RESUMO
Small-molecule fluorophores, such as fluorescein and rhodamine derivatives, are critical tools in modern biochemical and biological research. The field of chemical dyes is old; colored molecules were first discovered in the 1800s, and the fluorescein and rhodamine scaffolds have been known for over a century. Nevertheless, there has been a renaissance in using these dyes to create tools for biochemistry and biology. The application of modern chemistry, biochemistry, molecular genetics, and optical physics to these old structures enables and drives the development of novel, sophisticated fluorescent dyes. This critical review focuses on an important example of chemical biology-the melding of old and new chemical knowledge-leading to useful molecules for advanced biochemical and biological experiments.
Assuntos
Fluoresceínas/síntese química , Corantes Fluorescentes/síntese química , Sondas Moleculares/síntese química , Marcadores de Fotoafinidade/síntese química , Rodaminas/síntese química , Animais , Bactérias/metabolismo , Técnicas de Química Sintética , Fluoresceínas/história , Fluoresceínas/metabolismo , Corantes Fluorescentes/história , Corantes Fluorescentes/metabolismo , História do Século XIX , História do Século XX , História do Século XXI , Humanos , Microscopia de Fluorescência , Sondas Moleculares/história , Sondas Moleculares/metabolismo , Marcadores de Fotoafinidade/história , Marcadores de Fotoafinidade/metabolismo , Rodaminas/história , Rodaminas/metabolismoRESUMO
We present the design, synthesis, and applications of a new class of voltage-sensitive fluorescent indicators built on a modified carbofluorescein scaffold. Carbofluoresceins are an attractive target for responsive probes because they maintain oxygen substitution patterns at the 3' and 6' positions, similar to fluorescein, while simultaneously possessing excitation and emission profiles red-shifted nearly 50 nm compared to fluorescein. However, the high p Ka of carbofluorescein dyes, coupled with their tendency to cyclize to nonfluorescent configurations, precludes their use in voltage-imaging applications. Here, we overcome the limitations of carbofluoresceins via chlorination to lower the p Ka by 2 units to 5.2 and sulfonation to prevent cyclization to the nonabsorbing form. To achieve this, we devise a synthetic route to halogenated sulfonated carbofluoresceins from readily available, inexpensive starting materials. New, chlorinated sulfone carbofluoresceins have low p Ka values (5.2) and can be incorporated into phenylenevinylene molecular wire scaffolds to create carboVoltage-sensitive fluorophores (carboVF dyes). The best of the new carboVF dyes, carboVF2.1(OMe).Cl, possesses excitation and emission profiles of >560 nm, displays high voltage sensitivity (>30% Δ F/ F per 100 mV), and can be used in the presence of other blue-excited fluorophores such as green fluorescent protein. Because carboVF2.1(OMe).Cl contains a phenolic oxygen, it can be incorporated into fluorogenic labeling strategies. Alkylation with a sterically bulky cyclopropylmethyl-derived acetoxymethyl ether renders carboVF weakly fluorescent; we show that fluorescence can be restored by the action of porcine liver esterase both in vitro and on the surface of living cells and neurons. Together, these results suggest chlorinated sulfone carbofluoresceins can be promising candidates for hybrid chemical-genetic voltage imaging at wavelengths beyond typical fluorescein excitation and emission.
