Studies on bio-antioxidants--micellar effects on the reduction of nitroxides by vitamin C

The kinetics of reduction of nitroxides including 4-hydroxy-TEMPO, 4-methoxy-TEMPO and 4-hexanoyloxy-TEMPO, which are of different lipophilicities, by vitamin C in cationic, non-ionic and anionic micelles, i.e. CTAB, Triton X-100 and SDS, respectively, have been studied by FSR spectroscopy by a stopped-flow technique. A mechanism for the reaction conducted in micelles is proposed and the rate constants for the elementary reactions are evaluated. It is found that the rates of single electron transfer reactions involving the nitroxides are dependent on the nature of the micelle and the lipophilicity of the nitroxide. The rates are increased in CTAB, decreased in SDS, whereas unaffected in Triton X-100. And the greater the lipophilicity of the nitroxide, the more pronounced the rate variation. As high as a 3600-fold increase in the rate was observed for 4-hexanoyloxy-TEMPO in CTAB over that in SDS. The micellar effects are rationalized on the basis of analysis of parameters and line shape of the ESR spectra for the nitroxides in the micelles.     

位阻效应对N-(4-脱氢苯基)吡啶离子与氮氧自由基反应的理论计算研究

应用量子化学密度泛函理论(DFT),在B3LYP/cc-pVDZ基组水平上,对N-(4-脱氢苯基)吡啶离子与不同结构的氮氧自由基反应进行了热动力学研究.优化了反应通道上反应物、中间体、过渡态和产物的几何构型并计算出它们的零点振动能( Ezpv)和焓值,分析数据研究位阻效应对反应的影响.研究表明3类氮氧自由基均与N-(4...     

STUDIES ON BIO-ANTIOXIDANTS——MICELLAR EFFECTS ON THE REDUCTION OF NITROXIDES BY VITAMIN C

The kinetics of reduction of nitroxides including 4-hydroxy-TEMPO, 4-methoxy-TEMPOand 4-hexanoyloxy-TEMPO, which are of different lipophilicities, by vitamin C in cationic,non-ionic and anionic micelles, i. e. CTAB, Triton X-100 and SDS, respectively, have beenstudied by ESR spectroscopy by a stopped-flow technique. A mechanism for the reactionconducted in micelles is proposed and the rate constants for the elementary reactions areevaluated. It is found that the rates of single electron transfer reactions involving the ni-troxides are dependent on the nature of the micelle and the lipophilicity of the nitroxide.The rates are increased in CTAB, decreased in SDS, whereas unaffected in Triton X-100.And the greater the lipophilicity of the nitroxide, the more pronounced the rate variation.As high as a 3600-fold increase in the rate was observed for 4-hexanoyloxy-TEMPO in CTABover that in SDS. The micellar effects are rationalized on the basis of analysis of parame-ters and line shape of the ESR spectra for     

Energy transfer in polymers and nitroxide decay during photolysis

When polymeric materials doped with nitroxides of the 2,2,6,6-tetramethylpiperidine type are exposed to light, the nitroxide concentration decreases. The two mechanisms for the decrease of the nitroxide are the reaction of nitroxide with free radicals produced during photolysis of the polymer to form amino ethers and the abstraction of hydrogen atoms by excited-state nitroxides to form hydroxyl amines. Excited-state nitroxides can be formed in two ways: by direct absorption and by energy transfer. In this paper, the effect of energy transfer on the rate of decay of the nitroxide signal is studied, and measurements of nitroxide decay are used to probe energy transfer in crosslinked polymeric coatings. A simple kinetic scheme is used to interpret nitroxide decay during photolysis of both solutions and polymers containing benzophenone. These results are used to show that the slope of the line relating nitroxide decay rate to nitroxide concentration is essentially determined by energy transfer from a coating-based chromophore to nitroxide. The nitroxide decay assay is also used to study the effectiveness of a benzotriazole ultraviolet light absorber as a quencher.     

