Fluorescent Chromophore Research Articles

Structural basis for activity of highly efficient RNA mimics of green fluorescent protein.

GFP and its derivatives revolutionized the study of proteins. Spinach is a recently reported in vitro-evolved RNA mimic of GFP, which as genetically encoded fusions makes possible live-cell, real-time imaging of biological RNAs without resorting to large RNA-binding protein-GFP fusions. To elucidate the molecular basis of Spinach fluorescence, we solved the cocrystal structure of Spinach bound to its cognate exogenous chromophore, showing that Spinach activates the small molecule by immobilizing it between a base triple, a G-quadruplex and an unpaired G. Mutational and NMR analyses indicate that the G-quadruplex is essential for Spinach fluorescence, is also present in other fluorogenic RNAs and may represent a general strategy for RNAs to induce fluorescence of chromophores. The structure guided the design of a miniaturized 'Baby Spinach', and it provides a foundation for structure-driven design and tuning of fluorescent RNAs.

Two-Photon Absorption in Fluorescent Protein Chromophores: TDDFT and CC2 Results.

Two-photon spectroscopy of fluorescent proteins is a powerful bioimaging tool. Considerable effort has been made to measure absolute two-photon absorption (TPA) for the available fluorescent proteins. Being a technically involved procedure, there is significant variation in the published experimental measurements even for the same protein. In this work, we present a time-dependent density functional theory (TDDFT) study on isolated chromophores comparing the ability of four functionals (PBE0, B3LYP, CAM-B3LYP, and LC-BLYP) combined with the 6-31+G(d,p) basis set to reproduce averaged experimental TPA energies and cross sections. The TDDFT energies and TPA cross sections are also compared to corresponding CC2/6-31+G(d,p) results for excitation to S1 for the five smallest chromophores. In general, the computed TPA energies are less functional dependent than the TPA cross sections. The variation between functionals is more pronounced when higher-energy transitions are studied. Changes to the conformation of a chromophore are shown to change the TPA cross-section considerably. This adds to the difficulty of comparing an isolated chromophore to the one embedded in the protein environment. All functionals considered give moderate agreement with the corresponding CC2 results; in general, the TPA cross sections determined by TDDFT are 1.5-10 times smaller than the corresponding CC2 values for excitation to S1. LC-BLYP and CAM-B3LYP give erroneously large TPA cross sections in the higher-energy regions. On the other hand, B3LYP and PBE0 yield values that are of the same order of magnitude and in some cases very close to the averaged experimental data. Thus, based on the results reported here, B3LYP and PBE0 are the preferred functionals for screening chromphores for TPA. However, at best, TDDFT can be used to semiquantitatively scan chromophores for potential TPA probes and highlight spectroscopic peaks that could be present in the mature protein.

Grignard Metathesis Polymerization and Properties of 1,1-Disubstituted-2,5-dibromo-3,4-diphenylsiloles

Grignard metathesis polymerizations of 1,1-disubstituted-2,5-dibromo-3,4-diphenylsiloles such as 1,1dimethyl-2,5-dibromo-3,4-diphenylsilole, 1,1-diethyl-2,5-dibromo-3,4-diphenylsilole, 1,1-diisopropyl-2,5-dibromo-3,4-diphenylsilole, and 1,1-dihexyl-2,5-dibromo-3,4-diphenylsilole were performed to yield poly(1,1disubstituted-3,4-diphenyl-2,5-silole)s containing fluorescent aromatic chromophore groups such as phenyl and silole in the polymer main chain: poly(1,1-dimethyl-3,4-diphenyl-2,5-silole), poly(1,1-diethyl-3,4diphenyl-2,5-silole), poly(1,1-diisopropyl-3,4-diphenyl-2,5-silole), and poly(1,1-dihexyl-3,4-diphenyl-2,5silole), respectively. The obtained materials are highly soluble in common organic solvents such as chloroform and tetrahydrofuran. Fourier-transform infrared spectra of all the polymers have characteristic C=C stretching frequencies at 1620-1628 cm. The prepared organosilicon polymers exhibit strong absorption maximum peaks at 273-293 nm in the tetrahydrofuran solution, showing a redshift of 18-34 nm relative to those of the monomer, strong excitation maximum peaks at 276-303 nm, and strong fluorescence emission maximum bands at 350-440 nm. Thermogravimetric analysis shows that most of the polymers are stable up to 200 C with a weight loss of 6-16% in nitrogen.

