A New Application of Spin and Fluorescence Double-Sensor Molecules.

EPR imaging techniques are known to be successful tools for mapping living bodies, especially because of the high transparency of tissues in the microwave range. This technique assumes the presence of radicals whose in vivo transport is also controlled by serum albumins. Accordingly, in this study, the interactions between 3-hydroxymethyl-1-oxyl-4-(pyren-1-yl)-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrole radical and the human serum albumin molecules were investigated. To clarify the adsorption processes of this radical onto the surface of human serum albumin (HSA), the interaction of the OMe derivative of the radical was also examined parallel with the studies on the radical-HSA interactions. Considering the solubility issues and also to modulate the transport, inclusion complexes of the radical with a cavitand derivative were also studied. The latter interactions were observed through fluorescence spectroscopy, fluorescence polarization, and by EPR spectroscopy. As a double-sensor molecule, we found that the fluorophore nitroxide is a good candidate as it gave further information about host-guest interactions (fluorescence, fluorescence polarization, and EPR). We also found that in the presence of a cavitand, a complex with greater stability was formed between the sensor molecule and the human serum albumin. Based on these observations, we can conclude that applying this double-sensor (spin, fluorescent) molecule is useful in cases when different interactions can affect the EPR measurements.

Phase-Selective Synthesis of Anatase and Rutile TiO2 Nanocrystals and Their Impacts on Grapevine Leaves: Accumulation of Mineral Nutrients and Triggering the Plant Defense.

Titanium dioxide nanocrystals (TiO2 NCs), through their photocatalytic activity, are able to generate charge carriers and induce the formation of various reactive oxygen species (ROS) in the presence of O2 and H2O. This special feature makes TiO2 an important and promising material in several industrial applications. Under appropriate antioxidant balancing, the presence of ROS is crucial in plant growth and development, therefore, the regulated ROS production through the photocatalytic activity of TiO2 NCs may be also exploited in the agricultural sector. However, the effects of TiO2 NCs on plants are not fully understood and/or phase-pure TiO2 NCs are rarely used in plant experiments. In this work, we present a phase-selective synthesis of TiO2 NCs with anatase and rutile crystal phases. The nanomaterials obtained were characterized by means of X-ray diffraction (XRD), transmission electron microscopy (TEM), diffuse reflectance UV-Vis spectroscopy, and electron paramagnetic resonance spectroscopy (EPR). In field experiments, Vitis vinifera cv. Cabernet Sauvignon leaves developed under natural sunlight were treated with aqueous dispersions of TiO2 NCs at concentrations of 0.001, 0.01, 0.1, and 1 w/v%. The effect of the applied nanocrystals was characterized via leaf photochemistry, mineral nutrient contents, and pyridoxine levels. We found that stress responses of grapevine to anatase and rutile NCs treatments are different, which can be related to the different ROS profiles of the two polymorphs. Our results indicate that TiO2 NCs may be utilized not only for direct pathogen inactivation but also for eliciting plant defense mechanisms.

Comparative EPR Study on the Scavenging Effect of Methotrexate with the Isomers of Its Photoswitchable Derivative.

The scavenging effect of the antimetabolite dihydrofolate reductase inhibitor methotrexate (MTX) and the isomers of its photoswitchable derivate, cis- and trans-phototrexate (PHX), have been compared by ESR spectroscopy, with the application of a cyclic hydroxylamine spin probe. The results showed the most pronounced scavenging effect in the presence of trans-phototrexate (trans-PHX). At a low concentration (100 µM) cis-PHX also showed a greater scavenging effect than the parent molecule MTX. Direct antioxidant properties of the investigated molecules were measured by ABTS scavenging assay, which showed no significant difference between trans-PHX and cis-PHX, but both of the isomers of PHX showed a higher antioxidant capacity than MTX. These findings imply that trans-PHX may have more pronounced anti-inflammatory and tissue-protective effects than MTX, despite the lack of its cytotoxic, antineoplastic effect.

Syntheses and Study of a Pyrroline Nitroxide Condensed Phospholene Oxide and a Pyrroline Nitroxide Attached Diphenylphosphine.

