Encapsulated energy-transfer cassettes with extremely well resolved fluorescent outputs

This paper concerns the development of water-compatible fluorescent imaging probes with tunable photonic properties that can be excited at a single wavelength. Bichromophoric cassettes 1a-1c consisting of a BODIPY donor and a cyanine acceptor were prepared using a simple synthetic route, and their photophysical properties were investigated. Upon excitation of the BODIPY moiety at 488 nm the excitation energy is transferred through an acetylene bridge to the cyanine dye acceptor, which emits light at approximately 600, 700, and 800 nm, i.e., with remarkable dispersions. This effect is facilitated by efficient energy transfer that gives a "quasi-Stokes" shift between 86 and 290 nm, opening a huge spectral window for imaging. The emissive properties of the cassettes depend on the energy-transfer (ET) mechanism: the faster the transfer, the more efficient it is. Measurements of rates of ET indicate that a through-bond ET takes place in the cassettes 1a and 1b that is 2 orders of magnitude faster than the classical through-space, F?rster ET. In the case of cassette 1c, however, both mechanisms are possible, and the rate measurements do not allow us to discern between them. Thus, the cassettes 1a-1c are well suited for multiplexing experiments in biotechnological methods that involve a single laser excitation source. However, for widespread application of these probes, their solubility in aqueous media must be improved. Consequently, the probes were encapsulated in calcium phosphate/silicate nanoparticles (diameter ca. 22 nm) that are freely dispersible in water. This encapsulation process resulted in only minor changes in the photophysical properties of the cassettes. The system based on cassette 1a was chosen to probe how effectively these nanoparticles could be used to deliver the dyes into cells. Encapsulated cassette 1a permeated Clone 9 rat liver cells, where it localized in the mitochondria and fluoresced through the acceptor part, i.e., red. Overall, this paper reports readily accessible, cyanine-based through-bond ET cassettes that are lypophilic but can be encapsulated to form nanoparticles that disperse freely in water. These particles can be used to enter cells and to label organelles.     

Fluorescence energy transfer-sensitized photobleaching of a fluorescent label as a tool to study donor-acceptor distance distributions and dynamics in protein assemblies: studies of a complex of biotinylated IgM with streptavidin and aggregates of concanavalin A

A photokinetic method of detection of fluorescence resonance energy transfer (FRET) between special fluorescent labels is applied to study time-averaged spatial distribution of labeled proteins in protein assemblies. Prolonged irradiation of a sample at the absorption maximum of the energy donor initiates FRET-sensitized fluorescence photobleaching of the energy acceptor label, which was monitored by steady-state fluorimetric measurements. Kinetics of the acceptor photobleaching and kinetics of decreasing the efficiency of FRET from donors to unbleached acceptors were determined. The FRET efficiency was found from measuring sensitization of acceptor fluorescence. Analysis of the photokinetic data permits to estimate the time-averaged distribution of acceptors on donor-acceptor distances in the range of characteristic distances of FRET. Dynamic processes influencing donor-acceptor distances can be also investigated by the method. Application of the method is demonstrated by the studies of a complex of biotinylated IgM with streptavidin and aggregates composed of concanavalin A and sodium dodecyl sulphate. A new thiadicarbocyanine dye was used as the acceptor label. R-phycoerythrin and tetramethylrhodamine isothiocyanate were the donor labels. In the IgM-streptavidin complex, 16% of acceptors most contributed to FRET provided 90% of FRET efficiency, whereas acceptors made about the same time-averaged contribution to FRET in the concanavalin A aggregates.     

A novel fluorescent sensor for Sn4+ detection: Dark resonance energy transfer from silole to rhodamine

A novel dark resonance energy transfer (DRET) off–on cassette SR1 was constructed by coupling a silole donor with a rhodamine acceptor. Due to the intramolecular rotations of the phenyl rings, the silole fluorophore served as a dark donor in solution state and fluorescence leakage from the donor emission could be avoided. Binding with Sn⁴⁺ ion induced the ring‐opening of the rhodamine acceptor, thus increase the overlapping between the emission spectra of the donor and absorption spectra of the acceptor. DRET was turned on and energy was transferred from the silole donor to the rhodamine acceptor. Emission from the rhodamine acceptor was achieved with a large Stokes shift up to 198 nm. The sensor showed good sensitivity and selectivity towards Sn⁴⁺ to other metal ions in methanol aqueous solution through the formation of a 1:1 complex between SR1 and Sn⁴⁺. This research provides a new approach for the development of rhodamine‐based sensors towards metal ions with large Stokes shifts.     