Structure-activity relationship of cyclic nitroxides as SOD mimics and scavengers of nitrogen dioxide and carbonate radicals

Synthetic nitroxide antioxidants attenuate oxidative damage in various experimental models. Their protective effect reportedly depends on ring size and ring substituents and is greater for nitroxides having lower oxidation potential. The present study focuses on the kinetics and mechanisms of the reactions of piperidine, pyrrolidine and oxazolidine nitroxides with HO2*/O2*-, *NO2 and CO3*- radicals, which are key intermediates in many inflammatory and degenerative diseases. It is demonstrated that nitroxides are the most efficient scavengers of *NO2 at physiological pH (k = (3-9) x 10(8) M(-1) s(-1)) and among the most effective metal-independent scavengers of CO3*- radicals (k = (2 - 6) x 10(8) M(-1) s(-1)). Their reactivity toward HO2*, though not toward *NO2 and CO3*-, depends on the nature of the ring side-chain and particularly on the ring-size. All nitroxide derivatives react slowly with O2*- and are relatively inefficient SOD mimics at physiological pH. Even piperidine nitroxides, having the highest SOD-like activity, demonstrate a catalytic activity of about 1000-fold lower than that of native SOD at pH 7.4. The present results do not indicate any correlation between the kinetics of HO2*/O2*-, *NO2 and CO3*- removal by nitroxides and their protective activity against biological oxidative stress and emphasize the importance of target-oriented nitroxides, i.e., interaction between the biological target and specific nitroxides.     

The palladium-catalysed copper-free Sonogashira coupling of isoindoline nitroxides: a convenient route to robust profluorescent carbon-carbon frameworks

A series of novel acetylene-substituted isoindoline nitroxides were synthesised via palladium-catalysed copper-free Sonogashira coupling. These results demonstrate that the Sonogashira reaction is suitable for the generation of a wide range of aryl nitroxides of expanded structural variety. The novel aryl-iodide containing nitroxide, 5-iodo-1,1,3,3-tetramethylisoindolin-2-yloxyl, 3, was a key intermediate for this coupling, giving acetylene-substituted isoindoline nitroxides in high yield. Subsequent reaction of the deprotected ethynyl nitroxide 12 with iodinated polyaromatics furnished novel aromatic nitroxides with extended-conjugation. Such nitroxides have been described as profluorescent, as their quantum yields are significantly lower than those of the corresponding diamagnetic derivatives. The quantum yields of the naphthyl- and phenanthryl-acetylene isoindoline nitroxides (13 and 14) were found to be 200-fold and 65-fold less than the non-radical methoxyamine derivatives (23 and 24). Ethyne- and butadiyne-linked nitroxide dimers could also be synthesised by this cross coupling methodology.     

Kinetics and mechanism of peroxyl radical reactions with nitroxides

Cyclic nitroxides (>NO*) are stable radicals of diverse size, charge, lipophilicility, and cell permeability, which provide protection against oxidative stress via various mechanisms including SOD-mimic activity, oxidation of reduced transition metals and detoxification of oxygen- and nitrogen-centered radicals. However, there is no agreement regarding the reaction of nitroxides with peroxyl radicals, and many controversies in the literature exist. The question of whether nitroxides can protect by scavenging peroxyl radicals is important because peroxyl radicals are formed in biological systems. To further elucidate the mechanism(s) underlying the antioxidative effects of nitroxides, we studied by pulse radiolysis the reaction kinetics of piperidine, pyrrolidine, and oxazolidine nitroxides with several alkyl peroxyl radicals. It is demonstrated that nitroxides mainly reduce alkyl peroxyl radicals forming the respective oxoammonium cations (>N+=O). The most efficient scavenger of peroxyl radicals is 2,2,6,6-tetramethylpiperidine-N-oxyl (TPO), which has the lowest oxidation potential among the nitroxides tested in the present study. The rate constants of peroxyl reduction are in the order CH2(OH)OO*>CH3OO*>t-BuOO*, which correlate with the oxidation potential of these peroxyl radicals. The rate constants for TPO vary between 2.8x10(7) and 1.0x10(8) M-1 s-1 and for 3-carbamoylproxyl (3-CP) between 8.1x10(5) and 9.0x10(6) M-1 s-1. The efficacy of protection of nitroxides against inactivation of glucose oxidase caused by peroxyl radicals was studied. The results demonstrate a clear correlation between the kinetic features of the nitroxides and their ability to inhibit biological damage inflicted by peroxyl radicals.     

Electrospray ionization mass spectrometry of stable nitroxide free radicals and two isoindoline nitroxide dimers

Electrospray ionization (ESI) mass spectra were recorded for a range of substituted isoindoline nitroxides, two isoindoline nitroxide dimers and two piperidinyl nitroxides. In all cases the dominant molecular species arise from oxidation rather than protonation, an unusual process in ESI. Fragment ion spectroscopy was used to establish fragmentation mechanisms for the nitroxides under ESI conditions.     

