Literatura académica sobre el tema "Fluorogenic dyes"

Fluorescence imaging is an invaluable tool to study biological processes, and fluorogenic dyes are crucial to enhance cell permeability and target intracellular structures with high specificity. Polymethine dyes are vitally important fluorophores in single-molecule localization microscopy and in vivo imaging, but their use in live cells has been limited by high background fluorescence and low membrane permeability. Here, we present a general strategy to transform polymethine compounds into fluorogenic dyes by implementing a 5-exo-trig ring-closure. This method provides access to bright, fluorogenic polymethine dyes with emissions across the visible and near-infrared spectrum.

Here, we present a direct comparison of different dyes and assays for the determination of protein concentrations. We compared the classical Bradford assay with two modern assays based on the fluorogenic dyes QuDye and ProteOrange and showed that the Bradford reagent achieved excellent results in the determination of protein concentrations as compared with more modern rivals. We also showed that standard approaches for determining the limit of detection (LoD) and limit of quantification (LoQ) may not work correctly with the tested dyes. We proposed a new approach that extends the standard algorithm for LoD and LoQ determination. This approach works well with both classical colorimetric and fluorogenic dyes, as well as with nontrivial fluorescent probes.

A bromoaryltetrazole-modified uridine was synthesized as a new RNA building block for bioorthogonal, light-activated and postsynthetic modification with commercially available fluorescent dyes. It allows “photoclick”-type modifications by irradiation with light (300 nm LED) at internal and terminal positions of presynthesized RNA with maleimide-conjugated fluorophores in good yields. The reaction was evidenced for three different dyes. During irradiation, the emission increases due to the formation of an intrinsically fluorescent pyrazoline moiety as photoclick product. The fluorogenecity of the photoclick reaction was significantly enhanced by energy transfer between the pyrazoline as the reaction product (poor emitter) and the photoclicked dye as the strong emitter. The RNA-dye conjugates show remarkable fluorescent properties, in particular an up to 9.4 fold increase of fluorescence, which are important for chemical biology and fluorescent imaging of RNA in cells.

Fluorescent solvatochromic dyes and molecular rotors have attracted considerable attention as fluorogenic probes because of background-free detection of biomolecules in live cells in no-wash conditions.

The present PhD thesis entitled “Design, Synthesis and Evaluation of Chromofluorogenic Probes for Contaminating Species” is focused on the development of new chromo-fluorogenic sensors based on the principles of molecular recognition. The first part of this thesis is focused on the design and synthesis of suitable organic compounds as sensors for metal cations. The selected sensing paradigm was the binding site-signalling subunit approach. The synthetized receptors employs a chromophore (fluorescein or BODIPY) skeleton as signalling subunit and it is functionalized with aminoethoxy moieties as binging site; the metal coordination reduces the electron-donating ability of the nitrogen atom conjugated to the chromophore resulting in optical changes noticeable to the naked-eye. The sensing behavior is highly selective to trivalent cations (Fe3+, Al3+ and Cr3+) with remarkable limits of detection. The receptors based on BODIPYdyes retain the sensing abilities in mixed aqueous solutions. The remaining chapters of the thesis are centered in the detection and removal of nerve agents surrogates. The design, synthesis, characterization and application of new BODIPY chemosensors were studied. These chemosensors were designed containing different reactive sites in order to avoid interferences produced by acids or hydrolysis products, and also be able to distinguish between the different G-nerve agent mimics (DCNP and DFP). The BODIPY-probes allows screening of nerve agent surrogates with remarkable limits of detection and optical changes noticeable to the naked-eye. The sensing abilities are retained in solid support, allowing practical application in real-time monitoring by simple colorimetric tests. The displacement assay approach has been used to develop a selective sensor for V-nerve agent surrogates versus G-type. For this purpose, two Eu3+ and Au3+ BODIPY-complexes were prepared. In this case, V-surrogate is capable of coordinate the metallic center, releasing the BODIPY ligand. This causes a change in the optical properties visible to the naked-eye. Finally, the use of supramolecular-based organocatalyst for destruction of OP nerve agent surrogates was studied. Hydrolysis studies were performed in presence of 1,3-diindolylureas and thioureas, amines, aminoalcohol and glycols. Addition of catalyst enhances the electrophilic character of the P atom, and the final nucleophilic attack of water that results in the formation of the corresponding less toxic organophosphate derivatives, thus higher hydrolysis rates are obtained.
Barba Bon, A. (2014). Design, Synthesis and Evaluation of Chromo-fluorogenic Probes for Contaminating Species [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/48521
TESIS