A photochromic acceptor as a reversible light-driven switch in fluorescence resonance energy transfer (FRET)

A method has been developed for the quantitative determination of fluorescence resonance energy transfer (FRET) based on the modulation of donor fluorescence upon the reversible photoconversion of a photochromic acceptor. A model system was devised, consisting of Lucifer Yellow cadaverine (LYC, donor) conjugated to the photochromic molecule, 6-nitroBIPS (1′,3′-dihydro-1′-(2-carboxyethyl)-3′,3′-dimethyl-6-nitrospiro[2H-1-benzopyran-2,2′-(2H)-indoline]). Near-ultraviolet irradiation catalyzes the conversion of the colorless spiropyran (SP) to the colored merocyanine (MC) form of 6-nitroBIPS. Only the MC form absorbs at the emission wavelengths of the donor, thereby potentiating FRET, as demonstrated by quenching of the donor. Subsequent irradiation in the visible MC absorption band reverts 6-nitroBIPS to the SP form and FRET is inactivated. The acceptor exhibited high photostability under repeated cycles of alternating UV–Vis irradiation. In this model system, the intramolecular FRET efficiency was close to 100%. The observed maximal donor quenching of 34±3% was indicative of an equilibrium determined by the high quantum efficiency of forward conversion (SP→MC) induced by near-UV irradiation and a low but finite quantum efficiency of the back reaction resulting from excitation of the MC form directly as well as indirectly (by FRET via the donor). A quantitative formalism for the photokinetic scheme was developed. Photochromic FRET (pcFRET) permits repeated, quantitative, and non-destructive FRET determinations for arbitrary relative concentrations of donor and acceptor and thus offers great potential for monitoring dynamic molecular interactions in living cells over extended observation times by fluorescence microscopy.     

Correlations of structure and rates of energy transfer for through-bond energy-transfer cassettes

Fluorescent DNA-labeling cassettes are designed to have a common absorbing chromophore matched to a single exciting laser wavelength, but up to four different emitters. Experiments reported here have examined the energy-transfer rates and fluorescence polarization characteristics for two different types of cassette, involving three distinct relative orientations of the donor and acceptor transition moments and the axis of the rigid linker. Energy-transfer times range from <200 fs to approximately 20 ps, the fastest transfer times occurring when the transition moments of the donor and acceptor species are aligned parallel to the linker axis. Experimental evidence is presented that supports a through-bond energy-transfer mechanism, in contrast with a commercial DNA-labeling agent, which exhibits much slower transfer times controlled by FRET. These rigid cassettes also exhibit polarized fluorescence from the acceptor species, so that this particular type of DNA-labeling probe has some of the advantages of single-molecule probes such as rhodamine and coumarin dyes.     

Novel D − π − A dye sensitizers of polymeric metal complexes based on 8‐hydroxyquinoline and phenylethyl or fluorene units: synthesis,characterization and photovoltaic applications for dye‐sensitized solar cells

Four novel donor ? π‐bridge ? acceptor (D ? π ? A) polymeric metal complexes (P1–P4) based on 8‐hydroxyquinoline metal complexes were synthesized and tested for their performance in dye‐sensitized solar cells (DSSCs). The polymeric metal complexes dyes use alkoxy benzene or alkyl fluorene as the electron donor and C=C as π linker; the 8‐hydroxyquinoline derivative complex part was used as the electron acceptor and diaminomaleonitrile was used as ancillary ligand. The two strongly electron‐withdrawing cyano groups in the polymer structure can provide an efficient charge transport in the intramolecular between donor and acceptor parts. The thermal, photophysical, electrochemical and photovoltaic properties of these copolymers were investigated by TGA, differential scanning calorimetry, cyclic voltammetry and cureent density‐voltage curves, and the results showed that dye containing complex Zn(II) and alkoxy benzene unit benefited the generation of photocurrent and open‐circuit voltages, and a maximum power conversion efficiency of 1.91% (P2) was obtained, with an open‐circuit voltage of 0.71 V, a short‐circuit current density of 4.23 mA cm^?2, and a fill factor of 38.6% under AM1.5G irradiation. The study results also show that the four polymers exhibit good thermal stability, indicating that these polymeric metal complexes are suitable for the fabrication processes of optoelectronic devices. Copyright © 2013 John Wiley & Sons, Ltd.     

