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An effective, GFP-inspired fluorescent Zn2+ sensor is developed for two-photon microscopy and related biological application that features an 8-methoxyquinoline moiety. Excellent photophysical characteristics including a 37-fold fluorescence enhancement with excitation and emission maxima at 440â nm and 505â nm, respectively, as well as a high two-photon cross-section of 73â GM at 880â nm are reported. Based on the experimental data, the relationship between the structure and properties was elucidated and explained backed up by DFT calculations, particularly the observed PeT phenomenon for the turn-on process. Biological validation and detailed experimental and theoretical characterization of the free and the zinc-bound compounds are presented.
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Corantes Fluorescentes , Proteínas de Fluorescência Verde , Quinolinas , Zinco , Zinco/química , Corantes Fluorescentes/química , Quinolinas/química , Proteínas de Fluorescência Verde/química , Humanos , Teoria da Densidade Funcional , Microscopia de Fluorescência por Excitação Multifotônica/métodos , FótonsRESUMO
BACKGROUND: The organism-wide effects of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral infection are well studied, but little is known about the dynamics of how the infection spreads in time among or within cells due to the scarcity of suitable high-resolution experimental systems. It has been reported that SARS-CoV-2 infection pathways converge at calcium influx and subcellular calcium distribution changes. Imaging combined with a proper staining technique is an effective tool for studying subcellular calcium-related infection and replication mechanisms at such resolutions. METHODS: Using two-photon (2P) fluorescence imaging with our novel Ca-selective dye, automated image analysis and clustering analysis were applied to reveal titer and variant effects on SARS-CoV-2-infected Vero E6 cells. RESULTS: The application of a new calcium sensor molecule is shown, combined with a high-end 2P technique for imaging and identifying the patterns associated with cellular infection damage within cells. Vero E6 cells infected with SARS-CoV-2 variants, D614G or B.1.1.7, exhibit elevated cytosolic calcium levels, allowing infection monitoring by tracking the cellular changes in calcium level by the internalized calcium sensor. The imaging provides valuable information on how the level and intracellular distribution of calcium are perturbed during the infection. Moreover, two-photon calcium sensing allowed the distinction of infections by two studied viral variants via cluster analysis of the image parameters. This approach will facilitate the study of cellular correlates of infection and their quantification depending on viral variants and viral load. CONCLUSIONS: We propose a new two-photon microscopy-based method combined with a cell-internalized sensor to quantify the level of SARS-CoV-2 infection. We optimized the applied dye concentrations to not interfere with viral fusion and viral replication events. The presented method ensured the proper monitoring of viral infection, replication, and cell fate. It also enabled distinguishing intracellular details of cell damage, such as vacuole and apoptotic body formation. Using clustering analysis, 2P microscopy calcium fluorescence images were suitable to distinguish two different viral variants in cell cultures. Cellular harm levels read out by calcium imaging were quantitatively related to the initial viral multiplicity of infection numbers. Thus, 2P quantitative calcium imaging might be used as a correlate of infection or a correlate of activity in cellular antiviral studies.
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COVID-19 , Cálcio , Corantes Fluorescentes , SARS-CoV-2 , Chlorocebus aethiops , Células Vero , Cálcio/metabolismo , Cálcio/análise , Animais , COVID-19/virologia , COVID-19/metabolismo , Corantes Fluorescentes/química , Humanos , FótonsRESUMO
The few commercially available chemosensors and published probes for in vitro Zn2+ detection in two-photon microscopy are compromised by their flawed spectroscopic properties, causing issues in selectivity or challenging multistep syntheses. Herein, we present the development of an effective small molecular GFP chromophore-based fluorescent chemosensor with a 2,2'-bipyridine chelator moiety (GFZnP BIPY) for Zn2+ detection that has straightforward synthesis and uncompromised properties. Detailed experimental characterizations of the free and the zinc-bound compounds within the physiologically relevant pH range are presented. Excellent photophysical characteristics are reported, including a 53-fold fluorescence enhancement with excitation and emission maxima at 422 nm and 492 nm, respectively. A high two-photon cross section of 3.0 GM at 840 nm as well as excellent metal ion selectivity are reported. In vitro experiments on HEK 293 cell culture were carried out using two-photon microscopy to demonstrate the applicability of the novel sensor for zinc bioimaging.
