Profluorescent nitroxides: Sensors and stabilizers of radical-mediated oxidative damage

In this study, three profluorescent nitroxides 1,1,3,3-tetramethyldibenzo[e,g]isoindolin-2-yloxyl (TMDBIO), 1,1,3,3-tetramethyl-2,3-dihydro-2-azaphenalene-2-yloxyl (TMAO) and 5-[2-(4-methoxycarbonyl-phenyl)-ethenyl]1,1,3,3-tetramethylisoindoline-2-yloxyl (MeCSTMIO) were tested as probes for radical-mediated damage in polypropylene arising from both UV and thermally initiated sources. These nitroxides possess a very low fluorescence quantum yield due to quenching by the nitroxide group; however, when the free-radical moiety is removed by reaction with alkyl radicals (to give an alkoxyamine), strong fluorescence is observed. The results obtained from this profluorescent nitroxide trapping technique compare favourably with other methods of monitoring degradation, provided the appropriate probe is chosen for the conditions of oxidation, signalling an indication of damage well before other techniques show any response. The technique was also applied to the monitoring of crosslinked polyester coating resins. Differentiation in the oxidative stability of the resins was evident after as little as 200 min where other monitoring techniques require up to 300 h of accelerated degradation. This highlights the sensitivity of this method as well as demonstrating the scope of this technique to assess polymer stability.     

Investigation of polypropylene degradation during melt processing using a profluorescent nitroxide probe: A laboratory-scale study

Degradation of polypropylene (PP) during melt processing was studied using a novel profluorescence technique. The profluorescent nitroxide probe, 1,1,3,3-tetramethyldibenzo[e,g]isoindolin-2-yloxyl (TMDBIO) was added to PP during melt processing to act as a sensor for carbon-centred radicals. Trapping of carbon-centred radicals, formed during degradation of PP, led to an increase in fluorescence emission from TMDBIO adducts. Through analysis of viscosity changes during processing cumulative chain scission degradation was estimated. At processing temperatures of 210 °C or below, fluorescence emission from TMDBIO adducts could be correlated with cumulative chain scissions when the number of chain scissions was small. At higher temperatures, a correlation was not observed most probably due to radical-trap instability rather than decomposition of the TMDBIO. Thus, TMDBIO may be used as a profluorescent sensor for degradation of PP during melt processing when the processing temperature is 210 °C or below.     

Polyaromatic Profluorescent Nitroxide Probes with Enhanced Photostability

Novel profluorescent mono‐ and bis‐isoindoline nitroxides linked to napthalimide and perylene diimide structural cores are described. These nitroxide‐fluorophore probes display strongly suppressed fluorescence in comparison to their corresponding non‐radical diamagnetic methoxyamine derivatives. The perylene‐based probe possessing two isoindoline systems tethered through ethynyl linkages was shown to be the most photostable in solution, demonstrating significantly enhanced longevity over the 9,10‐bis(phenylethynyl)anthracene fluorophore used in previous profluorescent nitroxide probes.     

Two-photon fluorescence microscopy imaging of cellular oxidative stress using profluorescent nitroxides

A range of varying chromophore nitroxide free radicals and their nonradical methoxyamine analogues were synthesized and their linear photophysical properties examined. The presence of the proximate free radical masks the chromophore's usual fluorescence emission, and these species are described as profluorescent. Two nitroxides incorporating anthracene and fluorescein chromophores (compounds 7 and 19, respectively) exhibited two-photon absorption (2PA) cross sections of approximately 400 G.M. when excited at wavelengths greater than 800 nm. Both of these profluorescent nitroxides demonstrated low cytotoxicity toward Chinese hamster ovary (CHO) cells. Imaging colocalization experiments with the commercially available CellROX Deep Red oxidative stress monitor demonstrated good cellular uptake of the nitroxide probes. Sensitivity of the nitroxide probes to H(2)O(2)-induced damage was also demonstrated by both one- and two-photon fluorescence microscopy. These profluorescent nitroxide probes are potentially powerful tools for imaging oxidative stress in biological systems, and they essentially "light up" in the presence of certain species generated from oxidative stress. The high ratio of the fluorescence quantum yield between the profluorescent nitroxide species and their nonradical adducts provides the sensitivity required for measuring a range of cellular redox environments. Furthermore, their reasonable 2PA cross sections provide for the option of using two-photon fluorescence microscopy, which circumvents commonly encountered disadvantages associated with one-photon imaging such as photobleaching and poor tissue penetration.     