Fluorescent proton sensors based on energy transfer

Photophysical data and orbital energy levels (from electrochemistry) were compared for molecules with the same BODIPY acceptor part (red) and perpendicularly oriented xanthene or BODIPY donor fragments (green). Transfer of energy, hence the photophysical properties of the cassettes, including the pH dependent fluorescence in the xanthene-containing molecules, correlates with the relative energies of the frontier orbitals in these systems. Intracellular sensing of protons is often achieved via sensors that switch off completely at certain pH values, but probes of this type are not easy to locate inside cells in their "off-state". A communication from these laboratories (J. Am. Chem. Soc., 2009, 131, 1642-3) described how the energy transfer cassette 1 could be used for intracellular imaging of pH. This probe is fluorescent whatever the pH, but its exact photophysical properties are governed by the protonation states of the xanthene donors. This work was undertaken to further investigate correlations between structure, photophysical properties, and pH for energy transfer cassettes. To achieve this, three other cassettes 2-4 were prepared: another one containing pH-sensitive xanthene donors (2) and two "control cassettes" that each have two BODIPY-based donors (3 and 4). Both the cassettes 1 and 2 with xanthene-based donors fluoresce red under slightly acidic conditions (pH < ～6) and green when the medium is more basic (>～7), whereas the corresponding cassettes with BODIPY donors give almost complete energy transfer regardless of pH. The cassettes that have BODIPY donors, by contrast, show no significant fluorescence from the donor parts, but the overall quantum yields of the cassettes when excited at the donor (observation of acceptor fluorescence) are high (ca. 0.6 and 0.9). Electrochemical measurements were performed to elucidate orbital energy level differences between the pH-fluorescence profiles of cassettes with xanthene donors, relative to the two with BODIPY donors. These studies confirm energy transfer in the cassettes is dramatically altered by analytes that perturb relative orbital levels. Energy transfer cassettes with distinct fluorescent donor and acceptor units provide a new, and potentially useful, approach to sensors for biomedical applications.     

A new glycosylation method part 3: study of microwave effects at low temperatures to control reaction pathways and reduce byproducts

The efficiency of microwave irradiation at low temperature for glycosylations is described. Although oligosaccharide synthesis usually requires reactive donors for glycosylations, which have leaving groups on the anomer positions, i.e., trichloroacetoimidates, halogenates, thioalkyl glycosides, etc., the suitable donors in our microwave supported synthesis of Lewis X oligosaccharide were very stable acetate derivatives. Regarding glycosylation with a fucosyl acetate donor and a glucosamine acceptor, microwave irradiation with simultaneous cooling improved yields. Moreover, further synthesis to Lewis X derivatives was achieved only with microwave irradiation at low temperatures. Without microwave irradiation, we could only obtain byproducts and none of the designed product at any reaction temperature.     

Time-resolved measurements of intramolecular energy transfer in single donor/acceptor dyads

We have investigated electronic excitation energy transfer in a specifically designed bichromophoric donor/acceptor dyad in which the donor (perylenediimide) and acceptor (terrylenediimide) are linked by a rigid heptaphenyl-spacer. Because of the choice of the bridge, which defines the distance and orientation of the two chromophores, donor as well as acceptor emission is observed. The significantly smaller photostability of the donor allows for time-resolved measurements of the acceptor emission at the single-molecule level with and without energy transfer from the donor. By analyzing the differences of the rise/decay profiles for both pathways, we could determine time constants of energy transfer with high accuracy for single dyads. The results show that the experimental approach presented here works even for situations in which the energy transfer times are smaller than the temporal resolution of the detection system.     

A new chemical probe for the detection of the cancer-linked galectin-3

A chemical probe was developed for the detection of the emerging cancer marker galectin-3. The probe contains a benzophenone moiety which covalently attaches itself to the protein upon binding and irradiation. Introduction of a fluorescent label via'click' chemistry allows the labelled proteins to be visualized in a gel. With the probe, selective visualization of galectin-3 in protein mixtures was shown and remarkably even in cell lysates.     

