Photophysics of Halogenated Fluoresceins: Involvement of Both Intramolecular Electron Transfer and Heavy Atom Effect in the Deactivation of Excited States

The fluorescence quantum yield (Φ_f), fluorescence lifetime (τ_f), intersystem crossing quantum yield (Φ_isc) and redox potentials of seven halogenated fluoresceins in their dianion forms were measured and compared in methanol to get a deep insight into the effect of halogeno atoms on their photophysics. It is found that the heavy atom effect alone cannot explain the experimental results, as (1) Φ_f for chlorinated dyes exceeds that of fluorescein and close to unity, (2) the sum of Φ_f and Φ_isc for brominated and iodinated xanthene dyes is remarkably less than unity. The observations can be rationalized by the involvement of intramolecular photoinduced electron transfer, in which the benzoate acts as the electron donor while the xanthene moiety is the acceptor. The more negative reduction potential of excited singlet state for chlorinated fluoresceins results in their much smaller k_et, and hence higher Φ_f.     

Environmental effects on the aggregation of some xanthene dyes used in lasers

The effect of solvents, halo substituents and surfactants on the aggregation of xanthene dyes (fluorescein and eosin Y) has been investigated. It has been found that polar protic solvents promote the aggregation of both the dyes while polar aprotic solvents hinder the aggregation process. Apolar solvents can disintegrate the aggregates previously formed in aqueous medium. The nature of the driving forces for aggregation in the two dyes is different. Surfactants bearing charge opposite to the dye molecules facilitate aggregation. These observations bear direct relevance to the possible use of fluorescein and eosin Y as quantum yield standards, photosensitizers, laser dyes and biological labels.     

Trianionic 1,3,2-Dioxaborine-Containing Polymethines: Bright Near-Infrared Fluorophores

Trianionic polymethines of the A′-π-A-π-A-π-A′ type comprising dioxaborine rings (A) and different electron-accepting end groups (A′) have been synthesized. The obtained dyes absorb and emit light in the near-infrared region with remarkably high molar attenuation coefficients (ϵ up to 495 000 M⁻¹ cm⁻¹ in DMF) and fluorescence quantum yields (Φ_f up to 0.73 in DMF). Thus, the novel trianionic dyes stand among the brightest individual fluorophores known to date – with a magnitude of fluorescence brightness (ϵ⋅Φ_f) of 313 000 M⁻¹ cm⁻¹ in DMF. The synthesized dyes demonstrate a minor negative solvatochromism and small Stokes shifts. X-ray data reveal the nearly planar geometry of the trianionic chromophore. All the obtained compounds are stable in the solid state and in a solution, although the relative stability is much higher in polar aprotic than in protic solvents.     

Study of fluorescent aggregates of polymethine dyes

Absorption, fluorescence, and fluorescence excitation spectra have been studied for a number of anionic, cationic, and cationic-anionic polymethine dyes in low-polar and nonpolar solvents, as well as in binary mixtures of solvents differing in polarity. For most of the dyes studied, fluorescent aggregates have been found to form. Their broad fluorescence bands are located in the long-wave region with respect to those of the initial dyes. The quantum yield of the aggregate fluorescence is normally higher than that of the initial dyes. Fluorescence excitation spectra of some cationic-anionic dyes in nonpolar solvents disagree with their absorption spectra because of contact and solvent-separated ion pairs simultaneously present in the solution.Translated fromIzvestiya Akademii Nauk, Seriya Khimicheskaya, No. 1, pp. 69–75, January, 1993.     

THE PHOTOPHYSICAL CONSTANTS OF SEVERAL FLUORESCENT DYES PERTAINING TO ULTRASENSITIVE FLUORESCENCE SPECTROSCOPY

Abstract— The successful implementation of ultrasensitive fluorescence spectroscopy of biological and chemical species depends upon certain photophysical parameters associated with the fluorescent dye used in the investigation. These parameters include the fluorescence quantum efficiency, photodestruction quantum efficiency, absorption cross section and fluorescence lifetime. These photophysical constants were measured for several fluorescent dyes that are used for the tagging of biological species. Three different solvents, ethanol, water and a cationic surfactant used above its critical micelle concentration, were studied. The effective photon yield (ratio of the fluorescence quantum yield to the photodestruction quantum efficiency) for the dyes is nearly 100 times greater in ethanol than it is in water because of the superior photostabilities of these dyes in ethanol solvents. The implications of these parameters for the design of an ultrasensitive fluorescence experiment are discussed.     