Profluorescent verdazyl radicals – synthesis and characterization

The synthesis and characterization of various 6-oxo-verdazyl radicals and their diamagnetic styryl radical trapping products are presented. It is shown that styryl radical trapping products derived from N-phenyl verdazyls show fluorescence whereas the N-methyl congeners are non-fluorescent. In the parent N-phenyl verdazyls fluorescence is fully quenched which renders these compounds highly valuable profluorescent radical probes.     

Profluorescent nitroxides: Thermo-oxidation sensors for stabilised polypropylene

The profluorescent nitroxide, 1,1,3,3-tetramethyldibenzo[e,g]isoindolin-2-yloxyl (TMDBIO) was investigated as a probe for the radical-mediated degradation of stabilised polypropylene. TMDBIO has been previously shown to be a sensitive probe for free-radical degradation during the thermo-oxidation of unstabilised polypropylene. Here we report on the effect that adding hindered phenol or phosphite stabilisers to polypropylene has on the free-radical sensing ability of TMDBIO during thermo-oxidation. In addition, novel dual-functional, hindered phenol containing profluorescent nitroxides, 5-[2-(4-hydroxy-3,5-di-tert-butylphenyl)ethenyl]-1,1,3,3-tetramethylisoindolin-2-yloxyl (HSTMIO) and its derivatives were investigated as probes for the radical-mediated degradation of polypropylene. These dual-functional probes were shown to be efficient stabilisers for polypropylene during thermo-oxidation at 150 °C in oxygen and sensors of thermo-oxidation during its early stages, in the so-called “induction period”.     

Regulating Degradation Pathways of Polymers by Radical-Triggered Luminescence

Understanding the radical behaviours during polymer degradation is beneficial to unveil and regulate the degradation pathways of polymers to achieve a sustainable polymer development. However, it is a long-standing challenge to study radical behaviours owing to the ultra-short lifetime of the transient radicals generated during the polymer degradation. In this contribution, we have proposed the radical-triggered luminescence to monitor the radical behaviours during polymer degradation without/with the addition of inorganic additives. It was disclosed that the pure polymers showed a single sigmoidal dynamic curve from peroxy radicals (ROO⋅) emissions, leading to the exponential proliferation for the degradation evolution. Alternatively, the degradation pathways with the addition of additives, layered double hydroxides (LDHs) with positively charged Al centers, could be modulated into a double sigmoidal dynamics, involving the main product of alkoxy radicals (RO⋅) with the activation energy of 40.2 kJ/mol and a small amount of ROO⋅ with 76.3 kJ/mol, respectively. Accordingly, the polymers with the additive-regulated pathways could exhibit prominently anti-degradation behaviours. This work is beneficial for the deep understanding of the radical mechanisms during polymer degradation, and for the rational design of anti-degradation polymers.     

Sensitive luminescence techniques to study the early stages of polymer oxidation

This paper describes recent developments in the use of chemiluminescence (CL) and profluorescent nitroxides (PFNs) in probing the “induction period” of polymer oxidation. CL measures the instantaneous rate of reaction of hydroperoxides responsible for initiating degradation and the spreading of oxidation, while PFNs can be used to measure the concentration of alkyl radicals produced in oxidation events and thus provide an integrating sensor for the extent of cumulative damage. The PFN additive acts as an oxidation retarder by competing with oxygen to scavenge the alkyl radicals that generate chain carrying peroxy radicals and so mirrors the performance of hindered amine stabilisers (HAS) in one part of their stabilisation cycle. Using the example of polypropylene (PP) and cis-polyisoprene (PIP) as substrates which can rapidly spread oxidative damage, the factors controlling the reaction of PFNs can be determined from CL and fluorescence as well as infra-red (IR) spectroscopy through the detection of oxidation products as measured by the carbonyl index. Matrix effects on the reactivity are demonstrated using a polyethylene-norbornene copolymer (TOPAS) as carrier for both PIP and the PFN and it is seen that the PFN is a radical scavenger only above T_g of the carrier. When PIP alone is oxidized, the PFN is an integrating sensor for free radical production under ambient conditions for up to twelve months while also stabilizing the polymer. Critically, it is thus able to determine the underlying rate of radical production in the oxidation induction period.     

