Green Fluorescent Compounds Research Articles

Synthesis and studies of covalently linked pyrrolyl bridged fluorescent dimers

A series of stable fluorescent compounds namely the pyrrolyl bridged bis-(BODIPY) and bis-(22-oxacorrole) dimers were synthesized using pyrrolyl bridged bis-(dipyrromethane) as key precursor. The pyrrolyl bridged bis-(dipyrromethane) was synthesized in two steps by using commercially available compounds and used for the synthesis of pyrrolyl bridged bis-(BODIPY). The chloro groups present at the bridging pyrrole ring of bis-(BODIPY) dimer were replaced with aryl groups by the treatment of two different aryl boronic acids under Suzuki Coupling conditions. In order to show a further use of the pyrrole bridged bis-(dipyrromethane) precursor, it was condensed with 16-oxatripyrrane which rendered the pyrrolyl bridged bis-(22-oxacorrole) dimer as a green fluorescent compound in 8% yield. The dimers were thoroughly characterized by NMR and mass and the properties were investigated by absorption, fluorescence, electrochemical and DFT/TD-DFT studies. DFT studies showed that the bis-(BODIPY) and bis-(22-oxacorrole) dimers were highly symmetric and TD-DFT studies were in agreement with the experimental absorption and redox properties.

ChemFLuo: a web-server for structure analysis and identification of fluorescent compounds.

Fluorescent detection methods are indispensable tools for chemical biology. However, the frequent appearance of potential fluorescent compound has greatly interfered with the recognition of compounds with genuine activity. Such fluorescence interference is especially difficult to identify as it is reproducible and possesses concentration-dependent characteristic. Therefore, the development of a credible screening tool to detect fluorescent compounds from chemical libraries is urgently needed in early stages of drug discovery. In this study, we developed a webserver ChemFLuo for fluorescent compound detection, based on two large and high-quality training datasets containing 4906 blue and 8632 green fluorescent compounds. These molecules were used to construct a group of prediction models based on the combination of three machine learning algorithms and seven types of molecular representations. The best blue fluorescence prediction model achieved with balanced accuracy (BA) = 0.858 and area under the receiver operating characteristic curve (AUC) = 0.931 for the validation set, and BA = 0.823 and AUC = 0.903 for the test set. The best green fluorescence prediction model achieved the prediction accuracy with BA = 0.810 and AUC = 0.887 for the validation set, and BA = 0.771 and AUC = 0.852 for the test set. Besides prediction model, 22 blue and 16 green representative fluorescent substructures were summarized for the screening of potential fluorescent compounds. The comparison with other fluorescence detection tools and theapplication to external validation sets and large molecule libraries have demonstrated the reliability of prediction model for fluorescent compound detection. ChemFLuo is a public webserver to filter out compounds with undesirable fluorescent properties, which will benefit the design of high-quality chemical libraries for drug discovery. It is freely available at http://admet.scbdd.com/chemfluo/index/.

Conversion of 3,6‐O‐Dimethylfluorescein to Fluorescein‐Based Xanthylium Derivative: Characterization and Covalent Attachment to Bovine Serum Albumin

AbstractXanthene dyes, such as fluorescein and rhodamine, have played crucial rules in biological sciences especially covalent attachment of these organic compounds to biomolecules. 3‐O‐methylfluorescein and 3,6‐O‐dimethylfluorescein were synthesized and purified using a facile procedure. 3,6‐O‐dimethylfluorescein, which lacks fluorescence, was treated with sodium hydroxide and sulfuric acid solutions, giving rise to yellow and green fluorescent compounds, respectively. In both basic and acidic conditions, donor‐acceptor structures were formed. The reaction of 3,6‐O‐dimethylfluorescein with acids, in a reversible manner, emitted green fluorescence, whereas its reaction with hydroxide ion, irreversibly shifted the fluorescence to the yellow region. Using MS spectrometry, the product identity was determined as a xanthylium ion derivative. Stokes shift and quantum yield for xanthylium ion were measured. Various nucleophiles were examined for studying the mechanism of the xanthylium ion in aqueous solutions; the results confirmed that hydroxide ion was mediatory for the emission of the fluorescence. In addition, 3‐O‐methyl‐5(6)‐carboxyfluorescein and 3,6‐O‐dimethyl‐5(6)‐carboxyfluorescein were synthesized that can potentially be used in bioorthogonal reactions for labeling proteins. The synthesized products were used in covalent attachment to bovine serum albumin (BSA) and their unique fluorescences were detected.

