Electron Spin Resonance Spin-trapping Experiments Research Articles

Triphenylamine based oxime ester photoinitiator for LED-induced free radical photopolymerization and 3D printing

With the development of light-emitting diode (LED), it is increasingly important to design and prepare photoinitiators that can be used for LED excitation. We designed and synthesized four new LED oxime ester photoinitiators (TOXEs) based on the triphenylamine group. Among them, the maximum absorption wavelengths of TOXE-3 and TOXE-4 are red-shifted to 402 nm and 397 nm, and have high molar absorption coefficients (ε) of 24315 M-1cm−1 and 29915 M-1cm−1, respectively, which are higher than that of ethenone, 1-[9-ethyl-6-(2-methylbenzoyl)-9H-carbazol-3-yl] ethanone 1-(O-acetyloxime) (OXE02, 342 nm, 21836 M-1cm−1), indicating that it can be used in LED polymerization. Investigating the photopolymerization properties of the TOXEs/α, ω-diacryloyl poly(ethylene glycol) (PEGDA) system, under LED@365 nm irradiation, the double bond conversion rate (DC) of TOXE-1 and TOXE-2 were 83 % and 92 %, which were higher than OXE02 (80 %). Under LED@405 nm irradiation, the DC of TOXE-3 and TOXE-4 are 80 % and 78 % respectively, which is higher than that of OXE02 (52 %). To improve the photoinitiation ability of TOXEs, a two-component photoinitiation system using bis(4-tert-butylphenyl) iodonium (Iod) as an additive was tested. The DC of TOXEs has been improved under different light sources. Under LED@480 nm irradiation, the final DC of TOXE-4/Iod/PEGDA is 83 %. In addition, TOXE-3 and TOXE-4 can be applied to the laser cladding deposition (LCD) 3D printers. At the same time, we conduct research and verification on the polymerization mechanism of TOXEs through steady-state photolysis and electron spin resonance spin trapping experiments (ESR-ST). In addition, cytotoxicity assay and thermal stability testing showed that TOXEs exhibited excellent biocompatibility and thermal stability.

Novel chalcone-based phenothiazine derivative photoinitiators for visible light induced photopolymerization with photobleaching and good biocompatibility

Two novel visible light photoinitiators 4-(10-methyl-10H-phenothiazin-3-yl) but-3-en-2-one (PBO) and (1E,4E)-1,5-bis(10-methyl-10H-phenothiazin-3-yl)-1,4-pentadien-3-one (BMPPO) were successfully designed and synthesized. PBO and BMPPO have broad absorption range from 350 to 500 nm with the maximum absorption wavelength at 400 nm and 440 nm respectively. Three-component photoinitiating system of BMPPO/iodonium/amine was developed for free radical polymerization of acrylates. The results demonstrated that the photoinitiating system displayed rapid polymerization and high final vinyl group conversions under the irradiation of UV-LED (365 nm), blue LED (440) nm and solar light resource. Moreover, photopolymerization kinetics was studied by real-time FT-IR spectrum, the double band conversion was achieved up to 90% for BMPPO. The photocuring mechanism was identified via steady state photolysis and electron spin resonance spin-trapping experiments. The possible photoinduced mechanism of PBO and BMPPO in the presence of a co-initiator occurred through electron transfer. In addition, BMPPO possesses rapid photobleaching and good biocompatibility performance tested by photoinduced degradation and cytotoxicity experiments respectively, which exhibited that BMPPO has potential for light-color coatings and food packaging field.

DFT calculations as a powerful tool for ESR spin trapping experiments

A combined electron spin resonance (ESR) and density functional theory study has been carried out to obtain deeper insight in the reaction mechanism of the photorearrangement of 3-hydroxyindolic nitrones. Also, in this case, the combination of these techniques constitutes a powerful tool when discriminating between different reaction pathways. In particular, density functional theory calculations played an important role for unraveling the overlapping of signals coming from different species in the ESR spin trapping experiments. In the present study, the computed energies and the corresponding ESR parameters of each possible isomer hypothesized have been considered, taking also into account their different possibilities of intramolecular H-bond formation. However, the results obtained indicate that the possibility of intramolecular H-bonding did not play a determinant role in this case. Copyright © 2016 John Wiley & Sons, Ltd.

