Employed Electron Paramagnetic Resonance Research Articles

Non-thermal plasma elicits ferrous chloride-catalyzed DMPO-OH

Non-thermal plasma (NTP) induces the generation of reactive oxygen species (ROS) and reactive nitrogen species, such as hydroxyl radicals (•OH), hydrogen peroxide (H2O2), singlet oxygen, superoxide, ozone, and nitric oxide, at near-physiological temperatures. These molecules promote blood coagulation, wound healing, disinfection, and selective cancer cell death. Based on these evidences, clinical trials of NTP have been conducted for treating chronic wounds and head and neck cancers. Although clinical applications have progressed, the stoichiometric quantification of NTP-induced ROS remains unclear in the liquid phase in the presence of FeCl2 or FeCl3 in combination with biocompatible reducing agents, which may modulate the final biological effects of NTP. In this study, we employed electron paramagnetic resonance spectroscopy to quantify ROS using spin-trapping probe, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and H2O2, using luminescent probe in the presence of FeCl2 or FeCl3. NTP-induced DMPO-OH levels were elevated 10–100 µM FeCl2 or 500 and 1000 µM FeCl3. NTP-induced DMPO-OH with 10 µM FeCl2 or FeCl3 was significantly scavenged by ascorbate, α-tocopherol, dithiothreitol, reduced glutathione, or oxidized glutathione, whereas dehydroascorbate was ineffective in 2 mM DMPO. NTP-induced H2O2 was significantly degraded by 100 µM FeCl2 and FeCl3 in an iron-dependent manner. Meanwhile, decomposition of H2O2 by catalase decayed DMPO-OH efficiently in the presence of iron, indicating iron causes DMPO-OH production and degradation simultaneously. These results suggest that NTP-induced DMPO-OH is generated by the H2O2-consuming, iron-dependent Fenton reaction and ferryl intermediates. The potential iron-mediated ROS production by NTP is also discussed to clarify the interaction between NTP-induced ROS and biomolecules.

Non-thermal plasma-induced DMPO-OH yields hydrogen peroxide

Recent developments in electronics have enabled the medical applications of non-thermal plasma (NTP), which elicits reactive oxygen species (ROS) and reactive nitrogen species (RNS), such as hydroxyl radical (●OH), hydrogen peroxide (H2O2), singlet oxygen (1O2), superoxide (O2●-), ozone, and nitric oxide at near-physiological temperatures. In preclinical studies or human clinical trials, NTP promotes blood coagulation, eradication of bacterial, viral and biofilm-related infections, wound healing, and cancer cell death. To elucidate the solution-phase biological effects of NTP in the presence of biocompatible reducing agents, we employed electron paramagnetic resonance (EPR) spectroscopy to quantify ●OH using a spin-trapping probe, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO); 1O2 using a fluorescent probe; and O2●- and H2O2 using luminescent probes in the presence of thiols or tempol. NTP-induced ●OH was significantly scavenged by dithiothreitol (DTT), reduced glutathione (GSH), and oxidized glutathione (GSSG) in 2 or 5 mM DMPO. NTP-induced O2●- was significantly scavenged by 10 μM DTT and GSH, while 1O2 was not efficiently scavenged by these compounds. GSSG degraded H2O2 more effectively than GSH and DTT, suggesting that the disulfide bonds reacted with H2O2. In the presence of 1–50 mM DMPO, NTP-induced H2O2 quantities were unchanged. The inhibitory effect of tempol concentration (50 and 100 μM) on H2O2 production was observed in 1 and 10 mM DMPO, whereas it became ineffective in 50 mM DMPO. Furthermore, DMPO-OH did not interact with tempol. These results suggest that DMPO and tempol react competitively with O2●-. Further studies are warranted to elucidate the interaction between NTP-induced ROS and biomolecules.