Effect of TiO2 nanoparticles on the structure and activity of catalase

TiO2 nanoparticles are the most widely used metal oxide nanoparticles and have oxidative toxicity. Catalase is an important antioxidant enzyme. Here the understanding of an effect of TiO2 nanoparticles on the activity and structure of catalase is crucial to characterize the toxicity of TiO2 nanoparticles. These experimental data revealed that TiO2 nanoparticles could bind to catalase by the electrostatic and hydrogen bonding forces. On binding TiO2 nanoparticles, catalase got destabilized with the decrease of α-helices content, the solvent polarity of environment around the fluorescence chromophores on catalase were also affected. In addition, TiO2 nanoparticles also affected the activity of catalase. TiO2 nanoparticles acted as an activator of catalase activity at a low molar concentration and as an inhibitor at a higher molar concentration. With regard to human health, the present study could provide a better understanding of the potential nanotoxicity of TiO2 nanoparticles.

Competition between photodetachment and autodetachment of the 2(1)ππ* state of the green fluorescent protein chromophore anion.

Using a combination of photoelectron spectroscopy measurements and quantum chemistry calculations, we have identified competing electron emission processes that contribute to the 350-315 nm photoelectron spectra of the deprotonated green fluorescent protein chromophore anion, p-hydroxybenzylidene-2,3-dimethylimidazolinone. As well as direct electron detachment from S0, we observe resonant excitation of the 2(1)ππ* state of the anion followed by autodetachment. The experimental photoelectron spectra are found to be significantly broader than photoelectron spectrum calculated using the Franck-Condon method and we attribute this to rapid (∼10 fs) vibrational decoherence, or intramolecular vibrational energy redistribution, within the neutral radical.

Perylene-derived single-component organic nanoparticles with tunable emission: efficient anticancer drug carriers with real-time monitoring of drug release.

An organic nanoparticle-based drug delivery system with high drug loading efficacy (∼79 wt %) was developed using a perylene-derived photoremovable protecting group, namely, perylene-3,4,9,10-tetrayltetramethanol (Pe(OH)4). The anticancer drug chlorambucil was protected by coupling with Pe(OH)4 to form photocaged nanoparticles (Pe(Cbl)4). The photorelease mechanism of chlorambucil from the Pe(Cbl)4 conjugate was investigated experimentally by high-resolution mass spectrometry and theoretically by density functional theory calculations. The Pe(Cbl)4 nanoparticles perform four important roles: (i) a nanocarrier for drug delivery, (ii) a phototrigger for drug release, (iii) a fluorescent chromophore for cell imaging, and (iv) a photoswitchable fluorophore for real-time monitoring of drug release. Tunable emission of the perylene-derived nanoparticles was demonstrated by comparing the emission properties of the Pe(OH)4 and Pe(Cbl)4 nanoparticles with perylene-3-ylmethanol. These nanoparticles were subsequently employed in cell imaging for investigating their intracellular localization. Furthermore, the in vivo toxicity of the Pe(OH)4 nanoparticles was investigated using the mouse model. Histological tissue analysis of five major organs, i.e., heart, kidney, spleen, liver, and lung, indicates that the nanoparticles did not show any obvious damage to these major organs under the experimental conditions. The current research presents a successful example of integrating multiple functions into single-component organic nanoparticles for drug delivery.

