Photophysical properties of the prefluorescent nitroxide probes QT and C343T

The photophysical properties of the nitroxide prefluorescent probes 4-(3-hydroxy-2-methyl-4-quinolinoyloxy)-2,2,6,6-tetramethyl-piperidin-4-yl) ester free radical (QT) and 2,3,4,6,7,8-hexahydro-quinolizino [1,10,9-gh] coumarin-3-carboxylic acid (1-oxyl-2,2,6,6-tetramethyl-piperidin-4-yl) ester free radical (C343T) were evaluated as a function of pH and solvent properties. The absorbance of QT showed high pH sensitivity. The pKa values for the different ionization forms involved in the acid-base equilibrium of the quinoline chromophore were determined in the ground and excited states. The fluorescence lifetimes of QT, and N-hydroxylamine (QTH) and quinoline methyl ester (QMe) derivatives, showed that the intramolecular quenching efficiency by the nitroxide moiety is independent of the quinoline ionization form. The fluorescence and absorbance of C343T were highly sensitive to solvent polarity in agreement with a charged transfer excited state of the chromophore. However, we noted a decrease in the intramolecular fluorescence quenching efficiency by the nitroxide moiety when changing the polarity of the solvent from hexane to water. This behavior has been attributed to a suppression of an energy transfer mechanism in the nitroxide quenching process in very polar solvents. The results obtained in micelles allow us to propose QT and C343T as sensors for pH and micropolarity, respectively, in addition to their role as monitors for free radicals or hydrogen transfer from phenols.     

Investigation of polar and stereoelectronic effects on pure excited-state hydrogen atom abstractions from phenols and alkylbenzenes

The fluorescence quenching of singlet-excited 2,3-diazabicyclo[2.2.2]oct-2-ene (DBO) by 22 phenols and 12 alkylbenzenes has been investigated. Quenching rate constants in acetonitrile are in the range of 10(8)-10(9) M(-1)s(-1) for phenols and 10(5)-10(6) M(-1)s(-1) for alkylbenzenes. In contrast to the quenching of triplet-excited benzophenone, no exciplexes are involved, so that a pure hydrogen atom transfer is proposed as quenching mechanism. This is supported by (1) pronounced deuterium isotope effects (kH/kD ca 4-6), which were observed for phenols and alkylbenzenes, and (2) a strongly endergonic thermodynamics for charge transfer processes (electron transfer, exciplex formation). In the case of phenols, linear free energy relationships applied, which led to a reaction constant of rho = -0.40, suggesting a lower electrophilicity of singlet-excited DBO than that of triplet-excited ketones and alkoxyl radicals. The reactivity of singlet-excited DBO exposes statistical, steric, polar and stereoelectronic effects on the hydrogen atom abstraction process in the absence of complications because of competitive exciplex formation.     

Study of rare encounters in a membrane using quenching of cascade reaction between triplet and photochrome probes with nitroxide radicals

Measurements of active encounters between molecules in native membranes containing ingredients, including proteins, are of prime importance. To estimate rare encounters in a high range of rate constants (rate coefficients) and distances between interacting molecules in membranes, a cascade of photochemical reactions for molecules diffusing in multilamellar liposomes was investigated. The sensitised cascade triplet cis-trans photoisomerisation of the excited stilbene involves the use of a triplet sensitiser (Erythrosin B), a photochrome stilbene-derivative probe (4-dimethylamino-4'-aminostilbene) exhibiting the phenomenon of trans-cis photoisomerisation, and nitroxide radicals (5-doxyl stearic acid) to quench the excited triplet state of the sensitiser. Measurement of the phosphorescence lifetime of Erythrosin B and the fluorescence enhancement of the stilbene-derivative photochrome probe, at various concentrations of the nitroxide probe, made it possible to calculate the quenching rate constant k(q)= 1.1 x 10(15) cm2 M(-1) s(-1) and the rate constant of the triplet-triplet energy transfer between the sensitiser and stilbene probe k(T)= 1.0 x 10(12) cm2 M(-1) s(-1). These values, together with the data on diffusion rate constant, obtained by methods utilising various theoretical characteristic times of about seven orders of magnitude and the experimental rate constants of about five orders of magnitude, were found to be in good agreement with the advanced theory of diffusion-controlled reactions in two dimensions. Because the characteristic time of the proposed cascade method is relatively large (0.1 s), it is possible to follow rare collisions between molecules and free radicals in model and biological membranes with a very sensitive fluorescence spectroscopy technique, using a relatively low concentration of probes.     