Electrostatic properties of inner nanopore surfaces of anodic aluminum oxide membranes upon high temperature annealing revealed by EPR of pH-sensitive spin probes and labels

Anodic aluminum oxide (AAO) membranes are versatile nanomaterials that combine the chemically stable and mechanically robust properties of ceramics with homogeneous nanoscale organization that can be tuned to desirable pore diameters and lengths. The AAO substrates feature high surface area that is readily accessible to large and small molecules, making these nanostructures uniquely suited for many possible applications. Examples include templated self-assembly of macroscopically aligned biological membranes and substrates for heterogeneous catalysis. For further development of such applications, one would like to characterize and tune the electrostatic properties of the inner pore surface as well as the local acidity within the nanochannels. Here, we employed electron paramagnetic resonance (EPR) spectroscopy of a small molecule – ionizable nitroxide – as a reporter of the average local acidity in the nanochannels and the local electrostatic potential in the immediate vicinity of the pore surface. The former was achieved by measuring EPR spectra of this molecular probe diffusing in an aqueous phase confined in the AAO nanochannels while for the latter the nitroxide was covalently attached to the hydroxyl group of the alumina surface. We show that the local acidity within the nanochannels is increased by as much as ≈1.48 pH units vs. the pH of bulk solution by decreasing the pore diameter down to ca. 31 nm. Furthermore, the positive surface charge of the as-prepared AAO could be decreased and even switched to a negative surface charge upon annealing the membranes first to 700 °C and then to 1200 °C. For as-prepared AAO, the local electrostatic potential reaches ψ= (163 ± 5) mV for the nitroxide label covalently attached to AAO and located about 0.5 nm away from the surface. Overall, we demonstrate that the acid-based properties of the aqueous volume confined by the AAO nanopores pores can be tuned by either changing the pore diameter from ca. 71 to 31 nm or by thermal annealing to switch the sign of the surface charge. These observations provide a simple and robust means to tailor these versatile high-surface-area nanomaterials for specific applications that depend on acid-base equilibria.

The pivotal role of MBD4-ATP7B in the human Cu(i) excretion path as revealed by EPR experiments and all-atom simulations.

Copper's essentiality and toxicity require a meticulous mechanism for its acquisition, cellular distribution and excretion, which remains hitherto elusive. Herein, we jointly employed electron paramagnetic resonance spectroscopy and all-atom simulations to resolve the copper trafficking mechanism in humans considering the route travelled by Cu(i) from the metallochaperone Atox1 to the metal binding domains 3 and 4 of ATP7B. Our study shows that Cu(i) in the final part of its extraction pathway is most likely mediated by binding of Atox1 monomer to MBD4 of ATP7B. This interaction takes place through weak metal-stabilized protein-protein interactions.

Electron Paramagnetic Resonance Study of Al-incorporated ZnO:Mn Diluted Magnetic Semiconductors

We have employed electron paramagnetic resonance spectroscopy and magnetization measurements in order to study the effect of Al-incorporation on the magnetic interactions in ZnO:Mn diluted magnetic semiconductors. Al-doping is shown to decrease the antiferromagnetic correlation and to increase the ferromagnetic interaction, which is attributed to the hydrogen-mediated ferromagnetic Mn complexes in our Mn-doped ZnO samples.

Proton Activity in Nanochannels Revealed by Electron Paramagnetic Resonance of Ionizable Nitroxides: A Test of the Poisson–Boltzmann Double Layer Theory

Chemical and physical processes occurring within the nanochannels of mesoporous materials are known to be determined by both the chemical nature of the solution inside the pores/channels as well as the channel surface properties, including surface electrostatic potential. Such properties are important for numerous practical applications such as heterogeneous catalysis and chemical adsorption including chromatography. However, for solute molecules diffusing inside the pores, the surface potential is expected to be effectively screened by counter ions for the distances exceeding the Debye length. Here, we employed electron paramagnetic resonance spectroscopy of ionizable nitroxide spin probes to experimentally examine the conditions for the efficient electrostatic surface potential screening inside the nanochannels of chemically similar silica-based mesoporous molecular sieves (MMS) filled with water at ambient conditions and a moderate ionic strength of 0.1 M. Three silica MMS having average channel diamet...

Direct rate assessment of laccase catalysed radical formation in lignin by electron paramagnetic resonance spectroscopy.

