Fluorescent Chromophore Research Articles

Pyridine analogue of fluorescent protein chromophore: Fluorogenic dye suitable for mitochondria staining

In this paper, we present a novel fluorogenic analogue of the GFP chromophore with atypical solvent-dependent variations of the fluorescence quantum yield. This compound can be used as the selective dye for fluorescent staining of whole mitochondria in live cells regardless of their metabolic status.

Development of new and robust LC-MS method for simultaneous quantification of polyphenols from Sambucus ebulus fruits

A new and simple LC-MS method for analysis of flavonoids from Sambucus ebulus berry extracts was developed and validated. Successfully were quantitated seven polyphenols: epicatechin, epigallocatechin gallate, rutin, resveratrol, myricetin, quercetin, and kaempferol.Two detectors, working in parallel, were used: photodiode-array and single quadrupole mass-detector. The mass detection was used for identification and quantification of the analytes, while the diode-array detector was as confirmation tool. The following m/z were tracked: 457.15 (epigallocatechin gallate); 289.06 (epicatechin); 609.13 (rutin); 227.05 (resveratrol); 317.0 (myricetin); 301.02 (quercetin); 285.02 (kaempferol). For optimization the chromatographic separation three wavelengths 205 nm, 305 nm, 272 nm were monitored. The method was capable to detect in one run compounds with no UV or fluorescence chromophore and with very similar structures, such as plant polyphenols. The linearity was from 0.05 mg/L to 50 mg/L (R2 0.9962–0.9987). The recoveries for all tested analytes were between 81.6% and 104.7%.The method was applied for analysis of crude extract of Sambucus ebulus ripe fruits. Three major polyphenols – epicatechin (0.84 mg/100gFW), quercetin (0.15 mg/100gFW) and kaempferol (0.05 mg/100gFW) were identified and quantified.The proposed method could be successfully used for routine analysis of epigallocatechin gallate, epicatechin, rutin, resveratrol, myricetin, quercetin, and kaempferol in Sambucus ebulus extracts.

Synthesis of the New Green Fluorescent Protein Chromophore Analogue Starting from a Cinnamic Aldehyde Derivative

A novel derivative of green fluorescent protein chromophore (Z)-5-((E)-3-(4-hydroxyphenyl)allylidene)-2,3-dimethyl-3,5-dihydro-4H-imidazol-4-one was synthesised. The desired compound was prepared by the synthetic approach based on the use of carboxyimidate. Comparison of optical properties of the obtained compound and of the classical GFP chromophore analogue (Z)-5-(4-hydroxybenzylidene)-2,3-dimethyl-3,5-dihydro-4H-imidazol-4-one showed the shift of absorption and emission maxima to the long-wavelength region induced by the increase of the conjugated π-system in the benzylidene fragment.

A highly fluorescent adenine derivative and its interaction with iron ions

In this paper, an adenine-derived fluorescent chromophore (HCC-APA) was synthesized to detect its interaction with iron ions. The results showed that a large stokes shift (υ = 10981.5 cm−1) fluorescence of the HCC-APA was appeared. The fluorescence quantum yields of HCC-APA in DMF and THF were 3.49% and 17.54%, respectively, demonstrating its higher fluorescence performance than unmodified adenine. Besides, the fluorescence quenching of HCC-APA with iron ions was further detected. Fluorescence titration experiments confirmed that the detection limit of HCC-APA for Fe3+ was 3.69 μM. The interaction between iron ions and HCC-APA was investigated by continuous variable method, indicating that the binding ratio is 1:1. And the interaction site was deeply explored by 1H NMR and molecular simulation.

A photoelectron imaging and quantum chemistry study of the deprotonated cyan fluorescent protein chromophore anion

Cyan fluorescent protein (CFP), a blue-shifted analogue of green fluorescent protein, is used widely as a fluorescence resonance energy transfer donor in live cell fluorescence imaging. Here, we use a combination of anion photoelectron spectroscopy experiments and quantum chemistry calculations to probe the electronic structure of the CFP chromophore in its deprotonated anionic form. The vertical detachment energy measured as the maximum in the photoelectron spectrum is eV, which is an improvement on our earlier measurement. We see evidence for competing internal conversion following resonant excitation of electronically excited states of the anion lying in the detachment continuum. We find that the first electronically excited valence state of the anion lies very close to the detachment threshold, supporting the conclusions of gas-phase photodestruction action spectroscopy measurements, and that the second electronically excited valence state lies around 1.25 eV above the detachment threshold. These electronic states have shape resonance character and mixed excited shape and Feshbach resonance character, respectively.

