Structure elucidation of laser dye coumarin-540A by joint application of X-ray diffraction, X-ray fluorescence, prompt fluorescence, UV and visible spectroscopy

X-ray, ultraviolet, and visible light induced photophysical changes of coumarin-540A in ethanol have been studied by the joint applications of X-ray, ultraviolet, and visible spectroscopy. Some impurities were found by X-ray fluorescence measurements. During the high power optical pumping, coumarin showed photochemical changes. Photoproduct emission spectra characteristics showed that photoproduct molecules can also be used as a laser dye at a different emission frequency in the ultraviolet region.     

Non-covalent interactions of coumarin dyes with cucurbit[7]uril macrocycle: modulation of ICT to TICT state conversion

Non-covalent interaction of coumarin laser dyes, namely coumarin-1 (C1), coumarin-481 (C481) and coumarin-6H (C6H), with a versatile macrocyclic host molecule cucurbit[7]uril (CB7), has been investigated in aqueous solution using photophysical methods. Steady-state and time-resolved fluorescence studies illustrate significant enhancements/modifications in the fluorescence yields, lifetimes and spectral features of C1, C481 and C6H on interaction with CB7, and are assigned to 1 : 1 complex formation between the dyes and the CB7 host. The complex formation is mainly driven by charge-dipole interaction, as evident from the binding constant values (K ~ 10(4)-10(5) M(-1)). The large changes in the excited state behaviour of C1 and C481 as compared to C6H in the presence of CB7 indicate that CB7 binds C1 and C481 through the encapsulation of the 7-N,N'-diethylamino group of the dyes and the structural rigidity imposed by this interaction dramatically alters the excited state properties of the dyes by preventing the conversion of their emissive intramolecular charge transfer (ICT) state to the non-radiative twisted intramolecular charge transfer (TICT) state. The present results direct towards the probable supramolecular approach using water soluble macrocyclic CB7, in the development of aqueous dye laser systems in the blue-green region.     

4—甲基—7—羟基香豆素的发光特性研究

对比考察了多种溶剂介质中不同浓度４－甲基－７－羟基香豆素溶液的荧光光谱特性，发现其谱形状与峰位不仅随溶剂性质而异，且随浓度而变，在某些氢键溶剂中，激发光谱随浓度有很大变化，且发射光谱间存在某种内在联系。基于溶剂本身的性质及４－甲基－７－羟基香豆素在氢键溶剂中的二聚作用讨论了这种现象，本文还考察了这种香豆素衍生物在滤纸及聚酰胺膜基质上，以Ｐｂ（Ａｃ）２或Ｐｂ（ＮＯ３）２作重原子微扰剂时的室温磷光特性     

Intriguing H-aggregate and H-dimer formation of coumarin-481 dye in aqueous solution as evidenced from photophysical studies

Photophysical properties of coumarin-481 (C481) dye in aqueous solution show intriguing presence of multiple emitting species. Concentration and wavelength dependent fluorescence decays and time-resolved emission spectra and area-normalized emission spectra suggest the coexistence of dye monomers, dimers, and higher aggregates (mostly trimers) in the solution. Because of the efficient intramolecular charge transfer (ICT) state to twisted intramolecular charge transfer (TICT) state conversion, the dye monomers show very short fluorescence lifetime of ~0.2 ns. Fluorescence lifetimes of dimers (~4.1 ns) and higher aggregates (~1.4 ns) are relatively longer due to steric constrain toward ICT to TICT conversion. Observed results indicate that the emission spectra of the aggregates are substantially blue-shifted compared to monomers, suggesting H-aggregation of the dye in the solution. Temperature-dependent fluorescence decays in water and time-resolved fluorescence results in water-acetonitrile solvent mixtures are also in support of the dye aggregation in the solution. Though dynamic light scattering studies could not recognize the dye aggregates in the solution due to their small size and low concentration, fluorescence up-conversion measurements show a relatively higher decay tail in water than in water-acetonitrile solvent mixture, in agreement with higher dye aggregation in aqueous solution. Time-resolved fluorescence results with structurally related non-TICT dyes, especially those of coumarin-153 dye, are also in accordance with the aggregation behavior of these dyes in aqueous solution. To the best of our knowledge, this is the first report on the aggregation of coumarin dyes in aqueous solution. Present results are important because coumarin dyes are widely used as fluorescent probes in various microheterogeneous systems where water is always a solvent component, and the dye aggregation in these systems, if overlooked, can easily lead to a misinterpretation of the observed results.     