Assuntos
Fenômenos Eletrofisiológicos , Fluoresceínas/química , Fluoresceínas/síntese química , Imagem Molecular/métodos , Ácidos Sulfônicos/química , Técnicas de Química Sintética , Fluoresceínas/metabolismo , Células HEK293 , HumanosRESUMO
Methods for optical measurement of voltage dynamics in living cells are attractive because they provide spatial resolution surpassing traditional electrode-based measurements and temporal resolution exceeding that of widely used Ca2+ imaging. Chemically synthesized voltage-sensitive dyes that use photoinduced electron transfer as a voltage-sensing trigger offer high voltage sensitivity and fast-response kinetics, but targeting chemical indicators to specific cells remains an outstanding challenge. Here, we present a new family of readily functionalizable, fluorescein-based voltage-sensitive fluorescent dyes (sarcosine-VoltageFluors) that can be covalently attached to a genetically encoded cell surface receptor to achieve voltage imaging from genetically defined neurons. We synthesized four new VoltageFluor derivatives that possess carboxylic acid functionality for simple conjugation to flexible tethers. The best of this new group of dyes was conjugated via a polyethylene glycol (PEG) linker to a small peptide (SpyTag, 13 amino acids) that directs binding and formation of a covalent bond with its binding partner, SpyCatcher (15 kDa). The new VoltageSpy dyes effectively label cells expressing cell-surface SpyCatcher, display good voltage sensitivity, and maintain fast-response kinetics. In cultured neurons, VoltageSpy dyes enable robust, single-trial optical detection of action potentials at neuronal soma with sensitivity exceeding genetically encoded voltage indicators. Importantly, genetic targeting of chemically synthesized dyes enables VoltageSpy to report on action potentials in axons and dendrites in single trials, tens to hundreds of micrometers away from the cell body. Genetic targeting of synthetic voltage indicators with VoltageSpy enables voltage imaging with low nanomolar dye concentration and offers a promising method for allying the speed and sensitivity of synthetic indicators with the enhanced cellular resolution of genetically encoded probes.
Assuntos
Membrana Celular/metabolismo , Fluoresceínas/química , Corantes Fluorescentes/química , Potenciais da Membrana/fisiologia , Neurônios/metabolismo , Potenciais de Ação/fisiologia , Membrana Celular/química , Fluoresceínas/síntese química , Corantes Fluorescentes/síntese química , Marcação de Genes , Células HEK293 , Humanos , Peptídeos/síntese química , Peptídeos/química , Peptídeos/genética , Sarcosina/análogos & derivados , Sarcosina/síntese químicaRESUMO
In this article, we designed an amphiphilic lipopeptide molecule, 5(6)-carboxyfluorescein-KKKKKKSKTK-Cys(C12H25)-OMe (FAM-lipopeptide-C12), and studied its assembly behavior at the 4-cyano-4'-pentylbiphenyl (5CB)-aqueous interface. The ordering transitions of liquid crystals (LCs) revealed that FAM-lipopeptide-C12 can assemble at the LC-aqueous interface (both planar and curved interfaces). The assembly can be destroyed by adding trypsin, which catalyzes the hydrolysis of lipopeptides. Fluorescence measurements further confirmed the assembly and deassembly behavior of FAM-lipopeptide-C12 at the LC-aqueous interface. Overall, our work provides a general method for the construction of a biointerface by directly assembling amphiphilic lipopeptides at the LC-aqueous interface, which can potentially be used in selectively detecting the activity of specific enzymes and other biomolecular interactions.
Assuntos
Fluoresceínas/química , Lipopeptídeos/química , Cristais Líquidos/química , Tensoativos/química , Compostos de Bifenilo/química , Fluoresceínas/síntese química , Hidrólise , Lipopeptídeos/síntese química , Nitrilas/química , Tensoativos/síntese química , Tripsina/química , Água/químicaRESUMO
Cancer is a global health issue and a leading cause of death. The discrimination of cancer cells from normal cells is of significant importance for the early diagnosis of cancers. As one of the useful biomarkers for developing cancer diagnosis and chemotherapy resistance systems, biothiols not only play an essential role in physiological and pathological processes but also exhibit cytoprotective effects in the susceptibility to carcinogenesis. It would be highly desirable to explore near-infrared biothiol-specific fluorescent probes for cancer diagnosis with outstanding specificity. In this study, a novel near-infrared fluorescent probe BPO-THAZ decorated with thiazole as a recognition site was presented for sensitive and selective detection of endogenous biothiols. BPO-THAZ can be used to not only evaluate the biothiol level in living HeLa cells upon treatment with H2O2 or anti-cancer drugs but also assess endogenous biothiols in stem cells. Furthermore, BPO-THAZ was successfully utilized to discriminate cancer cells from normal cells showing great promise for cancer diagnosis.
