Two-photon uncaging of bioactive thiols in live cells at wavelengths above 800 nm.

Photoactivatable protecting groups (PPGs) are useful for a broad range of applications ranging from biology to materials science. In chemical biology, induction of biological processes via photoactivation is a powerful strategy for achieving spatiotemporal control. The importance of cysteine, glutathione, and other bioactive thiols in regulating protein structure/activity and cell redox homeostasis makes modulation of thiol activity particularly useful. One major objective for enhancing the utility of photoactivatable protecting groups (PPGs) in living systems is creating PPGs with longer wavelength absorption maxima and efficient two-photon (TP) absorption. Toward these objectives, we developed a carboxyl- and dimethylamine-functionalized nitrodibenzofuran PPG scaffold (cDMA-NDBF) for thiol photoactivation, which has a bathochromic shift in the one-photon absorption maximum from λmax = 315 nm with the unfunctionalized NDBF scaffold to λmax = 445 nm. While cDMA-NDBF-protected thiols are stable in the presence of UV irradiation, they undergo efficient broad-spectrum TP photolysis at wavelengths as long as 900 nm. To demonstrate the wavelength orthogonality of cDMA-NDBF and NDBF photolysis in a biological setting, caged farnesyltransferase enzyme inhibitors (FTI) were prepared and selectively photoactivated in live cells using 850-900 nm TP light for cDMA-NDBF-FTI and 300 nm UV light for NDBF-FTI. These experiments represent the first demonstration of thiol photoactivation at wavelengths above 800 nm. Consequently, cDMA-NDBF-caged thiols should have broad applicability in a wide range of experiments in chemical biology and materials science.

Screening of Drug-Induced Steatosis and Phospholipidosis Using Lipid Droplet-Selective Two-Photon Probes.

Lipid droplets (LDs) are organelles that play a major role in regulating the storage of neutral lipids. Dysregulation of LDs is associated with metabolic disorders, such as fatty liver diseases, obesity, diabetes, and atherosclerosis. We have developed LD-selective small-molecule fluorescence probes (probes 3 and 4) that are available for both one- and two-photon microscopy, employing live or fixed cells. We found that probes 3 and 4 sensitively detect the increased LDs in response to oleic acid or endoplasmic reticulum stress, both in cells and tissues of the liver. The narrow absorption and emission bands of probes 3 and 4 allow multicolor imaging for the study of the role of LDs in pathophysiology and LD-associated signaling by the coapplication of the probes for different organelles or antibodies against specific proteins. In addition, we show here, for the first time, that two-photon microscopy imaging using our LD-selective probes with LysoTracker provides a novel method for screening drugs to potentially induce steatosis and/or phospholipidosis.

Identification of photodegraded derivatives of usnic acid with improved toxicity profile and UVA/UVB protection in normal human L02 hepatocytes and epidermal melanocytes.

This study focused on the investigation of photodegradation of usnic acid (UA) which is a natural UV absorbing agent in lichens. Despite years of historical use in food supplement, traditional medicine or cosmetic products in many countries, liver toxicity has been found to be one of the severe and life threatening adverse effects in early 2000's. Such severe side effect has limited UA or its synthesized derivatives for further use clinically or commercially. In this study, extracted UA from Usnea longissima in methanol was exposed to natural sunlight for 21 days. Five photodegraded derivatives (1 to 5) with two new and three previously explored compounds were isolated and purified by column chromatography and preparative liquid chromatography. The structures of these derivatives were identified based on the data of nuclear magnetic resonance spectrum, mass spectrum, optical rotation, infrared spectrum, X-ray crystallography and/or electronic circulation dichroism. The cytotoxicity of (+)-UA and 2 to 5 in liver L02 cells and melanocytes were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Experimental results indicated that IC50 of (+)-UA in liver L02 cells and melanocytes were 24.4 and 6.9 µM respectively, while compound 2 to 5 have lower cytotoxicity with IC50 of 326.7, 1085.0, 62.7 and 152.4 µM in L02 cells and 87.7, 297.7, 60.2 and 85.0 µM in melanocytes respectively. Besides, (+)-UA and these derivatives were exposed to fix dosed of UVA or UVB. The anti-UVA/UVB activity was determined via Hoechst33342/propidium iodide double staining method, and quantified by computer linked fluorescence microscope equipped with CellsSense Dimension system. Based on analysis, Compound 2 to 5 captured prominent UVA/UVB protection capacity in both hepatocytes and melanocytes (p < .001). In addition, the effects of chemicals on tyrosinase were evaluated via Western Blot analysis. In terms of tyrosinase expression, only 2 showed significant stimulating effect (p < .05). However, the safe use of these derivatives cutaneously should be further studied. In conclusion, the photodegraded derivatives (2 to 5) of extracted UA have lower hepatotoxicity than (+)-UA and captured significant UV protection activities.

