Mitochondria- and nucleolus-targeted copper(i) complexes with pyrazole-linked triphenylphosphine moieties for live cell imaging.

The labelling and imaging of mitochondria and nucleolus have attracted great attention because of the involvement of these cellular organelles in critical cellular activities. Therefore, a large number of mitochondria- or nucleolus-labelling probes have been developed throughout the world. However, in the current study, we successfully developed two pyrazole-based, copper-linked triphenylphosphine-coupled emissive metallo-complexes (C1 and C2) for the simultaneous visualization of mitochondria and nucleolus in a single run. These complexes were very inexpensive and could be synthesized by a simple one-pot multicomponent reaction scheme. The complexes were very specific, and only a small concentration of 5 µM was found to be sufficient to probe both the organelles efficiently. Additionally, even under a shorter incubation period (half hour), the fluorescence intensity from the cells was appreciable. Also, both the compounds were found to be photostable when torched with 10% of a 100 mW laser for up to 10 min. All these results indicate that both the complexes may contribute towards the future development of cell imaging tools. To the best of our knowledge, this is the first report on the development of multifunctional live cell imaging tools for simultaneous mitochondria and nucleolus imaging and within the shortest incubation time of about 30 minutes.

Integration of a photocleavable element into DNA nanoswitches.

Reconfigurable DNA nanostructures can be designed to respond to external stimuli such as nucleic acids, pH, small molecules and enzymes. In this study, we incorporated photocleavable linkers in DNA strands that trigger a conformational change in binary DNA nanoswitches. We demonstrate control of the output using UV light, with potential applications in biosensing and molecular computation.

A water soluble donor-acceptor-donor conjugated oligomer as a photosensitizer for mitochondria-targeted photodynamic therapy.

We designed a water soluble benzotriazole-cored donor-acceptor-donor molecule bearing four triphenylphosphonium moieties for mitochondria targeting. The organic molecule showed strong singlet oxygen sensitizing capability (ΦΔ 0.64), minimal dark cytotoxicity and high photocytotoxicity. The molecule is efficient in photodynamic killing of tumour cells under normal and hypoxia conditions using a very low light flux, and has potential in near-infrared two-photon imaging and photodynamic therapy.

Mitochondrial Specific H2S n Fluorogenic Probe for Live Cell Imaging by Rational Utilization of a Dual-Functional-Photocage Group.

Reactive sulfur species play a very important role in modulating neural signal transmission. Hydrogen polysulfides (H2S n, n > 1) are recently suggested to be the actual signaling molecules. There are still few spatiotemporal controllable-based probes to detect H2S n. In this work, for the first time, we proposed the photocleavage product of the common photoremovable protecting group (2-nitrophenyl moiety) capable of trapping H2S n. Taking advantage of this, we constructed the probe H1 containing a photocontrollable group, a mitochondrial directing unit and a signal reporter fluorescein dye. H1 exhibited excellent fluorescence enhancement (50-fold) in response to H2S n under the aqueous buffer only after UV irradiation. H1 also showed high selectivity and sensitivity for H2S n over other reactive sulfur species, reactive oxygen species, and other analytes, especially biothoils including hydrogen sulfide, cysteine, homocysteine, and glutathione. We showed the utility of H1 to image H2S n in living cells with high spatiotemporal resolution.

Tuning the Hydrophobicity of a Mitochondria-Targeted NO Photodonor.

A few compounds in which the nitric oxide (NO) photodonor N-[4-nitro-3-(trifluoromethyl)phenyl]propane-1,3-diamine is joined to the mitochondria-targeting alkyltriphenylphosphonium moiety via flexible spacers of variable length were synthesized. The lipophilicity of the products was evaluated by measuring their partition coefficients in n-octanol/water. The obtained values, markedly lower than those calculated, are consistent with the likely collapsed conformation assumed by the compounds in solution, as suggested by molecular dynamics simulations. The capacity of the compounds to release NO under visible light irradiation was evaluated by measuring nitrite production by means of the Griess reaction. The accumulation of compounds in the mitochondria of human lung adenocarcinoma A549 cells was assessed by UPLC-MS. Interestingly, compound 13 [(9-((3-((4-nitro-3-(trifluoromethyl)phenyl)amino)propyl)amino)-9-oxononyl) triphenylphosphonium bromide] displayed both the highest accumulation value and high toxicity toward A549 cells upon irradiation-mediated NO release in mitochondria.