Laccases (EC 1.10.3.2) catalyse removal of an electron and a proton from phenolic hydroxyl groups, including phenolic hydroxyls in lignins, to form phenoxy radicals during reduction of O2. We employed electron paramagnetic resonance spectroscopy (EPR) for real time measurement of such catalytic radical formation activity on three types of lignin (two types of organosolv lignin, and a lignin rich residue from wheat straw hydrolysis) brought about by two different fungal laccases, derived from Trametes versicolor (Tv) and Myceliophthora thermophila (Mt), respectively. Laccase addition to suspensions of the individual lignin samples produced immediate time and enzyme dose dependent increases in intensity in the EPR signal with g-values in the range 2.0047-2.0050 allowing a direct quantitative monitoring of the radical formation and thus allowed laccase enzyme kinetics assessment on lignin. The experimental data verified that the laccases acted upon the insoluble lignin substrates in the suspensions. When the action on the lignin substrates of the two laccases were compared on equal enzyme dosage levels (by activity units on syringaldazine) the Mt laccase exerted a significantly faster radical formation than the Tv laccase on all three types of lignin substrates. When comparing the equal laccase dose rates on the three lignin substrates the enzymatic radical formation rate on the wheat straw lignin residue was consistently higher than those of the organosolv lignins. The pH-temperature optimum for the radical formation rate in organosolv lignin was determined by response surface methodology to pH 4.8, 33°C and pH 5.8, 33°C for the Tv laccase and the Mt laccase, respectively. The results verify direct radical formation action of fungal laccases on lignin without addition of mediators and the EPR methodology provides a new type of enzyme assay of laccases on lignin.

Structural Transitions in Myosin I Detected by Conventional and Pulsed EPR of a Bifunctional Spin Label

We have employed electron paramagnetic resonance (EPR) of a bifunctional spin label (BSL) to develop high-resolution constraints for the myosin catalytic domain in the presence and absence of actin. Two complementary EPR techniques were employed to measure protein orientation (continuous-wave EPR, CW-EPR) and intra-protein distances (double electron-electron resonance, DEER). The use of BSL greatly enhances the resolution of both techniques, by virtue of its strongly immobilized and stereospecific bifunctional attachment to the protein backbone at two engineered Cys residues. Crucially, both techniques utilized here permit the elucidation of myosin structure while in complex with actin, generating relevant constraints for the refinement of actomyosin structural models, and providing insight for structural changes induced by formation of the complex. In the current work, Dictyostelium myosin II was used as our model system. We measured nucleotide-dependent structural transitions of three key helices within the myosin CD. Three double-Cys sites were engineered, with Cys pairs located on the relay helix, helix HK (upper 50kDa domain) and helix HW (lower 50kDa domain), respectively. BSL on a construct with one of these pairs was used to measure myosin orientation relative to oriented actin; BSL on a construct with two pairs was used to measure interprobe distances. The effect of MgADP binding was clearly detected by EPR, and subsequently modeled using the orientation and distance measurements as constraints. This work was funded by grants from NIH (R01 AR32961, T32 AR07612, P30 AR0507220).

Evidence That Compound I Is the Active Species in Both the Hydroxylase and Lyase Steps by Which P450scc Converts Cholesterol to Pregnenolone: EPR/ENDOR/Cryoreduction/Annealing Studies.