Controlled radical polymerization of styrene in the presence of nitronyl nitroxides

Bulk radical polymerization of styrene in the presence of nitronyl nitroxides (2-(4-substituted phenyl)-4,4,5,5-tetramethyl-4,5-dihydroimidazolyl-1-oxyl 3-oxide) was studied. All nitronyl nitroxides, like other nitroxyl radicals such as 2,2,6,6-tetramethylpiperidine 1-oxyl radical (TEMPO), act as reversible radical scavengers. The efficiency of controlling the polymerization is affected by the substituent at the 4′-position. The efficiency increases with electron donating strength of 4′-substituents, at least at the beginning of the reaction. However, the thermal stability of nitronyl nitroxides decreases in the same order. Thus, TEMPO is more suitable than nitronyl nitroxides for controlled/“living” radical polymerization of styrene.     

Synthesis of the tetraethyl substituted pH-sensitive nitroxides of imidazole series with enhanced stability towards reduction

The synthesis of 2,2,5,5-tetraethylimidazole nitroxides from 3-ethylpent-2-ene is described. The newly synthesized nitroxides, namely 4-methyl-2,2,5,5-tetraethyl-2,5-dihydro-1H-imidazol-1-yloxy (1), 3,4-dimethyl-2,2,5,5-tetraethylperhydroimidazol-1-yloxy (2) and 2,2,5,5-tetraethyl-4-pyrrolidin-1-yl-2,5-dihydro-1H-imidazol-1-oxyl (3), were found to be pH sensitive spin probes, with pK values of 1.2, 4.95 and 7.4, respectively. The most important finding was the fact that these new nitroxides were 20-30 times more stable in the presence of ascorbate and had significantly longer halflifes in rat blood as compared to 2,2,5,5-tetramethyl analogs. The latter observation provides a unique advantage for the application of tetraethyl substituted imidazole nitroxides as functional EPR probes.     

TBARS法测定胰岛素口腔喷雾剂中脂质过氧化物研究

采用硫代巴比妥酸反应物法(ThiobarbituricAcidReactiveSubstanceAssay,TBARS)对胰岛素口腔喷雾剂(InsulinBuccalSpray,IBS)中脂质过氧化程度进行了测定,并对IBS前处理方法、稀释倍数、加热作用亦进行了探究。发现在使用丙二醛(Malondialdehyde,MDA)测试盒测量IBS中脂质过氧化物(LipidPeroxide,LPO)实验中,样品应该先用有机溶剂稀释或用含0.8%(m/V)TritonX 100的0.01mol·L-1HCl溶液破乳;而乳剂测量前以稀释10倍为宜。对不同时期的IBS制剂检测,发现在IBS的一年保质期内,其脂质过氧化程度变化不大。     

Chemically induced dynamic electron polarization generated through the interaction between singlet molecular oxygen and nitroxide radicals

An absorptive chemically induced dynamic electron polarization (CIDEP) was generated by the quenching of singlet oxygen by nitroxide radicals (TEMPO derivatives). The spin polarization decay time of the nitroxide (measured by time-resolved EPR) correlates with the lifetime of singlet oxygen (measured by singlet oxygen phosphorescence spectroscopy). In addition, a deuterium isotope effect on the spin polarization decay time was observed, a signature of singlet oxygen involvement. With use of isotope labeled nitroxides (15N, 14N), the relative spin polarization efficiencies of TEMPO, 4-oxo-TEMPO, and 4-hydroxy-TEMPO by singlet oxygen were determined. The relative spin polarization efficiencies (per quenching event) decrease in the order 4-hydroxy-TEMPO > TEMPO > 4-oxo-TEMPO, whereas an opposite trend was observed for the total quenching rate constants of singlet oxygen by the nitroxides where the order is 4-hydroxy-TEMPO < TEMPO < 4-oxo-TEMPO.     

