Biomedical subjects

Tomofumi Santa

Publications and source records attributed to Tomofumi Santa.

33 records · Page 2Linked to original sources

PubMed2003

Fluorescence on-off switching mechanism of benzofurazans.

Many fluorescent reagents with a benzofurazan (2,1,3-benzoxadiazole) skeleton have been developed and widely used in bio-analyses. In this study, we try to elucidate the fluorescence on-off switching mechanism of three fluorogenic reagents and their derivatives. Ten 4,7-disubstituted benzofurazans were used for this purpose and the measurements of their fluorescence, phosphorescence, photolysis, and time-resolved thermal lensing signal in acetonitrile were obtained in order to understand the relaxation processes of these compounds. These results indicate that the competition of fluorescence with a fast intersystem crossing or fast photoreaction plays a key role in the fluorescence on-off switching. Semi-empirical molecular orbital calculations show that the existence of the triplet n pi* state is responsible for the fast intersystem crossing while the proximity of the reactive second single pi pi* state to the first singlet pi pi* state contributes to the fast photoreaction in the excited states.

Journal Article↗

PubMed2003

Recent progress in derivatization methods for LC and CE analysis.

The derivatization procedure with a suitable fluorescence or chemiluminescence reagent is performed for the purpose of increasing the detection sensitivity and selectivity, in high-performance liquid chromatography (HPLC) and/or capillary electrophoresis (CE). In this article, recent derivatization methods and their applications to biosamples are described. In HPLC, femto mol order of mass detection limits are obtained by derivatization. Regarding the fluorescence reagents, the use of water-soluble reagents has been effective to avoid an undesired adsorption in the process of determination of peptides. In CE, the advantages of having extremely low mass detection limits (ranging from atto to yocto mol level) and requiring only a very short analysis time (less than a few minutes) are made possible by using laser-induced fluorescence or near infra-red detections.

Chromatography, High Pressure Liquid↗

PubMed2003

A study of the relationship between the chemical structures and the fluorescence quantum yields of coumarins, quinoxalinones and benzoxazinones for the development of sensitive fluorescent derivatization reagents.

To develop new fluorescent derivatization reagents, we investigated the relationship between the chemical structures and the fluorescence quantum yields (phi(f)) of coumarins, quinoxalinones and benzoxadinones. Forty-six compounds were synthesized and their fluorescence spectra were measured in n-hexane, ethyl acetate, methanol and water. The energy levels of these compounds were calculated by combination of the semi-empirical AM1 and INDO/S (CI = all) methods. The deltaE(Tn(n,pi*), S1(pi,pi*)) (the energy gap between the Tn(n,pi*) and S1(pi,pi*) states) values were well correlated with the phi(f) values, which enables us to predict the phi(f) values from their chemical structures. Based on this relationship, 3-phenyl-7-N-piperazinoquinoxalin-2(1H)-one (PQ-Pz) and 7-(3-(S)-aminopyrrolidin-1-yl)-3-phenylquinoxalin-2-(1H)-one (PQ-APy) were developed as fluorescent derivatization reagents for carboxylic acids. The derivatives of the carboxylic acids with PQ-Pz and PQ-APy showed large phi(f) values even in polar solvents, suggesting that these reagents are suitable for the microanalysis of biologically important carboxylic acids by reversed phase HPLC.

Benzoxazoles↗

PubMed2003

Study on interactions of endocrine disruptors with estrogen receptor using fluorescence polarization.

In this study, we examined the affinities of many (21) compounds such as hormones, pharmaceuticals, industrial chemicals, and phytoestrogens to the estrogen receptor (ER) by ER binding assay using fluorescence polarization (FP). This method is based on the competitive binding assay using fluorescein-labeled estradiol (F-E2), in which the polarization values decreased with the addition of the compounds (competitors). The obtained sigmoidal inhibition curves were transformed into the pseudo-Hill plots, and the concentrations at 50% inhibition (IC50) and Hill coefficients were obtained from the regression equations. We examined the relationship between the chemical structures and estrogenic activities, and finally classified the tested compounds into three categories, agonists, partial agonists and antagonists based on their Hill coefficients.

Binding, Competitive↗

PubMed2003

Study on interactions of endocrine disruptors with estrogen receptor-beta using fluorescence polarization.