Fluorescent Canthin-6-one Alkaloids from Simaba bahiensis: Isolation, Identification, and Cell-Labeling Properties.

Canthin-6-one alkaloids, which are present in plants of the genus Simaba, are natural compounds that are capable of acting as fluorescent probes. However, the chemical composition and fluorescent properties of most species of this genus have not been analyzed. The objective of this study was to characterize the fluorescent properties of an extract of S. bahiensis and identify the chemical entities responsible for these properties. In addition, the cell-labeling properties of the fluorescent dye from A and of the isolated compounds were characterized by confocal fluorescence microscopy and flow cytometry. One quassinoid and three fluorescent alkaloids were isolated from S. bahiensis, all compounds were identified by using NMR spectroscopy and high-resolution mass spectrometry. Staining experiments and HPLC-FL analysis shown that canthin-6-one alkaloids are the main green fluorescent compounds in the analyzed dyes. All compounds evaluated showed a cytoplasmic marker with a residence time of 24 h. The present study is the first to describe the presence of canthin-6-one alkaloids in S. bahiensis, in addition to demonstrating promising cell-labeling properties of fluorescent compounds from S. bahiensis with broad emission wavelengths.

Cd, Fe, and light sensitivity: interrelationships in Cd-treated populus.

Cadmium is a toxic heavy metal causing iron deficiency in the shoot and light sensitivity of photosynthetic tissues that leads to decreased photosynthetic performance and biomass production. Light intensity had strong impact on both photosynthetic activity and metal accumulation of cadmium-treated plants. At elevated irradiation, cadmium accumulation increased due to the higher dry mass of plants, but its allocation hardly changed. A considerable amount of iron accumulated in the roots, and iron concentration was higher in leaves developed at moderate rather than low irradiation. At the same time, the higher the irradiation the lower the maximal photochemical quantum efficiency. The decreased photochemical efficiency, however, started to recover after a week of Cd treatment at moderate light without substantial change in metal concentrations but following the accumulation of green fluorescent compounds. Both cadmium treatment and higher light caused the accumulation of flavonoids in leaf mesophyll vacuoles/chloroplasts, but accumulation of flavonols, fluorescing at 510 nm, was characteristic to cadmium stress. Therefore, flavonoids, which may act by scavenging reactive radicals, chelating Cd, and shielding against excess irradiation, play an important part in Cd stress tolerance of Populus, and may have special impact on its phytoremediation capacity.

Riboflavin concentration within ABCG2-rich extracellular vesicles is a novel marker for multidrug resistance in malignant cells

We have previously shown that overexpression of the multidrug resistance (MDR) efflux transporter ABCG2 in the membrane of novel extracellular vesicles that are confined to breast cancer cell–cell attachment zones confers mitoxantrone resistance and mediates a marked intravesicular concentration of an unknown endogenous green fluorescent compound (I. Ifergan, G.L. Scheffer, Y.G. Assaraf, Novel extracellular vesicles mediate an ABCG2-dependent anticancer drug sequestration and resistance, Cancer Res. 65 (2005) 10952–10958). Here we identified the latter as riboflavin (vitamin B2) and further demonstrated that the marked intravesicular concentration of riboflavin in ABCG2-overexpressing breast and lung cancer cells tightly correlates with the extent of ABCG2 overexpression and its differential localization to the vesicular membrane and not to the plasma membrane surrounded by growth medium. We hence propose that the ABCG2-dependent concentration of riboflavin in these intercellular compartments may serve as a novel, sensitive, and non-cytotoxic (i.e. based on vitamin accumulation) functional marker for the quantification of the levels of MDR mediated by ABCG2-rich extracellular vesicles in multiple malignant cells.

Sub-cellular trafficking of phytochemicals explored using auto-fluorescent compounds in maize cells