Fluorescent polyaspartic acid with an enhanced inhibition performance against calcium phosphate

Fluorescent polyaspartic acid (FPASP) used as a novel scale inhibitor was synthesized with partially ethanolamine-modified polysuccinimide, p-toluenesulfonyl chloride and 3-amino-9-ethyl carbazole. The products were characterized by 1H NMR, gel permeation chromatography and fluorescent spectra, respectively. The results show that carbazole groups, the fluorescent chromophore, were successfully incorporated into PASP. The relationship between the fluorescence intensity and the concentration of FPASP is linear. This provides a prerequisite for an automatic dosing of PASP by determining the concentration in situ using a fluorescence spectrometer with the help of computer technology. In addition, when the concentration was 12mgL−1, the inhibition efficiency of Ca3(PO4)2 was improved from 31.3% of PASP to 93.3% of FPASP, which were determined using the static scale inhibition method. This improvement originates from the hydroxyl groups introduced into PASP.

Multichannel-emissive V-shaped boryl-BODIPY dyads: synthesis, structure, and remarkably diverse response toward fluoride.

Three new V-shaped boryl-BODIPY dyads (1-3) were synthesized and structurally characterized. Compounds 1-3 are structurally close molecular siblings differing only in the number of methyl substituents on the BODIPY moiety that were found to play a major role in determining their photophysical behavior. The dyads show rare forms of multiple-channel emission characteristics arising from different extents of electronic energy transfer (EET) processes between the two covalently linked fluorescent chromophores (borane and BODIPY units). Insights into the origin and nature of their emission behavior were gained from comparison with closely related model molecular systems and related photophysical investigations. Because of the presence of the Lewis acidic triarylborane moiety, the dyads function as highly selective and sensitive fluoride sensors with vastly different response behaviors. When fluoride binds to the tricoordinate borane center, dyad 1 shows gradual quenching of its BODIPY-dominated emission due to the ceasing of the (borane to BODIPY) EET process. Dyad 2 shows a ratiometric fluorescence response for fluoride ions. Dyad 3 forms fluoride-induced nanoaggregates that result in fast and effective quenching of its fluorescence intensity just for ∼0.3 ppm of analyte (i.e., 0.1 equiv ≡ 0.26 ppm of fluoride). The small structural alterations in these three structurally close dyads (1-3) result in exceptionally versatile and unique photophysical behaviors and remarkably diverse responses toward a single analyte, i.e., fluoride ion.

Constructing polyatomic potential energy surfaces by interpolating diabatic Hamiltonian matrices with demonstration on green fluorescent protein chromophore

Simulating molecular dynamics directly on quantum chemically obtained potential energy surfaces is generally time consuming. The cost becomes overwhelming especially when excited state dynamics is aimed with multiple electronic states. The interpolated potential has been suggested as a remedy for the cost issue in various simulation settings ranging from fast gas phase reactions of small molecules to relatively slow condensed phase dynamics with complex surrounding. Here, we present a scheme for interpolating multiple electronic surfaces of a relatively large molecule, with an intention of applying it to studying nonadiabatic behaviors. The scheme starts with adiabatic potential information and its diabatic transformation, both of which can be readily obtained, in principle, with quantum chemical calculations. The adiabatic energies and their derivatives on each interpolation center are combined with the derivative coupling vectors to generate the corresponding diabatic Hamiltonian and its derivatives, and they are subsequently adopted in producing a globally defined diabatic Hamiltonian function. As a demonstration, we employ the scheme to build an interpolated Hamiltonian of a relatively large chromophore, para-hydroxybenzylidene imidazolinone, in reference to its all-atom analytical surface model. We show that the interpolation is indeed reliable enough to reproduce important features of the reference surface model, such as its adiabatic energies and derivative couplings. In addition, nonadiabatic surface hopping simulations with interpolation yield population transfer dynamics that is well in accord with the result generated with the reference analytic surface. With these, we conclude by suggesting that the interpolation of diabatic Hamiltonians will be applicable for studying nonadiabatic behaviors of sizeable molecules.