Design and use of β-phosphorus nitroxides and alkoxyamines in controlled/“living” free radical polymerizations

Persistent nitroxides and their corresponding alkoxyamines are important regulators of living radical polymerization. Here we describe the synthesis of β-phosphorus nitroxides bearing a β-hydrogen, that present very interesting properties for the control of the radical polymerization of styrenes, acrylates and other monomers. A large series of alkoxyamines derived from these nitroxides was prepared, and Electron Spin Resonance (ESR) was used to determine both the temperature (T_c) and the rate constant (k_d) for their homolysis. For the whole series of alkoxyamines (27 compounds), a very good linear correlation was found between T_c and logk_d. Satisfactory linear correlations were found between T_c and calculated (PM3 method) Bond Dissociation Energy (BDE) of the NO-C bond, for series of alkoxyamines with the same type of leaving radical. The characteristics of free radical polymerization of styrene carried out in the presence of these new nitroxides and alkoxyamines will be discussed.     

Reversed-phase thin-layer chromatography of various pesticides in the presence of water-soluble beta-cyclodextrin polymer

Summary The modification of the hydrophobicity of 28 commercial pesticides with a water-soluble -cyclodextrin polymer (SCDP) in the presence of aqueous NaCl has been studied by reversed-phase thin-layer chromatography. The pesticides formed inclusion complexes with SCDP and these complexes are less lipophilic than the parent pesticides. The sodium chloride exerted a typical saltingout effect, the retention of each pesticide increased with increasing concentration of the salt in the eluent. This effect can be tentatively explained by the suppression of the dissociation of the polar groups in the solute molecules resulting in increased apparent lipophilicity. The correlation between lipophilicity and salting-out effect was found to be significant, that is the salting-out effect increases with increasing polarity (lower lipophilicity) of the pesticides, whereas the lipophilicity has negligible influence on the inclusion-forming capacity of non-homologous series of pesticides.     

Studies on the chemical modification of monensin. IV. Synthesis, sodium ion permeability, and biological activity of 7-O-acyl- and 7-O-alkylmonensins.

7-O-Acyl-(4a-e) and 7-O-alkylmonensins (5a-d) were prepared from monensin (1). Their lipophilicity, sodium ion permeability in human erythrocytes, antibacterial activity and effect on rat tail artery were examined. There was a correlation between lipophilicity and sodium ion permeability as well as between lipophilicity and antibacterial activity. We also found that the compound having larger sodium ion permeability, showed stronger contraction of rat tail artery. 7-O-Benzylmonensin (5c) exhibited higher lipophilicity and larger sodium ion permeability than monensin (1) among the tested monensin derivatives. In addition, antibacterial activity and contractile effect on rat tail artery of 5c were comparable to those of 1.     

Synthesis of profluorescent isoindoline nitroxides via palladium-catalysed Heck alkenylation

The synthesis of a new structural class of isoindoline nitroxides (aminoxyls), accessible via the palladium-catalysed Heck reaction, is presented. Reaction of the aryl bromoamine, 5-bromo-1,1,3,3-tetramethylisoindoline (4) or dibromoamine, 5,6-dibromo-1,1,3,3-tetramethylisoindoline (5) or the analogous bromonitroxides 6 and 7 with methyl acrylate gives the acrylate substituted tetramethylisoindoline amines 8 and 10 and nitroxides 12 and 14. Similarly, the reaction of the aryl bromides and dibromides 4-7 with methyl 4-vinylbenzoate gives the carboxystyryl substituted tetramethylisoindoline amines 9 and 11 and the analogous nitroxides 13 and 15. The carboxystyryl tetramethylisoindoline nitroxides demonstrate strongly suppressed fluorescence, which is revealed upon removal of the free radical by reduction or radical coupling.     

Kinetics and mechanism of hydroxyl radical and OH-adduct radical reactions with nitroxides and with their hydroxylamines