Adsorption and photochemical behaviors of the novel cationic xanthene derivative on the clay surface

Novel tetra-cationic xanthene derivative (Flu) was synthesized. Its adsorption and photochemical behaviors on the clay surface were investigated. Fluorescence quantum yield (?_f) and fluorescence lifetime were 0.50 and 2.9 ns for Flu/clay complex. ?_f of Flu was enough high (>0.1) even at high density conditions (0.080 molecules nm⁻²). It is supposed that the strong interaction between clay and Flu by the ‘Size-Matching Effect’ realizes the highly emissive clay complexes at high density adsorption condition by a suppression of a molecular aggregation, which tends to decrease the photochemical activity.     

PHOTOPHYSICAL AND PHOTOSENSITIZING PROPERTIES OF BENZOPORPHYRIN DERIVATIVE MONOACID RING A (BPD-MA)*

The photophysical properties of benzoporphyrin derivative monoacid ring A (BPD-MA), a second-generation photosensitizer currently in phase II clinical trials, were investigated in homogeneous solution. Absorption, fluorescence, triplet-state, singlet oxygen (O₂(¹Δ_g)) sensitization studies and photobleaching experiments are reported. The ground state of this chlorin-type molecule shows a strong absorbance in the red (λ≈ 688 nm, ?≈ 33 000 M^?1 cm^?1 in organic solvents). For the singlet excited state the following data were determined in methanol: energy level, E_s= 42.1 kcal mol^?1, lifetime, Φ_f= 5.2 ns and fluorescence quantum yield, Φ_f= 0.05 in air-saturated solution. The triplet state of BPD-MA has a lifetime, τ_f >. 25 ns, an energy level, E_T= 26.9 kcal mol^?1 and the molar absorption coefficient is ?_T= 26 650 M^?1 cm^?1 at 720 nm. A dramatic effect of oxygen on the fluorescence (φ_f) and intersystem crossing (φ_T) quantum yields has been observed. The BPD-MA presents rather high triplet (φ_T= 0.68 under N₂-saturated conditions) and singlet oxygen (φ_Δ= 0.78) quantum yields. On the other hand, the presence of oxygen does not significantly modify the photobleaching of this photostable compound, the photodegradation quantum yield (φ_Pb) of which was found to be on the order of 5 × 10^?5 in organic solvents.     

Photophysics of Glycosylated Derivatives of a Chlorin,Isobacteriochlorin and Bacteriochlorin for Photodynamic Theragnostics: Discovery of a Two‐photon‐absorbing Photosensitizer

The photophysical properties of a chlorin, isobacteriochlorin and bacteriochlorin built on a core tetrapentafluorophenylporphyrin (TPPF₂₀) and the nonhydrolyzable para thioglycosylated conjugates of these chromophores are presented. The photophysical characterization of these compounds was done in three different solvents to correlate with different environments in cells and tissues. Compared with TPPF₂₀ other dyes have greater absorption in the red region of the visible spectrum and greater fluorescence quantum yields. The excited state lifetimes are from 3 to 11 ns. The radiative and nonradiative rate constants for deactivation of the excited state were estimated from the fluorescence quantum yield and excited state lifetime. The data indicate that the bacteriochlorin has strong absorption bands near 730 nm and efficiently enters the triplet manifold. The isobacteriochlorin has a 40–70% fluorescence quantum yield depending on solvent, so it may be a good fluorescent tag. The isobacteriochlorins also display enhanced two‐photon absorption, thereby allowing the use of 860 nm light to excite the compound. While the two‐photon cross section of 25 GM units is not large, excitation of low chromophore concentrations can induce apoptosis. The glycosylated compounds accumulate in cancer cells and a head and neck squamous carcinoma xenograft tumor model in mice. These compounds are robust to photobleaching.     

