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).     

An improved method for proteomics studies in C. elegans by fluorogenic derivatization, HPLC isolation, enzymatic digestion and liquid chromatography--tandem mass spectrometric identification

An improved method for proteomics studies, which includes the fluorogenic derivertization of protein mixtures with 7-chloro-4-(dimethylaminoethylaminosulfonyl)-2,1,3-benzoxadiazole (DAABD-Cl), followed by HPLC isolation, enzymatic digestion and identification of the derivatized proteins by HPLC-electrospray ionization (ESI)-MS/MS with the probability-based protein identification algorithm, identified 103 proteins in the soluble extract (10 microg protein) of Caenorhabditis elegans.     

Existence of low-molecular-weight thiols in Caenorhabditis elegans demonstrated by HPLC-fluorescene detection utilizing 7-chloro-N-[2-(dimethylamino)ethyl]-2,1,3-benzoxadiazole-4-sulfonamide

A highly sensitive and simple method using HPLC-fluorescence detection with 7-chloro-N-[2-(dimethylamino)ethyl]-2,1,3-benzoxadiazole-4-sulfonamide (DAABD-Cl) as a fluorogenic reagent demonstrated the existence of the low-molecular-weight thiols in the extract of Caenorhabditis elegans (C. elegans). The method includes derivatization of the thiols with DAABD-Cl at 40 degrees C for 10 min in borate buffer (pH 9.0) containing TCEP, CHAPS and EDTA, separation of the derivatives on an ODS column and fluorometric determination of the derivatives at 510 +/- 15 nm with excitation at 400 +/- 15 nm. The identification of the thiols was made by HPLC-electrospray ionization mass spectrometry (LC-MS) following isolation of the derivatives using HPLC-fluorescence detection. Low-molecular-weight thiols were found to exist in the extract of C. elegans, such as cysteine, cysteinylglycine, gamma-glutamylcysteine, reduced glutathione and two other unidentified thiol compounds, confirming the existence of the 'glutathione cycle' in C. elegans similar to the mammalian body.     

4-Azidocinnoline—Cinnoline-4-amine Pair as a New Fluorogenic and Fluorochromic Environment-Sensitive Probe

A new type of fluorogenic and fluorochromic probe based on the reduction of weakly fluorescent 4-azido-6-(4-cyanophenyl)cinnoline to the corresponding fluorescent cinnoline-4-amine was developed. We found that the fluorescence of 6-(4-cyanophenyl)cinnoline-4-amine is strongly affected by the nature of the solvent. The fluorogenic effect for the amine was detected in polar solvents with the strongest fluorescence increase in water. The environment-sensitive fluorogenic properties of cinnoline-4-amine in water were explained as a combination of two types of fluorescence mechanisms: aggregation-induced emission (AIE) and excited state intermolecular proton transfer (ESPT). The suitability of an azide–amine pair as a fluorogenic probe was tested using a HepG2 hepatic cancer cell line with detection by fluorescent microscopy, flow cytometry, and HPLC analysis of cells lysates. The results obtained confirm the possibility of the transformation of the azide to amine in cells and the potential applicability of the discovered fluorogenic and fluorochromic probe for different analytical and biological applications in aqueous medium.     

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.     

Postcolumn derivatization of proteins in capillary sieving electrophoresis/laser‐induced fluorescence detection

The separation methods for proteins with high resolution and sensitivity are absolutely important in the field of biological sciences. Capillary sieving electrophoresis (CSE) is an excellent separation technique for DNA and proteins with high resolution, while LIF permits the most sensitive detection in CSE. Therefore, proteins have to be labeled with fluorescent or fluorogenic reagent to produce fluorescent derivatives. Both precolumn and oncolumn derivatization have been employed for the labeling of proteins in CSE. However, there is no report on the postcolumn derivatization due to the limitation in the use of a standard migration buffer, despite it being a promising method for sensitive detection of proteins. Here, we show a novel postcolumn derivatization method for protein separation by CSE, using a tertiary amine as a buffer component in the running buffer. Tris, which is commonly used as a base in CSE separation buffers, was substituted by tertiary amines, 2‐(diethylamino)ethanol and triethanolamine. A buffer solution containing 2‐(diethylamino)ethanol or triethanolamine can be used for the CSE separation followed by the postcolumn derivatization of proteins, since both reagents are unreactive toward a fluorogenic labeling reagent, naphthalene‐2,3‐dicarbaldehyde. Thus, LIF detection using the postcolumn derivatization permits significant reduction in the LOD (by a factor of 2.4–28) of proteins, compared with conventional absorbance detection.     