A note on second-order nonlinear optical response of high-spin bis-TEMPO diradicals with possible application

In this note, we have theoretically evaluated second-order nonlinear optical (NLO) responses of two structural isomeric forms of three different green fluorescent protein chromophores (viz. cyan fluorescent protein (CFP), orange variant of Discosoma coral (DsRed) and Zoanthus sp. (zFP)) coupled bis-TEMPO based photomagnetic high-spin diradicals by measuring the second-order effect of first molecular hyperpolarizabilities. The magnetic character and certain biological applicabilities of these diradicals are investigated theoretically in one of our recent papers (Bhattacharya et al., 2013). The second-order total molecular hyperpolarizabilities of these molecules are correlated with their respective HOMO–LUMO energy gaps (ΔEH-L) and also with the extent of the π-network of the couplers. Relations between ΔEH-L, chemical hardness, NLO response and exchange energy (Bhattacharya et al., 2013) are made. The applicability of these diradicals in the light of NLO properties are also highlighted in this note. Here these resulting fluoroprotein coupled diradicals can switch back and forth between two isomeric states of different colors upon irradiation by light of appropriate wavelength. Nonetheless, the magnitude of second-order NLO responses is different for different isomeric forms; hence we made a correlation between the color codes with the magnitude of second-order NLO responses. Thence with just simple visual inspection one can understand the extent of second-order of NLO response for these diradicals.

A New Highly Fluorescent and Symmetric Pyrrole–BF2 Chromophore: BOPHY

The new fluorescent chromophore BOPHY can be readily synthesized in two steps from commercially available reagents via the coupling of pyrrole-2-carboxaldehyde with hydrazine followed by reaction with BF3. The resultant symmetric and dimeric tetracycle is composed of two BF2 units in six-membered chelate rings appended with pyrrole units on the periphery. The quantum yields of fluorescence for the unmodified compound and the tetramethyl variant are near unity, with values of 95 and 92%, respectively, in CH2Cl2. We have probed the electronic structure of this compound via cyclic voltammetry and density functional theory analysis.

Synthesis of Cyano‐Substituted Diaryltetracenes from Tetraaryl[3]cumulenes

A versatile, two-step synthesis of highly substituted, cyano-functionalized diaryltetracenes has been developed, starting from easily accessible tetraaryl[3]cumulenes. This unprecedented transformation is initiated by [2+2] cycloaddition of tetracyanoethylene (TCNE) to the proacetylenic central double bond of the cumulenes to give an intermediate zwitterion, which after an electrocyclization cascade and dehydrogenation yields 5,5,11,11-tetracyano-5,11-dihydrotetracenes in a one-pot procedure. A subsequent copper-assisted decyanation/aromatization provided the target 5,11-dicyano-6,12-diaryltetracene derivatives. All of the postulated structures were confirmed by X-ray crystallography. The new chromophores are thermally highly stable and feature promising fluorescence properties for potential use in optoelectronic devices. They are selective chemosensors for Cu(I) ions, which coordinate to one of the CN substituents and form a 1:1 complex with an association constant of Ka =1.5×10(5) L mol(-1) at 298 K.

Tunable Two-color Luminescence and Host–guest Energy Transfer of Fluorescent Chromophores Encapsulated in Metal-Organic Frameworks

Co-assembly of chromophore guests with host matrices can afford materials which have photofunctionalities different from those of individual components. Compared with clay and zeolite materials, the use of metal–organic frameworks (MOFs) as a host structure for fabricating luminescent host–guest materials is still at an early stage. Herein, we report the incorporation of a laser dye, 4-(dicyanomethylene)-2-methyl-6-(4-dimethylaminostyryl)-4H-pyran (DCM), into stilbene-based and naphthalene-based MOF systems. The resulting materials exhibit blue/red two-color emission, and the intensity ratio of blue to red fluorescence varies in different planes within the MOF crystal as detected by 3D confocal fluorescence microscopy. The observed changes in ratiometric fluorescence suggest the occurrence of energy transfer from MOF host to DCM molecules, which can be further confirmed by periodic density functional theoretical (DFT) calculations. Moreover, selective changes in luminescence behavior are observed on treating the guest@MOF samples with volatile organic compounds (methanol, acetone and toluene), indicating that these host–guest systems have potential applications as fluorescence sensors. It can be expected that by rational selection of MOF hosts and guest chromophores with suitable emissive colors and energy levels, a wide variety of multi-color luminescent and energy-transfer systems can readily be prepared in a similar manner.