Generation and reactivity toward oxygen of carbon-centered radicals containing indane,indene, and fluorenyl moieties

Resonance-stabilized radicals containing indane, indene, and fluorenyl moieties exhibit attenuated reactivity toward oxygen. Rate constants of approximately 10(5) M(-1) s(-1) were observed for the most stabilized radicals. The dependence of k(OX) (rate constant for radical trapping by oxygen) on the corresponding bond dissociation energies revealed that stereoelectronic effects are more important than steric effects in determining the low radical reactivity with oxygen. Scavenging by the nitroxide TEMPO was also examined, and revealed that in this case steric effects are more important than in the case of oxygen. The rate constants for the hydrogen abstraction by cumyloxyl and tert-butoxyl radicals generated thermally and photochemically have been determined in benzene, and were in the range of ca. (1-13) x 10(6) M(-1) s(-1), showing that benzylic stabilization has a modest effect on substrate reactivity as a hydrogen donor toward alkoxyl radicals.     

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.     

Free radical sensor based on CdSe quantum dots with added 4-amino-2,2,6,6-tetramethylpiperidine oxide functionality

The association and resulting fluorescence quenching of CdSe quantum dots by 4-amino-2,2,6,6-tetramethylpiperidine oxide (4-amino-TEMPO), a persistent nitroxide, have been examined using electron paramagnetic resonance (EPR) and fluorescence spectroscopy. EPR data suggest binding constants around (8 +/- 4) x 10(6) M(-1) for green (2.4-2.5 nm) nanoparticles, and the application of Job's method indicates that the preferred mode of binding involves one or two quencher molecules per quantum dot, although more quenchers could bind at high concentrations of 4-amino-TEMPO. Fluorescence quenching by 4-amino-TEMPO is at least 3 orders of magnitude more efficient than by TEMPO itself, reflecting the strong binding confirmed by the EPR data. Stern-Volmer plots are nonlinear and in light of the EPR data probably reflect ready accessibility of the CdSe surface to one or two 4-amino-TEMPO molecules, while additional quenchers can only bind if they displace trioctylphosphine oxide ligands. Quantum dot-4-amino-TEMPO complexes can be used as free radical sensors, since the fluorescence (quenched by the nitroxide) is readily restored when radicals are trapped to form alkoxyamines.     

Quenching of cascade reaction between triplet and photochrome probes with nitroxide radicals

We proposed a new method for the study of molecular dynamics and fluidity of the living and model biomembranes and surface systems. The method is based on the measurements of the sensitized photoisomerization kinetics of a photochrome probe. The cascade triplet cis-trans photoisomerization of the excited stilbene derivative sensitized with the excited triplet Erythrosin B has been studied in a model liposome membrane. The photoisomerization reaction is depressed with nitroxide radicals quenching the excited triplet state of the sensitizer. The enhanced fluorescence polarization of the stilbene probe incorporated into liposome membranes indicates that the stilbene molecules are squeezed in a relatively viscous media of the phospholipids. Calibration of the "triple" cascade system is based on a previously proposed method that allows the measurement of the product of the quenching rate constant and the sensitizer's triplet lifetime, as well as the quantitative detection of the nitroxide radicals in the vicinity of the membrane surface. The experiment was conducted using the constant-illumination fluorescence technique. Sensitivity of the method using a standard commercial spectrofluorimeter is about 10(-12) mol of fluorescence molecules per sample and can be improved using an advanced fluorescence technique. The minimal local concentration of nitroxide radicals or any other quenchers being detected is about 10(-5) M. This method enables the investigation of any chemical and biological surface processes of microscopic scale when the minimal volume is about 10(-3) microL or less.     