BackgroundLittle is known regarding the trafficking mechanisms of small molecules within plant cells. It remains to be established whether phytochemicals are transported by pathways similar to those used by proteins, or whether the expansion of metabolic pathways in plants was associated with the evolution of novel trafficking pathways. In this paper, we exploited the induction of green and yellow auto-fluorescent compounds in maize cultured cells by the P1 transcription factor to investigate their targeting to the cell wall and vacuole, respectively.ResultsWe investigated the accumulation and sub-cellular localization of the green and yellow auto-fluorescent compounds in maize BMS cells expressing the P1 transcription factor from an estradiol inducible promoter. We established that the yellow fluorescent compounds accumulate inside the vacuole in YFBs that resemble AVIs. The green fluorescent compounds accumulate initially in the cytoplasm in large spherical GFBs. Cells accumulating GFBs also contain electron-dense structures that accumulate initially in the ER and which later appear to fuse with the plasma membrane. Structures resembling the GFBs were also observed in the periplasmic space of plasmolized cells. Ultimately, the green fluorescence accumulates in the cell wall, in a process that is insensitive to the Golgi-disturbing agents BFA and monensin.ConclusionsOur results suggest the presence of at least two distinct trafficking pathways, one to the cell wall and the other to the vacuole, for different auto-fluorescent compounds induced by the same transcription factor in maize BMS cells. These compartments represent two of the major sites of accumulation of phenolic compounds characteristic of maize cells. The secretion of the green auto-fluorescent compounds occurs by a pathway that does not involve the TGN, suggesting that it is different from the secretion of most proteins, polysaccharides or epicuticular waxes. The yellow auto-fluorescent compounds accumulate in a vacuolar compartment, in structures that resemble the AVIs present in many cells accumulating anthocyanins. Together, our studies suggest that the accumulation of auto-fluorescent compounds can provide a powerful tool to dissect the trafficking of phytochemicals, knowledge necessary for the efficient engineering of plant metabolism.

Expression and function of P-glycoprotein and absence of multidrug resistance-related protein in rat and beige mouse peritoneal mast cells.

To clarify the function of the multidrug transporter P-glycoprotein in mast cells we used the green fluorescent compound Bodipy-FL-verapamil, which is a substrate of P-glycoprotein. This compound is also transported by Multidrug Resistance-related Protein (MRP), another membrane transport protein expressed in many tumour resistant cells as well as in normal cells. When rat peritoneal mast cells were incubated with Bodipy-verapamil, a rapid uptake of this compound was observed. Pretreatment with modulators of P-glycoprotein activity, such as verapamil and vinblastine, increased Bodipy-verapamil intracellular concentrations. In addition, Bodipy-verapamil efflux from these cells was rapid and also inhibited by verapamil and vinblastine. In contrast, no effect was observed when cells were treated with agents, such as probenecid and indomethacin, that are known inhibitors of MRP. Methylamine and monensin, substances that modify the pH values in the granules, were able to lower the concentrations of Bodipy-verapamil. Microscopical observations, conducted in both rat and beige mouse mast cells, demonstrated that the fluorochrome accumulated in the cytoplasmic secretory granules. RT-PCR performed on rat peritoneal mast cells revealed the presence of MDR1a and MDR1b mRNAs; on the contrary, MRP mRNA was not expressed. Mast cells were further treated with the fluorescent probe LysoSensor Blue, a weak base that becomes fluorescent when inside acidic organelles. This substance accumulated in mast cell granular structures and its fluorescence was reduced either by treatment with P-glycoprotein modulators or with agents that disrupt pH gradients. In conclusion, these data further confirm the presence of an active P-glycoprotein, but not of MRP, in rat peritoneal mast cells. These findings, coupled with previous ultrastructural data, lend further support to the assumption that this protein is located on the mast cell perigranular membrane. The functional role of P-glycoprotein in these cells is at present unclear, but a possible involvement in the transport of molecules from the granules to the cytosol can be hypothesized. Alternatively, this protein might be indirectly implicated in changes of pH values inside secretory granules.

The application of fluorescence polarisation for the enhanced detection of green fluorescent protein (GFP) in the presence of cellular auto-fluorescence and other green fluorescent compounds

The jellyfish green fluorescent protein (GFP) is a versatile biological marker and reporter. Here we demonstrate that marked polarisation in GFP fluorescence, upon excitation with plane polarised light, can be exploited to enhance the detection of GFP when in the presence of cellular auto-fluorescence and other fluorescent compounds that emit at the same wavelength. The development of flow-though instrumentation dedicated to the sensitive detection of GFP by fluorescence polarisation is described, and used in both a continuous flow and flow-injection format. The intensity, spectral properties and extent of polarisation in the auto-fluorescence of various yeast strains and growth media are investigated. The application of fluorescence polarisation is shown to enhance the measurement of the induction of GFP in yeast cells, genetically modified to produce GFP as an indicator of DNA damage, compared with a conventional fluorescence method.