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2,2'-Dipiridil , Compostos Heterocíclicos , Humanos , Células HEK293 , Microscopia de Fluorescência , Quelantes , Zinco , Corantes Fluorescentes/química , Espectrometria de FluorescênciaRESUMO
An asymmetric cyanine-type fluorescent dye was designed and synthesized via a versatile, multi-step process, aiming to conjugate with an Her2+ receptor specific antibody by an azide-alkyne click reaction. The aromaticity and the excitation and relaxation energetics of the fluorophore were characterized by computational methods. The synthesized dye exhibited excellent fluorescence properties for confocal microscopy, offering efficient applicability in in vitro imaging due to its merits such as a high molar absorption coefficient (36 816 M-1 cm-1), excellent brightness, optimal wavelength (627 nm), larger Stokes shift (26 nm) and appropriate photostability compared to cyanines. The conjugated cyanine-trastuzumab was constructed via an effective, metal-free, strain-promoted azide-alkyne click reaction leading to a regulated number of dyes being conjugated. This novel cyanine-labelled antibody was successfully applied for in vitro confocal imaging and flow cytometry of Her2+ tumor cells.
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Azidas , Corantes Fluorescentes , Carbocianinas , Anticorpos , Alcinos , Microscopia ConfocalRESUMO
A tremendous number of solvents, either as liquids or vapors, contaminate the environment on a daily basis worldwide. Olefin metathesis, which has been widely used as high-yielding protocols for ring-opening metathesis polymerization (ROMP), ring-closing metathesis (RCM), and isomerization reactions, is typically performed in toxic and volatile solvents such as dichloromethane. In this study, the results of our systematic experiments with the Grubbs G1, G2, and Hoveyda-Grubbs HG2 catalysts proved that benzotrifluoride (BTF) can replace dichloromethane (DCM) in these reactions, providing high yields and similar or even higher reaction rates in certain cases. The ROMP of norbornene resulted not only in high yields but also in polynorbornenes with a high molecular weight at low catalyst loadings. Ring-closing metathesis (RCM) experiments proved that, with the exception of the G1 catalyst, RCM occurs with similar high efficiencies in BTF as in DCM. It was found that isomerization of (Z)-but-2-ene-1,4-diyl diacetate with the G2 and HG2 catalysts proceeds at significantly higher initial rates in BTF than in DCM, leading to rapid isomerization with high yields in a short time. Overall, BTF is a suitable solvent for olefin metathesis, such as polymer syntheses by ROMP and the ring-closing and isomerization reactions.
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Alcenos , Cloreto de Metileno , Polimerização , FluorbenzenosRESUMO
Novel poly(dithiophosphate)s (PDTPs) were successfully synthesized under mild conditions without any additive in the presence of THF or toluene diluents at 60 °C by a direct, catalyst-free reaction between the abundant phosphorus pentasulfide (P4S10) and glycols such as ethylene glycol (EG), 1,6-hexanediol (HD) and poly(ethylene glycol) (PEG). GPC, FTIR, 1H and 31P NMR analyses proved the formation of macromolecules with dithiophosphate coupling groups having P=S and P-SH pendant functionalities. Surprisingly, the ring-opening of THF by the P-SH group and its pendant incorporation as a branching point occur during polymerization. This process is absent with toluene, providing conditions to obtain linear chains. 31P NMR measurements indicate long-time partial hydrolysis and esterification, resulting in the formation of a thiophosphoric acid moiety and branching points. Copolymerization, i.e., using mixtures of EG or HD with PEG, results in polymers with broadly varying viscoelastic properties. TGA shows the lower thermal stability of PDTPs than that of PEG due to the relatively low thermal stability of the P-O-C moieties. The low Tgs of these polymers, from -4 to -50 °C, and a lack of PEG crystallites were found by DSC. This polymerization process and the resulting novel PDTPs enable various new routes for polymer synthesis and application possibilities.