Recent progress in the development of derivatization reagents having a benzofurazan structure

Chemical derivatization is often used to enhance the detectability of the target compounds and to improve the separation efficiency in high-performance liquid chromatography (HPLC). In this review, we describe the recent progress in the development of derivatization reagents having a benzofurazan structure, namely, the fluorogenic reagents, water-soluble reagents, reagents for the analysis of peptides and proteins, and reagents for mass spectrometric detection. The application of these reagents to bio-samples is also briefly described.     

New Chemodosimetric Reagents as Ratiometric Probes for Cysteine and Homocysteine and Possible Detection in Living Cells and in Blood Plasma

In this work, we have rationally designed and synthesized two new reagents ( L₁ and L₂ ), each bearing a pendant aldehyde functionality. This aldehyde group can take part in cyclization reactions with β‐ or γ‐amino thiols to yield the corresponding thiazolidine and thiazinane derivatives, respectively. The intramolecular charge‐transfer (ICT) bands of these thiazolidine and thiazinane derivatives are distinctly different from those of the molecular probes ( L₁ and L₂ ). Such changes could serve as a potential platform for using L₁ and L₂ as new colorimetric/fluorogenic as well as ratiometric sensors for cysteine (Cys) and homocysteine (Hcy) under physiological conditions. Both reagents proved to be specific towards Cys and Hcy even in the presence of various amino acids, glucose, and DNA. Importantly, these two chemodosimetric reagents could be used for the quantitative detection of Cys present in blood plasma by using a pre‐column HPLC technique. Such examples are not common in contemporary literature. MTT assay studies have revealed that these probes have low cytotoxicity. Confocal laser scanning micrographs of cells demonstrated that these probes could penetrate cell membranes and could be used to detect intracellular Cys/Hcy present within living cells. Thus, the results presented in this article not only demonstrate the efficiency and specificity of two ratiometric chemodosimeter molecules for the quantitative detection of Cys and Hcy, but also provide a strategy for developing reagents for analysis of these vital amino acids in biological samples.     

Fluorogenic Strain‐Promoted Alkyne–Diazo Cycloadditions

Fluorogenic reactions, in which non‐ or weakly fluorescent reagents produce highly fluorescent products, are attractive for detecting a broad range of compounds in the fields of bioconjugation and material sciences. Herein, we report that a dibenzocyclooctyne derivative modified with a cyclopropenone moiety (Fl‐DIBO) can undergo fast strain‐promoted cycloaddition reactions under catalyst‐free conditions with azides, nitrones, nitrile oxides, as well as mono‐ and disubstituted diazo‐derivatives. Although the reaction with nitrile oxides, nitrones, and disubstituted diazo compounds gave cycloadducts with low quantum yield, monosubstituted diazo reagents produced 1H‐pyrazole derivatives that exhibited an approximately 160‐fold fluorescence enhancement over Fl‐DIBO combined with a greater than 10 000‐fold increase in brightness. Concluding from quantum chemical calculations, fluorescence quenching of 3H‐pyrazoles, which are formed by reaction with disubstituted diazo‐derivatives, is likely due to the presence of energetically low‐lying (n,π*) states. The fluorogenic probe Fl‐DIBO was successfully employed for the labeling of diazo‐tagged proteins without detectable background signal. Diazo‐derivatives are emerging as attractive reporters for the labeling of biomolecules, and the studies presented herein demonstrate that Fl‐DIBO can be employed for visualizing such biomolecules without the need for probe washout.     

Semmler-Wolff aromatization of 4-hydroxyimino-4,5,6,7-tetrahydro-[2,1,3]benzoxadiazole and 4-hydroxyimino-4,5,6,7-tetrahydro[2,1,3]benzoxadiazole 1-oxide and some properties of the obtained amines

The Semmler-Wolff aromatization of 4-hydroxyimino-4,5,6,7-tetrahydro[2,1,3]benzoxadiazole and its 1-oxide derivative in sulfuric acid and in acetic anhydride gave 4-amino[2,1,3]benzoxadiazole and 4-acetamido[2,1,3]benzoxadiazole 1-oxide. Some reactions of the resultant amines were studied.     

Two-dye and one- or two-quencher DNA probes for real-time PCR assay: synthesis and comparison with a TaqMan? probe

A typical TaqMan? real-time PCR probe contains a 5'-fluorescent dye and a 3'-quencher. In the course of the amplification, the probe is degraded starting from the 5'-end, thus releasing fluorescent dye. Some fluorophores (including fluorescein) are known to be prone to self-quenching when located near each other. This work is aimed at studying dye-dye and dye-quencher interactions in multiply modified DNA probes. Twenty-one fluorogenic probes containing one and two fluoresceins (FAM), or a FAM-JOE pair, and one or two BHQ1 quenchers were synthesized using non-nucleoside reagents and "click chemistry" post-modification on solid phase and in solution. The probes were tested in real-time PCR using an ~300-bp-long natural DNA fragment as a template. The structural prerequisites for lowering the probe background fluorescence and increasing the end-plateau fluorescence intensity were evaluated and discussed.     