Light-induced transformations of hematoporphyrin diacetate and hematoporphyrin

Illumination of hematoporphyrin diacetate (HP-Diac) and hematoporphyrin (HP) in phosphate-buffered solutions causes the formation of a stable photoproduct absorbing at 636 nm. Simultaneously the photo-oxidation of HP-Diac and HP takes place. These phototransformations depend on the illumination dose and the concentration of the HP-Diac or HP solution, and cause qualitatively the same spectral changes independently of the light source used.     

Determination of resveratrol in wine by photochemically induced second-derivative fluorescence coupled with liquid–liquid extraction

Basic studies on the photochemical behaviour of trans-resveratrol and its photoproduct are reported. Photometrically and fluorimetrically calculated acidity constants of the former were determined. The usefulness of the determination of resveratrol by photochemically induced fluorescence and second-derivative photochemically induced fluorescence was also examined. The very weakly fluorescent trans-resveratrol is converted into a highly fluorescent photoproduct by irradiating hydroethanolic solutions of trans-resveratrol containing 40% v/v of ethanol for 60 s with intense UV radiation. The photoproduct presents excitation and emission maxima centred at 260 nm, and 364 and 382 nm, respectively. Under these conditions, a linear relationship between fluorescence intensity and trans-resveratrol concentration was found between 6.6 and 66 ng mL⁻¹. Optimum conditions for the extraction of trans-resveratrol from an aqueous phase at pH 5.0 with diethylether were a phase ratio (aqueous/organic) of 2, a shaking time of 60 s and a buffer concentration of 0.15 mol L⁻¹. An extraction recovery of 100% was reached under these conditions. The optimized extraction procedure was applied to the analysis of resveratrol in wine samples, employing the amplitude between 356 and 364 nm of the second-derivative photoinduced emission spectrum as analytical signal. It was found that there is not matrix effect and recoveries around 100% were obtained at different fortification levels.     

Synthesis of Novel Coumarin-7,8-cyclophosphoramide Analogs

Coumarin-7,8-cyclophosphoramide derivatives were conveniently prepared by a facile method. 7-Hydroxycoumarin was aminomethylated regioselectively at the 8-C position using the Mannich reaction. The newly formed 8-(N-alkylaminomethyl) coumarins were then coupled with bis-β-chloroethyl dichlorophosphamide to form coumarin-7,8-cyclophosphoramide analogs. An efficient, highly regioselective method to synthesize coumarin-7,8-cyclophosphoramide derivatives is provided, and the approach has the merits of mild reaction conditions.     

Calculation of singlet oxygen dose using explicit and implicit dose metrics during benzoporphyrin derivative monoacid ring A (BPD-MA)-PDT in vitro and correlation with MLL cell survival

Photodynamic therapy (PDT) oxygen consumption, clonogenic cell survival, fluorescence photobleaching and photoproduct formation were investigated during benzoporphyrin derivative monoacid (BPD-MA)-PDT of MAT-LyLu cells in vitro. Cells were incubated with BPD-MA concentrations of 0.1, 0.5 or 2.5 μg mL(-1) for 2 h and then treated with 405 nm light under oxygenated and hypoxic conditions. Fluorescence spectra were acquired during treatment, and photobleaching and photoproduct generation were quantified using singular value decomposition of the spectra. Cell survival was measured at set times during the treatment using a colony-forming assay. The amount of oxygen consumed by PDT per photon absorbed decreased with BPD-MA intracellular concentration. Survival was correlated with the total amount of oxygen consumed by PDT per unit volume, which is assumed to be equivalent to the amount of singlet oxygen that reacted. A photobleaching-based singlet oxygen dose metric was also found to predict survival independent of intracellular BPD-MA concentration. The BPD-MA photoproduct was bleached during the treatment. Two singlet oxygen dose metrics based on photoproduct kinetics could not be correlated with cell survival over the full range of intracellular BPD-MA concentrations used.     