Double strong exciton-plasmon coupling in gold nanoshells infiltrated with fluorophores

We report on the broadband resonant energy transfer processes observed in dye doped gold nanoshells, consisting of spherical particles with a dielectric core (SiO2) covered by a thin gold shell. The silica core has been doped with rhodamine B molecules in order to harness a coherent plasmon-exciton coupling between chromophores and plasmonic shell. This plasmon-exciton interplay depends on the relative spectral position of their bands. Here, we present a simultaneous double strong coupling plasmon-exciton and exciton-plasmon. Indeed, experimental observations reveal of a transmittance enhancement as function of the gain in a wide range of optical wavelengths (about 100 nm), while scattering cross sections remains almost unmodified. These results are accompanied by an overall reduction of chromophore fluorescence lifetimes that are a clear evidence of nonradiative energy transfer processes. The increasing of transmission in the range of 630–750 nm is associated with a striking enhancement of the extinction cross-section in the 510–630 nm spectral region. In this range, the system assumes super-absorbing features. This double behavior, as well as the broadband response of the presented system, represents a promising step to enable a wide range of electromagnetic properties and fascinating applications of plasmonic nanoshells as building blocks for advanced optical materials.

Molecular properties of excited electronic state: formalism, implementation, and applications of analytical second energy derivatives within the framework of the time-dependent density functional theory/molecular mechanics.

This work extends our previous works [J. Liu and W. Z. Liang, J. Chem. Phys. 135, 014113 (2011); J. Liu and W. Z. Liang, J. Chem. Phys. 135, 184111 (2011)] on analytical excited-state energy Hessian within the framework of time-dependent density functional theory (TDDFT) to couple with molecular mechanics (MM). The formalism, implementation, and applications of analytical first and second energy derivatives of TDDFT/MM excited state with respect to the nuclear and electric perturbations are presented. Their performances are demonstrated by the calculations of adiabatic excitation energies, and excited-state geometries, harmonic vibrational frequencies, and infrared intensities for a number of benchmark systems. The consistent results with the full quantum mechanical method and other hybrid theoretical methods indicate the reliability of the current numerical implementation of developed algorithms. The computational accuracy and efficiency of the current analytical approach are also checked and the computational efficient strategies are suggested to speed up the calculations of complex systems with many MM degrees of freedom. Finally, we apply the current analytical approach in TDDFT/MM to a realistic system, a red fluorescent protein chromophore together with part of its nearby protein matrix. The calculated results indicate that the rearrangement of the hydrogen bond interactions between the chromophore and the protein matrix is responsible for the large Stokes shift.

Aggregation-induced emission of GFP-like chromophores via exclusion of solvent–solute hydrogen bonding

The fluorescence of GFP-like chromophores in aqueous solutions is turned on upon forming aggregates or embedment in cell membranes as a result of exclusion of solvent-solute H-bonding.

Detailed Photoisomerization Dynamics of a Green Fluorescent Protein Chromophore Based Molecular Switch

With density-functional-based nonadiabatic molecular dynamics simulations,trans-to-cisandcis-to-transphotoisomerizations of a green fluorescent protein chromophore based molecule 4-benzylidene-2-methyloxazol-5(4H)-one (BMH) induced by the excitation to itsS1excited state were performed. We find a quantum yield of 32% for thetrans-to-cisphotoisomerization of BMH and a quantum yield of 33% for itscis-to-transphotoisomerization. For those simulations that did producetrans-to-cisisomerization, the averageS1excited state lifetime oftrans-BMH is about 1460 fs, which is much shorter than that ofcis-BMH (3100 fs) in those simulations that did producecis-to-transisomerization. For both photoisomerization processes, rotation around the central C2=C3 bond is the dominant reaction mechanism. Deexcitation occurs at an avoided crossing near theS1/S0conical intersection, which is near the midpoint of the rotation.