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Fósforo , Polímeros , Polímeros/química , Polietilenoglicóis/química , EnxofreRESUMO
A general, scalable two-step regio- and diastereoselective method has been described for the synthesis of versatile alkaloid-type azetidines from simple building blocks with excellent overall yields. In the kinetically controlled reaction, only the formation of the strained four-membered ring can be achieved instead of the thermodynamically favorable five-membered rings under appropriate conditions. Remarkable functional group tolerance has also been demonstrated. In this paper, we give a new scope of Baldwin's rules by density functional theory (DFT) calculations with an explicit solvent model, confirming the proposed reaction mechanisms and the role of kinetic controls in the stereochemical outcome of the reported transition-metal-free carbon-carbon bond formation reactions.
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The present study reports regio- and highly diastereoselective preparative methods for the synthesis of versatile alkaloid-type compounds from oxiranylmethyl tetrahydroisoquinolines. 2,5-Methanobenzo[ c]azepines or azetidine-fused heterocycles were synthesized in tandem reactions depending on the absence or presence of a BF3 co-reagent. A high functional group tolerance has also been demonstrated. DFT calculations with an explicit solvent model confirmed the proposed reaction mechanisms and the role of kinetic controls on the stereochemical outcome of the reported new methods.
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Not all amide bonds are created equally. The purpose of the present paper is the reinterpretation of the amide group by means of two concepts: amidicity and carbonylicity. These concepts are meant to provide a new viewpoint in defining the stability and reactivity of amides. With the help of simple quantum-chemical calculations, practicing chemists can easily predict the outcome of a desired process. The main benefit of the concepts is their simplicity. They provide intuitive, but quasi-thermodynamic data, making them a practical rule of thumb for routine use. In the current paper we demonstrate the performance of our methods to describe the chemical character of an amide bond strength and the way of its activation methods. Examples include transamidation, acyl transfer and amide reductions. Also, the method is highly capable for simple interpretation of mechanisms for biological processes, such as protein splicing and drug mechanisms. Finally, we demonstrate how these methods can provide information about photo-activation of amides, through the examples of two caged neurotransmitter derivatives.
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Amidas/química , Modelos Químicos , Algoritmos , Amidas/síntese química , Técnicas de Química Sintética , TermodinâmicaRESUMO
A novel family of julolidine-containing fluorescent rhodols equipped with a wide variety of substituents was synthesized in a versatile two-step process. The prepared compounds were fully characterized and exhibited excellent fluorescence properties for microscopy imaging. The best candidate was conjugated to the therapeutic antibody trastuzumab through a copper-free strain-promoted azide-alkyne click reaction. The rhodol-labeled antibody was successfully applied for in vitro confocal and two-photon microscopy imaging of Her2+ cells.
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We evaluated a year-long treatment regime testing synthetic, 10-component, honey bee, Apis mellifera L. (Hymenoptera: Apidae), brood pheromone (SuperBoost; Contech Enterprises Inc., Delta, BC, Canada) on the productivity and vigor of package bee colonies in the lower Fraser Valley of British Columbia, Canada. Fifty-eight newlyestablished 1.3-kg (3-lb) colonies treated three times with SuperBoost at 5-wk intervals starting 30 April 2009 were compared with 52 untreated control colonies. Treated colonies produced 84.3% more honey than untreated control colonies. By 8 September 2009, SuperBoost-treated colonies had 35.4% more adults than untreated colonies. By 28 September, net survival of treated and control colonies was 72.4 and 67.3%, respectively. On 5 October, treated and control colonies were divided into two additional groups, making up four cohorts: SuperBoost-treated colonies treated again during fall and spring build-up feeding with pollen substitute diet (BeePro, Mann Lake Ltd., Hackensack, MN; TIT); controls that remained untreated throughout the year (CCC); colonies treated with SuperBoost in spring-summer 2009 but not treated thereafter (TCC); and original control colonies treated with SuperBoost during the fall and spring build-up feeding periods (CTT). There was no difference among cohorts in consumption of BeePro during fall feeding, but TTT colonies (including daughter colonies split off from parent colonies) consumed 50.8% more diet than CCC colonies during spring build-up feeding. By 21 April, the normalized percentages of the original number of colonies remaining (dead colonies partially offset by splits) were as follows: CCC, 31.4%; CTT, 43.8%; TCC, 53.59%; and TTT, 80.0%. The net benefit of placing 100 newly established package bee colonies on a year-long six-treatment regime with SuperBoost would be US$6,202 (US$62.02 per colony). We conclude that treatment with SuperBoost enhanced the productivity and survival of package bee colonies and hypothesize that similar results could be achieved with established colonies.