In this study, we developed a rapid, simple and homogeneous human recombinant estrogen receptor-beta (hrER-beta) binding assay method using fluorescence polarization (FP) by applying a fluorescent ligand, fluorescein-labeled estradiol (F-E2). A Scatchard plot and a Hill plot analysis of the saturation binding assay using F-E2 and hrER-beta were studied. F-E2 showed a high affinity for hrER-beta, the dissociation constant was 5.53 nM, indicating that F-E2 is applicable to the hrER-beta binding assay. Competitive binding assays using F-E2, in which the FP values decreased upon the addition of compounds (competitors) were carried out to evaluate the binding characteristics of compounds with and without biological activities to hrER-beta. Twenty-one compounds, such as hormones, pharmaceuticals, industrial chemicals and phytoestrogens, were examined. The obtained sigmoidal inhibition curves were transformed into pseudo-Hill plots and the concentrations at 50% inhibition (IC50) and Hill coefficients, the degree of cooperativity in ER-ligand binding, were obtained from the regression equations. Antagonists exhibited larger Hill coefficients than agonists, showing the correlation between the Hill coefficients and the estrogenic/antiestrogenic activities.

Binding, Competitive↗

PubMed2002

Development of hydrophilic fluorogenic derivatization reagents for thiols: 4-(N-acetylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole and 4-(N-trichloroacetylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole.

Sensitive, reactive, and hydrophilic fluorogenic reagents for thiols with the benzofurazan skeleton, 4-(N-acetylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (AcABD-F) and 4-(N-trichloroacetylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (TCAcABD-F) have been developed. These reagents reacted with thiols within 10 min at 60 degrees C. AcABD-F and TCAcABD-F themselves do not fluoresce but are strongly fluorescent after the reaction with thiol compounds. The generated derivatives were highly water-soluble, since they dissociated a proton and ionized in the neutral pH region. The derivatives with four biologically important thiol compounds were separated on a reversed-phase HPLC column and detected fluorometrically at 504 nm with excitation at 388 nm. The detection limit attained for homocysteine with AcABD-F was 25 fmol on column (11 nM) (signal-to-noise ratio = 3), and that for glutathione with TCAcABD-F was 45 fmol on column (20 nM).

Benzoxazoles↗

PubMed2002

Estrogen receptor binding assay method for endocrine disruptors using fluorescence polarization.

A rapid, simple and nonhazardous assay method for endcrine disruptors was developed using an estrogen receptor (ER) and fluorescence polarization (FP). Among the fluorescent compounds, the 17alpha-fluorescein-labeled estradiol derivative was selected as the most suitable ligand for the ER binding assay, since it showed the highest affinity to ER. In the Scatchard plot analysis, its convex curve exhibited a positive cooperative binding, indicating the induction of a conformational change of the ER with the binding of the ligand to form a dimer and to increase the affinity for the additional ligand. On the basis of the Hill plot analysis, its dissociation constant and Hill coefficient were 10.4 nM and 1.63, respectively. A competitive binding assay with an unlabeled 17beta-estradiol (E2) yielded an IC50 value of 2.82 nM and a Hill coefficient of 1.67, thus providing a Ki value of 0.65 nM. In the same manner, the Hill coefficients for estrone, estriol, diethylstilbestrol, and tamoxifen were determined to be 0.99, 1.17, 1.59, and 2.44, respectively.

Animals↗

PubMed2002

A photoinduced electron-transfer reagent for peroxyacetic acid, 4-ethylthioacetylamino-7-phenylsulfonyl-2,1,3-benzoxadiazole, based on the method for predicting the fluorescence quantum yields.

To develop new photoinduced electron-transfer (PET) reagents, we established a method for predicting the fluorescence quantum yields (phi) of the benzofurazan compounds bearing an aliphatic substituent group having an n-electron. The PET process occurred sufficiently to reduce the phi values in the benzofurazan compounds bearing an aliphatic moiety, which had a high quenching ability. The quenching ability was estimated by the molecular orbital calculation and Stern-Volmer plotting. The phi values of the benzofurazan compounds could be controlled by changing the quenching ability of a substituent group. We succeeded in designing a PET reagent for peroxyacetic acid (PAA), 4-ethylthioacetylamino-7-phenylsulfonyl-2,1,3-benzoxadiazole (EPB), using the established method for predicting the phi values. EPB and its oxidized derivative were separated by reversed-phase HPLC and fluorometrically detected at 479 nm with excitation at 362 nm. The attained detection limit for PAA was 105 fmol (S/N = 3) and the cross-reactivity toward hydrogen peroxide was very low, indicating EPB is a highly sensitive and selective reagent for PAA.