The absolute yield of bacteriochlorophyll fluorescence in vivo

Abstract— –The method of Weber and Teale for determining absolute fluorescence quantum yield of dyes in solution was modified for determination of the yield of bacteriochlorophyll fluorescence from chromatophores and whole cells of photosynthetic bacteria. Measured yields ranged from about 1–6 per cent. The yield depended on intensity and wavelength of the exciting light. The higher yield at higher light intensity was interpreted as due to saturation of photosynthesis. The lower yield in some strains when excited at 810 nm was attributed to preferential excitation of the reaction center pigment P800. From this study and the lifetime measurements of others, the relation τ=Q.τ₀ was substantiated for the fluorescence of bacteriochlorophyll in vivo, τ being the actual lifetime, τ₀ the intrinsic lifetime as estimated from the absorption band area, and Q the quantum yield of fluorescence.     

Viscosity dependence of the fluorescence lifetimes of cryptocyanine,pinacyanol and DDI

Viscosity dependence of the fluorescence lifetimes of cryptocyanine, pinacyanol and I, I'-diethyl-2,2'-dicarbocyanine iodide (DDI) has been studied by making relative quantum yield measurements in five solvents. It has also been established. by means of flash kinetic spectroscopy, that, in rigid solvents, φ^o_F+φ^o_IC ≈ 1 for all three dyes, where φ^o_F and φ^o_IC stand for the intrinsic quantum yields of fluorescence emission and internal conversion, respectively. Values of the intrisic rate constant of internal conversion are also presented.     

Spectroscopic Properties of Fluorescein and Rhodamine Dyes Attached to DNA

We report the spectroscopic properties of fluorescein, x-rhodamine, tetramethyl-rhodamine, attached to single strand, duplex DNA, and to the digestion products by DNAse I. The properties reported include: molar absorptivity, quantum yield, absorbance and fluorescence spectra, fluorescence lifetime, intrinsic lifetime (τ⁰), static quenching (S) and the Förster critical distances (R₀) between fluorescein and x-rhodamine or tetramethyl-rhodamine (acceptors). These spectroscopic properties depend strongly on the local dye environment. Fluorescein was studied: (1) attached to biotin (BF), (2) BF bound to avidin; and attached to two positions in DNA. X-rhodamine and tetramethyl-rhodamine were studied as free dyes and attached at the 5′-end of DNA. We propose a general method to determine the molar absorptivity and τ⁰ of a dye attached to DNA based on the reaction of a biotinylated and dye-labeled oligomer with standardized avidin. The molar absorptivity of a second dye attached to a DNA duplex can be obtained by comparing spectra of doubly and singly labeled sequences. S, arising from dye–DNA interactions can then be determined. R₀ for free and attached dyes showed differences from 1.1 to 4.2 Å. We present evidence for the direct interaction of dyes attached to the termini of various single-stranded DNA sequences.     

Quantitative Detection of G‐Quadruplex DNA in Live Cells Based on Photon Counts and Complex Structure Discrimination

G‐quadruplex DNA show structural polymorphism, leading to challenges in the use of selective recognition probes for the accurate detection of G‐quadruplexes in vivo. Herein, we present a tripodal cationic fluorescent probe, NBTE , which showed distinguishable fluorescence lifetime responses between G‐quadruplexes and other DNA topologies, and fluorescence quantum yield (Φ_f) enhancement upon G‐quadruplex binding. We determined two NBTE ‐G‐quadruplex complex structures with high Φ_f values by NMR spectroscopy. The structures indicated NBTE interacted with G‐quadruplexes using three arms through π–π stacking, differing from that with duplex DNA using two arms, which rationalized the higher Φ_f values and lifetime response of NBTE upon G‐quadruplex binding. Based on photon counts of FLIM, we detected the percentage of G‐quadruplex DNA in live cells with NBTE and found G‐quadruplex DNA content in cancer cells is 4‐fold that in normal cells, suggesting the potential applications of this probe in cancer cell detection.     