四个香豆素磺酰氯衍生物的合成及其荧光性能的研究

四个香豆素磺酰氯衍生物的合成及其荧光性能的研究曹秋娥,徐其亨（云南大学化学系,昆明,６５００９１）关键词香豆素磺酰氯衍生物,荧光试剂,荧光性能香豆素衍生物是一类较好的荧光试剂,本文以香豆素为母体,合成了４个香豆素磺酰氯衍生物：４－甲基－７－羟基香豆素...     

Evaluation of capillary liquid chromatography-electrospray ionization ion mobility spectrometry with mass spectrometry detection

Due to the proteomics revolution, multi-dimensional separation and detection instruments are required to evaluate many peptides and proteins in single samples. In this study, electrospray ionization (ESI) ion mobility spectrometry (IMS) was evaluated as an additional separation after HPLC separations. Common HPLC mobile phase compositions (solvents, acid modifiers, and buffers) were assessed for the effect on ESI-IMS response. Up to 5 mM sodium phosphate, a non-volatile buffer, was able to be electrosprayed into the IMS without degradation of the instrumental performance. Due to the rapid separation times of IMS, multiple IMS spectra were obtained within a single HPLC peak. A five-peptide mixture was separated in a capillary HPLC column under isocratic conditions within 3 min. Coelution of two peaks due to non-optimal HPLC conditions occurred and these two peaks could not be distinguished by HPLC with UV detection. In contrast, the single ion mobility chromatograms provided separation of each peptide as well as providing a second degree of analyte identification (HPLC retention time and IMS mobility). Furthermore, IMS-MS analysis of the five peptides and comparison with HPLC retention times showed that each peptide had a unique retention time-ion mobility-mass to charge value. This work showed that IMS could be employed for direct separation and detection of HPLC eluents and also could be combined with HPLC-MS for three unique dimensions of separation.     

A new method for assessing the effect of residual silanol groups on total retention in reversed-phase HPLC

A new approach is proposed to assess the effect of residual silanol groups (hydrophilicity) on the retention of polar substances in reversed-phase high-performance liquid chromatography (HPLC). It is shown that the numerical characteristic of the relative position of trend lines in the coordinates logk(B) vs. logk(A) of the relative retention parameters (at different mobile phase compositions) for any stationary phase relative to the stationary phase selected as a standard is preferable to the separation selectivity of the same pair of substances. Full information can be obtained by determining the retention of corresponding adsorbates with two different mobile phase compositions for each column under study. It was shown that the pair of adsorbates p-toluidine-p-cresol exhibits higher sensitivity to a change in the hydrophilicity of stationary phases as compared to the pair aniline-phenol. However, the use of a nonpolar substance as a reference compound is more informative.     

Synthesis of benzofurazan derivatization reagents for short chain carboxylic acids in liquid chromatography/electrospray ionization‐tandem mass spectrometry (LC/ESI‐MS/MS)

Benzofurazan derivatization reagents, 4‐[2‐(N,N‐dimethylamino)ethylaminosulfonyl]‐7‐(2‐aminopentylamino)‐2,1,3‐benzoxadiazole (DAABD‐AP) and 4‐[2‐(N,N‐dimethylamino) ethylaminosulfonyl]‐7‐(2‐aminobutylamino)‐2,1,3‐benzoxadiazole (DAABD‐AB), for short‐chain carboxylic acids in liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI‐MS/MS) were synthesized. These reagents reacted with short chain carboxylic acids in the presence of the condensation reagents at 60°C for 60 min. The generated derivatives were separated on the reversed‐phase column and detected by ESI‐MS/MS with the detection limits of 0.1–0.12 pmol on column. Upon collision‐induced dissociation, a single and intense product ion at m/z 151 was observed. These results indicated that DAABD‐AP and DAABD‐AB are suitable as the derivatization reagents in LC/ESI‐MS/MS analysis. Copyright © 2008 John Wiley & Sons, Ltd.     

Determination of rat hepatocellular glutathione by reversed-phase liquid chromatography with fluorescence detection and cytotoxicity evaluation of environmental pollutants based on the concentration change.