Engineering Secondary Metabolism in Maize Cells by Ectopic Expression of Transcription Factors

Manipulation of plant natural product biosynthesis through genetic engineering is an attractive but technically challenging goal. Here, we demonstrate that different secondary metabolites can be produced in cultured maize cells by ectopic expression of the appropriate regulatory genes. Cell lines engineered to express the maize transcriptional activators C1 and R accumulate two cyanidin derivatives, which are similar to the predominant anthocyanin found in differentiated plant tissues. In contrast, cell lines that express P accumulate various 3-deoxy flavonoids. Unexpectedly, P-expressing cells in culture also accumulate phenylpropanoids and green fluorescent compounds that are targeted to different subcellular compartments. Two endogenous biosynthetic genes (c2 and a1, encoding chalcone synthase and flavanone/dihydroflavonol reductase, respectively) are independently activated by ectopic expression of either P or C1/R, and there is a dose-response relationship between the transcript level of P and the degree to which c2 or a1 is expressed. Our results support a simple model showing how the gene encoding P may act as a quantitative trait locus controlling insecticidal C-glycosyl flavone level in maize silks, and they suggest how p1 might confer a selective advantage against insect predation in maize.

On the fluorescence and photoreduction of methyl viologens

The fluorescence of methyl viologen (MV2+) has been reinvestigated. The green emission has been attributed to impurities whose formation from MV2+ can be induced thermally or photochemically. One of the green fluorescent compounds has been found to act as an efficient photosensitizer for the reduction of MV2+ to MV+. In contrast to MV2+, the 1,1′,2,2′,6,6′-hexamethyl-4,4′-bipyridinium dication was found to be fluorescent. The fluorescence properties of 2,2′,6,6′-tetramethyl-4,4′-bipyridine and its protonated cations are also reported. The values of *pKa(S1) of a 4,4′-bipyridine have been obtained for the first time.

Isolation of 4-(1'-D-ribitylamino)-5-amino-2,6-dihydroxypyrimidine from a riboflavin-adenine-deficient mutant of Bacillus subtilis.

Studies were carried out to determine possible intermediates involved in the biosynthetic pathway of riboflavin, using resting cells of a riboflavin-adenine-deficient mutant, Bacillus subtilis AJ1988. The cells excreted 6,7-dimethyl-8-ribityllumazine, the end product in the biosynthetic pathway, into the incubation medium in large amounts. The addition of glyoxal caused a large accumulation of a green fluorescent compound; an inverse relation was observed between the formation of the lumazine and the concentration of glyoxal. Furthermore, added [2-14C]guanine effectively incorporated into the lumazine and the fluorescent compound in the same specific activity during incubation. The fluorescent compound was isolated, purified, and identified by paper chromatographic, fluorometric, and spectrophotometric analyses. It was proved to be 8-(1'-D-ribityl)lumazine, which appeared to have been formed by a reaction between glyoxal and a possible intermediate in the cells. Accordingly, 4-(1'-D-ribitylamino)-5-amino-2,6-dihydroxypyrimidine was concluded to be an immediate precursor of 6,7-dimethyl-8-ribityllumazine.

Isolation and identification of green fluorescent compound accumulated in non-growing cells of Eremothecium ashbyii by the addition of glyoxal.

The addition of a trapping agent, glyoxal, to a non-growing cell medium with Eremothecium ashbyii brought about the accumulation of large quantities of a green fluorescent compound, with a simultaneous rigid inhibition of riboflavin formation. The fluorescent compound was isolated from the mycelia after non-growing cell incubation and highly purified to a crystalline form through various column chromatographic steps. The purified compound was identified as 8-ribityllumazine by comparison with a synthetized reference compound by means of spectrophotometric and fluorometric measurements. Furthermore, it was verified that glyoxal, at the added concentration, and 8-ribityllumazine, at the accumulated concentration, caused slight inhibition of riboflavin formation with riboflavin synthetase from E. ashbyii. Accordingly, it was concluded that the 8-ribityllumazine accumulated is a derivative compound of an intermediate in flavinogenesis which is 4-ribitylamino-5-amino-2,6-dihydroxypyrimidine based on the trapping action of glyoxal.

Examination of the structure of an unknown green fluorescent compound, compound G2, accumulated in non-growing cells of Eremothecium ashbyii by the addition of dimeric diacetyl.