A fluorescent nanoprobe for real-time monitoring of intracellular singlet oxygen during photodynamic therapy.

Sensing of intracellular singlet oxygen (1O2) is required in order to optimize photodynamic therapy (PDT). An optical nanoprobe is reported here for the optical determination of intracellular 1O2. The probe consists of a porous particle core doped with the commercial 1O2 probe 1,3-diphenylisobenzofuran (DPBF) and a layer of poly-L-lysine. The nanoparticle probes have a particle size of ~80 nm in diameter, exhibit good biocompatibility, improved photostability and high sensitivity for 1O2 in both absorbance (peak at 420 nm) and fluorescence (with excitation/emission peaks at 405/458 nm). Nanoprobes doped with 20% of DPBF are best suited even though they suffer from concentration quenching of fluorescence. In comparison with the commercial fluorescent 1O2 probe SOSG, 20%-doped DPBF-NPs (aged) shows higher sensitivity for 1O2 generated at an early stage. The best nanoprobes were used to real-time monitor the PDT-triggered generation of 1O2 inside live cells, and the generation rate is found to depend on the supply of intracellular oxygen. Graphical abstract A fluorescent nanoprobe featured with refined selectivity and improved sensitivity towards 1O2 was prepared from the absorption-based probe DBPF and used to real-time monitoring of the generation of intracellular 1O2 produced during PDT.

Two-Photon Tracer for Human Epidermal Growth Factor Receptor-2: Detection of Breast Cancer in a Live Tissue.

We have developed a two-photon fluorescent tracer (Pyr-affibody) that shows high selectivity for human epidermal growth factor receptor-2 (HER-2). Pyr-affibody showed absorption and emission maxima at 439 and 574 nm, respectively, with a two-photon absorption cross-section value of 40 × 10-50 cm4s/photon (GM) at 750 nm in aqueous buffer solution. The effective two-photon action cross-section value measured in HeLa cells was 600 GM at 730 nm, a value sufficient to obtain bright two-photon microscopy (TPM) images. Using Pyr-affibody, it was possible to detect HER-2 overexpressing cells and breast cancers at a depth of 90-130 µm in live mouse tissue by TPM.

Destruction kinetic of PCDDs/Fs in MSWI fly ash using microwave peroxide oxidation.

Microwave peroxide oxidation is a less greenhouse gas emission and energy-efficient technology to destroy toxic organic compounds in hazardous waste. The research novelty is to adopt the innovative microwave peroxide oxidation in H2SO4/HNO3 solution to efficiently destroy the polychlorinated dibenzo-p-dioxins (PCDDs)/Fs in municipal solid waste incineration fly ash. The major objective of this paper is to study dynamic destruction of PCDDs/Fs using the microwave peroxide oxidation. Almost all PCDDs/Fs in the raw fly ash can be destructed in 120 min at a temperature of 423 K using the microwave peroxide oxidation treatment. It was found that the microwave peroxide oxidation provides the potential to destruct the PCDDs/Fs content in municipal solid waste incinerator (MSWI) fly ash to a low level as a function of treatment time. A useful kinetic correlation between destruction efficiency and treatment conditions is proposed on the basis of the experimental data obtained in this study. The significance of this work in terms of practical engineering applications is that the necessary minimum treatment time can be solved using a proposed graphic illustration method, by which the minimum treatment time is obtained if the desired destruction efficiency and treatment temperature are known. Because of inorganic salt dissolution, the temperature would be a critical factor facilitating the parts of fly ash dissolution. Material loss problem caused by the microwave peroxide oxidation and the effects of treatment time and temperature are also discussed in this paper.

Lichens--photophysical studies of potential new sunscreens.