Les colorants à la fluorescéine sont largement utilisés dans différents brunchs de la chimie et des sciences connexes, tout d'abord en raison de leurs propriétés fluorescentes uniques. Parmi ces composés, les dérivés nitrés sont beaucoup moins explorés. Ce travail visait à divulguer les principales propriétés protolytiques et spectrales dans des solutions non aqueuses de ces colorants.Une série de nitrofluorescéines et de plusieurs dérivés aminés, 22 composés au total, a été étudiée en détention principalement par des méthodes spectroscopiques.Le comportement de ces composés diffère significativement de celui d'autres colorants à base de fluorescéine, par exemple des dérivés halogénés très populaires. Le déplacement de l'équilibre tautomère des formes neutres des colorants est déplacé vers la lactone. La particularité des colorants portant quatre groupes NO2 dans la partie xanthène de la molécule est la formation de lactones anioniques, provoquée par l'éloignement de la densité électronique de l'atome de carbone nodal. Une autre caractéristique est la facilité de rupture du cycle pyrane dans des solutions fortement alcalines.Les valeurs des colorants ont été déterminées par la méthode spectrophotométrique dans des solutions tampons dans du DMSO et dans plusieurs cas dans de l'acétonitrile contenant 4 % en masse de DMSO, à 25 oC.L'interprétation des valeurs nécessite une compréhension de l'état des équilibres tautomères. Par exemple, la différence entre les s de dissociation par étapes des colorants, dans le DMSO varie de 1,2 pour la 5'-nitrofluorescéine à 7,6 pour la 4,5-dinito-2,7-dibromofluorescéine. L'analyse des spectres d'absorption dans le visible permet d'identifier les structures moléculaires et ioniques des colorants. L'évaluation des fractions des tautomères a permis de calculer les constantes d'équilibre dites microscopiques, décrivant la dissociation des tautomères simples. Ces derniers coïncident avec ceux des composés modèles : les esters, l'éther et un analogue sulfo.Une étude spéciale a été consacrée à révéler la transmission des effets électroniques dans la molécule de fluorescéine en examinant l'influence des substituants NO2 et NH2 dans le résidu d'acide phtalique sur le 's des groupes OH dans la partie xanthène.L'introduction d'un groupe nitro dans la partie phtalique de la fluorescéine éteint l'émission. Étonnamment, les colorants avec quatre groupes NO2 dans la partie xanthènes présentent une fluorescence brillante dans le DMSO et aprotic. En conséquence, un nouvel indicateur fluorescent indépendant du pH, l'ester méthylique de la 2,4,5,7-tétranitrofluorescéine, très sensible à la capacité du solvant à former des liaisons hydrogène, est proposé. En outre, les conditions des anions à charge simple et double de la 5'-aminofluorescéine dans des solutions de nature différente sont élucidées.L'étude d'un mélange de solvants aprotiques et donneurs de liaisons hydrogène avec des composés modèles (BODIPY) nous permettra d'étudier plus en détail le mécanisme de l'effet des liaisons hydrogène sur les dérivés de la fluorescéine
Fluorescein dyes are widely used in different brunches of chemistry and related sciences, first of all owing to their unique fluorescent properties. Among these compounds, nitro derivatives are much less explored. This work was aimed to disclose the main protolytic and spectral properties in non-aqueous solutions of these dyes.A series of nitrofluoresceins and several amino derivatives, 22 compounds in total, was studied in detain mainly by spectroscopic methods.The behavior of these compounds differs significantly from that of other fluorescein dyes, e.g., very popular halogen derivatives. The shift of the tautomeric equilibrium of the neutral forms of the dyes is shifted toward the lactone. The peculiar feature of the dyes bearing four NO2 groups in the xanthenes portion of the molecule is formation of anionic lactones, caused by the pulling electron density away from the nodal carbon atom. Another feature is the ease of the rupture of the pyran ring in highly alkaline solutions.The values of the dyes were determined by the spectrophotometric method in buffer solutions in DMSO and in several cases in acetonitrile that contains 4 mass % DMSO, at 25 oC.Interpreting the values require an understanding of the state of tautomeric equilibria. For instance, the difference between the values of stepwise dissociation of the dyes, , in DMSO vary from 1.2 for 5'-nitrofluorescein to 7.6 to 4,5-dinito-2,7-dibromofluorescein. The analysis of the absorption spectra in the visible region allows identifying the molecular and ionic structures of the dyes. The evaluation of the fractions of the tautomers made it possible to calculate the so-called microscopic equilibrium constants, describing the dissociation of single tautomers. The last-named coincide with those of model compounds: esters, ether, and a sulfo analogue.A special study was devoted to revealing the transmittance of the electronic effects in the fluorescein molecule by examining the influence of NO2 and NH2 substituents in the phthalic acid residue on the s of the OH groups in the xanthenes part.Introduction of a nitro group in the phthalic part of fluorescein quenches the emission. Surprisingly, the dyes with four NO2 groups in the xanthenes part exhibit bright fluorescence in DMSO and aprotic solvents. As result, a new pH-independent fluorescent indicator, methyl ester of 2,4,5,7-tetranitrofluorescein, very sensitive to the ability of the solvent to hydrogen bonding, is proposed. Also, the conditions of single- and double-charged anions of 5'-aminofluorescein in solutions of different nature are elucidated.The study of a mixture of aprotic and hydrogen bond donor solvents with model compounds (BODIPY) will allow us to further study in more detail the mechanism of the effect of hydrogen bonds on fluorescein derivatives