フェノール性二量体の抗酸化活性と抗菌活性

We studied the antioxidant and antibacterial activities of monomeric and dimeric phenol compounds. Dimeric compounds had higher antioxidant activities than monomeric compounds. Electron spin resonance spin-trapping experiments showed that phenol compounds with an allyl substituent on their aromatic rings directly scavenged superoxide, and that only eugenol trapped hydroxyl radicals. We developed a generation system of the hydroxyl radical without using any metals by adding L-DOPA and DMPO to PBS or MiliQ water in vitro. We found that eugenol trapped hydroxyl radicals directly and is metabolized to a dimer. On the other hand, dipropofol, a dimer of propofol, has strong antibacterial activity against Gram-positive bacteria. However, it lacks solubility in water and this property is assumed to limit its efficacy. We tried to improve the solubility and found a new solubilization method of dipropofol in water with the addition of a monosaccharide or ascorbic acid.

Mechanism of UVA-dependent DNA Damage Induced by An Antitumor Drug Dacarbazine in Relation to its Photogenotoxicity

It has been reported that dacarbazine (DTIC) is photogenotoxic. The purpose of this study is to clarify the mechanism of photogenotoxicity induced by DTIC. We examined DNA damage induced by UVA-irradiated DTIC using 32P-5'-end-labeled DNA fragments obtained from human genes. Formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) in calf thymus DNA was measured by high performance liquid chromatograph with an electrochemical detector. Electron spin resonance (ESR) spin-trapping experiments were performed to detect radical species generated from UVA-irradiated DTIC. UVA-irradiated DTIC caused DNA damage at guanine residues, especially at the 5'-GGT-3' sequence in the presence of Cu(II) and also induced 8-oxodG generation in calf thymus DNA. DTIC-induced photodamage to DNA fragments was partially inhibited by catalase, whereas 8-oxodG formation was significantly increased by catalase. NaN3, a carbene scavenger, inhibited DNA damage and 8-oxodG formation in a dose-dependent manner, suggesting that carbene intermediates are involved. The ESR spin-trapping experiments demonstrated the generation of aryl radicals in the process of photodegradation of DTIC. Photoactivated DTIC generates the carbene and aryl radicals, which may induce both DNA adduct and 8-oxodG formation, resulting in photogenotoxicity. This study could provide an insight into the safe usage of DTIC.

Molecular mechanisms of DNA damage induced by procarbazine in the presence of Cu(II)

Procarbazine [ N-isopropyl-α-(2-methylhydrazino)- p-toluamide], a hydrazine derivative, which has been shown to have effective antineoplastic activity, induces cancer in some experimental animals and humans. To clarify a new mechanism for its carcinogenic effect, we examined DNA damage induced by procarbazine in the presence of metal ion, using 32 P -5′-end-labeled DNA fragments obtained from the human p53 tumor suppressor gene and the c-Ha- ras-1 protooncogene. Procarbazine plus Cu(II) induced piperidine-labile and formamidopyrimidine-DNA glycosylase-sensitive lesions at the 5′-A CG -3′ sequence, complementary to a hotspot of the p53 gene, and the 5′-TG-3′ sequence. Catalase partially inhibited DNA damage, suggesting that not only H 2O 2 but also other reactive species are involved. Procarbazine plus Cu(II) significantly increased the formation of 8-oxo-7,8-dihydro-2′-deoxyguanosine, which was completely inhibited by calatase. Electron spin resonance spin-trapping experiments revealed that methyl radicals were generated from procarbazine and Cu(II). On the basis of these findings, it is considered that procarbazine causes DNA damage through non-enzymatic formation of the Cu(I)–hydroperoxo complex and methyl radicals. In conclusion, in addition to alkylation, oxidative DNA damage may play important roles in not only antitumor effects but also mutagenesis and carcinogenesis induced by procarbazine.