The reaction of a diene nitroxide precursor with dichlorophenylphosphine in a McCormac procedure afforded 1,1,3,3-tetramethyl-5-phenyl-1,2,3,4,5,6-hexahydrophospholo[3,4-c]pyrrole-5-oxide-2-oxyl. Lithiation of the protected 3-iodo-pyrroline nitroxide followed by treatment with chlorodiphenylphosphine after deprotection afforded (1-oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl)diphenylphosphine oxide, and after reduction, (1-oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl)diphenylphosphine was realized, which was also supported by X-ray single crystal diffraction measurements. This pyrroline diphenylphosphine derivative was converted to hexadecylphosphonium salt, which is an analogue of antineoplastic agent, MITO-CP.

Photocatalytic Inactivation of Plant Pathogenic Bacteria Using TiO2 Nanoparticles Prepared Hydrothermally.

Exploitation of engineered nanomaterials with unique properties has been dynamically growing in numerous fields, including the agricultural sector. Due to the increasing resistance of phytopathogenic microbes, human control over various plant pathogens in crop production is a big challenge and requires the development of novel antimicrobial materials. Photocatalytic active nanomaterials could offer an alternative solution to suppress the plant pathogens. In this work, titanium dioxide nanoparticles (TiO2 NPs) with high photocatalytic activity were synthesized by hydrothermal post-treatment of amorphous titania at different temperatures (250 °C or 310 °C) without using any additives or doping agents. The obtained samples were investigated through X-ray diffraction, N2-sorption measurements, diffuse reflectance UV-Vis spectroscopy, transmission electron microscopy, electron paramagnetic resonance spectroscopy, and X-ray photoelectron spectroscopy. The applied hydrothermal treatment led to the formation of TiO2 nanocrystallites with a predominant anatase crystal phase, with increasing crystallinity and crystallite size by prolonging treatment time. The photocatalytic activity of the TiO2 NPs was tested for the photo-degradation of phenol and applied for the inactivation of various plant pathogens such as Erwinia amylovora, Xanthomonas arboricola pv. juglandis, Pseudomonas syringae pv. tomato and Allhorizobium vitis. The studied bacteria showed different susceptibilities; their living cell numbers were quickly and remarkably reduced by UV-A-irradiated TiO2 NPs. The effectiveness of the most active sample prepared at 310 °C was much higher than that of commercial P25 TiO2. We found that fine-tuning of the structural properties by modulating the time and temperature of the hydrothermal treatment influenced the photocatalytic properties of the TiO2 NPs considerably. This work provides valuable information to the development of TiO2-based antimicrobial photocatalysts.

Syntheses and Reactions of Pyrroline, Piperidine Nitroxide Phosphonates.

Organophosphorus compounds occupy a significant position among the plethora of organic compounds, but a limited number of paramagnetic phosphorus compounds have been reported, including paramagnetic phosphonates. This paper describes the syntheses and further transformations of pyrroline and piperidine nitroxide phosphonates by well-established methods, such as the Pudovik, Arbuzov and Horner-Wadsworth-Emmons (HWE) reactions. The reaction of paramagnetic a-bromoketone produced a vinylphosphonate in the Perkow reaction. Paramagnetic a-hydroxyphosphonates could be subjected to oxidation, elimination and substitution reactions to produce various paramagnetic phosphonates. The synthesized paramagnetic phosphonates proved to be useful synthetic building blocks for carbon-carbon bond-forming reactions in the Horner-Wadsworth-Emmons olefination reactions. The unsaturated compounds achieved could be transformed into various substituted pyrroline nitroxides, proxyl nitroxides and paramagnetic polyaromatics. The Trolox® equivalent antioxidant capacity (TEAC) of new phosphonates was also screened, and tertiary a-hydroxyphosphonatate nitroxides exhibited remarkable antioxidant activity.

Selective Induction of Cellular Toxicity and Anti-tumor Efficacy by N-Methylpiperazinyl Diarylidenylpiperidone and its Pro-nitroxide Conjugate through ROS-mediated Mitochondrial Dysfunction and G2/M Cell-cycle Arrest in Human Pancreatic Cancer.