UV/H2O2 advanced oxidation for abatement of organophosphorous pesticides and the effects on various toxicity screening assays.

Advanced oxidation processes (AOPs) are utilized due to their ability to treat emerging contaminants with the fast reacting and non-selective hydroxyl radical (OH). Organophosphorous insecticides are common drinking water contaminants, with 12 different compounds of this class being found on the US EPA's most recent Candidate Contaminant List (CCL4). The use of the AOP UV/H2O2 for the treatment of organophosphorous insecticides was explored in this study, by coupling biological and analytical tools to follow the abatement of the target compounds. Four insecticides were explored for advanced oxidation treatment: acephate, dicrotophos, fenamiphos, and methamidophos. All four compounds were fast reacting with OH, all reacting with second order rate constants ≥5.5 × 109 M-1s-1. Three major endpoints of toxicity were studied: estrogenicity, genotoxicity (mutagenicity) and neurotoxicity. None of the target compounds showed any estrogenic activity, while all compounds showed an active genotoxic (mutagenic) response (AMES II assay) and most compounds had some level of neurotoxic activity. AOP treatment did not induce any estrogenic activity, and reduced the compounds' neurotoxicity and genotoxicity in all but one case. Methamidophos degradation by UV/H2O2 resulted in an increase in genotoxicity, likely due to the formation of toxic transformation products. The increase in toxicity gradually decreased with time, possibly due to hydrolysis of the transformation products formed. This study provides insights into parent compound abatement and the changes in toxicity due to transformation products.

Can activated sludge treatments and advanced oxidation processes remove organophosphorus flame retardants?

This study aims to determine the occurrence of 10 OPFRs (including chlorinated, nonchlorinated alkyl and aryl compounds) in influent, effluent wastewaters and partitioning into sludge of 5 wastewater treatment plants (WWTP) in Catalonia (Spain). All target OPFRs were detected in the WWTPs influents, and the total concentration ranged from 3.67 µg L(-1) to 150 µg L(-1). During activated sludge treatment, most OPFRs were accumulated in the sludge at concentrations from 35.3 to 9980 ng g(-1) dw. Chlorinated compounds tris(2-chloroethyl) phosphate (TCEP), tris(2-chloroisopropyl) phosphate (TCIPP) and tris(2,3-dichloropropyl) phosphate (TDCPP) were not removed by the conventional activated sludge treatment and they were released by the effluents at approximately the same inlet concentration. On the contrary, aryl compounds tris(methylphenyl) phosphate (TMPP) and 2-ethylhexyl diphenyl phosphate (EHDP) together with alkyl tris(2-ethylhexyl) phosphate (TEHP) were not detected in any of the effluents. Advanced oxidation processes (UV/H2O2 and O3) were applied to investigate the degradability of recalcitrant OPFRs in WWTP effluents. Those detected in the effluent sample (TCEP, TCIPP, TDCPP, tributyl phosphate (TNBP), tri-iso-butyl phosphate (TIBP) and tris(2-butoxyethyl) phosphate (TBOEP)) had very low direct UV-C photolysis rates. TBOEP, TNBP and TIBP were degraded by UV/H2O2 and O3. Chlorinated compounds TCEP, TDCPP and TCIPP were the most recalcitrant OPFR to the advanced oxidation processes applied. The study provides information on the partitioning and degradability pathways of OPFR within conventional activated sludge WWTPs.

Microwave-assisted preparation of nucleoside-phosphoramidites.

Microwave-assisted phosphitylation of sterically hindered nucleosides is demonstrated to be an efficient method for the preparation of corresponding phosphoramidites (otherwise onerous under standard conditions) and is shown to be general in its applicability.

Determination of carbamate and organophosphorus pesticides in vegetable samples and the efficiency of gamma-radiation in their removal.