Assuntos
Corantes Fluorescentes/química , Compostos de Sulfidrila/análise , Tiazóis/química , Animais , Biomarcadores Tumorais/análise , Linhagem Celular Tumoral , Células-Tronco Embrionárias , Fluoresceínas/síntese química , Fluoresceínas/química , Fluoresceínas/toxicidade , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/toxicidade , Humanos , Limite de Detecção , Camundongos , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Células NIH 3T3 , Células-Tronco Pluripotentes , Tiazóis/síntese química , Tiazóis/toxicidadeRESUMO
The superoxide anion (O2Ë-) plays a crucial role in several physiological processes and many human diseases. Developing new methods for O2Ë- detection in biological systems is very important. A FRET-based two-photon (TP) fluorescent probe with a ratiometric signal, TFR-O, was developed. A naphthalene derivative based TP fluorescent group was selected as the energy donor group, and a rhodol fluorescent group was chosen as the energy acceptor; the trifluoromethanesulfonate group was chosen as the recognition moiety. After reacting with O2Ë-, the recognition moiety was removed and the fluorophore was released, leading to a fluorescence intensity decrease at the wavelength of 425 nm and a significant enhancement of the fluorescence intensity at 550 nm. The fluorescence intensity ratio between 550 and 425 nm (I550/I425) varied from 0.15 to 6.72, with the O2Ë- concentration increasing from 0 to 50 µM. The detection limit of the TFR-O was 83 nM. Moreover, TFR-O was applied for detecting and imaging O2Ë- in cells and liver tissues.
Assuntos
Fluoresceínas/química , Corantes Fluorescentes/química , Mesilatos/química , Naftalenos/química , Superóxidos/análise , Animais , Fluoresceínas/síntese química , Fluoresceínas/efeitos da radiação , Fluoresceínas/toxicidade , Fluorescência , Transferência Ressonante de Energia de Fluorescência/métodos , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/efeitos da radiação , Corantes Fluorescentes/toxicidade , Limite de Detecção , Fígado/metabolismo , Mesilatos/síntese química , Mesilatos/efeitos da radiação , Mesilatos/toxicidade , Camundongos , Naftalenos/síntese química , Naftalenos/efeitos da radiação , Naftalenos/toxicidade , Fótons , Células RAW 264.7 , Superóxidos/metabolismoRESUMO
Dying cell clearance is critical for myriad biological processes such as tissue homeostasis. We herein report an enzyme-activated fluorescence cell labeling approach and its use for multicolor imaging of dying cell clearance. Diacetylated 4-hydroxymandelic acid (DHA)-conjugated dyes give rise to reactive quinone methides upon deacetylation in live cells, which in turn covalently labels cellular proteins. With partner cells tagged with distinct fluorescence, apoptotic cell clearance by Raw 264.7 macrophages and epithelial HeLa cells was captured by confocal microscopy, showing the potential of DHA-based cell labeling for investigating cell-cell interactions.