Fluorescence lifetimes and quantum yields of rhodamine derivatives: new insights from theory and experiment

Although lifetimes and quantum yields of widely used fluorophores are often largely characterized, a systematic approach providing a rationale of their photophysical behavior on a quantitative basis is still a challenging goal. Here we combine methods rooted in the time-dependent density functional theory and fluorescence lifetime imaging microscopy to accurately determine and analyze fluorescence signatures (lifetime, quantum yield, and band peaks) of several commonly used rhodamine and pyronin dyes. We show that the radiative lifetime of rhodamines can be correlated to the charge transfer from the phenyl toward the xanthene moiety occurring upon the S(0) ← S(1) de-excitation, and to the xanthene/phenyl relative orientation assumed in the S(1) minimum structure, which in turn is variable upon the amino and the phenyl substituents. These findings encourage the synergy of experiment and theory as unique tool to design finely tuned fluorescent probes, such those conceived for modern optical sensors.     

Fluorescent Properties of Some Thia- and Oxacarbocyanine Dyes in the Presence of DNA

Spectral and fluorescent properties of some thia- and oxacarbocyanine dyes were studied in the presence of DNA. Interaction of the dyes with DNA led to complex formation. A significant increase in the fluorescence quantum yield of the dyes in the presence of DNA was observed. The influence of a heteroatom and a substituent in the meso-position of the polymethine chain on the fluorescence growth was investigated. The stoichiometry of the strongly fluorescent dye–DNA complex was determined.     

A new class of laser dyes, 2-oxa-bicyclo[3.3.0]octa-4,8-diene-3,6-diones, with unity fluorescence yield

A new class of highly fluorescent dyes, 4,8-diphenyl-2-oxa-bicyclo[3.3.0]octa-4,8-diene-3,6-diones (1a-c), have been synthesized, they all exhibit unity fluorescence quantum yield and short radiative lifetime (< 4 ns) in common organic solvents and have demonstrated remarkable amplified spontaneous emission with a gain efficiency of > 10.     

Spectral luminescence properties of 1- and 2-alkyl(cycloalkyl) derivatives of benzo[f]quinoline

The absorption and fluorescence spectra of alcohol solutions of 3-aryl-substituted benzo[f]-quinolines were investigated, and the fluorescence quantum yields were measured. It was established that the indicated compounds have intense absorption in the UV region and fluorescence at 350–450 nm. The fluorescence quantum yields range from 5 to 70%, depending on the substituents. The introduction of aromatic substituents in the 1 and 3 positions of benzo-[f]quinoline ring raises the fluorescence quantum yields. A methyl group in the 2 position of the molecule leads to a decrease in the fluorescence quantum yield. Benzo[f]quinoline derivatives that contain a cyclopentene ring in the 1 and 2 positions fluoresce intensely (=40–60%), while cyclohexene and cycloheptene condensed in the same positions cause a decrease in the fluorescence yield to 7–13%; this is associated with the three-dimensional structure of these molecules.Translated from Khimiya Geterotsiklicheskikh Soedinenii, No. 9, pp. 1237–1240, September, 1977.     

Effect of halogenated fluorescent compounds on bioluminescent reactions

The paper investigates an application of luminescent bioassays to monitor the toxicity of organic halides. Effects of xanthene dyes (fluorescein, eosin Y, and erythrosin B), used as model compounds, on bioluminescent reactions of firefly Luciola mingrelica, marine bacteria Photobacterium leiognathi, and hydroid polyp Obelia longissima were studied. Dependence of bioluminescence quenching constants on the atomic weight of halogen substituents in dye molecules was demonstrated. Bacterial bioluminescence was shown to be most sensitive to heavy halogen atoms involved in molecular structure; hence, it is suitable for construction of sensors to monitor toxicity of halogenated compounds. Mechanisms of bioluminescence quenching—energy transfer processes, collisional interactions, and enzyme–dye binding—were considered. Changes of bioluminescence (BL) spectra in the presence of the dyes were analyzed. Interactions of the dyes with enzymes were studied using fluorescence characteristics of the dyes in steady-state and time-resolved experiments. The dependences of fluorescence anisotropy of enzyme-bound dyes, the average fluorescence lifetime, and the number of exponential components in fluorescence decay on the atomic weight of halogen substituents were demonstrated. The results are discussed in terms of “dark effect of heavy halogen atom” in the process of enzyme–dye binding; hydrophobic interactions were assumed to be responsible for the effect.     