Particulate and soluble Eu(III)-chelates as donor labels in homogeneous fluorescence resonance energy transfer based immunoassay

Many well-established homogeneous separation free immunoassays rely on particulate label technologies. Particles generally contain a high concentration of the embedded label and they have a large surface area, which enables conjugation of a large amount of protein per particle. Eu(III)-chelate dyed nanoparticles have been successfully used as labels in heterogeneous and homogeneous immunoassays. In this study, we compared the characteristics of two homogeneous competitive immunoassays using either soluble Eu(III)-chelates or polystyrene particles containing Eu(III)-chelates as donors in a fluorescence resonance energy transfer based assay. The use of the particulate label significantly increased the obtained sensitized emission, which was generated by a single binding event. This was due to the extremely high specific activity of the nanoparticle label and also in some extent the longer Förster radius between the donor and the acceptor. The amount of the binder protein used in the assay could be decreased by 10-fold without impairing the obtainable sensitized emission, which subsequently led to improved assay sensitivity. The optimized assay using particulate donor had the lowest limit of detection (calculated using 3 × S.D. of the 0 nM standard) 50 pM of estradiol in the assay well, which was approximately 20-fold more sensitive than assays using soluble Eu(III)-chelates.     

Glycosidation with a disarmed glycosyl iodide: promotion and scope

[reaction: see text] Glucuronyl iodide 1 has been studied in detail as a "disarmed" glycosyl donor. In a model reaction, using N-iodosuccinimide (NIS) as a promoter and 2-phenylethanol as acceptor, best results were obtained using NIS with I(2), followed by trimethylsilyltrifluoromethanesulfonate (TMSOTf). When a series of primary and secondary alcohols was glycosylated using these conditions, yields of 60-83% of beta-glucuronides were obtained. Various "nonheavy" metal salts also effectively catalyzed the model reaction but led to significant amounts of alpha-product with less reactive secondary alcohols.     

[2.2]- and [3.3]Paracyclophane as Bridging Units in Organic Dyads for Visible-Light-Driven Dye-Sensitized Hydrogen Production

Novel donor–acceptor dyads containing [2.2]- and [3.3]paracyclophane (PCP) as the bridging moiety were synthesized and used to effectively fabricate dye-sensitized hydrogen production systems. All the prepared compounds had a phenothiazine and a cyanoacrylic acid/pyridinyl acrylonitrile moiety acting as an electron donor and acceptor, respectively. Although cyclic voltammetry measurements showed similar electron-donating properties among all the synthesized dyads, the lowest absorption energy of the [2.2]PCP moiety was lower than that of the [3.3]PCP one; this was due to its shorter distance between benzene rings, which could effectively drive the charge transfer between the donor and acceptor chromophores. Under visible light (>395 nm), a dyad-loaded photocatalyst in a 0.5 M aqueous glycerol solution generated detectable hydrogen gases. The optimal turnover number and photocurrent order exhibited the same trend as the hydrogen production rate since the suggested number of excited photons played a critical role in hydrogen production.     

Detection of Explosives Using Field‐Effect Transistors

The gate surfaces of ion‐sensitive field‐effect transistor (ISFET) devices were functionalized with the π‐donor units, 6‐hydroxydopamine ( 1 ) or 4‐aminothiophenol ( 2 ). Concentration of trinitrotoluene, TNT, on the gate via π‐donor‐acceptor interactions yields charge‐transfer complexes that alter the gate potential. This enables the label‐free analysis of TNT with a detection limit corresponding to 1×10^?7 M.     

Supramolecular control of the template-induced selective photodimerization of 4-methyl-7-O-hexylcoumarin

A symmetric ditopic molecular receptor (3), containing two identical hydrogen-bonding recognition subunits, was designed and synthesized. These subunits are capable of binding substrates with complementary donor and acceptor sites to form a supramolecular complex through hydrogen bonding. Receptor 3 was designed to accept two guest molecules, which are held in close proximity within the supramolecular species. The substrate molecule, 4-methyl-7-O-hexylcoumarin (1 c), forms a 2:1 complex with a binding constant of 150 m(-1) for the second substrate, passing first through a 1:1 complex with an affinity constant of 510 m(-1). The orientation of two molecules of 1 c when bound to the template leads to the selective formation of the trans-syn [2+2] photoproduct 2 cB upon irradiation. Other photoproducts typically produced in the absence of the template are suppressed.     