Assuntos
Apoptose , Corantes Fluorescentes/química , Ácidos Mandélicos/química , Necrose , Animais , Bovinos , Linhagem Celular Tumoral , Cumarínicos/síntese química , Cumarínicos/química , Cumarínicos/toxicidade , Esterases/química , Fluoresceínas/síntese química , Fluoresceínas/química , Fluoresceínas/toxicidade , Fluorescência , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/toxicidade , Humanos , Ácidos Mandélicos/síntese química , Ácidos Mandélicos/toxicidade , Camundongos , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Estudo de Prova de Conceito , Células RAW 264.7 , Rodaminas/síntese química , Rodaminas/química , Rodaminas/toxicidade , Coloração e Rotulagem/métodos , SuínosRESUMO
Fluorescein diester which is conjugated with cell membrane permeable Arg9 peptide was proposed as probe for ester prodrug stability and drug release study in living cells. α-Amino protected d-Val and l-Ala which bear differently hindered side chains were used to afford model diesters of 5-maleimide-fluorescein. Such fluorescein diesters were further conjugated with a Cys containing cell membrane permeable Arg9 peptide via thiol-ene crosslink reaction. The resulted conjugates of fluorescein diester and Arg9 peptide were purified with HPLC and characterized with MALDI-TOF MS. Upon incubation with cultured cells, the fluorescein diesters were delivered into the cells, the following hydrolysis of fluorescein diesters and release of fluorescein inside living cells were observed by monitoring the fluorescence accumulation. Fluorescence microscopic imaging studies of HeLa cells treated with fluorescein l-Ala diester show strong fluorescence accumulation in 30â¯min indicating fast hydrolysis of fluorescein diester and fluorescein release; in contrast d-Val diester remains stable inside cells evidenced by margin fluorescence formation. Further flowcytometry studies on the fluorescein diester-Arg9 conjugate treated cells show that the hydrolysis t1/2 for l-Ala diester is 15â¯min. The results also show that Arg9 peptide not only transports the ester probes into cell efficiently but also can retain and concentrate hydrolytic product fluorescein inside cells so that the accumulated fluorescence can be accurately quantified. This fluorogenic probe approach provides feasible applications in dynamic studies on ester prodrug hydrolysis and release, facilitating screening and optimization of prodrug structures in living cell settings.
Assuntos
Peptídeos Penetradores de Células/química , Ésteres/química , Fluoresceínas/química , Corantes Fluorescentes/química , Pró-Fármacos/química , Peptídeos Penetradores de Células/síntese química , Peptídeos Penetradores de Células/metabolismo , Liberação Controlada de Fármacos , Ésteres/síntese química , Ésteres/metabolismo , Citometria de Fluxo , Fluoresceínas/síntese química , Fluoresceínas/metabolismo , Fluorescência , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/metabolismo , Células HeLa , Humanos , Hidrólise , Microscopia de Fluorescência , Pró-Fármacos/síntese química , Pró-Fármacos/metabolismo , Estudo de Prova de ConceitoRESUMO
Light-driven phase change materials (PCMs) have received significant attention due to their capacity to convert visible light into thermal energy, storing it as latent heat. However, continuous photo-thermal conversion can cause the PCMs to reach high thermal equilibrium temperatures after phase transition. In our study, a novel light-driven phase change material system with temperature-control properties was constructed using a thermochromic compound. Thermochromic phase change materials (TC-PCMs) were prepared by introducing 2-anilino-6-dibutylamino-3-methylfluoran (ODB-2) and bisphenol A (BPA) into 1-hexadecanol (1-HD) in various proportions. Photo-thermal conversion performance was investigated with solar radiation (low power of 0.09 W/cm2) and a xenon lamp (at a high power of 0.14 W/cm2). The TC-PCMs showed a low equilibrium temperature due to variations in absorbance. Specifically, the temperature of TC-PCM180 (ODB-2, bisphenol A and 1-HD ratio 1:2:180) could stabilize at 54 °C approximately. TC-PCMs exhibited reversibility and repeatability after 20 irradiation and cooling cycles.