Nitroxides scavenge myeloperoxidase-catalyzed thiyl radicals in model systems and in cells

Nitroxide radicals possess important antioxidant activity in live tissues because of their ability to scavenge reactive radicals. Despite the fact that, in cells, damaging free radicals are primarily quenched by glutathione (GSH) with subsequent formation of harmful glutathionyl radical (GS(*)), interactions of nitroxide radicals with GS(*) and thiols have not been studied in detail. In addition, intracellular metabolic pathways leading to the formation of secondary amines from nitroxides are unknown. Here we report that GS(*) radicals react efficiently and irreversibly with nitroxides to produce secondary amines. We developed a sensitive method for the detection of GS(*) based on their specific interaction with Ac-Tempo, a nonfluorescent conjugate of fluorogenic acridine with paramagnetic nitroxide Tempo, and used it to characterize interactions between nitroxide and thiyl radicals generated through phenoxyl radical recycling by peroxidase. During reaction of Ac-Tempo with GS(*), Tempo EPR signals decayed and acridine fluorescence concurrently increased. DMPO and PBN, spin traps for GS(*), inhibited this interaction. Using combined HPLC and mass spectrometry, we determined that 90% of the Ac-Tempo was converted into fluorescent acridine (Ac)-piperidine; GSH was primarily oxidized into sulfonic acid. In myeloperoxidase-rich HL-60 cells, Ac-piperidine fluorescence was observed upon stimulation of GS(*) generation by H(2)O(2) and phenol. Development of fluorescence was prevented by preincubation of cells with the thiol-blocking reagent N-ethylmaleimide as well as with peroxidase inhibitiors. Furthermore, Ac-Tempo preserved intracellular GSH and protected cells from phenol/GS(*) toxicity, suggesting a new mechanism for the free-radical scavenging activity of nitroxides in live cells.     

Degradation of poly(vinyl chloride) by u.v. radiation—II: Mechanism

The mechanism of the light-induced degradation of solid poly(vinyl chloride) (PVC) has been investigated, and an overall reaction scheme has been developed, based on values of the quantum yields for the primary photoproducts. Only a very small fraction (0.2%) of the excited polyenes induces the degradation of PVC, primarily by photocleavage of the allylic CCl bond. The high instability of β-chloroalkyl radicals is responsible for the chain dehydrochlorination that leads to formation of polyenes. In the absence of O₂, chain scissions and crosslinking are postulated to originate mainly from α-chloroalkyl radicals through β-cleavage of CC bonds and radical coupling, respectively. In the presence of O₂, the chain dehydrochlorination still proceeds, together with an oxidative chain process which yields, via peroxy and alkoxy radicals, hydroperoxides, ketones and peroxide crosslinks. Cleavage of the polymer backbone results most probably from the decomposition of tertiary alkoxy radicals by a carbon-carbon β-scission process.     

A DFT study of H-isomerisation in alkoxy-, alkylperoxy- and alkyl radicals: Some implications for radical chain reactions in polymer systems

Intramolecular 1-n H-shift (n = 2, 3… 7) reactions in alkoxy, alkyl and peroxy radicals were studied by density functional theory (DFT) at the B3LYP/6-311+G∗∗ level and compared with respective intermolecular H-transfers. It was found that starting from 1 to 3 H-shift the barrier heights stepwise decrease with increasing n reaching minimum for 1-5 and 1-6 H-shifts. This dependence can be ascribed to the decrease of the strain with increasing transition state (TS) ring size, which is minimal in six- and seven-member rings. The barrier heights of H-shifts in alkyl radicals are systematically larger than those in alkoxy radicals: the respective activation energies (E_a) of 1-5 and 1-6 H-shifts are about 59-67 kJ/mol for alkyl radical and 21-34 kJ/mol for alkoxy radicals. Further increase of the TS ring size in 1-7 H-shifts leads to the increase of the barrier to 44 kJ/mol in the hexyloxy radical and 84 kJ/mol for n-heptyl radical. We have also found that intermolecular H-transfer reactions in all three types of free radicals have smaller barriers than respective intramolecular 1-5 or 1-6 H-shifts by 4-25 kJ/mol. The mentioned difference can be explained in terms of enhanced nonbonding repulsion interaction in the cyclic TS structures compared to respective intermolecular TS. B3LYP/6-311+G∗∗ geometric parameters and imaginary frequencies for 1-n H-shifts TS are consistent with respective calculated barrier heights. Reactivity of some other radicals compared to alkoxy, peroxy and alkyl radicals as well as other factors influencing their reactivity (π-conjugation, steric effect and ring strain in cyclic TS, etc.) are also briefly discussed in relation to free radical reactions in polymer systems.     