Pancreatic adenocarcinoma is an aggressive cancer with poor clinical prognosis and limited therapeutic options. There is a significant lack of effective, safe, and targeted therapies for successful treatment of pancreatic cancer. In this report, we describe the anticancer efficacy of two novel compounds, N-methylpiperazinyl diarylidenylpiperidone (L-2663) and its pro-nitroxide conjugate (HO-4589) evaluated on human pancreatic adenocarcinoma (AsPC-1) cell line and xenograft tumor in mice. Using flow cytometry, we determined the effect of the L-2663 and HO-4589 drugs in inducing mitochondrial toxicity, triggering cell-cycle arrest, and apoptosis. EPR spectroscopy was used to quantify cellular uptake, metabolic conversion and stability of HO-4589 in cells and in vivo monitoring of tumor oxygenation as a function of growth. The results established different antiproliferative efficacy of the L-2663 and HO-4589 compounds, with a targeted action on cancer cells while being less toxic to noncancerous cells. The study may have important implications in the future designs of safe and effective chemotherapeutic agents for the treatment of pancreatic cancer.

Synthesis of Spin-Labelled Bergamottin: A Potent CYP3A4 Inhibitor with Antiproliferative Activity.

Bergamottin (BM, 1), a component of grapefruit juice, acts as an inhibitor of some isoforms of the cytochrome P450 (CYP) enzyme, particularly CYP3A4. Herein, a new bergamottin containing a nitroxide moiety (SL-bergamottin, SL-BM, 10) was synthesized; chemically characterized, evaluated as a potential inhibitor of the CYP2C19, CYP3A4, and CYP2C9 enzymes; and compared to BM and known inhibitors such as ketoconazole (KET) (3A4), warfarin (WAR) (2C9), and ticlopidine (TIC) (2C19). The antitumor activity of the new SL-bergamottin was also investigated. Among the compounds studied, BM showed the strongest inhibition of the CYP2C9 and 2C19 enzymes. SL-BM is a more potent inhibitor of CYP3A4 than the parent compound; this finding was also supported by docking studies, suggesting that the binding positions of BM and SL-BM to the active site of CYP3A4 are very similar, but that SL-BM had a better ∆Gbind value than that of BM. The nitroxide moiety markedly increased the antitumor activity of BM toward HeLa cells and marginally increased its toxicity toward a normal cell line. In conclusion, modification of the geranyl sidechain of BM can result in new CYP3A4 enzyme inhibitors with strong antitumor effects.

Thermodynamic Characterization of the Interaction between the Antimicrobial Drug Sulfamethazine and Two Selected Cyclodextrins.

Sulfamethazine is a representative member of the sulfonamide antibiotic drugs; it is still used in human and veterinary therapy. The protonation state of this drug affects its aqueous solubility, which can be controlled by its inclusion complexes with native or chemically-modified cyclodextrins. In this work, the temperature-dependent (298-313 K) interaction of sulfamethazine with native and randomly methylated ß-cyclodextrins have been investigated at acidic and neutral pH. Surprisingly, the interaction between the neutral and anionic forms of the guest molecule and cyclodextrins with electron rich cavity are thermodynamically more favorable compared to the cationic guest. This property probably due to the enhanced formation of zwitterionic form of sulfamethazine in the hydrophobic cavities of cyclodextrins. Spectroscopic measurements and molecular modeling studies indicated the possible driving forces (hydrophobic interaction, hydrogen bonding, and electrostatic interaction) of the complex formation, and highlighted the importance of the reorganization of the solvent molecules during the entering of the guest molecule into the host's cavity.

Antiproliferative Effect of a Novel 4,4'-Disulfonyldiarylidenyl Piperidone in Human Colon Cancer Cells.

The synthesis and antiproliferative effect of a novel curcumin analog, 4,4'-disulfonyldiarylidenyl piperidone, are reported. The design of the molecule is based on the fusion of an antiproliferative segment, namely diarylidenyl piperidone (DAP), with N-hyroxypyrroline, which is known to metabolically convert to nitroxide and protect healthy cells. Cellular uptake, metabolic conversion, cytotoxicity and antiproliferative effect of the DAP derivative against HCT-116 human colon cancer cells have been determined. Based on cell viability and proliferation assays as well as western-blot analysis of major transcription factors and inhibitory proteins, it is determined that the DAP compound is cytotoxic by inhibiting cell survival and proliferation pathways. The findings may have important implications in the design and development of effective anticancer agents.