La détection et l'imagerie par fluorescence de systèmes biologiques nécessite la mise en place d'outils efficaces, robustes et simples d'utilisation. Les sondes fluorogéniques conventionnelles, actuellement utilisées en microbiologie manquent de précision puisqu'elles résultent de la simple modification chimique de la structure d'un fluorophore déjà formé, conduisant souvent à une extinction incomplète de sa fluorescence intrinsèque. Mes travaux de thèse ont pour but de développer de nouveaux substrats fluorogéniques d'enzymes basés sur le principe du "covalent assembly". Cette approche de synthèse chimique in situ d'un cœur fluorescent à partir d'un précurseur dit "cagé" non-fluorescent, met en jeu des réactions domino caractérisées par la formation et la rupture de liaisons covalentes, déclenchées par une réaction enzymatique déclenchée par les bactéries à identifier. Les avantages d'une telle approche sont nombreux (meilleur rapport signal sur bruit, exemplification aisée à un large éventail d'analytes, …). Dans ce contexte, le premier objectif de ma thèse a été de synthétiser des fluorophores de type hétéro-xanthène originaux afin de valider leur stabilité dans des conditions physiologiques. De nouvelles sulfone- et Si-pyronines ont été obtenus. La synthèse des précurseurs "cagés" correspondant aux composés les plus stables en milieux aqueux, a été entreprise pour pouvoir les utiliser comme sondes à peptidases. L'ensemble de ces travaux sont décrits dans le chapitre I et II de ce manuscrit. Les difficultés rencontrées lors de la mise en œuvre de l'approche "covalent assembly" avec ces hétéro-xanthènes, nous ont conduit à explorer une nouvelle famille de fluorophores, les quinoxalinones. La formation in-situ de ces hétérocycles fluorescents, faisant suite à un stimuli enzymatique d'un précurseur "cagé" est présenté dans le chapitre III. Enfin le dernier chapitre traite de la synthèse d'une sonde conventionnelle dérivée d'une bis-sulfonyle-bis-aniline récemment publiée dans la littérature
Detection and fluorescence imaging of biologic systems requires the implementation of efficient, robust and easy-to-use tools. Conventional fluorogenic probes currently used in microbiology lack efficiency since they are based on the single chemical modification of a fluorophore bearing an optically tunable reactive group, which often leads to incomplete fluorescence quenching. The main goal of my Ph.D thesis was to develop novel fluorogenic enzymatic substrates based on the "covalent assembly" principle. This approach also named "in situ synthesis" is based on the use of domino reactions to form a fluorescent moiety starting from a "caged" non-fluorescent molecule. In our case, the bioanalyte that triggers the reaction is present in bacteria, that we want to detect. This strategy provides many advantages (improved signal-to-noise ratio, easy exemplification to a wide range of analytes, …). In this context, the first goal of my thesis was to synthesize original fluorescent hetero-xanthene dyes to assess their stability under physiological conditions. Novel sulfone- and Si-pyronin derivatives were obtained. Synthesis of the corresponding "caged" precursors to the most stable compounds was then undertaken for their use as a peptidase-responsive probes. This work is described in the first and second chapter of this manuscript. Faced with difficulties to implement "covalent assembly" probe design principle to Si-pyronins, another class of fluorophores, namely quinoxalinone was explored. In situ formation of these fluorescent heterocycles triggered by an enzyme is presented in the third chapter. Finally, the last chapter was devoted to the synthesis of a conventional probe derived from a bis-sulfonyl-bis-aniline recently reported in the literature