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.     

A new approach for the detection of carbon-centered radicals in enzymatic processes using prefluorescent probes

In this communication we propose a novel application for prefluorescent probes in the detection of free carbon-centered radicals in enzymatic processes. Prefluorescent probes combine a fluorescent moiety tethered to a paramagnetic nitroxide that acts as a fluorescence quencher. Trapping of a radical by the nitroxide group restores the fluorescence properties. The increase in fluorescence intensity with time reflects the formation and quenching of carbon-centered radicals and can be used for the quantitative evaluation of yields and kinetics. As a test system we used horseradish peroxidase, an oxidoreductase that is widely accepted to operate by a radical-mediated mechanism. We used the prefluorescent probe (quinoline-TEMPO), where a quinoline moiety has been tethered to 2,2,6,6-tetramethylpiperidin-1-oxyl.     

Electrochemical and spectroelectrochemical properties of nitroxyl radical species in PTMA,an organic radical polymer. Influence of the microstructure

In this work, an electrochemical and ESR-spectrochemical study of the oxidation properties for nitroxide radicals contained in the structure of poly(4-methacryloyloxy-2,2,6,6-tetramethylpiperidin-N-oxyl) (PTMA) in acetonitrile solution was performed. Results revealed that this polymer contains two types of nitroxide radical species, the first associated to a multielectronic and reversible electron transfer process. Remaining radical species are irreversibly oxidized at more positive potential values. Proportions obtained for the respective amount of each oxidized species, 78% and 22% respectively, are similar to the values expected due to the difference in syndiotactic:heterotactic triads in the polymer and reported values for the experimental charge/discharge efficiencies of batteries constructed with this material. These effects also influence electron transfer kinetics during the oxidation of each type of radical species in the polymer. These findings suggest that the detailed understanding of the oxidation processes of different microstructures in radical polymers is useful to analyze performance based in these materials.     

A long-lived luminescence and EPR bimodal lanthanide-based probe for free radicals

We developed a novel spin-labeled terbium complex Tb(3+)/cs124-DTPA-TEMPO (1) by covalently labeling a nitroxide radical on the terbium complex for monitoring free radicals of various areas. This lanthanide complex probe shows a high EPR signal which resulted from the nitroxide radical moiety, and is weakly luminescent which resulted from the intramolecular quenching effect of the nitroxide radical on sensitised terbium luminescence. The intensity of both the EPR and luminescence can be modulated by eliminating the paramagnetism of the nitroxide radical through recognition of a carbon-centered radical analyte and thus gives a quantification of the analyte. We have preliminarily applied this probe in the luminescent detection of model carbon-centered radicals and hydroxyl radicals (·OH). This probe is water-soluble and contains lanthanide-luminescence properties, favorable for the time-resolved luminescence technique. The investigation of the intramolecular quenching process has showed that the labeled nitroxide radical quenches multiple excited states of the terbium complex, resulting in highly efficient quenching of terbium luminescence. This probe is the first example of intramolecular modulation of lanthanide luminescence by a nitroxide radical.     