Synthesis and spectroscopic characterization of novel GFP chromophore analogues based on aminoimidazolone derivatives.

In order to improve the fluorescence properties of the green fluorescent protein chromophore, p‑HOBDI ((5‑(4‑hydroxybenzylidene)‑2,3‑dimethyl‑3,5‑dihydro‑4H‑imidazol‑4‑one), 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 p‑HOBDI as a reference. The new compounds all exhibited significantly stronger fluorescence than p‑HOBDI, 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.

Red emission cysteine probe with high selectivity based on fluorescent protein chromophores and turn-on fluorescence in cell cultures

Cysteine (Cys) plays a vital role in many physiological processes, and also is closely related to various diseases, thus the development of novel high selectivity and sensitivity fluorescent probes for in situ monitoring of biothiol (Cys) is crucial. Herein, we present a fluorogenic method to detect Cys in vitro and live cells based on fluorescent protein chromophores, which have been modulated by chemical reaction. Probe (RFP) is a fluorescence turn on probe with red emission wavelength and large Stokes shift (127 nm). This Michael-addition reaction-based probe exhibits low detection limit (18.7 μM), fast response time, and low cytotoxicity. RFP can differentiate the Cys and Hcy, using the difference of reaction time between probe and Cys or Hcy. Together, this probe exhibited excellent cell permeability, and can be successfully applied to detect the endogenous Cys in living cells.

Resonant enhancement of the nanocrystals fluorescence near the plasmonic film surface

Effective enhancement of the fluorescence signal of chromophores adsorbed directly onto plasmonic films can be observed under conditions of strong spectral resonance between plasmon and chromophore absorptions. This effect seems to contradict the established mechanisms of complete quenching of the fluorescence of chromophores under their adsorbtion directly onto the metal surface. However, under certain conditions, enhancement of the fluorescence signal is observed for both inorganic and organic chromophores. To understand the effect and conditions of its observation, we propose to use the quantum concept of virtual photon exchange in the near optical field - dressed photons. This concept is borrowed from the physics of elementary particles and is already well adapted to the problems of nanophotonics by M. Otsu. In this paper, we discuss exclusively the key factors responsible for enhancement of fluorescence of CdSe/ZnS nanocrystals and the effective dressed photons exchange: the size of nanoparticles, the distance between them, and the presence of spectral overlap indicating the possibility of resonant interactions between plasmons and chromophores. Communicated by Academician Sergei V. Gaponenko

Therapeutic ultrasound for improving the tropical corneal delivery of macromolecules

The goal of this project is to facilitate the delivery of topical drugs into the cornea and anterior segment of the eye using therapeutic ultrasound which could present a promising treatment for keratoconus and other corneal diseases. Each cornea is dissected and placed in a diffusion cell. smURFP-blue, a blue fluorescent chromophore, was used as the drug. The experimental groups of corneas were treated with 1 and 0.8 W/cm2 continuous ultrasound for 5 min at frequencies of 400 kHz and 600 kHz, respectively, then left in the diffusion cell for another 55 min. Fluorescence images of the fixed corneas were obtained to determine the relative amount of smURFP-blue that remained in the tissue. Safety of ultrasound application was tested by comparing the damage in the corneal layers. Spectroscopy measurements indicated no statistical difference in the presence of the chromophore in the receiver compartment in ultrasound- and sham-treatment groups. Preliminary results showed greater fluorescent intensity in the cornea when smURFP-blue is delivered with ultrasound compared to smURFP-blue added without ultrasound. The histology studies did not show any significant damage in ultrasound-treated corneas. This work may allow for the development of an inexpensive, clinically applicable, and minimally invasive ultrasound method for corneal drug delivery.

Dynamics of a family of cyan fluorescent proteins probed by incoherent neutron scattering.