In the present study, the residual pesticide levels were determined in eggplants (Solanum melongena) (n = 16), purchased from four different markets in Dhaka, Bangladesh. The carbamate and organophosphorus pesticide residual levels were determined by high performance liquid chromatography (HPLC), and the efficiency of gamma radiation on pesticide removal in three different types of vegetables was also studied. Many (50%) of the samples contained pesticides, and three samples had residual levels above the maximum residue levels determined by the World Health Organisation. Three carbamates (carbaryl, carbofuran, and pirimicarb) and six organophosphates (phenthoate, diazinon, parathion, dimethoate, phosphamidon, and pirimiphos-methyl) were detected in eggplant samples; the highest carbofuran level detected was 1.86 mg/kg, while phenthoate was detected at 0.311 mg/kg. Gamma radiation decreased pesticide levels proportionately with increasing radiation doses. Diazinon, chlorpyrifos, and phosphamidon were reduced by 40-48%, 35-43%, and 30-45%, respectively, when a radiation strength of 0.5 kGy was utilized. However, when the radiation dose was increased to 1.0 kGy, the levels of the pesticides were reduced to 85-90%, 80-91%, and 90-95%, respectively. In summary, our study revealed that pesticide residues are present at high amounts in vegetable samples and that gamma radiation at 1.0 kGy can remove 80-95% of some pesticides.

Zinc(II) phthalocyanines immobilized in mesoporous silica Al-MCM-41 and their applications in photocatalytic degradation of pesticides.

In the present study the authors investigated a set of three new zinc(II) phthalocyanines (zinc(II) tetranitrophthalocyanine (ZnTNPc), zinc(II) tetra(phenyloxy)phthalocyanine (ZnTPhOPc) and the tetraiodide salt of zinc(II)tetra(N,N,N-trimethylaminoethyloxy) phthalocyaninate (ZnTTMAEOPcI)) immobilized into Al-MCM-41 prepared via ship-in-a-bottle methodology. The samples were fully characterized by diffuse reflectance-UV-vis spectroscopy (DRS-UV-vis), luminescence, thermogravimetric analysis (TG/DSC), N(2) adsorption techniques and elemental analysis. A comparative study was made on the photocatalytic performance upon irradiation within the wavelength range 320-460nm of these three systems in the degradation of pesticides fenamiphos and pentachlorophenol. ZnTNPc@Al-MCM-41 and ZnTTMAEOPcI@Al-MCM-41 were found to be the most active systems, with the best performance observed with the immobilized cationic phthalocyanine, ZnTTMAEOPcI@Al-MCM-41. This system showed high activity even after three photocatalytic cycles. LC-MS product characterization and mechanistic studies indicate that singlet oxygen ((1)O(2)), produced by excitation of these immobilized photosensitizers, is a key intermediate in the photocatalytic degradation of both pesticides.

Phototransformation of selected organophosphorus pesticides: roles of hydroxyl and carbonate radicals.

The phototransformation of two organophosphorus pesticides, parathion and chlorpyrifos, by hydroxyl radicals and carbonate radicals in aqueous solution were studied. Addition of hydrogen peroxide increased the UV degradation rates of both pesticides and data were simulated through kinetic modeling. The second-order rate constants of parathion and chlorpyrifos with hydroxyl radical were determined to be 9.7 +/- 0.5 x 10(9) and 4.9 +/- 0.1 x 10(9) M(-1) s(-1), respectively. The presence of bi/carbonate ions reduced the pesticide degradation rates via scavenging of hydroxyl radical but the formation of carbonate radical also contributed to the degradation of the pesticides with second-order reaction rate constants of 2.8 +/- 0.2 x 10(6) and 8.8 +/- 0.4 x 10(6) M(-1) s(-1) for parathion and chlorpyrifos, respectively. The dual roles of bicarbonate ion in UV/H2O2 treatment systems, i.e., scavenging of hydroxyl radicals and formation of carbonate radicals, were examined and discussed using a simulative kinetic model. The transformation of pesticides by carbonate radicals at environmentally relevant bi/carbonate concentrations was shown to be a significant contributor to the environmental fate of the pesticides and it reshaped the general phototransformation kinetics of both pesticides in UV/H2O2 systems.

Removal of high concentration dimethyl methylphosphonate in the gas phase by repeated-batch reactions using TiO2.

The aim of our study is to develop apparatuses that use TiO(2) for effective decontamination of air contaminated by Sarin gas. We performed photocatalytic decomposition of gaseous dimethyl methylphosphonate (DMMP) by TiO(2) and identified the oxidization products. The high activity of TiO(2) (0.01 g) was observed under UV-light irradiation and high concentration DMMP (33.5 microM) was removed rapidly. On the other hand, DMMP was not decreased under UV-light irradiation without TiO(2). This indicates that photocatalytic treatment is very effective for the removal of DMMP. Methanol, formaldehyde, formic acid, methyl formate, CO, CO(2) and H(2)O were detected as the primary products. In the gas phase, no highly poisonous substances were detected. In order to examine the performance of photocatalytic activity during long-term reactions, we performed photocatalytic decomposition by repeated-batch reactions using TiO(2). High photocatalytic activities decreased gradually. Meanwhile, the strong adsorption of TiO(2) against DMMP was observed as photocatalytic activities decreased. During the repeated-batch reactions with the sample scaled up (TiO(2): 0.1g), the total amount of removed DMMP reached 968.5 microM by both photocatalytic decomposition and the strong adsorption of TiO(2). These results suggest the possibility of removing large amounts of DMMP.