Preparation and in vitro properties of redox-responsive polymeric nanoparticles for paclitaxel delivery

Rice-like polymeric nanoparticles (NPs) composed of a new redox-responsive polymer, poly(ethylene glycol)-b-poly(lactic acid) (MPEG-SS-PLA), were prepared to carry paclitaxel (PTX) for glutathione (GSH)-regulated drug delivery. The PTX-loaded MPEG-SS-PLA NPs were fabricated using an optimized oil-in-water emulsion/solvent evaporation method. The size and morphology of the prepared NPs were characterized by scanning electron microscopy (SEM). The SEM results demonstrate that the NPs were dispersed as individual particles and were rice-shaped. The PTX loading efficiency, in vitro release, and stability of the NPs were analyzed by high-performance liquid chromatography (HPLC). The HPLC results revealed that the NPs released almost 90% PTX within 96 h when GSH presented at intracellular concentrations, whereas only a very small PTX amount was released at plasma GSH levels. The in vitro cytotoxicities of the NPs against A549, MCF-7, and HeLa carcinoma cells were assessed using a standard methyl thiazolyl tetrazoliun (MTT) assay. The MTT assay results show that the NPs caused concentration- and time-dependent changes in cell viability. To investigate the cellular uptake of the PTX-loaded NPs, visual endocytosis assay was performed using the fluorescent dye coumarin-6 as a model drug. The endocytosis assay results reveal rapid penetration and intracellular accumulation of coumarin-6-loaded NPs, as well as rapid coumarin-6 dispersion from the NPs. Overall, these findings establish that the NPs containing the synthesized redox-responsive polymer MPEG-SS-PLA can be used as potential carrier systems for antitumor drug delivery.     

Effects of a high-affinity antibody fragment on DNA polymerase reactions near a (6-4) photoproduct site

Pyrimidine (6-4) pyrimidone photodimers are major photoproducts that have mutagenic and carcinogenic consequences. One major reason for these biological effects of (6-4) photoproducts may be base mispairing/DNA replication errors due to hydrogen bonding to bases opposite these damaged sites. We synthesized a modified 41-mer DNA containing a (6-4) photoproduct using a preformed building block, then employed it as a template for primer extension reactions catalyzed by Klenow fragment and DNA polymerases alpha, beta and delta (pol alpha, pol beta and pol delta). None of these DNA polymerases were able to bypass the (6-4) photoproduct and elongation terminated at or near the 3'-pyrimidone of the photoproduct, depending on the dNTP concentration. When a single-chain Fv (scFv) with high affinity for the (6-4) photoproduct was included in the polymerization reaction, DNA synthesis was inhibited at base positions four, six, eight or eight nucleotides prior to the 3'-pyrimidone by Klenow fragment, pol alpha, pol beta or pol delta, respectively. These results suggest that the scFv can bind to the template DNA containing a (6-4) photoproduct and inhibit extension reactions by polymerases.     

EFFECT OF BASE SEQUENCE ON THE ULTRAVIOLET IRRADIATION PRODUCTS OF DOUBLE-STRANDED POLYNUCLEOTIDES CONTAINING BROMOURACIL AND ADENINE