Three methods for the determination of rat hepatocellular thiols by high-performance liquid chromatography (HPLC) with fluorescence (FL) detection have been developed. The thiols in the cells were tagged with three fluorogenic reagents, SBD-F, ABD-F and DBD-F. These reagents could permeate into cells and effectively reacted with thiols to produce highly fluorescent derivatives. These derivatives fluoresced in the long wavelength region at around 530 nm (excitation at around 380 nm). The five biological thiols tagged were perfectly separated by reversed-phase liquid chromatography and were sensitively and selectively detected without any interference from endogenous substanaces. The main thiol in the cells was reduced GSH and the concentration was at the mM level. The proposed procedures were applied to the determination of hepatocellular GSH after treatment of environmental pollutants such as volatile organic compounds (VOC) and endocrine disrupting chemicals (EDC). From the comparison of intracellular GSH concentration, the test compounds were classified into four groups: compounds of strong depletion (eg triphenyltin chloride, hexachlorocyclohexene, nonylphenol, bromoacetic acid, 4-chlorobenzyl chloride and 1,3-dichloropropene), slight decrease (eg bisphenol A, benzo[a]pylene, carbon tetrachloride and benzene), slight increase (eg bromoform and toluene), and no effect (eg 1,1,1-trichloroethane, 1,1,2-trichloroethane and 1,2-dichloroethane). Although the decrease of GSH concentration does not reflect the cytotoxicity of chemicals, the proposed procedure utilizing isolated rat hepatpcytes seems to be useful for investigating the bioactivation of VOC, and EDC, etc.     

Mechanism‐Based Fluorogenic trans‐Cyclooctene–Tetrazine Cycloaddition

The development of fluorogenic reactions which lead to the formation of fluorescent products from two nonfluorescent starting materials is highly desirable, but challenging. Reported herein is a new concept of fluorescent product formation upon the inverse electron‐demand Diels–Alder reaction of 1,2,4,5‐tetrazines with particular trans ‐cyclooctene (TCO) isomers. In sharp contrast to known fluorogenic reagents the presented chemistry leads to the rapid formation of unprecedented fluorescent 1,4‐dihydropyridazines so that the fluorophore is built directly upon the chemical reaction. Attachment of an extra fluorophore moiety is therefore not needed. The photochemical properties of the resulting dyes can be easily tuned by changing the substitution pattern of the starting 1,2,4,5‐tetrazine. We support the claim with NMR measurements and rationalize the data by computational study. Cell‐labeling experiments were performed to demonstrate the potential of the fluorogenic reaction for bioimaging.     

Fluorescent high-performance liquid chromatography of folic acid and its derivatives using permanganate as a fluorogenic reagent

Summary A fluorescent high-performance liquid chromatography (HPLC) analysis of folic acid (pteroylglutamic acid) and its derivatives using permanganate as a fluorogenic reagent has been developed. In the present work, the various analytical conditions in which the fluorogenic reaction with permanganate could be applied to the HPLC of folic acid in human serum, have been studied. The present method may be employed for the rapid clinical analysis of small amounts of folic acid (150 ppb), without interference from coexisting natural fluorophors; the precision of the present method for folic acid was 6.6% (n=6) (coefficient of variation).Dedicated to Professor Dr. Wilhelm Fresenius on the occasion of his 80th birthdayA part of this paper was presented at the 12th International Symposium on Microchemical Techniques, Cordoba (Spain), September 1992     

HPLC Determination of Chlorpropamide in Human Serum by Fluorogenic Derivatization Based on the Suzuki Coupling Reaction with Phenylboronic Acid

A fluorogenic derivatization method for the determination of chlorpropamide in human serum was developed by means of high-performance liquid chromatography (HPLC) with fluorescence detection. The Suzuki coupling reaction with a non-fluorescent reagent, phenylboronic acid (PBA), was employed to convert chlorpropamide into highly fluorescent biphenyl derivative. Chlorpropamide was extracted from human serum by liquid–liquid extraction with toluene after addition of hydrochloric acid, and subsequently reacted with PBA. Because the fluorogenic derivatization was highly selective for aryl halide, the proposed method allowed sensitive and selective detection of chlorpropamide with a detection limit (at a signal to noise ratio of 3) of 0.5 ng mL^?1. The sensitivity of our method was from 4 to 100 times better than HPLC–UV, gas chromatography, and LC-mass spectrometry.     

HPLC Determination of Chlorpropamide in Human Serum by Fluorogenic Derivatization Based on the Suzuki Coupling Reaction with Phenylboronic Acid

A fluorogenic derivatization method for the determination of chlorpropamide in human serum was developed by means of high-performance liquid chromatography (HPLC) with fluorescence detection. The Suzuki coupling reaction with a non-fluorescent reagent, phenylboronic acid (PBA), was employed to convert chlorpropamide into highly fluorescent biphenyl derivative. Chlorpropamide was extracted from human serum by liquid–liquid extraction with toluene after addition of hydrochloric acid, and subsequently reacted with PBA. Because the fluorogenic derivatization was highly selective for aryl halide, the proposed method allowed sensitive and selective detection of chlorpropamide with a detection limit (at a signal to noise ratio of 3) of 0.5 ng mL⁻¹. The sensitivity of our method was from 4 to 100 times better than HPLC–UV, gas chromatography, and LC-mass spectrometry.