Journal Article↗

PubMed2002

An automated fluorescence protein sequencer using 7-methylthio-4-(2,1,3-benzoxadiazolyl) isothiocyanate (MTBD-NCS) as an Edman reagent.

An automated fluorescence protein sequencer using 7-methylthio-5-(2,1,3-benzoxadiazolyl) isithiocyanate (MTBD-NCS), a fluorescent Edman reagent, is developed by the modification of a commercial protein sequencer. The generated MTBD-thiohydantoin amino acids fluoresced strongly, whereas the by-products such as MTBD-thiocarbamoyl amino acids and MTBD-carbamoly amino acids did not fluoresce. A few interfering peaks were observed in the chromatogram and amino acid sequence was easily determined. The coupling and cyclization/cleavage reaction conditions and extraction conditions of generated MTBD-thiazolinone amino acids were optimized using an autonalyzer. Finally, the sequence of a synthetic peptide (25 pmol), leucine-enkephalin-Thr-amide, was determined and up to six residues were successively analyzed.

Fluorescent Dyes↗

PubMed2002

Design and synthesis of a hydrophilic fluorescent derivatization reagent for carboxylic acids, 4-N-(4-N-aminoethyl)piperazino-7-nitro-2,1,3-benzoxadiazole (NBD-PZ-NH2), and its application to capillary electrophoresis with laser-induced fluorescence detection.

A hydrophilic fluorescent derivatization reagent for fatty acids, 4-N-(4-N-aminoethyl)piperazino-7-nitro-2,1,3-benzoxadiazole (NBD-PZ-NH(2)), was designed and synthesized. NBD-PZ-NH(2) possesses not only a fluorophore and a reacting group but also a positive charge group and, thus, was hydrophilic and suitable for application to capillary electrophoresis. NBD-PZ-NH(2) reacted with fatty acids in the presence of triphenylphosphine (TPP) and 2,2'-dipyridyl disulfide (DPDS) at room temperature within 10 min. The derivatives were strongly fluoresced and were positively charged at pH below 3. The derivatives of C4-C20 fatty acids were separated within 10 min in 50% acetonitrile in water containing 30 mM ammonium acetate and 1.0 M acetic acid by capillary electrophoresis with laser-induced fluorescence (CE-LIF) detection. The detection limits attained were 6.5 nM (signal-to-noise ratio of 3). It is proposed that NBD-PZ-NH(2) is a prominent derivatization reagent for fatty acids which is suitable for CE-LIF application.

Carboxylic Acids↗

PubMed2002

An automated analyzer for methylated arginines in rat plasma by high-performance liquid chromatography with post-column fluorescence reaction.

A fully automated analyzer for methylated L-arginine metabolites [N,N-dimethyl-L-arginine (ADMA), N-methylarginine (NMMA) and N,N'-dimethyl-L-arginine (SDMA)] by high-performance liquid chromatography with post-column fluorescence derivatization was developed. This system consists of an on-line extraction, a separation on a reversed phase ion-pair chromatograph, a post-column derivatization by o-phthaladehyde (OPA) and thiol reaction, and fluorescence detection. NMMA, ADMA and SDMA were separated in 40 min with isocratic elution by a combination of octanoate and cyclohexane carboxylate as ion-pair reagents. The eluate was monitored at 450 nm with excitation at 337 nm. The calibration curves for NMMA, ADMA and SDMA showed linearity over the range from 0.05 micromol l(-1) (0.5 pmol on column) to 5.0 micromol l(-1) (50 pmol on column). This method does not require any time-consuming pre-treatment and requires only 10 microl of plasma sample for assay.

Animals↗

PubMed2002

Development of 7-(N,N-dimethylaminosulfonyl)-5-N-(4-N-aminoethyl)piperazino-2,1,3-benzoxadiazole as a water-soluble fluorogenic reagent for the sensitive liquid chromatographic determination of saturated carboxylic acids.