Abstract —The effects of ultraviolet irradiation of double-stranded synthetic polynucleotides containing BrU and A have been investigated. Homopolymer pairs and alternating copolymers composed of either ribo- or deoxyribo-nucleotides were prepared and were irradiated with either 313 nm or ˜ 285 nm light. Strand separation and a modest amount of strand breakage followed irradiation of the homopolymer pairs. Changes in the ultraviolet absorption spectra of the polymers during irradiation reflected the sum of hyperchromic increases caused by progressive strand separation and loss of absorbance caused by photoproduct formation. Extensive debromination occurred. An RNase digest of irradiated poly(rA)–poly¹⁴C(rBrU), analysed by column chromatography, showed components similar to those found previously upon irradiation of single-stranded poly(rBrU). Little photoproduct was released by RNase digestion as mononucleotides. The major photoproduct was in the dinucleotide fraction, and may be 5,5'-diuracil. Base sequence had a profound effect on the sensitivity of the polynucleotides. Irradiation of alternating copolymers with doses of light comparable to those that produced major photochemical changes in the homopolymer pairs brought about little if any change in the copolymers of alternating base sequence.     

Structure determination of an interstrand-type cis-anti cyclobutane thymine dimer produced in high yield by UVB light in an oligodeoxynucleotide at acidic pH

UVB irradiation of DNA produces photodimers in adjacent DNA bases and on rare occasions in nonadjacent bases. UVB irradiation (312 nm) of d(GTATCATGAGGTGC) gave rise to an unknown DNA photoproduct in approximately 40% yield at acidic pH of about 5. This product has a much shorter retention time in reverse phase HPLC compared to known dipyrimidine photoproducts of this sequence. A large upfield shift of two thymine H6 NMR signals and photoreversion to the parent ODN upon irradiation with 254 nm light indicates that the photoproduct is a cyclobutane thymine dimer. Exonuclease-coupled MS assay establishes that the photodimer forms between T2 and T7, which was confirmed by tandem mass spectrometric MS/MS identification of the endonuclease P1 digestion product pd(T2[A3])=pd(T7[G8]). Acidic hydrolysis of the photoproduct gave a product with the same retention time on reverse phase HPLC and the same MS/MS fragmentation pattern as authentic Thy[ c,a]Thy. 2D NOE NMR data are consistent with a cis-anti cyclobutane dimer between the 3'-sides of T2 and T7 in anti glycosyl conformations that had to have arisen from an interstand type reaction. In addition to pH dependency, the photoproduct yield is highly sequence specific and concentration dependent, indicating that it results from a higher order folded structure. The efficient formation of this interstrand-type photoproduct suggests the existence of a new type of folding motif and the possibility that this type of photoproduct might also form in other folded structures, such as G-quadruplexes and i-motif structures which can be now studied by the methods described.     

Synthetic and Structural Studies on Novel 4,3′-Bicoumarins

Abstract

A series of directly linked 4-3′ bicoumarins have been synthesized by both Knoevenagel and Perkin reactions. This single-step transformation was accomplished by the reaction of coumarin-4-acetates with substituted salicylaldehydes in presence of piperidine using ethanol as solvent and by the reaction of coumarin-4-acetic acids with substituted salicylaldehydes in the presence of sodium hydride in acetic anhydride. Greater yields have been obtained in the sodium hydride and acetic anhydride condition. An intermediate chalcone with an ortho-hydroxyl group, which is a precursor for lactone formation, has been isolated and its structure has been confirmed by x-ray analysis. The nonplanar S-cis arrangement of two C4-C3’ double bonds has been confirmed by nuclear Overhauser spectroscopy (NOE) and x-ray studies. The UV-fluorescence studies support the formation of a conjugated bicoumarin system.     

PhI(OAc)2/I2-Mediated Decarboxylative C4-Amination of Coumarin-3-Carboxylic Acids via Csp2−H Dehydrogenative C−N Cross-Coupling under Ambient Conditions

An efficient and straightforward protocol for accessing a new series of functionalized 4-aminocoumarins from PIDA/I₂-mediated decarboxylative C₄-amination of coumarin-3-carboxylic acids via direct C_sp2−H dehydrogenative C−N cross-coupling with secondary amines under ambient conditions has been accomplished. The notable advantages of this protocol are the tolerance of diverse functional groups, mild reaction conditions at ambient temperature, moderate to good yields, short reaction times (in minutes), high regioselectivity, gram-scale applicability, and eco-friendliness. This is the first report of decarboxylative C_sp2−H cross-dehydrogenative C−N coupling of coumarin-3-carboxylic acids for synthesizing 4-aminocoumarins.     