Cytochrome P450scc (CYP 11A1) catalyzes the conversion of cholesterol (Ch) to pregnenolone, the precursor to steroid hormones. This process proceeds via three sequential monooxygenation reactions: two hydroxylations of Ch first form 22(R)-hydroxycholesterol (HC) and then 20α,22(R)-dihydroxycholesterol (DHC); a lyase reaction then cleaves the C20-C22 bond to form pregnenolone. Recent cryoreduction/annealing studies that employed electron paramagnetic resonance (EPR)/electron nuclear double resonance (ENDOR) spectroscopy [Davydov, R., et al. (2012) J. Am. Chem. Soc. 134, 17149] showed that compound I (Cpd I) is the active intermediate in the first step, hydroxylation of Ch. Herein, we have employed EPR and ENDOR spectroscopy to characterize the intermediates in the second and third steps of the enzymatic process, as conducted by 77 K radiolytic one-electron cryoreduction and subsequent annealing of the ternary oxy-cytochrome P450scc complexes with HC and DHC. This procedure is validated by showing that the cryoreduced ternary complexes of oxy-cytochrome P450scc with HC and DHC are catalytically competent and during annealing generate DHC and pregnenolone, respectively. Cryoreduction of the oxy-P450scc-HC ternary complex trapped at 77K produces the superoxo-ferrous P450scc intermediate along with a minor fraction of ferric hydroperoxo intermediates. The superoxo-ferrous intermediate converts into a ferric-hydroperoxo species after annealing at 145 K. During subsequent annealing at 170-180 K, the ferric-hydroperoxo intermediate converts to the primary product complex with the large solvent kinetic isotope effect that indicates Cpd I is being formed, and (1)H ENDOR measurements of the primary product formed in D2O demonstrate that Cpd I is the active species. They show that the primary product contains Fe(III) coordinated to the 20-O(1)H of DHC with the (1)H derived from substrate, the signature of the Cpd I reaction. Hydroperoxo ferric intermediates are the primary species formed during cryoreduction of the oxy-P450scc-DHC ternary complex, and they decay at 185 K with a strong solvent kinetic isotope effect to form low-spin ferric P450scc. Together, these observations indicated that Cpd I also is the active intermediate in the C20,22 lyase final step. In combination with our previous results, this study thus indicates that Cpd I is the active species in each of the three sequential monooxygenation reactions by which P450scc catalytically converts Ch to pregnenolone.

The destructive effect of botulinum neurotoxins on the SNARE protein: SNAP-25 and synaptic membrane fusion.

Synaptic exocytosis requires the assembly of syntaxin 1A and SNAP-25 on the plasma membrane and synaptobrevin 2 (VAMP2) on the vesicular membrane to bridge the two opposite membranes. It is believed that the three SNARE proteins assemble in steps along the dynamic assembly pathway. The C-terminus of SNAP-25 is known to be the target of botulinum neurotoxins (BoNT/A and BoNT/E) that block neurotransmitters release in vivo. In this study, we employed electron paramagnetic resonance (EPR) spectroscopy to investigate the conformation of the SNAP-25 C-terminus in binary and ternary SNARE complexes. The fluorescence lipid mixing assay shows that the C-terminal of SNAP-25 is essential for membrane fusion, and that the truncated SNAP-25 mutants cleaved by BoNT/A and BoNT/E display different inhibition effects on membrane fusion: SNAP-25E (Δ26) abolishes the fusion activity of the SNARE complex, while SNAP-25A (Δ9) loses most of its function, although it can still form a SDS-resistant SNARE complex as the wild-type SNAP-25. CW-EPR spectra validate the unstable structures of the SNARE complex formed by SNAP-25 mutants. We propose that the truncated SNAP-25 mutants will disrupt the assembly of the SNARE core complex, and then inhibit the synaptic membrane fusion accordingly.

TROL-FNR interaction reveals alternative pathways of electron partitioning in photosynthesis.

In photosynthesis, final electron transfer from ferredoxin to NADP+ is accomplished by the flavo enzyme ferredoxin:NADP+ oxidoreductase (FNR). FNR is recruited to thylakoid membranes via integral membrane thylakoid rhodanase-like protein TROL. We address the fate of electrons downstream of photosystem I when TROL is absent. We have employed electron paramagnetic resonance (EPR) spectroscopy to study free radical formation and electron partitioning in TROL-depleted chloroplasts. DMPO was used to detect superoxide anion (O2.−) formation, while the generation of other free radicals was monitored by Tiron. Chloroplasts from trol plants pre-acclimated to different light conditions consistently exhibited diminished O2.− accumulation. Generation of other radical forms was elevated in trol chloroplasts in all tested conditions, except for the plants pre-acclimated to high-light. Remarkably, dark- and growth light-acclimated trol chloroplasts were resilient to O2.− generation induced by methyl-viologen. We propose that the dynamic binding and release of FNR from TROL can control the flow of photosynthetic electrons prior to activation of the pseudo-cyclic electron transfer pathway.