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Criação de Abelhas/métodos , Abelhas/fisiologia , Mel , Feromônios/farmacologia , Animais , Criação de Abelhas/economia , Abelhas/crescimento & desenvolvimento , Oregon , Dinâmica Populacional , Estações do AnoRESUMO
Sex pheromones are commonly used in traps to monitor populations and movements of male click beetles, but to date few attractants have been identified for females. Notable exceptions are plant-derived kairomones for female Agriotes brevis and A. ustulatus, allowing the monitoring of both males and females of these species with lures containing both pheromones and plant volatiles. The attractiveness of these plant volatiles for two congeners, A. obscurus and A. lineatus, which are agricultural pests in Europe and North America, was evaluated in the current study. Both the four-component MINIM plant-derived lure for A. brevis, and the blend of (E)-anethol and (E)-cinnamaldehyde for A. ustulatus, were not attractive to A. obscurus and A. lineatus, and instead appeared to reduce captures-both when compared to blank controls, and when blended with and compared to the sex pheromones of these species. This was most pronounced in A. obscurus, where (E)-anethol and (E)-cinnamaldehyde reduced male captures by 43 and 37%, respectively. Combining the pheromones of A. obscurus and A. lineatus reduced captures of these species by 77 and 19%, respectively, compared to these pheromones singly. This suggests that attractants for female click beetles can be highly species-specific, and that the blending of pheromones of congeneric species with each other, or with plant volatiles, can reduce captures. Further research into developing such attractants for economic species is urgently needed.
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We conducted a series of nine laboratory experiments testing the response of "vinegar flies," Drosophila melanogaster Meigen (Diptera: Drosophilidae), released in bioassay chambers to experimental traps and lures. These experiments showed that an effective trap could be constructed from a clear 225-ml screw-cap jar fitted with a hollow 8-mm-diameter cylindrical cross bridge. Flies could enter the trap from either end of the cylindrical "gate" and in turn could enter the interior chamber of the trap through a cut out portion at mid-span of the cylinder. The experiments also showed that a natural-component lure could be made using a teabag containing freeze-dried banana powder, yeast, and carrageenan gum powder as a humectant. When dipped in water for 10-15 s and then placed in the bottom of a trap, the teabag provided effective attraction for at least 7 d. Captured flies were immobilized on a sticky card placed in the trap, allowing them to be easily seen. Unlike other traps that cannot be opened and have liquid lures, the cylindrical-gate trap can be reused repeatedly if the teabag and sticky card are replaced. A final two experiments showed that the prototype operational cylindrical-gate trap with a teabag lure captured 3.3 and 2.3 times more released flies, respectively, than the next best of three commercially available traps.