Nanostructured TiO2-induced photocatalytic stress enhances the antioxidant capacity and phenolic content in the leaves of Vitis vinifera on a genotype-dependent manner.

Over the past decades, nanotechnology has received great attention and brought revolutionary solutions for a number of challenges in scientific fields. Industrial, agricultural and medical applications of engineered nanomaterials have increased intensively. The ability of titanium dioxide nanoparticles (TiO2 NPs) to produce reactive oxygen species (ROS), when excited by ultra-violet (UV) light, makes them useful for effectively inactivate various pathogens. It is known that ROS also have signalling role in living organisms, therefore, TiO2 NPs-induced ROS can influence both enzymatic and non-enzymatic defence systems, and could play a role in the resistance of plants to pathogens. Herein, we studied the photocatalytic stress responses of grapevine (Vitis vinifera L.) as model plant, when exposed to a well-known photocatalyst, Degussa P25 TiO2 NPs. The photocatalytically produced ROS such as superoxide anion, hydroxyl radical and singlet oxygen were confirmed by electron paramagnetic resonance spectroscopy. Foliar exposure of five red cultivars (Cabernet sauvignon, Cabernet franc, Merlot, Kékfrankos and Kadarka) was carried out in blooming phenophase under field condition where plants are exposed to natural sunlight with relatively high UV radiation (with a maximum of ~ 45 W m-2). After two weeks of exposure, the effects of photogenerated ROS on the total phenolic content, antioxidant capacity, flavonol profile and the main macro-, microelements of the leaves were studied in detail. We found that foliar application of TiO2 NPs boosted the total phenolic content and biosynthesis of the leaf flavonols depending on the grapevine variety. Photocatalytically active TiO2 NPs also increased K, Mg, Ca, B and Mn levels in the leaves as shown by ICP-AES measurements.

Interaction of Chrysin and Its Main Conjugated Metabolites Chrysin-7-Sulfate and Chrysin-7-Glucuronide with Serum Albumin.

Chrysin (5,7-dihydroxyflavone) is a flavonoid aglycone, which is found in nature and in several dietary supplements. During the biotransformation of chrysin, its conjugated metabolites chrysin-7-sulfate (C7S) and chrysin-7-glucuronide (C7G) are formed. Despite the fact that these conjugates appear in the circulation at much higher concentrations than chrysin, their interactions with serum albumin have not been reported. In this study, the complex formation of chrysin, C7S, and C7G with human (HSA) and bovine (BSA) serum albumins was investigated employing fluorescence spectroscopic, ultrafiltration, and modeling studies. Our major observations/conclusions are as follows: (1) Compared to chrysin, C7S binds with a threefold higher affinity to HSA, while C7G binds with a threefold lower affinity; (2) the albumin-binding of chrysin, C7S, and C7G did not show any large species differences regarding HSA and BSA; (3) tested flavonoids likely occupy Sudlow's Site I in HSA; (4) C7S causes significant displacement of Sudlow's Site I ligands, exerting an even stronger displacing ability than the parent compound chrysin. Considering the above-listed observations, the high intake of chrysin (e.g., through the consumption of dietary supplements with high chrysin contents) may interfere with the albumin-binding of several drugs, mainly due to the strong interaction of C7S with HSA.

Synthesis and Biological Evaluation of Curcumin-Nitroxide-Based Molecular Hybrids as Antioxidant and Anti-Proliferative Agents.