EPR Spectroscopy of MOLB2C2-A Reveals Mechanism of Transport for A Type II Molybdate Importer

ABC importers are embedded in the plasma membrane of all bacteria. There they facilitate the uptake of several vital nutrients and cofactors. Due to differences in structure and activity, ABC importers are divided into two types. Type I importers have been well studied, but most of what we know about Type II importers has been observed in studies of the vitamin B12 importer, BtuC2D2. MolB2C2 (formally known as HI1470/71) is also a Type II importer, but its substrate, molybdate, is about 10x smaller than vitamin B12. To understand mechanistic differences among Type II importers, we focused our studies on MolBC where alternative conformations may be required to transport its relatively small substrate. To investigate the mechanism of MolBC, we employed electron paramagnetic resonance (EPR) spectroscopy with the transporter imbedded in detergent micelles and liposomes (spherical lipid bilayers). The use of both detergent and lipid have allowed us to identify conformational changes in MolBC and the impact of the lipid environment. We observed that nucleotide binding and hydrolysis shift the conformation of MolBC at both the cytoplasmic and periplasmic gates. Mechanistic differences between MolBC and other Type II transporters highlight potential effects of substrate size on transport. Combining our results, we propose a mechanism for MolBC-A embedded in a lipid bilayer, which gives new insight in the transport of small substrates.

Pheomelanin in the skin of Hymenochirus boettgeri (Amphibia: Anura: Pipidae)

Pheomelanin is supposed to be the first type of melanin found in vertebrates, in contrast to the main type - eumelanin. Our study aimed at detecting pheomelanin in the skin of Hymenochirus boettgerii. We employed electron paramagnetic resonance (EPR) spectroscopy, and transmission electron microscopy (TEM), supplemented with standard histology and immunochemistry. We identified pheomelanin in the dorsal skin of adult frogs (not only in the dermis, but also in the epidermis) and in the dorsal tadpole. Our work identifies Hymenochirus boettgerii as a model in the basic study on the mechanism, evolution and role of melanogenesis in animals, including human.

On the Radiation Stability of Crown Ethers in Ionic Liquids

Crown ethers (CEs) are macrocyclic ionophores used for the separation of strontium-90 from acidic nuclear waste streams. Room temperature ionic liquids (ILs) are presently being considered as replacements for traditional molecular solvents employed in such separations. It is desirable that the extraction efficacy obtained with such solvents should not deteriorate in the strong radiation fields generated by decaying radionuclides. This deterioration will depend on the extent of radiation damage to both the IL solvent and the CE solute. While radiation damage to ILs has been extensively studied, the issue of the radiation stability of crown ethers, particularly in an IL matrix, has not been adequately addressed. With this in mind, we have employed electron paramagnetic resonance (EPR) spectroscopy to study the formation of CE-related radicals in the radiolysis of selected CEs in ILs incorporating aromatic (imidazolium and pyridinium) cations. The crown ethers have been found to yield primarily hydrogen loss radicals, H atoms, and the formyl radical. In the low-dose regime, the relative yield of these radicals increases linearly with the mole fraction of the solute, suggesting negligible transfer of the excitation energy from the solvent to the solute; that is, the solvent has a "radioprotective" effect. The damage to the CE in the loading region of practical interest is relatively low. Under such conditions, the main chemical pathway leading to decreased extraction performance is protonation of the macrocycle. At high radiation doses, sufficient to increase the acidity of the IL solvent significantly, such proton complexes compete with the solvent cations as electron traps. In this regime, the CEs will rapidly degrade as the result of H abstraction from the CE ring by the released H atoms. Thus, the radiation dose to which a CE/IL system is exposed must be maintained at a level sufficiently low to avoid this regime.

Mechanism of Permanganate Chemiluminescence

Spectroscopic and synthetic methods have been exploited to deduce the mechanism for acidic potassium permanganate chemiluminescence. We have employed electron paramagnetic resonance (EPR) spectroscopy with a continuous flow assembly to monitor the formation of radical intermediates in real time generated from substrate oxidation by manganese(VII). These transient species react with manganese(III) in solution to produce the previously characterized manganese(II)* emission source. Using UV-vis, EPR, attenuated total reflection (ATR)-FT-IR, and chemiluminescence spectroscopies, we have established that there are two distinct enhancement mechanisms that in combination afford a 50-fold increase in emission intensity when the reaction is conducted in the presence of phosphate oligomers. In addition to preventing disproportionation of the manganese(III) precursor, the phosphate oligomers form protective "cagelike" structures around the manganese(II)* emitter, thus preventing nonradiative relaxation pathways.