A novel estrogenic compound transformed from fenthion under UV-A irradiation.

The photo-transformed products of fenthion well-known as one of the most photosensitive organophosphorus insecticides and their estrogenic activities were investigated using a yeast two-hybrid assay incorporating the human estrogen receptor alpha (hERalpha). We identified fenthion sulfoxide and 3-methyl-4-methylsulfinylphenol (MMS) as the major transformed products and 3-methyl-4-(methylthio)phenol (MMP) as the minor product under UV-A irradiation. Further, significant estrogenic activity was observed in the solution irradiated for 160 min; this activity was evaluated as 18 pM converted to 17beta-estradiol (E(2)) equivalent concentration. By using authentic standards, it was found that MMP possessed weak estrogenic activity; its activity was evaluated as 1.7x10(-6) times compared with that of E(2). However, it was also revealed that the activity due to MMP was only 13%. From high-performance liquid chromatography (HPLC) and nuclear magnetic resonance (NMR) spectroscopies, we newly identified a significant estrogenic compound transformed from fenthion, O,O-dimethyl S-[3-methyl-4-(methylthio)phenyl]phosphorothioate, S-aryl fenthion.

Photochemically induced RNA and DNA abasic sites.

Two phosphoramidite building blocks were synthesized that can easily be deprotected by UV light to reveal natural abasic sites in oligoribonucleotides as well as in oligodeoxyribonucleotides. Another building block which releases a 2'-O-methylated abasic site upon UV radiation is also described.

Model study on the pyridine-Dewar pyridine and some related photoisomerization reactions.

The potential energy surfaces corresponding to the photochemical reactions of pyridine, phosphinine, and arsabenzene have been investigated by employing the CAS(6,6)/6-311G(d,p) and MP2-CAS-(6,6)/6-311++G(3df,3pd)//CAS(6,6)/6-311G(d,p) methods. The thermal (or dark) reactions of these reactant species have also been examined using the same level of theory. The mechanisms of drastic structural change in the excited- and ground-state reactions of pyridine, phosphinine, and arsabenzene and the differences between them are elucidated. The theoretical investigations suggest that conical intersections play a crucial role in their photoisomerization reactions. The results obtained allow a number of predictions to be made.

Effect of microwave irradiation on phosphoramidite couplings on controlled pore glass.

The chain extension step in the synthesis of DNA oligomers on controlled pore glass was shown to be higher yielding when the reaction mixture is irradiated with microwaves. Both a commercial thymidine 3'-phosphoramidite building block and a 3'-phosphoramidite of protected 1'-aminomethylthymidine were coupled using dilute solutions that give only partial conversion. In either case, higher coupling yields were observed when microwaves were used. The results of our exploratory experiments suggest that microwave-assisted DNA syntheses might require fewer equivalents of phosphoramidites and/or shorter coupling times than those performed at room temperature.

Lipid-mediated light activation of a mechanosensitive channel of large conductance.

This paper describes the reversible activation of a mechanosensitive channel via a light-sensitive lipid mimic. For these experiments, the mechanosensitive channel of large conductance (MscL) protein from Lactococcus lactis and Escherichia coli was reconstituted in lipid bilayers composed of 80 mol % 1,2-dioleoyl-sn-glycero-3-phosphocholine and 20 mol % di-(5-[[4-(4-butylphenyl)azo]phenoxy]pentyl)phosphate (4-Azo-5P). Light-induced isomerization of the azobenzene moiety of 4-Azo-5P from trans to cis was used to activate MscL.

Analysis of violet-excited fluorochromes by flow cytometry using a violet laser diode.