Tryptophan Photolysis Leads to a UVB-lnduced 66 kDa Photoproduct of Ribulose-1,5 Bisphosphate Carboxylase/Oxygenase (Rubisco) In Vitro and In Vivo

Abstract— Proteins are vulnerable to environmental UVB (290-320 nm) because aromatic amino acids, particularly Trp, absorb in this spectral region. We have shown previously that UVB impacts on ribulose-l,5-bisphosphate carboxylase/oxygenase (Rubisco), resulting in the formation of a 66 kDa photoproduct in vivo (Wilson et al, Plant Physiol. 109,221–229, 1995). To determine if Trp photolysis is involved in the production of this specific protein photoproduct, the effects of UVB on a homogeneous preparation of Rubisco were examined. A UVB photoproduct of 66 kDa, identical to the in vivo product, was formed in vitro. The 66 kDa product was shown by immunological methods to be a cross-link between a large subunit (53 kDa) and a small subunit (14 kDa). Time-resolved Trp fluorescence was used to demonstrate that a Trp fluorescence signal is lost with kinetics that mirror the rate of formation of the 66 kDa photoproduct, indicating that a Trp photolysis step is involved in the mechanism of photoproduct formation. The relative rates of both Trp photolysis and 66 kDa photoproduct formation did not change with Rubisco concentration, consistent with a monomolecular reaction that would occur between sub-units within a Rubisco holoenzyme complex. Finally, formation of the 66 kDa photoproduct was found to be pH dependent.     

ABSORBANCE and CIRCULAR DICHROISM SPECTRA OF 1-C1S PHOTOPRODUCT FORMED BY IRRADIATING FROG RHODOPSIN

Abstract— We showed by spectrophotometry and HPLC that a photoproduct having 7-cis retinal (1-cis photoproduct) can be derived from the photoisomerization of frog lumirhodopsin (L) and metarhodopsin I (M I). The efficiency of the isomerization was higher in M I than in L. The absorption maximum of the 1-cis photoproduct at -20°C is at 455 nm, and its maximum absorbance 1.1 times as large as that of rhodopsin. The photoproduct exhibited two positive CD bands at 450 nm α-band) and 320 nm (β-band); the molecular ellipticity at a-band ([θ] = 73000) being larger than that of rhodopsin ([θ] = 61000). Re-examination of the absorption spectra of rhodopsin intermediates gave the absorption maxima of L. M 1 and M 111 to be 522, 482 and 475 nm, respectively.     

Photodegradation and Photobinding of Tiaprofenic Acid: in vitro Versus in vivo

Abstract— The photoreactivity of the photosensitizing nonsteroidal anti-inflammatory drug tiaprofenic acid (TA) and its photoproduct decarboxytiaprofenic acid (DTA) was studied both in the presence and in the absence of bovine serum albumin (BSA). The photoproduct DTA was found to be photostable in buffered aqueous solution at pH 7.4, but photodecomposed when BSA was present in the reaction medium. Both TA and DTA underwent irreversible photobinding to BSA in an almost quantitative way, as evidenced by radioactivity measurements using labeled (3H) compounds. In the case of TA, it has been proven that photobinding is mainly attributable to the phototoreactivity of in situ -generated DTA. The photo-degradation and photobinding of TA were also investigated in the epidermis in vivo. Rats were exposed to UVA after application of TA to their shaven dorsal skin. During the initial periods of irradiation, the amount of TA decreased sharply, and the yield of the corresponding photoproduct (DTA) reached a maximum. Prolonged irradiation led to photodegradation of DTA. In vivo photobinding was studied using 3H-TA. Photobinding took place slowly at the beginning, but its rate increased sharply after complete photoconversion of TA, when the photoproduct DTA reached the maximum concentration. Thereafter, the decrease of DTA was more pronounced than that of TA. This indicates thatalso in vivoDTA rather than TA is responsible for the photobinding to biomacromolecules in the viable layer of the epidermis. Overall, the above results suggest that irradiation of TA in buffered aqueous solution, in the presence of proteins, is a reasonable model system to study the photodegradation and photobinding behavior of this drug in vivo. From the qualitative point of view, the main conclusion is that DTA plays a key role both in vivo and in vitro: it is the major photoproduct, it undergoes further photodegradation upon prolonged irradiation, and it appears to be responsible for the photobinding process.     