Design and synthesis of an enzyme-cleavable sensor molecule for phosphodiesterase activity based on fluorescence resonance energy transfer

Ratiometric measurement is a technique that can provide precise data and even quantitative detection. To carry out ratiometric measurements, it is necessary that the sensor molecule exhibits a large shift in its emission or excitation spectrum after reaction with the target molecule. Fluorescence resonance energy transfer (FRET) is one mechanism used to obtain a large spectral shift. In this study, our aim was to develop a ratiometric fluorescent sensor molecule for phosphodiesterase activity based on FRET. We designed and synthesized CPF4 with a coumarin donor, a fluorescein acceptor, and two phenyl linkers having the phosphodiester moiety interposed between them. In the emission spectrum of CPF4 in aqueous buffer excited at 370 nm, the emission of the coumarin donor was strongly quenched and the emission of the fluorescein acceptor was observed. This emission spectrum demonstrates that energy transfer from the coumarin donor to the fluorescein acceptor proceeds efficiently. Addition of a phosphodiesterase to an aqueous solution of CPF4 resulted in an increase in the donor fluorescence and a decrease in the acceptor fluorescence. CPF4 exhibited a large shift in its emission spectrum after the hydrolysis of the phosphodiester group by the enzyme. This large shift of the emission spectrum indicates that ratiometric measurements can be made by using CPF4. The method described in this paper for designing enzyme-cleavable sensor molecules based on FRET should be readily applicable to other hydrolytic enzymes.     

Modification of D–A–π–A Configuration toward a High‐Performance Triphenylamine‐Based Sensitizer for Dye‐Sensitized Solar Cells: A Theoretical Investigation

In an attempt to shed light on how the addition of a benzothiadiazole (BTD) moiety influences the properties of dyes, a series of newly designed triphenylamine‐based sensitizers incorporating a BTD unit as an additional electron‐withdrawing group in a specific donor–acceptor–π‐acceptor architecture has been investigated. We found that different positions of the BTD unit provided significantly different responses for light absorption. Among these, it was established that the further the BTD unit is away from the donor part, the broader the absorption spectra, which is an observation that can be applied to improve light‐harvesting ability. However, when the BTD unit is connected to the anchoring group a faster, unfavorable charge recombination takes place; therefore, a thiophene unit was inserted between these two acceptors, providing redshifted absorption spectra as well as blocking unfavorable charge recombination. The results of our calculations provide valuable information and illustrate the potential benefits of using computation‐aided sensitizer design prior to further experimental synthesis.     

Unprecedented laser action from energy transfer in multichromophoric BODIPY cassettes

A cassette molecule, featuring direct integration of two donor BODIPY units to one acceptor BODIPY unit, was conveniently developed as the first highly "through-bond energy transfer" (TBET) laser dye. This multicolor absorbing dye exhibited highly efficient and photostable laser action under drastic pumping conditions.     

Donor-conformation-dependent energy transfer for dual-color fluorescent probe with high-resolution imaging

Herein, we presented a brand-new concept to construct the Forster resonance energy transfer(FRET) based cassette by integrating a vibration-induced emission(VIE) chromophore as the donor. Different from traditional donors only with a single emission, the VIE donor possessed well-separated dual emission bands by altering the excited state molecular configuration from the bent state to the planar state. By linking an acceptor such as a cyanine dye(Cy5), a novel VIE-FRET cassette(PPCy5) was prepared. The planar emission profile of the VIE donor moiety could fully cover the absorption of Cy5, and thus the complete FRET process enabled the excellent bimodal spectra difference of 142 nm and ultra-large pseudo-Stokes shift of up to 300 nm.Benefiting from the viscosity-dependent characteristic of the VIE donor, PPCy5 could clearly and intuitively reveal the different viscosity regions in vivo by dual-color and high-resolution imaging. The VIE-FRET paradigm provides an optional platform for developing donor-acceptor-based dual-color fluorescent probes with high-resolution imaging ability.