BACKGROUND: Low power violet laser diodes (VLDs) have been evaluated as potential replacements for water-cooled argon-ion and krypton-ion ultraviolet and violet lasers for DNA content analysis using the Hoechst dyes and 4,6-diamidino-2-phenylindole (Shapiro HMN, Perlmutter NG: Cytometry 44:133-136, 2001). In this study, we used a VLD to excite a variety of violet-excited fluorescent molecules important in biomedical analysis, including the fluorochromes Cascade Blue and Pacific Blue, the expressible fluorescent protein cyan fluorescent protein (CFP), and the fluorogenic alkaline phosphatase (AP) substrate 2-(5'-chloro-2'-phosphoryloxyphenyl)-6-chloro-4-(3H)-quinazoline (ELF-97; for endogenous AP detection and cell surface labeling with AP-conjugated antibodies). METHODS: Comparisons were made between VLD excitation and a krypton-ion laser emitting at 407 nm (both at higher power levels and with the beam attenuated at levels approximating the VLD) on the same FACSVantage SE stream-in-air flow cytometer. We evaluated a Power Technology 408-nm VLD (30 mW) equipped with circularization optics (18 mW maximum output, set to 15 mW) and a Coherent I-302C krypton-ion laser emitting at power levels ranging from 15 to 75 mW. RESULTS: Cascade Blue, Pacific Blue, and CFP showed comparable signal-to-noise ratios and levels of sensitivity with VLD excitation versus the krypton-ion laser at high and VLD-matched power outputs. Multicolor fluorescent protein analysis with 488-nm excitation of green fluorescent protein and DsRed and VLD excitation of CFP was therefore feasible and was demonstrated. Similar levels of excitation efficiency between krypton-ion and VLD sources also were observed for ELF-97 detection. CONCLUSIONS: These evaluations confirmed that VLDs may be cost- and maintenance-effective replacements for water-cooled gas lasers for applications requiring violet excitation in addition to DNA binding dyes.

Studies on the mechanisms of activation of indolequinone phosphoramidate prodrugs.

Previously a series of 2- and 3-substituted indolequinone phosphoramidate prodrugs was synthesized, and the compounds were shown to be nanomolar inhibitors of cell proliferation. The activation of these compounds following both one- and two-electron reduction has been investigated. (31)P NMR experiments demonstrated that both series of compounds undergo rapid activation following two-electron reduction. Additionally, the 3-series of compounds undergo rapid activation following one-electron reduction, while activation of the 2-series of compounds via this mechanism is very slow. The activation of these prodrugs by direct displacement using sulfur nucleophiles such as glutathione has been examined. Activation via this route is rapid for the 3-regioisomers, but is considerably slower for the 2-substituted analogues under similar conditions. Together these findings suggest that drug delivery via two-electron reduction from the 2-position is the more selective prodrug strategy.

A psoralen-conjugated triplex-forming oligodeoxyribonucleotide containing alternating methylphosphonate-phosphodiester linkages: synthesis and interactions with DNA.

A psoralen-conjugated oligodeoxyribopyrimidine (1443), PS-pTTTTCTTTTCTTCTT, where PS is trimethylpsoralen and C is 5-methyl-2'-deoxycytidine, that contains alternating methylphosphonate-phosphodiester internucleotide linkages was synthesized. The ability of 1443 to form triple-stranded complexes with a purine tract in a synthetic DNA duplex was studied. Irradiation of solutions containing the DNA target and 10 microM 1443 or 0.25 microM of a similar psoralen-conjugated oligodeoxyribonucleotide that contained all phosphodiester linkages, (1193), with long-wavelength UV light resulted in approximately 80% formation of interstrand cross-links at pH 7.0, 37 degrees C, in the presence of 20 mM magnesium chloride. The extent of triplex formation as monitored by photo-cross-linking decreased over the pH range 5.5-8.0, and the apparent pK of the 5-methylcytosines (C) in 1443 was approximately one-half of a pH unit less than that of the 5-methylcytosines in 1193. Oligomer 1443 formed triplexes in the absence of magnesium, and maximum triplex formation was observed in solutions containing 2.5 mM magnesium, whereas maximal triplex formation by the fully charged 1193 was not observed until the magnesium concentration was 10 mM or higher. Unlike the all-phosphodiester backbone of 1193, the alternating methylphosphonate-phosphodiester backbone of 1193 is resistant to hydrolysis by exonucleases in fetal calf serum. The nuclease resistance of 1443 and its ability to form triplexes at very low magnesium concentrations suggests that triplex-forming oligomers with alternating methylphosphonate-phosphodiester backbones may be good candidates for use as antigene reagents in cell culture.