Generation of profluorescent isoindoline nitroxides using click chemistry

Novel profluorescent nitroxides bearing a triazole linker between the coumarin fluorophore and an isoindoline nitroxide were prepared in good yields using the copper-catalyzed azide-alkyne 1,3-dipolar cycloaddition reaction (CuAAC). Nitroxides containing 7-hydroxy and 7-diethylamino substitution on their coumarin rings displayed significant fluorescence suppression, and upon reaction with methyl radicals, normal fluorescence emission was returned. The fluorescence emission for the 7-hydroxycoumarin nitroxide and its diamagnetic analogue was found to be strongly influenced by pH with maximal fluorescence emission achieved in basic solution. Solvent polarity was also shown to affect fluorescence emission. The significant difference in fluorescence output between the nitroxides and their corresponding diamagnetic analogues makes these compounds ideal tools for monitoring processes involving free-radical species.     

Aromatic inorganic acid radical

Stable radicals are challenging to prepare due to their intrinsic high reactivity. Herein, three trisphenolamine radicals were readily synthesized and exhibited unexpected thermal/electrochemical stability and semiconductor property. These three nitroxide radicals could be considered as a class of aromatized nitro groups or HNO_3 derivatives. The closed-shell nitro-like and open-shell nitroxide resonance structure contribute to their outstanding stability. Furthermore, the tunable ground states, extremely low band gap and p-type charge transport properties were systematically investigated. More importantly, the work presents the concept of aromatic inorganic acid radical(AIAR) and aggregation-induced radical(AIR) mechanism to understand the intrinsic structureproperty relationship of these radicals. In addition, we provide a novel strategy for the design of stable and low bandgap radicals for organic electronics, magnetics, spintronics, etc.     

Rotational mobility of nitroxyl radicals in polyesters

Rotational mobility of nitroxyl radicals in polyester films was investigated at temperatures from ?180 to +240°C by ESR spin-probe techniques. Evidence obtained indicated that mobility of radicals was related to polymer structure and reflected increased volume made available to the probe molecule by the polymer matrix. A single correlation time, which followed the form τ_C = τ₀ exp {E_a/RT}, was calculated for the temperature range of rapid reorientation (～10⁹ Hz) of nitroxyl radicals. E_a ranged from 5 to 25 kcal/mole for various systems. Uniaxially oriented semicrystalline polymer matrix restricted radical mobility to a greater extent than a semicrystalline biaxially oriented sample. Effective local viscosity encountered by a nitroxyl radical in several polymers was calculated as 9–13 poise.     

Mechanism and kinetics of the enzymatic hydrolysis of polyester nanoparticles by lipases

The degradation of several aliphatic and aromatic polyesters with lipases from Candida cylindracea (CcL) and Pseudomonas species (PsL) was investigated applying nanoparticles of the polymers. Nanoparticles (diameters 50 nm to 250 nm) of a particle concentration up to 6 mg/ml could be prepared by a precipitation technique without adding any stabilizing agents in the aqueous solutions. Using a titration system to monitor ester cleavage, enzymatic degradation experiments could be performed in the time scale of some minutes. A kinetic model is proposed which is based on a surface erosion process dependent on molar ester bond density and enzyme loading. Experimental evidence provided that degradation of the particles occurs uniformly at the surface after a Langmuir type adsorption of the enzyme. Rate constants and the maximal enzyme loadings of enzyme were estimated from the kinetic model for different polyesters and the rate constants correlate well with the length of the diacid component of the polyester. Comparison of degradation rates of polyester films and nanoparticles revealed that nanoparticles of aliphatic polyesters are in the amorphous state. Hence, differences of the rate constants reflect the direct influence of the polymer structure on the enzymatic hydrolysis not overlaid by effects of crystallinity.     

The role of hydroperoxides in photo-oxidative degradation of cis-1,4-polybutadiene

Hydroperoxides undergo various types of homolytic reactions on exposure to u.v. radiation. Free radicals formed from the photodecomposition of the hydroperoxide group (OOH) are oxy (HO.) and peroxy (HOO.) radicals which participate in further reactions. In cis-1,4-polybutadiene, they may initiate free radical oxidations. Cleavage of alkoxy (RO.) radicals and crosslinking of polymer radicals through polymer peroxides in the presence of air in solid film nearly balance. Most polymer radicals produced in the absence of oxygen undergo cross-linking but form peroxy radicals (POO.) in its presence. This paper presents results on the photodecomposition of tert-butyl hydroperoxide, cumyl hydroperoxide and 2,5-dimethyl-2,5-dihydroperoxyhexane in cis-1,4-polybutadiene in film and in solution.