Assuntos
Compostos de Anilina/síntese química , Compostos Benzidrílicos/síntese química , Álcoois Graxos/síntese química , Fluoresceínas/síntese química , Fenóis/síntese química , Compostos de Anilina/química , Compostos de Anilina/efeitos da radiação , Compostos Benzidrílicos/química , Compostos Benzidrílicos/efeitos da radiação , Álcoois Graxos/química , Álcoois Graxos/efeitos da radiação , Fluoresceínas/química , Fluoresceínas/efeitos da radiação , Temperatura Alta , Luz , Transição de Fase/efeitos da radiação , Fenóis/química , Fenóis/efeitos da radiação , TemperaturaRESUMO
Methods to detect low concentrations of small molecules are useful for a wide range of analytical problems including the development of clinical assays, the study of complex biological systems, and the detection of biological warfare agents. This paper describes a semisynthetic ion channel platform capable of detecting small molecule analytes with picomolar sensitivity. Our methodology exploits the transient nature of ion channels formed from gramicidin A (gA) nanopores and the frequency of observed single channel events as a function of concentration of free gA molecules that reversibly dimerize in a bilayer membrane. We initially use a protein (here, a monoclonal antibody) to sequester the ion channel activity of a C-terminally modified gA derivative. When a small molecule analyte is introduced to the electrical recording medium, it competitively binds to the protein and liberates the gA derivative, restoring its single ion channel activity. We found that monitoring the frequency of gA channel events makes it possible to detect picomolar concentrations of small molecule in solution. In part, due to the digital on/off nature of frequency-based analysis, this approach is 103 times more sensitive than measuring macroscopic membrane ion flux through gA channels as a basis for detection. This novel methodology, therefore, significantly improves the limit of detection of nanopore-based sensors for small molecule analytes, which has the potential for incorporation into miniaturized and low cost devices that could complement current established assays.
Assuntos
Técnicas Biossensoriais/métodos , Fluoresceínas/análise , Gramicidina/metabolismo , Canais Iônicos/metabolismo , Bicamadas Lipídicas/metabolismo , Anticorpos Monoclonais/imunologia , Fluoresceínas/síntese química , Fluoresceínas/química , Gramicidina/análogos & derivados , Gramicidina/síntese química , Haptenos/imunologia , Limite de Detecção , Bicamadas Lipídicas/química , NanoporosRESUMO
The tyrosine side chain is amphiphilic leading to significant variations in the surface exposure of tyrosine residues in the folded structure of a native sequence protein. This variability can be exploited to give residue-selective functionalization of a protein substrate by using a highly reactive diazonium group tethered to an agarose-based resin. This novel catch-and-release approach to protein modification has been demonstrated for proteins with accessible tyrosine residues, which are compared with a control group of proteins in which there are no accessible tyrosine residues. MS analysis of the modified proteins showed that functionalization was highly selective, but reactivity was further attenuated by the electrostatic environment of any individual residue. Automated screening of PDB structures allows identification of potential candidates for selective modification by comparison with the accessibility of the tyrosine residue in a benchmark peptide (GYG).
Assuntos
Proteínas/química , Tirosina/química , Sequência de Aminoácidos , Aminofenóis/síntese química , Aminofenóis/química , Compostos de Diazônio/química , Ferricianetos/química , Fluoresceínas/síntese química , Fluoresceínas/química , Oligopeptídeos/químicaRESUMO
The design, synthesis, and photophysical properties of organic fluorophores have attracted considerable research interest due to the utility of these compounds for various optoelectronic, analytical, and biological applications. In this study, we synthesized two novel π-extended red-emitting hybrid xanthene dyes, each of which has two spirolactone rings and combines a seminaphthofluorescein moiety and a seminaphthorhodafluor moiety in a single molecule. The photophysical properties of the dyes in methanol in the presence of acid, base, and metal cations were investigated. Mono-ring-opened seminaphthofluorescein and seminaphthorhodafluor forms of the dyes could be obtained by the addition of OH- or H+, respectively. Owing to the changes in the absorbance spectra of the mono-ring-opened forms induced by addition of H+ and OH-, the dyes could perform simultaneously the functions of an XOR gate and an INHIBIT gate, with the absorbances at 510 and 560 nm as outputs, respectively, and could act as half-subtractors with H+ and OH- as inputs. Furthermore, stepwise ring-opening could be induced by Hg2+ ions in methanol. In water, the dyes existed in double-ring-opened forms that emitted deep-red fluorescence and were mitochondria-targetable, suggesting that these chromophores might be useful as fluorescence tracers in biological applications. Because the absorption and fluorescence properties of these fluorophores can be regulated via their two spirolactone rings, we expect that these compounds will find utility in various optoelectronic, analytical, and biological applications.