Cyan fluorescent proteins (CFPs) are variants of green fluorescent proteins in which the central tyrosine of the chromophore has been replaced by a tryptophan. The increased bulk of the chromophore within a compact protein and the change in the positioning of atoms capable of hydrogen bonding have made it difficult to optimize their fluorescence properties, which took approximately 15 years between the availability of the first useable CFP, enhanced cyan fluorescent protein (ECFP), and that of a variant with almost perfect fluorescence efficiency, mTurquoise2. To understand the molecular bases of the progressive improvement in between these two CFPs, we have studied by incoherent neutron scattering the dynamics of five different variants exhibiting progressively increased fluorescence efficiency along the evolution pathway. Our results correlate well with the analysis of the previously determined X-ray crystallographic structures, which show an increase in flexibility between ECFP and the second variant, Cerulean, which is then hindered in the three later variants, SCFP3A (Super Cyan Fluorescent Protein 3A), mTurquoise and mTurquoise2. This confirms that increasing the rigidity of the direct environment of the fluorescent chromophore is not the sole parameter leading to brighter fluorescent proteins and that increased flexibility in some cases may be helpful.

Synthesis of Green Fluorescent Protein Chromophore Analogues for Interdisciplinary Learning for High School Students

Interdisciplinary outreach activities with easy-to-implement experiments have been developed to enhance high school students’ interest in chemistry and their inquisitive minds with active learning strategies. The synthesis of a model chromophore of the green fluorescent protein was employed as a key motif. Students simply enjoyed observing changes in the substances as well as witnessing a variety of fluorescent emissions. They gradually realized the importance of molecular design in the understanding and development of chemistry, biology, physics, and medicine.

Origin of the π-stacking induced shifts in absorption spectral bands of the green fluorescent protein chromophore

We present the results of computational studies of the shifts in absorption bands in the 4-hydroxybenzylidene-1,2-dimethylimidazolinone (HBDI), a synthetic analogue of the green fluorescent protein chromophore, induced by its π-stacking interaction with aromatic compounds. The set of model systems composed of HBDI in the anionic form π-stacked with the substituted derivatives of benzene is considered. Ground state equilibrium geometry parameters are obtained at the MP2/cc-pvdz level of theory and the vertical S0,min-S1 excitation energies are estimated using the XMCQDPT2/sa2-CASSCF(12/12) approach. It is shown that the bathochromic shifts are obtained for systems with smaller values of the dipole moment variation while the hypsochromic shifts correlate with larger values of this quantity. We also demonstrate that absolute values of the S0,min-S1 transition energy shift well correlate with the calculated bond interaction energy parameter of π-stacked complexes.

Template-Free Construction of Highly Ordered Monolayered Fluorescent Protein Nanosheets: A Bioinspired Artificial Light-Harvesting System.

Using biological materials for light-harvesting applications has attracted considerable attention in recent years. Such materials provide excellent environmental compatibility and often exhibit superior properties over synthetic materials. Herein, inspired by the outstanding energy transfer performance in coelenterates, we constructed a template-free, highly ordered two-dimensional light-harvesting system by covalent-induced coassembly of EBFP2 (donor) and EGFP (acceptor), in which the fluorescent chromophores were well distributed and adopted a fixed orientation. By introducing approximate square planar binding sites on the side surface of protein, assembly pattern was pin down and self-assembly extended in orthogonal directions to achieve monolayered and tessellated protein nanoarrays. The excellent antiself-quenching property of fluorescent proteins endowed the coassembled system with attractive light-harvesting capability. Even at high local concentrations, a low resonance energy transfer self-quenching was observed and, therefore, energy can be efficiently transferred. More importantly, the distance between adjacent chromophores is continuously adjustable. By making minor changes to the length of the inducing linker, we have achieved significant control over the size of the assembly. A micron-sized light-harvesting system with satisfactory energy transfer efficiency was finally obtained. This work developed a template-free light-harvesting system completely based on fluorescent proteins (FPs), which overcame the restriction of using templates. Not limited to this work, the special core-shell structure of FPs may be expected to direct the optimization of fluorescent dyes by cladding.