The addition of dimeric diacetyl to Eremothecium ashbyii caused simultaneous accumulation of two green fluorescent compounds with the inhibition of riboflavin formation in non-growing cells. One compound, referred to as Compound G1, was identified as 6, 7-dimethyl-8-ribityllumazine as reported previously and the other is referred to as Compound G2. The latter compound was considered to be 6-methyl-7-(2-hydroxy-2-methyl-3-oxobutyl)-8-ribityllumazine because a violet fluorescent compound, 6-methyl-7-hydroxy-8-ribityllumazine was derived from Compound G2 in the presence of p-quinone and because the known action mechanism of dimeric diacetyl as a trapping agent of possible intermediates in the biosynthetic pathway. The results indicate that an immediate intermediate to 6, 7-dimethyl-8-ribityllumazine in the biosynthetic pathway of riboflavin is 4-ribitylamino-5-amino-2, 6-dihydroxypyrimidine, whose pyrimidine portion is derived by the elimination of a dimeric diacetyl fragment from the molecule of the isolated lumazine.

Studies on the intermediates in the biosynthetic pathway of riboflavin. I. Identification of a green fluorescent compound, compound G1, accumulated in non-growing cells of Eremothecium ashbyii by the addition of dimeric diacetyl.

Effects of sugars and carbonyl compounds (1%) on ribo-flavin formation were examined with non-growing cells of Eremothecium ashbyii. During non-growing cell incubation for 18hr, the addition of glucose markedly stimulated the formation of riboflavin, but the addition of pentoses (ribose, xylose, arabinose), ribitol and acetoin indicated no effect on the production. The supplementation of pyruvate, acetate, di-acetyl, glyoxal and acetoacetate, on the other hand, noticeably inhibited the formation of riboflavin. The effects of diacetyl monomer, dieter and trimer on the formation of riboflavin were further examined. Dimeric diacetyl (0.5%) among the diacetyl derivatives caused the accumulation of two green fluorescent compounds inside and outside of the cells during the 18hr incubation. The fraction (Compound G1), which was first eluted in Dowex 50W×4 column chromatography, was accumulated in large amounts indicating an inverse relation to riboflavin formation. Compound G1 was isolated from the mycelia and highly purified through many purification procedures. The structure of compound G1 was studied by paper chromatographic comparison, by determination of ultraviolet, emission, excitation and infrared spectra, by following the change of the maximum fluorescence of the reaction mixture containing the isolated compound in the presence of p-quinone and by examining the possibility of it as a substrate for riboflavin synthetase. The results obtained indi-cated that the isolated compound G1 is 6, 7-dimethyl-8-(1'-D-ribityl) lumazine. We speculated that dimeric diacetyl or its derivative becomes a significant inhibitor for the riboflavin synthetase reaction to result in the accumu-lation of 6, 7-dimethyl-8-ribityllumazine although the true inhibitor remains to be solved in future.

Identification of the second product of the riboflavin synthetase reaction.

This study was concerned with the detailed identification of the second product involved in the riboflavin synthetase reaction with riboflavin synthetase from Eremothecium ashbyii and a trapping agent, glyoxal.Thus, a green fluorescent compound accumulated during the incubation. The compound was purified through various column chromatography steps, and was examined by UV, IR, excitation and emission spectra and paper chromatography to prove that the isolated compound was 8-ribityllumazine. Accordingly it was concluded that a second product in riboflavin synthetase reaction was 4-ribitylamino-5-amino-2,6-dihydroxypyrimidine, the fragment, except for C-6 and C-7 of 8-ribityllumazine, being an incorporated glyoxal portion.

Isolation of 4-ribitylamino-5-amino-2,6-dihydroxypyrimidine from a high flavinogenic mold Eremothecium ashbyii1.

The addition of glyoxal, a trapping agent, caused simultaneously the accumulation of a green fluorescent compound and the inhibition of riboflavin formation in non-growing cells of Eremothecium ashbyii. The fluorescent compound purified was identified as 8-ribityllumazine from the results of spectrophotometric and fluorometric analyses. Accordingly, the fragment, except for the glyoxal portion on the 8-ribityllumazine molecule, 4-ribitylamino-5-amino-2,6-dihydroxypyrimidine, is believed to be an intermediate in the riboflavin biosynthetic pathway.

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One hundred, and twenty six strains of the genus Aspergillus were examined for their productivity of aflatoxins in surface cultures. Most of the strains commonly produced a bluish green fluorescent compound similar to aflatoxin G in fluorescent color and Rf value on TLC under some experimental conditions. This compound was isolated from the culture filtrate of Aspergillus oniki 1784 and proved to be lumichrome.