Assuntos
Fluoresceínas/química , Corantes Fluorescentes/química , Espironolactona/química , Xantenos/química , Fluoresceínas/síntese química , Corantes Fluorescentes/síntese química , Células HeLa , Humanos , Imagem Óptica/métodos , Espectrometria de Fluorescência/métodos , Espironolactona/síntese química , Água/química , Xantenos/síntese químicaRESUMO
To examine the hydrophobic structure of PI polyamides on tumor accumulation in vivo, PI polyamide-fluorescein conjugates 1-5 with the distinct number of N-methylimidazole (Im) units were synthesized. There existed an inverse relationship between the Im unit number of the compounds and their hydrophobicity. Compound 1 with one Im unit and 3 with three Im units accumulated and retained preferentially in tumor tissues compared to 5 with five Im units. These results suggest the importance of a PI polyamide's primary structure, which partly contributes to its hydrophobic property, on its accumulation and/or retention in tumor tissues in vivo.
Assuntos
Imidazóis/metabolismo , Neoplasias/metabolismo , Nylons/metabolismo , Pirróis/metabolismo , Animais , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Feminino , Fluoresceínas/síntese química , Fluoresceínas/química , Fluoresceínas/metabolismo , Fluorescência , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Imidazóis/síntese química , Imidazóis/química , Camundongos Endogâmicos BALB C , Estrutura Molecular , Nylons/síntese química , Nylons/química , Pirróis/síntese química , Pirróis/química , Distribuição TecidualRESUMO
Oxidative stress, a state in which intra- or extracellular oxidant production outweighs the antioxidative capacity, lies at the basis of many diseases. DCFH2-DA (2',7'-dichlorodihydrofluorescein diacetate) is the most widely used fluorogenic probe for the detection of general oxidative stress. However, the use of DCFH2-DA, as many other fluorogenic redox probes, is mainly confined to the detection of intracellular oxidative stress in vitro. To expand the applicability of the probe, an alkaline hydrolysis and solvent extraction procedure was developed to generate high-purity DCFH2 (2',7'-dichlorodihydrofluorescein) from DCFH2-DA using basic laboratory equipment. Next, the utility of DCFH2 was exemplified in a variety of cell-free and in vitro redox assay systems, including oxidant production by transition metals, photodynamic therapy, activated macrophages, and platelets, as well as the antioxidative capacity of different antioxidants. In cells, the concomitant use of DCFH2-DA and DCFH2 enabled the measurement and compartmentalized analysis of intra- and extracellularly produced oxidants, respectively, using a single read-out parameter. Furthermore, hepatocyte-targeted liposomes were developed to deliver the carboxylated derivative, 5(6)-carboxy-DCFH2, to hepatocytes in vivo. Liposome-delivered 5(6)-carboxy-DCFH2 enabled real-time visualization and measurement of hepatocellular oxidant production during liver ischemia-reperfusion. The liposomal 5(6)-carboxy-DCFH2 can be targeted to other tissues where oxidative stress is important, including cancer.
Assuntos
Fluoresceínas/síntese química , Acetilação , Fluoresceínas/química , Fluoresceínas/isolamento & purificação , Estrutura Molecular , OxirreduçãoRESUMO
Two fluorescent galactofuranosides were synthesized and their biological activities evaluated on non-infected and Leishmania infected macrophages. Both tagged scaffolds were able to penetrate macrophages. Compared to the activity of the parent octyl galactofuranoside used as a reference, the fluorescein-conjugate showed altered biological properties while the rhodamine 6G one synergistically acted with the lipid chain to significantly increase antiparasitic activity.