The fluorescent toolbox is expanding, and Fluorogen Activating Protein Technology (FAP) is contributing by providing tools for collecting direct real-time, live cell protein trafficking measurements. Chapter I includes a brief overview of current fluorescent and fluorogenic labeling approaches. After the review of labeling methods, FAP Technology is presented as a well suited tool for cell surface protein trafficking investigations. Core strengths of the developed fluorogenic dyes are highlighted. This chapter is a discussion on using FAP labeling strategies to quantify cell surface protein translocation, particularly addressing the needs for a direct, quick, and high-throughput adaptable protein trafficking detection assay for GPCRs and Ion channels. Chapter I concludes with introducing new methods for assessing intracellular trafficking of protein pools in the cell. Chapter II entails the development of a new pH sensor tool that can quantify pH related changes associated with protein trafficking from the surface to the endosomal network, and how it was applied to observe B2AR differential agonist induced trafficking behavior. Chapter III describes the development of a high-throughput assay for identifying potential kinase targets that enhance VX-809 corrector ability to rescue the mutant ΔF508 cystic fibrosis transmembrane conductance regulator (CFTR). The screen was based on a FAP approach that uses both surface and total protein fluorescence measurements collected from a plate reader. Chapter IV presents the current work on creating a lysosomal FAP construct that will function as a test-bed for monitoring protein trafficking related to lysosomal dysfunction.

碩士
靜宜大學
應用化學研究所
97
Three new chemodosimeters 23-25 were prepared, and their chromogenic and fluorogenic behaviors toward various metal cations were investigated. Receptor 23-25 show exclusively response toward Hg2+ ion and also distinguish Hg2+from other metal cations by different color changes in DMSO aqueous solution (DMSO/H2O = 9/1, v/v). Among them, receptor 23 also exhibits a pronounced Hg2+-induced fluorescence enhancement. Thus, the receptor 23 can be use as a colormetric and fluorescent chemodosimeter for determination of Hg2+ ion. The use of the test strip of the receptor 23 to detect Hg2+ was also reported.

碩士
靜宜大學
應用化學系
100
Three new sensitive and selective cyanide chemosensors 15-17 were prepared, and their chromogenic and fluorogenic behaviours toward various anions were investigated. Among them, receptor 15 shows exclusive response toward CN- ion. When cyanide ions were added (EtOH/H2O = 3/7, v/v), the pronounced enhancement of the fluorescence, as well as the color change of 15 was observed so that micromolar concentrations of cyanide were detectable by the naked eye. The use of test strip of the receptor 15 to detect CN- ion was also reported.

碩士
靜宜大學
應用化學研究所
98
Part 1. A new chemodosimeter 1-(4-(4-trifluoromethyl phenyldiazenyl) phenyl)-3-(pyren-1-yl)thiourea (20) was prepared, and its chromogenic and fluorogenic behaviors toward various metal cations were investigated. Receptor 20 shows exclusive a response toward Hg2+ ion and also distinguished Hg2+ ion from other metal cations by color changes in DMSO aqueous solution (DMSO/H2O = 9/1, v/v). Additionally, receptor 20 also exhibited a pronounced Hg2+-induced fluorescence enhancement. Thus, the receptor 20 can be used as a colorimetric and fluorescent chemodosimeter for the determination of Hg2+ ion. Part 2. The passionvine mealybug, Planococcus minor is a serious pest of economically important crops in Taiwan. The effective control of this pest is limited by only using pesticides. Also the side effects such as the insect resistance, the pesticide residue and the environment contamination will be occurred. To avoid these side effects, the nontoxic sex pheromone of the passionvine mealybug is chosen instead. The sex pheromone of Planococcus minor was identified as (E)-2-isopropyl-5-methyl-2, 4-hexadienyl acetate (1). The synthesis of this sex pheromone would produce two isomers in the products. This caused some difficulties and troublesome. On our hand, due to the high cost of the starting material and the low yield of the product, the development of a cost-effective, high yield and operationally simple synthetic approach is necessary. In this report a series of new palladium reagents are used for the synthesis of the intermediate, 2-(5-Methylhex-4-en-2-ynyloxy)- tetrahydro -2H-pyran (8). In addition, the subsequent coupling reaction can also be successfully preceded by using FeCl3. This improved synthetic method can overcome the above limitations. Receutly, the effective control of this pest in the tests by using this pheromone is achieved. This indicates that the synthesized pheromone has strong attraetiveness for the passionvine mealybug.