Elucidation of the Mechanism by Which Catecholamine Stress Hormones Liberate Iron from the Innate Immune Defense Proteins Transferrin and Lactoferrin

The ability of catecholamine stress hormones and inotropes to stimulate the growth of infectious bacteria is now well established. A major element of the growth induction process has been shown to involve the catecholamines binding to the high-affinity ferric-iron-binding proteins transferrin (Tf) and lactoferrin, which then enables bacterial acquisition of normally inaccessible sequestered host iron. The nature of the mechanism(s) by which the stress hormones perturb iron binding of these key innate immune defense proteins has not been fully elucidated. The present study employed electron paramagnetic resonance spectroscopy and chemical iron-binding analyses to demonstrate that catecholamine stress hormones form direct complexes with the ferric iron within transferrin and lactoferrin. Moreover, these complexes were shown to result in the reduction of Fe(III) to Fe(II) and the loss of protein-complexed iron. The use of bacterial ferric iron uptake mutants further showed that both the Fe(II) and Fe(III) released from the Tf could be directly used as bacterial nutrient sources. We also analyzed the transferrin-catecholamine interactions in human serum and found that therapeutically relevant concentrations of stress hormones and inotropes could directly affect the iron binding of serum-transferrin so that the normally highly bacteriostatic tissue fluid became significantly more supportive of the growth of bacteria. The relevance of these catecholamine-transferrin/lactoferrin interactions to the infectious disease process is considered.

UVA/B-Induced Formation of Free Radicals from Decabromodiphenyl Ether

Polybrominated diphenyl ether (PBDE) flame retardants are ubiquitous in the environment and in humans. A deca-bromodiphenyl ether mixture (deca-BDE) is the dominating commercial PBDE product today. Deca-BDE is degraded by UV to PBDEs with fewer bromines. We hypothesized that photodegradation of deca-BDE results in the formation of free radicals. We employed electron paramagnetic resonance (EPR) with spin trap agents to examine the free radicals formed from UV irradiation of a deca-BDE mixture (DE-83R). The activating wavelength for deca-BDE photochemistry was in the UVA to UVB range. The yields of radicals from irradiated deca-BDE in tetrahydrofuran, dimethylformamide, and toluene were about 9-, 4-, and 7-fold higher, respectively, than from irradiated solvent alone. Radical formation increased with deca-BDE concentration and irradiation time. The quantum yield of radical formation of the deca-BDE mixture was higher than with an octa-BDE mixture (DE-79; approximately 2-fold), decabromobiphenyl (PBB 209; approximately 2-fold), decachlorobiphenyl (PCB 209; approximately 3-fold), and diphenyl ether (DE; approximately 6-fold), indicating the positive effects of bromine and an ether bond on radical formation. Analysis of hyperfine splittings of the spin adducts suggests that radical formation is initiated or significantly enhanced by debromination paired with hydrogen abstraction from the solvents. To our knowledge this is the first study that uses EPR to demonstrate the formation of free radicals during the photolytic degradation of PBDEs. Our findings strongly suggestthe potential of negative consequences due to radical formation during UV exposure of PBDEs in biological systems.