Interpolation of Potential Energy Surfaces for Nonadiabatic Simulations of Biological Systems

Simulations of photophysical processes in biological systems are essential for understanding the role of molecular environment regarding the utilization of the light energy. However, such simulations are challenging in part due to the associated high computational costs, especially with the often adopted ab initio calculations of excited state potential energy information. Here, an application of a scheme for interpolating diabatic Hamiltonian matrix will be presented. We will show that this method is applicable for a sizeable molecular system (with 21 atoms) with a capability of describing the conical intersection properly and with an extensibility to condensed phase situations, by testing it with a model analytic potential energy surface of the green fluorescent protein chromophore. A procedure for using high-level quantum chemical calculations in the interpolation of diabatic Hamiltonian will also be presented together with the results of trajectory hopping simulations. Finally, we will discuss how this approach can be applied to computational studies of other photobiological systems.

Blue electroluminescence from polyhedral oligomeric silsesquioxane containing spirobifluorene trimers

We synthesized and characterized a new polyhedral oligomeric silsesquioxane (POSS)-based deep blue light-emitting nanoparticle. A spirobifluorene trimer (SFL3) was used as the blue fluorescent chromophore. The allyl-functionalized SFL3 was introduced to octakis(dimethylsiloxy)silsesquioxane via hydrosilylation to afford POSS-SFL3. POSS-SFL3 was completely soluble in common organic solvents such as chloroform, tetrahydrofuran, and p-xylene, and formed a uniform thin film upon spin-coating. The UV–vis absorption and photoluminescence (PL) emission maxima of POSS-SFL3 were 356 and 425nm, respectively. An electroluminescent (EL) device with configuration ITO/PEDOT:PSS(40nm)/poly-TPD(50nm)/POSS-SFL3 (30nm)/TPBI (40nm)/LiF (0.5nm)/Al (100nm) was fabricated and showed a stable deep-blue electroluminescence with an electroluminescence (EL) maximum at 431nm and CIE chromaticity coordinates of (0.17, 0.10).

Microcrystals with Enhanced Emission Prepared from Hydrophobic Analogues of the Green Fluorescent Protein Chromophore via Reprecipitation

Certain synthetic analogues of the green fluorescent protein (GFP) chromophore are almost nonfluorescent in dilute solutions but are strongly light-emissive in the solid state, thus exhibiting aggregation-induced emission (AIE) behavior. In the present work, two such hydrophobic derivatives of the GFP chromophore known to be fluorescent in the crystalline state (p-hexyloxy- and p-dodecyloxybenzylideneimidazolinone) were used to prepare aqueous suspensions of particles via a mild solvent-exchange reprecipitation (RP) method. This evolution was monitored at various experimental conditions by UV-vis absorption and fluorescence spectroscopy, fluorescence microscopy, as well as electron transmission and scanning microscopy. Both compounds spontaneously produced platelet-like microcrystals, the size and shape of which were influenced by the experimental conditions. The dodecyl derivative also led to the concomitant formation of nanofibers, a tendency reinforced by addition of poly(acrylic acid) to the RP medium. The photoluminescence properties of the solids produced by RP were compared to pristine microcrystalline powders obtained by crystallization in an organic solvent. Significant differences in the emission properties were found and are discussed. This study illustrates the fact that AIE fluorescence is strongly dependent on the nature of the particles and hence on the preparation methods. Being aware of these variations is important for the preparation and subsequent use of AIE-active compounds as fluorescent materials.