BACKGROUND: Natural products and their derivatives are widely used to treat cancer and other diseases associated with ROS- and RNS-induced damages. METHODS: A series of paramagnetic modified curcumin analogs and 3,5-diarylidene-piperidones (DAP) have been designed, synthesized, and characterized on their anti-proliferative and antioxidant activity. RESULTS: Biological characterization of the new compounds supported the earlier results that incorporation of a nitroxide moiety or its precursor into curcumin or diarylidenylpiperidone (DAP) scaffolds resulted in anti-proliferative effect toward cancerous cell-lines in case of aryl hydroxy and/or methoxy substituent containing derivatives, suggesting their potential for targeted therapeutic applications. In case of basic side chain derivatives, nitroxide incorporation gave unambiguous results, however in tendency the more accessible DAP derivatives had stronger anti-proliferative effect. In most cases, the nitroxide incorporation increased the TEAC value (proton and electron donation capability) of DAP derivatives. CONCLUSIONS: Among the compounds synthesized and investigated the spin-labeled curcumin and 3,5-bis(4-hydroxy-3-methoxybenzylidene)piperidin-4-one derivatives were the most effective antiproliferative and antioxidant derivatives.

Pulmonary hypertension secondary to left-heart failure involves peroxynitrite-induced downregulation of PTEN in the lung.

Pulmonary hypertension (PH) that occurs after left-heart failure (LHF), classified as Group 2 PH, involves progressive pulmonary vascular remodeling induced by smooth muscle cell (SMC) proliferation. However, mechanisms involved in the activation of SMCs remain unknown. The objective of this study was to determine the involvement of peroxynitrite and phosphatase-and-tensin homolog on chromosome 10 (PTEN) in vascular SMC proliferation and remodeling in the LHF-induced PH (LHF-PH). LHF was induced by permanent ligation of left anterior descending coronary artery in rats for 4 weeks. MRI, ultrasound, and hemodynamic measurements were performed to confirm LHF and PH. Histopathology, Western blot, and real-time polymerase chain reaction analyses were used to identify key molecular signatures. Therapeutic intervention was demonstrated using an antiproliferative compound, HO-3867. LHF-PH was confirmed by significant elevation of pulmonary artery pressure (mean pulmonary artery pressure/mm Hg: 35.9±1.8 versus 14.8±2.0, control; P<0.001) and vascular remodeling. HO-3867 treatment decreased mean pulmonary artery pressure to 22.6±0.8 mm Hg (P<0.001). Substantially higher levels of peroxynitrite and significant loss of PTEN expression were observed in the lungs of LHF rats when compared with control. In vitro studies using human pulmonary artery SMCs implicated peroxynitrite-mediated downregulation of PTEN expression as a key mechanism of SMC proliferation. The results further established that HO-3867 attenuated LHF-PH by decreasing oxidative stress and increasing PTEN expression in the lung. In conclusion, peroxynitrite and peroxynitrite-mediated PTEN inactivation seem to be key mediators of lung microvascular remodeling associated with PH secondary to LHF.

Synthesis and study of new paramagnetic and diamagnetic verapamil derivatives.

New derivatives of verapamil (1) modified with nitroxides and their precursors were synthesized and screened for reactive oxygen species (ROS)-scavenging activities. The basic structure was modified by changing the nitrile group to an amide or the methyl substituent on tertiary nitrogen with nitroxides and their reduced forms (hydroxylamine and secondary amines). Among the new verapamil derivatives compound 16B [Mohan, I. K.; Kahn, M.; Wisel, S.; Selvendiran, K.; Sridhar, A.; Carnes, C.A.; Bognár, B.; Kálai, T.; Hideg, K.; Kuppusamy, P. Am. J. Physiol. Heart Circ. Physiol.2009, 296, 140], modified with hydroxylamine salt of 2,2,6,6-tetramethyl-1,2,3,6-tetrahydropyridine-1-yloxyl proved to be the best ROS scavenger in vitro and protected HSMC and CHO cells against H(2)O(2) induced damage.

Cardioprotection by HO-4038, a novel verapamil derivative, targeted against ischemia and reperfusion-mediated acute myocardial infarction.