Assuntos
Antiprotozoários/farmacologia , Fluoresceínas/farmacologia , Corantes Fluorescentes/farmacologia , Galactosídeos/farmacologia , Rodaminas/farmacologia , Antiprotozoários/síntese química , Antiprotozoários/toxicidade , Fluoresceínas/síntese química , Fluoresceínas/toxicidade , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/toxicidade , Galactosídeos/síntese química , Galactosídeos/toxicidade , Humanos , Leishmania donovani/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/parasitologia , Rodaminas/síntese química , Rodaminas/toxicidadeRESUMO
Mossy fiber termini in the hippocampus accumulate Zn(2+), which is released with glutamate from synaptic vesicles upon neural excitation. Understanding the spatiotemporal regulation of mobile Zn(2+) at the synaptic level is challenging owing to the difficulty of visualizing Zn(2+) at individual synapses. Here we describe the use of zinc-responsive fluorescent probes together with two-photon microscopy to image Zn(2+) dynamics mediated by NMDA receptor-dependent long-term potentiation induction at single mossy fiber termini of dentate gyrus neurons in adult mouse hippocampal slices. The membrane-impermeant fluorescent Zn(2+) probe, 6-CO2H-ZAP4, was loaded into presynaptic vesicles in hippocampal mossy fiber termini upon KCl-induced depolarization, which triggers subsequent endocytosis and vesicular restoration. Local tetanic stimulation decreased the Zn(2+) signal observed at individual presynaptic sites, indicating release of the Zn(2+) from vesicles in synaptic potentiation. This synapse-level two-photon Zn(2+) imaging method enables monitoring of presynaptic Zn(2+) dynamics for improving the understanding of physiological roles of mobile Zn(2+) in regular and aberrant neurologic function.
Assuntos
Neuroimagem Funcional/métodos , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Fibras Musgosas Hipocampais/metabolismo , Zinco/metabolismo , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Transporte de Cátions , Fluoresceínas/síntese química , Fluoresceínas/química , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/química , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras , Camundongos , Camundongos Knockout , Estrutura Molecular , Processos FotoquímicosRESUMO
Human G protein-coupled receptor 40 (hGPR40), with medium- and long-chain free fatty acids (FFAs) as its natural ligands, plays an important role in the enhancement of glucose-dependent insulin secretion. To date, information about the direct binding of FFAs to hGPR40 is very limited, and how carbon-chain length affects the activities of FFAs on hGPR40 is not yet understood. In this study, a fluorescein-fasiglifam analogue (F-TAK-875A) conjugate was designed and synthesized as a site-specific fluorescence probe to study the interaction of FFAs with hGPR40. hGPR40 was expressed in human embryonic kidney 293 cells and labeled with F-TAK-875A. By using flow cytometry, competitive binding of FFA and F-TAK-875A to hGPR40-expressed cells was measured. Binding affinities of 18 saturated FFAs, with carbon-chain lengths ranging from C6 to C23, were analyzed. The results showed that the binding potencies of FFAs to hGPR40 were dependent on carbon length. There was a positive correlation between length and binding potency for seven FFAs (C9-C15), with myristic acid (C15) showing the highest potency, 0.2% relative to TAK-875. For FFAs with a length of fewer than C9 or more than C15, they had very weak or no binding. Molecular docking results showed that the binding pocket of TAK-875 in hGPR40 could enclose FFAs with lengths of C15 or fewer. However, for FFAs with lengths longer than C15, part of the alkyl chain extended out of the binding pocket. This study provided insights into the structural dependence of FFAs binding to and activation of hGPR40.