A reversed-phase high-performance liquid chromatographic (HPLC) method for the femtomole determination of nine saturated carboxylic acids, n-butyric (C4), n-hexanoic (C6), n-caprylic (C8), n-decanoic (C10), lauric (C12), n-tetradecanoic (C14), palmitic (C16), stearic (C18) and arachidic (C20), based on the condensation reaction of these acids with a newly synthesized water-soluble benzofurazan fluorescent reagent, 7-(N,N-dimethylaminosulfonyl)-4-N-(4-N-aminoethyl)piperazino-2,1,3-benzoxadiazole (DBD-PZ-NH2), was developed. The derivatization reaction proceeds with 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide (EDC) in the presence of the catalyst 4-(dimethylamino)pyridine (DMAP). A model derivative of the reagent with n-caprylic acid (C8) was synthesized for fluorescence excitation/emission characterization. Depending on the solvents, including water, methanol, acetonitrile, 1,4-dioxane or N.N-dimethyformamide (DMF), the C8 derivative has a fluorescence emission with a fluorescence quantum yield (phi) ranging from 0.01 to 0.20 in the region from 545 to 580 nm. An exponential increase in phi was observed with increasing acetonitrile content. The calculated detection limits (signal-to-noise ratio = 3:1) of the proposed method for the above nine carboxylic acids were 9.1, 4.0, 2.5, 2.2, 2.0, 1.8, 1.2, 1.0 and 1.3 fmol, respectively. Biological samples including Intralipos 20% and rat plasma were analysed satisfactorily.

Carboxylic Acids↗

PubMedDate not supplied

The effects of spacer length on the fluorescence quantum yields of the benzofurazan compounds bearing a donor-acceptor system.

We studied the effects of spacer length on the fluorescence quantum yields (Phi) of photoinduced electron transfer (PET) reagents, using nitrobenzoxadiazole (NBD) derivatives that have the -NMe2 moiety and NBD-NH- fluorophore as electron donor (D) and electron acceptor (A), respectively. The Phi values were reduced as the spacer length became shorter (n < or = 4; n is the number of methylene units of the spacer) and the fluorescence recovered by suppression of the PET process. It is necessary for the useful PET reagents to link D and A with a short spacer to obtain a difference in the Phi values between fluorescent "off-state" and "on-state".

Benzoxazoles↗

PubMedDate not supplied

Alterations of plasma and cerebrospinal fluid glutamate levels in rats treated with the N-methyl-D-aspartate receptor antagonist, ketamine.

It has been reported that the repeated administration of a sub-anesthetic dose of an N-methyl-D-aspartate receptor antagonist, ketamine, can produce an animal model of schizophrenia. Since no information is available on the alterations of the amino acid levels in ketamine-treated rats, we investigated the amino acid composition in the plasma and cerebrospinal fluid of rats that were repeatedly administered with ketamine for 5 consecutive days (30 mg/kg/day). The plasma and cerebrospinal fluid amino acid compositions in the fifth week after cessation of repeated ketamine administration were determined by highperformance liquid chromatography with fluorescence detection using a pre-column fluorescence reagent, i.e. 4-fluoro-7nitro-2,1,3-benzoxadiazole. Among the amino acids investigated in the present study, the level of plasma glutamic acid increased significantly (p < 0.05), while that of the cerebrospinal fluid glutamic acid decreased significantly in the ketamine-treated rats as compared with these levels in control rats injected with saline (p < 0.05, n = 7). These alterations in the glutamic acid level in the plasma and cerebrospinal fluid resemble those in schizophrenic patients, suggesting that ketamine-treated rats may be a useful model for performing research on the pathophysiology of schizophrenia.

Animals↗

PubMedDate not supplied

Suppression of thiol exchange reaction in the determination of reduced-form thiols by high-performance liquid chromatography with fluorescence detection after derivatization with fluorogenic benzofurazan reagent, 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate and 4-aminosulfonyl-7-fluoro-2,1,3-benzoxadiazole.

The derivatization of the reduced-form thiols with SBD-F (7-fluoro-2,1,3-benzoxadiazole-4-sulfonate) and ABD-F (4-aminosulfonyl-7-fluoro-2,1,3-benzoxadiazole) was studied. The yields of the derivatives of the reduced-form thiols (cysteine, homocysteine, reduced-form glutathione) with SBD-F at 60 degrees C for 45 min in the borate buffer (pH 9.3) were significantly decreased in the presence of the oxidized-form thiols (cystine, homocystine, oxidized-form glutathione) because of the thiol exchange reaction between the reduced-form and the oxidized-form thiols. The use of ABD-F at low temperature enabled the suppression of these thiol exchange reactions, and the recommended conditions were below 5 degrees C for 90 min in borate buffer (pH 9.3). These results suggest that ABD-F is a preferred derivatization reagent for the accurate determination of the reduced-form thiols in samples containing the oxidized-form thiols. In addition, it was also suggested that the derivatization of the reduced-form thiols should also be performed at low temperature when derivatization reagents such as o-phthalaldehyde (OPA) and monobromobimane (BrB) are used.

Chromatography, High Pressure Liquid↗