Many cardiac interventional procedures, such as coronary angioplasty, stenting, and thrombolysis, attempt to reintroduce blood flow (reperfusion) to an ischemic region of myocardium. However, the reperfusion is accompanied by a complex cascade of cellular and molecular events resulting in oxidative damage, termed myocardial ischemia-reperfusion (I/R) injury. In this study, we evaluated the ability of HO-4038, an N-hydroxypiperidine derivative of verapamil, on the modulation of myocardial tissue oxygenation (Po(2)), I/R injury, and key signaling molecules involved in cardioprotection in an in vivo rat model of acute myocardial infarction (MI). MI was created in rats by ligating the left anterior descending coronary artery (LAD) for 30 min followed by 24 h of reperfusion. Verapamil or HO-4038 was infused through the jugular vein 10 min before the induction of ischemia. Myocardial Po(2) and the free-radical scavenging ability of HO-4038 were measured using electron paramagnetic resonance spectroscopy. HO-4038 showed a significantly better scavenging ability of reactive oxygen radicals compared with verapamil. The cardiac contractile functions in the I/R hearts were significantly higher recovery in HO-4038 compared with the verapamil group. A significant decrease in the plasma levels of creatine kinase and lactate dehydrogenase was observed in the HO-4038 group compared with the verapamil or untreated I/R groups. The left ventricular infarct size was significantly less in the HO-4038 (23 +/- 2%) compared with the untreated I/R (36 +/- 4%) group. HO-4038 significantly attenuated the hyperoxygenation (36 +/- 1 mmHg) during reperfusion compared with the untreated I/R group (44 +/- 2 mmHg). The HO-4038-treated group also markedly attenuated superoxide production, increased nitric oxide generation, and enhanced Akt and Bcl-2 levels in the reperfused myocardium. Overall, the results demonstrated that HO-4038 significantly protected hearts against I/R-induced cardiac dysfunction and damage through the combined beneficial actions of calcium-channel blocking, antioxidant, and prosurvival signaling activities.

A novel SOD-mimetic permeability transition inhibitor agent protects ischemic heart by inhibiting both apoptotic and necrotic cell death.

In ischemia-reperfusion injuries, elevated calcium and reactive oxygen species (ROS) induce mitochondrial permeability transition (mPT), which plays a pivotal role in mediating damages and cell death. Inhibition of mPT decreases necrotic cell death; however, during reperfusion, the continuous production of ROS may contribute to the temporary opening of the pore and thus the onset of the delayed apoptotic cell death. Based on amiodarone structure, we developed the first SOD-mimetic mPT inhibitor (HO-3538) that can eliminate ROS in the microenvironment of the permeability pore. In isolated mitochondria, HO-3538 inhibited mPT and the release of proapoptotic mitochondrial proteins. It had a ROS scavenging effect and antiapoptotic effect in a cardiomyocyte line and it diminished release of mitochondrial proapoptotic proteins. Furthermore, HO-3538 significantly enhanced the recovery of mitochondrial energy metabolism and functional cardiac parameters; decreased infarct size, lipid peroxidation, and protein oxidation; and suppressed necrotic as well as apoptotic cell death pathways in Langendorff-perfused hearts. In these respects it was somewhat superior to its two constituents, amiodarone and a pyrrol-derivative free radical scavenger. These data suggest that the SOD-mimetic mPT inhibitors are ideal candidates for drug development for the alleviation of postinfarct myocardial injuries.

Synthesis and evaluation of the permeability transition inhibitory characteristics of paramagnetic and diamagnetic amiodarone derivatives.

Several amiodarone analogues were synthesized varying the 2-substituent on the benzofuran ring and diethylaminoethyl side chain of phenolether by introducing 2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrole and 1,2,5,6-tetrahydropyridine nitroxides or their amino or hydroxylamino precursors. The new compounds were screened on isolated mitochondria and perfused heart and their toxicity was evaluated on WRL-68 liver cells and H9C2 cardiomyocytes. Most of the newly synthesized derivatives exerted uncoupling effect on the mitochondrial oxidative phosphorilation at higher concentrations, compared to amiodarone and one of the modified amiodarone analogues showed an effect similar to that of amiodarone on the mitochondrial permeability transition and on restoring of mitochondrial high-energy phosphate metabolites in perfused hearts. This amiodarone analogue can be new leading compound among the experimental amiodarone analogues with the same or enhanced efficiency of amiodarone, but with less side effects.