Light‐Controlled Conformational Switch of an Aromatic Oligoamide Foldamer

An aromatic oligoamide sequence composed of a light‐responsive diazaanthracene‐based aromatic β‐sheet flanked by two variable diameter helical segments was prepared. Structural investigations revealed that such oligomers adopt two distinct conformations: a canonical symmetrical conformation with the two helices stacked above and below the sheet, and an unanticipated unsymmetrical conformation in which one helix has flipped to directly stack with the first helix. Photoirradiation of the foldamer led to the quantitative, and thermally reversible, formation of a single photoproduct resulting from the [4+4] cycloaddition of two diazaanthracenes within the aromatic β‐sheet. NMR and crystallographic studies revealed a parallel arrangement of the diazaanthracene photoproduct and a complete conversion into a symmetrical conformation requiring a rearrangement of all unsymmetrical conformers. These results highlight the potential of foldamers, with structures more complex than isolated helices, for the design of photoswitches showing nontrivial nanometer scale shape changes.     

AN EXPERIMENTAL STUDY OF THE CHANGES IN PIGMENTATION IN HUMAN SKIN IN VIVO WITH VISIBLE AND NEAR INFRARED LIGHT

Abstract— The skin of the lower inner arm of volunteers was irradiated, with a 390–1700 nm light source, through a fiber optic bundle for times of up to 1.2 × 10⁴ s and with powers of up to 0.35 W/cm². Simultaneously with the irradiation, spectra (390–720 nm) of the remitted intensity were measured, while a 5.0 cm in diameter area of the skin around the fiber bundle was maintained at constant temperature, within 0.2°C. The generation of a photoproduct was observed and measured as changes in the remitted intensity within 600 s (10 min) of the start of irradiation.
The photoproduct formed was characterized by a weak absorption in the blue part of the spectrum (400–450 nm), leading to a bluish appearance in the irradiated area only. The color change appears as a two step process. It starts with a "soluble" photoproduct, which disappears, within 24 h after irradiation, and an "insoluble" photoproduct which appears with irradiation greater than 3 ×10³ s (50 min). No spectral differences were detected between the two photoproducts. The "insoluble" photoproduct persists for periods of up to 8 weeks. The color change in the skin is immediate and there is no erythema associated with this color change.     

Action spectra for survival and spore photoproduct formation of Bacillus subtilis irradiated with short-wavelength (200-300 nm) UV at atmospheric pressure and in vacuo.

Spores of Bacillus subtilis are approximately ten times less likely to survive UV light irradiation in a vacuum than under atmospheric conditions. Photoproduct formation was studied in spores irradiated under ultrahigh vacuum (UHV) conditions and in spores irradiated at atmospheric pressure. In addition to the "spore photoproduct" 5-thyminyl-5,6-dihydrothymine (TDHT), which is produced in response to irradiation at atmospheric pressure, two additional photoproducts, known as the cis-syn and trans-syn isomers of thymine dimer, are produced on irradiation in vacuo. The spectral efficiencies for photoproduct formation in spores are reduced under vacuum conditions compared with atmospheric conditions by a factor of 2-6, depending on the wavelength. Because formation of TDHT does not increase after irradiation in vacuo, TDHT cannot be responsible for the observed vacuum effect. Vacuum specific photoproducts may cause a synergistic response of spores to the simultaneous action of UV light and UHV. An increased quantum efficiency, destruction of repair systems and formation of irreparable lesions are postulated for the enhanced sensitivity of B. subtilis spores to UV radiation in vacuo.