins and sulfides to afford epoxides and sulfoxides, respectively, in high yields. The system in the presence of hydrogen halide effectively oxidized unactivated alkanes and arenes to give alcohols (or ketones) and p -quinones in high yields with high selectivity and an extremely high catalyst turnover number (up to 1.2 × 10 5 ). (3) A polypeptide-bound porphyrinatoiron complex was prepared. The polymer complex exhibited greater P450-like activity than non-bound Fe(TPP)Cl in the oxidation of olefin and aniline derivatives. (4) P450 mimics were applied to drug metabolism studies. These model systems were effective for one-step preparation of unstable metabolic intermediates, ‘candidate metabolites’, and for the discovery of novel modes of metabolism.

Remarkable axial thiolate ligand effect on the oxidation of hydrocarbons by active intermediate of iron porphyrin and cytochrome P450.

In order to examine the reactivity of active intermediate derived form iron porphyrins, competitive oxidations of alkane and alkene were carried out. It has been proposed that the first step of alkane hydroxylation is H atom abstraction and that of alkene is one-electron transfer. Therefore, it is expected that alkene-alkane competitive oxidation can be used as a probe for discrimination of differences in chemical properties among active species. Cytochrome P450 and SR complex, which is a stable thiolate-ligated iron porphyrin, mediated the oxidation of alkane much more preferentially than iron porphyrin coordinated by imidazole or chloride. These results indicate that thiolate coordination alters the reactivity of the two-electron-oxidized intermediate in a manner that is much more favorable to alkane hydroxylation than the case of chloride or imidazole coordination.

Highly efficient oxygen transfer reactions from various heteroaromatic N-oxides to olefins, alcohols, and sulfides catalyzed by ruthenium porphyrin

Abstract Ruthenium porphyrin catalyzed the oxygen transfer reactions from various heteroaromatic N-oxides to olefins, alcohols, and sulfides to afford epoxides, aldehydes, and sulfoxides, respectively, in satisfactory yield.

The selectivities and the mechanism on highly efficient epoxidation of olefins with 2,6-disubstituted pyridine N-oxides catalyzed by ruthenium porphyrin

Abstract Several remarkable selectivities in competitive epoxidations using a ruthenium porphyrin/ 2,6-disubstittuted pyridine N-oxide system were observed. The proposl that the active intermediate of this system differed from the trans -dioxo complex of ruthenium porphyrin was indicated.