Fundamental photophysical properties have been obtained for six polyaromatics, calycine, usnic acid, vicanicine, 1-Cl-pannerine and epiphorelic acids I and II, extracted from Antarctic lichens--potential future sunscreens. None of the lichen compounds produced a measurable amount of triplet states and the singlet oxygen quantum yield was also very low ranging from 0.003 to 0.06. However, three exhibited triplet energy levels which may be above that of thymine. The radical cations of calycine and usnic acid were generated via pulse radiolysis and were observed to be quenched by vitamin C, vitamin E and Trolox.

Chemistry and folding of photomodulable peptides--stilbene and thioaurone-type candidates for conformational switches.

Optimized synthetic strategies for the preparation of photoswitchable molecular scaffolds based on stilbene or on thioaurone chromophores and their conformationally directing properties, as studied by computations and by NMR spectroscopy, are addressed. For the stilbene peptidomimetics 1, 2 and 3, the length of connecting linkers between the chromophore and the peptide strands was varied, resulting in photochromic dipeptidomimetics with various flexibility. Building blocks of higher rigidity, based on para-substituted thioaurone (4 and 6) and meta-substituted thioaurone chromophores (5 and 7) are shown to have a stronger conformationally directing effect. Design, synthesis, theoretical and experimental conformational analyses are presented.

Aqueous photodegradation and toxicity of the polycyclic aromatic hydrocarbons fluorene, dibenzofuran, and dibenzothiophene.

Decay kinetics resulting from the application of UV and UV/H(2)O(2) to the polycyclic aromatic hydrocarbons (PAHs) fluorene, dibenzofuran and dibenzothiophene was studied. Batch experiments were conducted with both low-pressure monochromatic (253.7nm) and medium pressure polychromatic (200-300nm) UV sources alone or in the presence of up to 25mg/L hydrogen peroxide, in a quasi-collimated beam apparatus. Degradation of all three PAHs, by both UV and UV/H(2)O(2), exhibited pseudo-first-order reaction kinetics and low quantum yields ranging from 1.4x10(-3) to 1.8x10(-2)mol/E using both UV lamps. Toxicity testing using a bioluminesence inhibition bioassay was correlated to the decay in concentration of the PAHs as analyzed analytically using HPLC. Results demonstrated that treatment efficacy of oxidative PAH degradation measured by following the decay of the target compound is best complemented by also evaluating the toxicity of the treated water due to byproduct formation concerns.

Photodegradation of PCDD/Fs adsorbed on spruce (Picea abies (L.) Karst.) needles under sunlight irradiation.

Spruce (Picea abies (L.) Karst.) needles were exposed to exhaust gas containing polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) generated by combustion of polyvinyl chloride, wood, high-density polyethylene, and styrene. Photodegradation of PCDD/Fs adsorbed on spruce needles under sunlight irradiation was studied. The photodegradation of PCDD/Fs follows pseudo-first-order reaction kinetics, with photolysis half-lives ranging between 40 and 100 h. The photolysis rates of PCDF congeners are higher than PCDD congeners with the same chlorinated substitutions. Higher chlorinated PCDD/Fs tend to photolyze slowly. The wax components in spruce needles may act as proton donors and accelerate the photolysis rate. C-Cl cleavage through the addition of protons to PCDD/F molecules may be an important route for PCDD/Fs photodegradation.

Studies on the mechanism of the hematoporphyrin-sensitized photooxidation of 1,3-diphenylisobenzofuran in ethanol and unilamellar liposomes.

The 5 microM hematoporphyrin-sensitized photooxidation of 1,3-diphenylisobenzofuran (DPBF) was studied in homogeneous ethanolic solutions and in aqueous dispersions of unilamellar liposomes of dipalmitoylphosphatidylcholine; both the porphyrin and DPBF are embedded in the phospholipid bilayer. The rate and quantum yield of DPBF photooxidation were found to increase upon increasing the substrate concentration and were higher in the liposome system, while they were unaffected by the fluidity of the phospholipid bilayer. Time-resolved spectroscopic measurements showed that the photooxidation of DPBF in ethanol solution proceeds by a type II O2(1 delta g)-involving mechanism. In the liposomal vesicles the high local concentration of hematoporphyrin (Hp) and DPBF in the phospholipid bilayer (ca 2000-fold higher than the stoichiometric concentration) enhances the probability of energy transfer from triplet Hp to DPBF with generation of triplet DPBF; hence O2 (1 delta g) formation can be promoted by both triplet Hp and triplet DPBF. A minor fraction of triplet DPBF quenchings appears to generate radical species which propagate DPBF damage by chain reaction.