Phenoxyl Free Radical Formation during the Oxidation of the Fluorescent Dye 2′,7′-Dichlorofluorescein by Horseradish Peroxidase: POSSIBLE CONSEQUENCES FOR OXIDATIVE STRESS MEASUREMENTS

The oxidation of the fluorescent dye 2',7'-dichlorofluorescein (DCF) by horseradish peroxidase was investigated by optical absorption, electron spin resonance (ESR), and oxygen consumption measurements. Spectrophotometric measurements showed that DCF could be oxidized either by horseradish peroxidase-compound I or -compound II with the obligate generation of the DCF phenoxyl radical (DCF(.)). This one-electron oxidation was confirmed by ESR spin-trapping experiments. DCF(.) oxidizes GSH, generating the glutathione thiyl radical (GS(.)), which was detected by the ESR spin-trapping technique. In this case, oxygen was consumed by a sequence of reactions initiated by the GS(.) radical. Similarly, DCF(.) oxidized NADH, generating the NAD(.) radical that reduced oxygen to superoxide (O-(2)), which was also detected by the ESR spin-trapping technique. Superoxide dismutated to generate H(2)O(2), which reacted with horseradish peroxidase, setting up an enzymatic chain reaction leading to H(2)O(2) production and oxygen consumption. In contrast, when ascorbic acid reduced the DCF phenoxyl radical back to its parent molecule, it formed the unreactive ascorbate anion radical. Clearly, DCF catalytically stimulates the formation of reactive oxygen species in a manner that is dependent on and affected by various biochemical reducing agents. This study, together with our earlier studies, demonstrates that DCFH cannot be used conclusively to measure superoxide or hydrogen peroxide formation in cells undergoing oxidative stress.

Reactive Oxygen Species Generated from the Reaction of Copper(II) Complexes with Biological Reductants Cause DNA Strand Scission

The generation of hydroxyl radicals (•OH) from the reaction of Cu(II) complexes with biological reductants such as ascorbic acid, glutathione, acetylcysteine, and hydroquinone was confirmed by spin-trapping experiments using electron spin resonance (ESR).The following Cu(II) complexes were used: Cu(II)-(CyHH)2(CyHH, cyclo(l-histidyl-l-histidyl)), Cu(II)(OP)2(OP,o-phenanthroline), Cu(II)(HGG) (HGG,l-histidylglycylglycine), and Cu(II)(en)2(en, ethylenediamine). The methyl radical adduct of α-(pyridyl-4-N-oxide)-N-tert-butylnitrone (POBN–CH3) was obtained from the reaction of ascorbic acid with all Cu(II) complexes used here in the presence of a spin trap, POBN, and dimethyl sulfoxide, indicating the generation of•OH. Glutathione,N-acetylcysteine, and hydroquinone reacted with both Cu(II)(CyHH)2and Cu(II)(OP)2to generate POBN-CH3, while these reductants did not react with either Cu(II)(HGG) or Cu(II)(en)2. Interestingly, the formation of POBN–CH3in the reaction of Cu(II)-(CyHH)2with glutathione orN-acetylcysteine was found only at a Cu(II)(CyHH)2/glutathione or Cu(II)-(CyHH)2/N-acetylcysteine ratio of 1. The DNA strand scission caused by reaction mixtures of Cu(II) complexes with reductants was investigated under thesame conditions as the ESR spin-trapping experiments. Addition of ascorbic acid to mixtures of these four Cu(II) complexes and DNA resulted in DNA strand breakage. Hydroquinone plus Cu(II)(CyHH)2also caused DNA strand scission. In addition, DNA strand breakage was observed with the reaction of Cu(II)(OP)2with glutathione,N-acetylcysteine, and hydroquinone. In contrast, reaction mixtures of glutathione,N-acetylcysteine, or hydroquinone with Cu(II)-(HGG) or Cu(II)(en)2did not cause DNA strand scission within the concentration range used. The results obtained here suggest that there is a good correlation between POBN-CH3formation and DNA strand scission. Thus, DNA strand scission may be caused by•OH generated from the reaction of some Cu(II) complexes with biological reductants under aerobic conditions. Since ascorbic acid, glutathione, andN-acetylcysteine are present in living cells, some Cu(II) complexes may be capable of initiating DNA damage in the presence of these reductants.