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Drosophila melanogaster/efeitos dos fármacos , Controle de Insetos/métodos , Feromônios , Animais , Comportamento Animal/efeitos dos fármacos , Carragenina/química , Feminino , Alimentos , Controle de Insetos/instrumentação , Masculino , Musa/química , Odorantes , Pós/química , Distribuição Aleatória , Fermento Seco/químicaRESUMO
In this paper, we present an additional, new cage-GABA compound, called 4-amino-1-(4'-dimethylaminoisopropoxy-5',7'-dinitro-2',3'-dihydro-indol-1-yl)-1-oxobutane-γ-aminobutyric acid (iDMPO-DNI-GABA), and currently, this compound is the only photoreagent, which can be applied for GABA uncaging without experimental compromises. By a systematic theoretical design and successful synthesis of several compounds, the best reagent exhibits a high two-photon efficiency within the 700-760 nm range with excellent pharmacological behavior, which proved to be suitable for a complex epileptic study. Quantum chemical design showed that the optimal length of the cationic side chain enhances the two-photon absorption by 1 order of magnitude due to the cooperating internal hydrogen bonding to the extra nitro group on the core. This feature increased solubility while suppressing membrane permeability. The efficiency was demonstrated in a systematic, wide range of in vitro single-cell neurophysiological experiments by electrophysiological as well as calcium imaging techniques. Scalable inhibitory ion currents were elicited by iDMPO-DNI-GABA with appropriate spatial-temporal precision, blocking both spontaneous and evoked cell activity with excellent efficiency. Additionally, to demonstrate its applicability in a real neurobiological study, we could smoothly and selectively modulate neuronal activities during artificial epileptic rhythms first time in a neural network of GCaMP6f transgenic mouse brain slices.
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A variety of quaternary aryl amino acid derivatives can be synthesised using tandem SN2/Smiles rearrangement chemistry involving aryl sulfonamides and α-chloro carbonyl compounds. The reaction harnesses a sulfur dioxide extrusion pathway to construct a C-N and C-Caryl bond under simple conditions with no requirement for organometallics or transition metal catalysts. The reaction is also successful for alkenyl sulfonamides, producing sterically congested quaternary alkene amino acid derivatives.
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Vinyl alcohol (VA) copolymers having fine tunable polarities are emerging materials in drug delivery applications. VA copolymers rendering well-defined molecular architecture (C/OH ratioâ¯=â¯2, 4, 5 and 8) were used as carriers for model drug compound, fluorescein, which exhibited significantly different release characteristics depending on the polarity of the polymers. Based on the preliminary drug release tests the well-defined VA copolymer having C/OHâ¯=â¯5 ratio, poly(vinyl alcohol alt-propenylene) copolymer (PVA-5) was selected for nanocomposite synthesis. Sorafenib anticancer drug was embedded into PVA-5 (C/OHâ¯=â¯5 ratio) nanoparticles by nanoprecipitation resulting in nanoparticles exhibiting unusual cubic shape. The sorafenib-loaded nanocomposites showed continuous release during a day and concentration-dependant cytotoxicity on HT-29 cancer cells. This might be interpreted by the sustained release of the drug.
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Antineoplásicos , Portadores de Fármacos , Nanocompostos , Sorafenibe , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Portadores de Fármacos/administração & dosagem , Portadores de Fármacos/química , Liberação Controlada de Fármacos , Células HT29 , Humanos , Nanocompostos/administração & dosagem , Nanocompostos/química , Polímeros/administração & dosagem , Polímeros/química , Sorafenibe/administração & dosagem , Sorafenibe/químicaRESUMO
In order to improve the fluorescence properties of the green fluorescent protein chromophore, pHOBDI ((5(4hydroxybenzylidene)2,3dimethyl3,5dihydro4Himidazol4one), sixteen dihydroimidazolone derivates were synthesized from thiohydantoin and arylaldehydes. The synthesis developed is an efficient, novel, one-pot procedure. The study provides a detailed description of the spectroscopic characteristics of the newly synthesized compounds, using pHOBDI as a reference. The new compounds all exhibited significantly stronger fluorescence than pHOBDI, up to 28 times higher quantum yields. An experimental and theoretical investigation of the relationship of the fluorescence properties with the molecular structure was also carried out. A good correlation was found between the emission wavenumber and the Hammett constant of the functional group, which suggests the intermolecular charge transfer (ICT) mechanism between the aromatic groups.