PH sensor potential and antimicrobial activity of a new PPA dendrimer modified with benzanthrone fluorophores in solution and on viscose fabric

The paper reports on the influence of pH on the absorption and fluorescence characteristics of a new poly(propylene amine) dendrimer (BD) from second generation modified with eight fluorescent benzanthrone chromophore units. Spectral characteristics have been determined using various percentages of the DMF-water mixture. The antimicrobial activity of the newly synthesized dendrimer BD has been determined in vitro against Gram-positive bacteria Bacillus cereus, Gram-negative bacteria Pseudomonas aeruginosa, and the yeasts Candida lipolytica used as test cultures. Viewing production of antibacterial textile, the antimicrobial activity of the dendrimer deposited on the surface of viscous fabric has also been investigated.

Highly emissive excitons with reduced exchange energy in thermally activated delayed fluorescent molecules

Unlike conventional thermally activated delayed fluorescence chromophores, boron-centered azatriangulene-like molecules combine a small excited-state singlet-triplet energy gap with high oscillator strengths and minor reorganization energies. Here, using highly correlated quantum-chemical calculations, we report this is driven by short-range reorganization of the electron density taking place upon electronic excitation of these multi-resonant structures. Based on this finding, we design a series of π-extended boron- and nitrogen-doped nanographenes as promising candidates for efficient thermally activated delayed fluorescence emitters with concomitantly decreased singlet-triplet energy gaps, improved oscillator strengths and core rigidity compared to previously reported structures, permitting both emission color purity and tunability across the visible spectrum.

Near-IR Photochemistry for Biology: Exploiting the Optical Window of Tissue.

Photoactive molecules enable much of modern biology and biochemistry-a vast library of fluorescent chromophores is used to track and label cellular structures and macromolecules. However, photochemistry is better known to the synthetic or physical organic chemist as a "light switch" that turns on unusual excited-state reactivity, isomerization, or dynamic adjustment of structure. This review details a rapidly growing approach to biophotochemistry that uses low-energy near-IR wavelengths not only for imaging, but also for close spatial control over chemical switching events in biosystems. Emphasis is placed on topics of biomedical interest: release of gaseous biological messengers, uncaging of drugs, nano-therapeutics, and modification of biomaterials.

Excited-state locked amino analogues of the green fluorescent protein chromophore with a giant Stokes shift.

We design a novel class of excited-state locked GFP chromophores by introducing an amine group at the double exo-bond and a difluoroboryl bridge. We show that these chromophores intrinsically exhibit a very large Stokes shift of 1 eV. Further tuning through chemical modifications of their aryl substituents makes them environmentally sensitive.

Why does a para-amino group make the green fluorescent protein chromophore non-fluorescent: coherent intramolecular charge transfer reduces the Z/E-photoisomerization barrier.

A para-N-phenyl-amino group significantly increases the fluorescence quantum yield of N-phenyl-4-aminostilbene by the "amino conjugation effect", but, in contrast, a para-amino group in the para-amino analogue (p-ABDI) of the green fluorescent protein (GFP) chromophore makes p-ABDI non-fluorescent because the coherent photo-induced intramolecular charge transfer reduces the barrier of the Z/E-photoisomerization.

A supramolecular assembly bearing an organic TADF chromophore: synthesis, characterization and light-driven cooperative acceptorless dehydrogenation of secondary amines.

A noble-metal-free chromophore-catalyst supramolecular assembly, which is composed of an organic thermally activated delayed fluorescence (TADF) chromophore and cobaloximes, has been designed and synthesized for the first time for the efficient acceptorless dehydrogenation of secondary amines under blue light irradiation at room temperature. The TADF chromophore has a long lifetime of 17.4 μs with suitable redox potentials for the selective acceptorless dehydrogenation of secondary amines to afford imines and H2 through cooperative catalysis of the chromophore and cobaloximes in the supramolecular assembly. A high TON of 895 was obtained for the acceptorless dehydrogenation of 1,2,3,4-tetrahydroisoquinoline despite its high oxidation potential (+1.38 V vs. SCE).

Ratiometric temperature sensing with fluorescent thermochromic switches.

The connection of fluorescent chromophores to switchable heterocycles translates into molecular probes with ratiometric response to temperature. The opening and closing of their heterocyclic component equilibrates two emissive species with resolved fluorescence. Their relative emission intensities change monotonically with temperature to enable the visualization of thermal distributions at the microscale.