Evidence for a role of caveolin-1 in neurokinin-1 receptor plasma-membrane localization, efficient signaling, and interaction with β-arrestin 2

This study was focused on the relationship between the plasma-membrane localization of neurokinin-1 receptor (NK1-R) and its endocytic and signaling properties. First, we employed electron paramagnetic resonance (EPR) to study the domain structure of HEK-293 cells and NK1-R microlocalization. EPR spectra and the GHOST condensation routine demonstrated that NK1-R was distributed in a well-ordered domain of HEK-293 cells possibly representing lipid raft/caveolae microdomains, whereas the impairment of caveolae changed the NK1-R plasma-membrane distribution. Internalization and second messenger assays combined with bioluminescence resonance energy transfer were employed subsequently to evaluate the functional importance of the NK1-R microlocalization in lipid raft/caveolae microdomains. The internalization pattern was delineated through the use of dominant-negative mutants (DNM) of caveolin-1 S80E (Cav1 S80E), dynamin-1 K44A (Dyn K44A), and beta-arrestin (beta-arr 319-418) and by means of cell lines that expressed various endogenous levels of beta-arrestins. NK1-R displayed rapid internalization that was substantially reduced by DNMs of dynamin-1 and beta-arrestin and even more profoundly in cells lacking both beta-arrestin1 and beta-arrestin2. These internalization data were highly suggestive of the predominant use of the clathrin-mediated pathway by NK1-R, even though NK1-R tended to reside constitutively in lipid raft/caveolae microdomains. Evidence was also obtained that the proper clustering of the receptor in these microdomains was important for effective agonist-induced NK1-R signaling and for its interaction with beta-arrestin2.

Photochemistry of Naphthalene Diimides: EPR Study of Free Radical Formation via Photoredox Process¶

ABSTRACTEarlier studies have shown that on exposure to UVA, hydroperoxynaphthalene diimide (IA) generates hydroxyl radicals, induces DNA strand scission, and kills cells. Here we employed electron paramagnetic resonance (EPR) and spin trapping to investigate the free radical photochemistry of IA and that of related naphthalene diimides, which are devoid of the hydroperoxyl moiety (N,N′‐bis[2‐methyl]‐1,4,5,8‐naphthaldiimide [IB], N,N′‐bis[2‐thiomethyl‐2‐methoxyethyl]‐1,4,5,8‐naphthaldiimide [IC]) and therefore are unable to generate hydroxyl radicals. It is shown that on UV irradiation (>300 nm) in air‐free methanol or ethanol solutions all these naphthalene diimides undergo one‐electron reduction to corresponding anion radicals, positively identified by EPR. With EPR and a spin trap 5,5‐dimethyl‐1‐pyrroline N‐oxide (DMPO), we found that the photogeneration of the naphthalene diimide radicals is concomitant with the formation of radicals from the solvents, presumably through electron/hydrogen atom abstraction by photoactivated diimides. Irradiation of IA, IB or IC in the presence of oxygen generates superoxide, which was detected as a DMPO adduct. The high photoreactivity of IB and IC supports the notion that hydroperoxide IA can induce oxidative damage via photoprocesses that are independent of *OH generation. These observations could be pertinent to the application of naphthalene diimides as selective photonucleases, PDT anticancer agents or both.

Photochemistry of Naphthalene Diimides: EPR Study of Free Radical Formation via Photoredox Process¶

Earlier studies have shown that on exposure to UVA, hydroperoxynaphthalene diimide (IA) generates hydroxyl radicals, induces DNA strand scission, and kills cells. Here we employed electron paramagnetic resonance (EPR) and spin trapping to investigate the free radical photochemistry of IA and that of related naphthalene diimides, which are devoid of the hydroperoxyl moiety (N,N'-bis[2-methyl]-1,4,5,8-naphthaldiimide [IB], N,N'-bis[2-thiomethyl-2-methoxyethyl]-1,4,5,8-naphthaldiimide [IC]) and therefore are unable to generate hydroxyl radicals. It is shown that on UV irradiation (>300 nm) in air-free methanol or ethanol solutions all these naphthalene diimides undergo one-electron reduction to corresponding anion radicals, positively identified by EPR. With EPR and a spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO), we found that the photogeneration of the naphthalene diimide radicals is concomitant with the formation of radicals from the solvents, presumably through electron/hydrogen atom abstraction by photoactivated diimides. Irradiation of IA, IB or IC in the presence of oxygen generates superoxide, which was detected as a DMPO adduct. The high photoreactivity of IB and IC supports the notion that hydroperoxide IA can induce oxidative damage via photoprocesses that are independent of *OH generation. These observations could be pertinent to the application of naphthalene diimides as selective photonucleases, PDT anticancer agents or both.