Assuntos
Benzofuranos/metabolismo , Desenho de Fármacos , Ácidos Graxos não Esterificados/metabolismo , Fluoresceínas/metabolismo , Corantes Fluorescentes/metabolismo , Modelos Moleculares , Receptores Acoplados a Proteínas G/agonistas , Benzofuranos/síntese química , Benzofuranos/química , Sítios de Ligação , Ligação Competitiva , Ácidos Graxos não Esterificados/química , Citometria de Fluxo , Fluoresceína/química , Fluoresceína/metabolismo , Fluoresceínas/síntese química , Fluoresceínas/química , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Ligantes , Metilaminas/química , Metilaminas/metabolismo , Simulação de Acoplamento Molecular , Peso Molecular , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Propionatos/química , Propionatos/metabolismo , Conformação Proteica , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Sulfonas/química , Sulfonas/metabolismoRESUMO
Carbon monoxide (CO) is an important gasotransmitter in living systems and its fluorescent detection is of particular interest. However, fluorescent detection of CO in living cells is still challenging due to lack of effective probes. In this paper, a readily available fluorescein-based fluorescent probe was developed for rapid detection of CO. This probe can be used to detect CO in almost wholly aqueous solution under mild conditions and shows high selectivity and sensitivity for CO with colorimetric and remarkable fluorescent turn-on signal changes. The detection limit of this probe for CO is as low as 37 nM with a linear range of 0-30 µM. More importantly, this probe (1 µM dose) can be conveniently used for fluorescent imaging CO in living cells.
Assuntos
Monóxido de Carbono/análise , Monóxido de Carbono/metabolismo , Fluoresceínas/farmacologia , Corantes Fluorescentes/farmacologia , Microscopia de Fluorescência/métodos , Células A549 , Fluoresceínas/síntese química , Fluoresceínas/toxicidade , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/toxicidade , Humanos , Limite de Detecção , Compostos Organometálicos/química , Sensibilidade e EspecificidadeRESUMO
Two photo-crosslinking biarsenical (CrAsH-EDT2 )-modified probes were synthesized that are expected to be useful tools for tetracysteine-labeled proteins to facilitate the co-affinity purification of their DNA binding sequences and interacting proteins. In addition, improvements for the synthesis of CrAsH-EDT2 and N(1) -(4-azido-2-nitrophenyl)hexane-1,6-diamine are reported. Both photoprobes effectively entered HeLa cells (and the nucleus) and were dependent on the tetracysteine motif in recombinant DMRT1 (doublesex and Mab3-related transcription factor) to induce fluorescence, suggesting that their crosslinking abilities can be exploited for the identification of nucleic acids and proteins associated with a protein of interest.
Assuntos
Arsênio , Arsenicais/química , Azidas/química , Reagentes de Ligações Cruzadas/química , Diaminas/química , Diazometano/análogos & derivados , Diazometano/química , Fluoresceínas/química , Mercaptoetanol/análogos & derivados , Marcadores de Fotoafinidade/química , Arsenicais/síntese química , Azidas/síntese química , Diaminas/síntese química , Diazometano/síntese química , Fluoresceínas/síntese química , Células HeLa , Humanos , Mercaptoetanol/química , Marcadores de Fotoafinidade/síntese química , Relação Estrutura-Atividade , Fatores de Transcrição/metabolismoRESUMO
Hydrogen sulfide (H2S) is a critical gaseous signaling molecule emerging at the center of a rich field of chemical and biological research. As our understanding of the complexity of physiological H2S in signaling pathways evolves, advanced chemical and technological investigative tools are required to make sense of this interconnectivity. Toward this goal, we have developed an azide-functionalized O-methylrhodol fluorophore, MeRho-Az, which exhibits a rapid >1000-fold fluorescence response when treated with H2S, is selective for H2S over other biological analytes, and has a detection limit of 86 nM. Additionally, the MeRho-Az scaffold is less susceptible to photoactivation than other commonly used azide-based systems, increasing its potential application in imaging experiments. To demonstrate the efficacy of this probe for H2S detection, we demonstrate the ability of MeRho-Az to detect differences in H2S levels in C6 cells and those treated with AOAA, a common inhibitor of enzymatic H2S synthesis. Expanding the use of MeRho-Az to complex and heterogeneous biological settings, we used MeRho-Az in combination with light sheet fluorescence microscopy (LSFM) to visualize H2S in the intestinal tract of live zebrafish. This application provides the first demonstration of analyte-responsive 3D imaging with LSFM, highlighting the utility of combining new probes and live imaging methods for investigating chemical signaling in complex multicellular systems.