Photocytotoxicity of the Fluorescent Nonsteroidal Androgen Receptor Ligand TDPQ†

1,2,3,4‐tetrahydro‐2,2‐dimethyl‐6‐(trifluoromethyl)‐8‐pyridono[5,6‐g]quinoline (TDPQ), a selective nonsteroidal androgen receptor (AR) ligand, is a fluorescent compound. We characterized its spectral properties in comparison with the structural precursor carbostyril 151 (C151) and with its racemic structural isomer 4‐ethyl‐1,2,3,4‐tetrahydro‐6‐(trifluoromethyl)‐8‐pyridino[5,6‐g]quinoline (ETPQ). The absorption maximum in CH₃CN of either TDPQ or ETPQ is 400 nm whereas that of C151 is 350 nm. The fluorescence lifetimes (τ) and quantum yields (?_f) in CH₃CN are typical of fluorescent dyes: TDPQ (4.2 ns, 0.8) and ETPQ (4.6 ns, 0.76). C151 showed lower τ and ?_f of 0.2 ns and 0.02, respectively. TDPQ can function as a fluorescent label at (sub)micromolar concentrations. We detected TDPQ fluorescence in human breast tumor cells using confocal microscopy. While the fluorescence maxima of the compounds were solvent insensitive, the ?_f for ETPQ decreased in aqueous solutions regardless of the presence of albumin or DNA. The ?_f of TDPQ was less affected. The quantum yield of singlet oxygen (¹O₂) photosensitization (?_so) by TDPQ and ETPQ was about 7% in CH₃CN, sufficient to induce photocytotoxicity. TDPQ was photocytotoxic in AR‐positive MDA‐MB‐453 breast cancer cells but not in AR‐negative MDA‐MB‐231 cells. The combination of AR selectivity with photocytotoxicity makes TDPQ a promising candidate for selective targeting of AR‐positive cells during photodynamic therapy.     

Quantitative Detection of G-Quadruplex DNA in Live Cells Based on Photon Counts and Complex Structure Discrimination

G-quadruplex DNA show structural polymorphism, leading to challenges in the use of selective recognition probes for the accurate detection of G-quadruplexes in vivo. Herein, we present a tripodal cationic fluorescent probe, NBTE , which showed distinguishable fluorescence lifetime responses between G-quadruplexes and other DNA topologies, and fluorescence quantum yield (Φ_f) enhancement upon G-quadruplex binding. We determined two NBTE -G-quadruplex complex structures with high Φ_f values by NMR spectroscopy. The structures indicated NBTE interacted with G-quadruplexes using three arms through π–π stacking, differing from that with duplex DNA using two arms, which rationalized the higher Φ_f values and lifetime response of NBTE upon G-quadruplex binding. Based on photon counts of FLIM, we detected the percentage of G-quadruplex DNA in live cells with NBTE and found G-quadruplex DNA content in cancer cells is 4-fold that in normal cells, suggesting the potential applications of this probe in cancer cell detection.     

Deuterium effects on radiationless electronic transitions of xanthene dyes in aqueous and alcoholic solutions

Solvent deuteration effects on internal conversion (S₁ ～→ S₀) and intersystem crossing (S₁ ～→ T₁) in 6-hydroxy-9-phenylfluoron and fluorescein were determined from fluorescence and triplet quantum yield measurements in alkaline solutions of H₂O, D₂O, CH₃OH, CH₃OD, C₂H₅OH, and C₂D₅OD. Deuterium substitution of the oxygen-bonded hydrogens of these solvenis reduces both the internal conversion (by approximately 1.6) and the intersystem crossing (by approximately 1.2) rate constants; the additional deuteration of the alkyl groups of the alcohols does not produce any further effect. The effect of solvent deuteration was negligible for fluorescein. Deuteration of the xanthene groups of the dyes does not influence the radiationless transition probabilities to any significant extent.