Stable nitroxide radicals are potent antioxidants and are among the most effective non-thiol radioprotectants, although they react with hydroxyl radicals more slowly than typical phenolic antioxidants or thiols. Surprisingly, the reduced forms of cyclic nitroxides, cyclic hydroxylamines, are better reductants yet have no radioprotective activity. To clarify the reason for this difference, we studied the kinetics and mechanisms of the reactions of nitroxides and their hydroxylamines with (*)OH radicals and with OH-adducts by using pulse radiolysis, fluorimetric determination of phenolic radiation products, and electron paramagnetic resonance spectrometric determination of nitroxide concentrations following radiolysis. Competition kinetics with phenylalanine as a reference compound in pulse radiolysis experiments yielded rate constants of (4.5 +/- 0.4) x 10(9) M(-1) s(-1) for the reaction of (*)OH radical with 2,2,6,6-tetramethylpiperidine-N-oxyl (TPO), 4-hydroxy-TPO (4-OH-TPO), and 4-oxo-TPO (4-O-TPO), (3.0 +/- 0.3) x 10(9) M(-1) s(-1) for deuterated 4-O-TPO, and (1.0 +/- 0.1) x 10(9) M(-1) s(-1) for the hydroxylamine 4-OH-TPO-H. The kinetic isotope effect suggests the occurrence of both (*)OH addition to the aminoxyl moiety of 4-O-TPO and H-atom abstraction from the 2- or 6-methyl groups or from the 3- and 5-methylene positions. This conclusion was further supported by final product analysis, which demonstrated that (*)OH partially oxidizes 4-O-TPO to the corresponding oxoammonium cation. The rate constants for the reactions of the nitroxides with the OH-adducts of phenylalanine and terephthalate have been determined to be near 4 x 10(6) M(-1) s(-1), whereas the hydroxylamine reacted at least 50 times slower, if at all. These findings indicate that the reactivity toward (*)OH does not explain the differences between the radioprotective activities of nitroxides and hydroxylamines. Instead, the radioprotective activity of nitroxides, but not of hydroxylamines, can be partially attributed to their ability to detoxify OH-derived secondary radicals.     

Determination of the lipophilicity of some anti-hypoxia drugs: Comparison of TLC and HPLC methods

Summary The lipophilicity of 14 anti-hypoxia drugs has been determined by reversed phase thin-layer (RPTLC) and reversed phase high performance liquid chromatography (RPHPLC) in eluent systems containing different concentrations of acetonitrile and potassium dihydrogen phosphate. There was significant correlation between lipophilicity and the specific hydrophobic surface area of the drugs in RPTLC, indicating that the drugs behave as an homologous series of compounds. In RPTLC the concentration of buffer has a negligible effect on the retention of the drugs whereas in RPHPLC the buffer concentration influenced the retention. This discrepancy can be explained by the lower sensitivity of RPTLC. There was strong correlation between lipophilicity values determined by both methods, proving that both are suitable for the determination of molecular lipophilicity.     

Evaluation of analgesic activities of tremetone derivatives isolated from the Chilean altiplano medicine Parastrephia lepidophylla

Parastrephia lepidophylla, family Asteraceae, has ancient use in traditional medicine in the region of Tarapacá, Chile. Bioguided fractionation of extracts of this plant was undertaken in the search for compounds with analgesic and antioxidant activity. Two benzofuran derivatives were isolated as the major components of this plant, identified as tremetone 1 and methoxytremetone 6. Remarkably, neither of these showed antioxidant activity, but tremetone 1 exhibited a morphine-like analgesic property. Reduction of this analgesic effect by naloxone suggests a direct effect on opiate receptors as a possible signaling pathway. However, both the low diffusion across lipid membranes (PAMPA assay) and the lipophilicity (Log P) shown by tremetone 1 make elusive the mechanism explaining its induced analgesia.     

The vibrational group frequency of the N-O* stretching band of nitroxide stable free radicals

The group frequency of the N-O radical stretching vibration has received scant attention in the literature. The few existing treatments of the vibrational spectroscopy of nitroxides are incomplete at best and potentially misleading to workers in the field. To close this gap in the available knowledge, the existing literature on the vibrational spectra of nitroxide stable free radicals is critically reviewed with particular reference to the wavenumber position of the N-O stretching vibration, nu(N-O). Poor evidentiary bases for the assignment nu(N-O) were found in many instances. Ab initio Density Field Theory calculations using a model chemistry of UB3LYP at the 6-311++G(d,p) level were performed to obtain a theoretical band position of nu(N-O) for comparison with the published data. Large discrepancies between the theoretical and experimental values were found for the radical 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrrolin-1-yloxyl, which currently sets the lower limit of the accepted wavenumber range of nu(N-O), as well as for the nitronyl and iminyl nitroxides. The wavenumber position of nu(N-O) was found to occur in the range 1450-1420cm(-1) for 5-membered cyclic nitroxides and 1395-1340cm(-1) for 6-membered cyclic and acyclic nitroxides. In nitronyl nitroxides, the symmetric stretching vibration occurs in the region 1470cm(-1), but coupling to other modes makes specific band assignments problematic for the nitronyl nitroxide group.