Carotenoid reaction with free radicals in acetone and toluene at different oxygen partial pressures

The influence of eight commonly occurring carotenoids on (1) the production of spin adducts, (2) the hyperfine splitting constants aH and aN of the spin adduct electron spin resonance (ESR) spectra, and (3) the production of the oxidized N-tert-butyl-α-phenylnitrone (PBN) derivative N-benzoyl-N-tert-butylnitroxide (PBNOx) was investigated in ESR spin-trapping experiments, in which radicals were thermally generated from 2,2′-azobis(2,4-dimethylvalero nitrile) (AMVN) and trapped by PBN. In the polar solvent acetone under air, all carotenoids except β-carotene were shown to diminish the number of spin adducts formed by about 20%. A prooxidative effect of β-carotene was even demonstrated at a higher concentration. In the less polar solvent toluene under air, most carotenoids lowered the amount of spin adducts, but effects were not as large as in acetone, and differences were found between (1) the more effective carotenoids with two cyclohexene rings conjugated to the polyene backbone (and lycopene), and (2) carotenoids with zero or one conjugated cyclohexene ring. The formation of PBNOx in toluene, which was essentially due to breakdown of peroxyl spin adducts, was significantly inhibited by astaxanthin, canthaxanthin and lycopene. Throughout, only alkoxyl radical adducts of PBN were observed. However, a significant difference was found between the aH values of zeaxanthin and the carbonyl carotenoid astaxanthin, indicating structural differences of the spin adducts.

Requirement of endogenous iron for cytotoxicity caused by hydrogen peroxide in Euglena gracilis

It is widely thought that redox-active metals in cells such as iron or copper catalyze the reduction of hydrogen peroxide to toxic hydroxyl radicals or their equivalent. However, this has not been directly demonstrated in vivo. To probe this requirement, the freshwater microorganism Euglena gracilis was used. Its intracellular iron content can be modulated by the concentration of iron in the defined growth medium without effect on the proliferation rate of the cells. E. gracilis contains two large storage pools of cytosolic iron which can be monitored to assess cellular iron status. The toxicity of H 2O 2 in E. gracilis was inversely related to the amount of iron in the extracellular medium. At high levels of external iron, the metal carried out the Fenton reaction with H 2O 2 outside the cell, producing hydroxyl radicals as detected by electron-spin resonance spin-trapping experiments. This reaction reduced the amount of H 2O 2 that could diffuse into cells to cause toxicity. When cells with different intracellular iron content were placed in iron-deficient media, the the toxicity of H 2O 2, measured by inhibition of proliferation, was directly related to the concentration of internal iron. Using cells deficient in iron, this oxidant did not inhibit proliferation at low concentration but was somewhat effective at higher concentrations. Although the iron chelating agents, 1,10-phenanthroline and desferrioxamine, also depressed cytosolic iron, they were cytotoxic to E. gracilis and so could not be used unambiguously to examine the role of intracellular iron in the toxic effects of hydrogen peroxide.

Detection of singlet (1O2) oxygen phosphorescence during chloroperoxidase-catalyzed decomposition of ethyl hydroperoxide

Evidence for the production of singlet molecular oxygen (1O2) during the chloroperoxidase-catalyzed decomposition of ethyl hydroperoxide has been obtained through the use of optical spectroscopy, oxygen electrode experiments, and electron spin resonance (ESR). ESR spin-trapping experiments with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) demonstrate the production of the ethyl peroxyl free radical during the chloroperoxidase/ethyl hydroperoxide reaction. Oxygen and acetaldehyde concentrations suggest that the production of ethyl peroxyl radicals constitutes less than 2% of the decomposition of ethyl hydroperoxide at the concentrations of reactants used. The phosphorescence of 1O2 at 1268 nm was observed during the chloroperoxidase-catalyzed decomposition of ethyl hydroperoxide in deuterium oxide buffer. Chloroperoxidase also catalyzes the decomposition of tert-butyl hydroperoxide to its corresponding peroxyl radical. Alkoxyl and alkyl-DMPO spin adducts were also detected. A much lower yield of 1O2 phosphorescence was observed during the chloroperoxidase-catalyzed decomposition of tert-butyl hydroperoxide. This phosphorescence probably arises through secondary production of alkyl peroxyl radicals. These results suggest that the initial enzyme-dependent production of ethyl peroxyl radicals is followed by enzyme-independent reaction of two peroxyl radicals through the tetroxide intermediate, as originally proposed by Russell (Russell, G. A. (1957) J. Am. Chem. Soc. 79, 3871-3877), to form acetaldehyde, ethyl alcohol, and molecular oxygen.