Synthesis and Properties of Isoindoline Nitroxide‐containing Porphyrins

Isoindoline nitroxide‐containing porphyrins were synthesized by the reaction of 5‐phenyldipyrromethane and 5‐(4′‐nitrophenyl)‐dipyrromethane with 5‐formyl‐1,1,3,3‐tetramethylisoindolin‐2‐yloxyl using the Lindsey method. These spin‐labeled porphyrins were further characterized by MS, UV, FTIR, ¹H‐NMR, cyclic voltammetry, electron paramagnetic resonance (EPR), and fluorescence spectroscopy. The electrochemical assay demonstrated that these isoindoline nitroxides‐containing porphyrins had similar electrochemical and redox properties as 5‐carboxy‐1,1,3,3‐tetramethylisoindolin‐2‐yloxyl. Electron paramagnetic resonance test exhibited these porphyrins possessed the hyperfine splittings and characteristic spectra of isoindoline nitroxides, with typical nitroxide g‐values and nitrogen isotropic hyperfine coupling constants. Fluorescence spectroscopy revealed that these porphyrins indicated fluorescence suppression characteristic of nitroxide–fluorophore systems. Moreover, their reduced isoindoline nitroxide‐containing porphyrins eliminated the fluorescence suppression and displayed strong fluorescence. Thus, these isoindoline nitroxide‐containing porphyrins may be considered as the potential fluorescent and EPR probes.     

Electron spin exchange in isoviscous solvent mixtures

Electron spin exchange rate constants and hyperfine coupling constants have been measured for two nitroxide spin probes in a number of isoviscous mixed solvents. Collision rate constants, normalized to 1 cP, are lower for solvents in which the major component is water. Further anomalies in the coupling constant for the systemtert-butanol-water are explained in terms of the marked concentration fluctuations known to occur in this solvent mixture. Substitution of a hydrophobic spin probe by one containing an –OH group leads to lowering of the spin exchange rate, possibly due to solvation of the probe.     

Hydrogen atom transfer from iron(II)-tris[2,2'-bi(tetrahydropyrimidine)] to TEMPO: a negative enthalpy of activation predicted by the Marcus equation

The transfer of a hydrogen atom from iron(II)-tris[2,2'-bi(tetrahydropyrimidine)], [FeII(H2bip)3]2+, to the stable nitroxide, TEMPO, was studied by stopped-flow UV-vis spectrophotometry. The products are the deprotonated iron(III) complex [FeIII(H2bip)2(Hbip)]2+ and the hydroxylamine, TEMPO-H. This reaction can also be referred to as proton-coupled electron transfer (PCET). The equilibrium constant for the reaction is close to 1; thus, the reaction can be driven in either direction. The rate constants for the forward and reverse reactions at 298 K are k1 = 260 +/- 30 M-1 s-1 and k-1 = 150 +/- 20 M-1 s-1. Interestingly, the rate constant for the forward reaction decreases as reaction temperature is increased, implying a negative activation enthalpy: DeltaH1 = -2.7 +/- 0.4 kcal mol-1, DeltaS1 = -57 +/- 8 cal mol-1 K-1. Marcus theory predicts this unusual temperature dependence on the basis of independently measured self-exchange rate constants and equilibrium constants: DeltaHcalcd = -3.5 +/- 0.5 kcal mol-1, DeltaScalcd = -42 +/- 10 cal mol-1 K-1. This result illustrates the value of the Marcus approach for these types of reactions. The dominant contributor to the negative activation enthalpy is the favorable enthalpy of reaction, DeltaH1 degrees = -9.4 +/- 0.6 kcal mol-1, rather than the small negative activation enthalpy for the H-atom self-exchange between the iron complexes.     

Origins of selectivity in a colorimetric charge-transfer sensor for diols

Bispyridyl hydrogen bonding receptor 1 forms colored charge transfer (CT) complexes with complementary phenols and naphthols. Despite its low association constants of approximately 10(1) M(-1), receptor 1 was highly selective forming CT complexes of varying color and intensity with different diol guests. The selectivity of 1 was correlated with the ability of its CT band to simultaneously yield information about the association constant and the electronic structure of the phenols and naphthols.     

Transfer of a hydrogen atom to acridine in the triplet state

Conclusions The quenching of triplet acridine by substituted phenols, thiophenols, and anilines is accomplished with high rate constants by hydrogen atom transfer with the quantitative formation of the corresponding neutral radicals.The solvation of the reagents by ethanol molecules or a decrease in the electron donor capacity of the quencher is accompanied by a decrease in the reaction rate constants.Translated from Izvestiya Akademii Nauk SSSR, Seriya Khimicheskaya, No. 1, pp. 178–181, January, 1989.     

Perylene-based profluorescent nitroxides for the rapid monitoring of polyester degradation upon weathering: An assessment

A profluorescent nitroxide possessing an isoindoline nitroxide moiety linked to a perylene fluorophore was developed to monitor radical mediated degradation of melamine-formaldehyde crosslinked polyester coil coatings in an industry standard accelerated weathering tester. Trapping of polyester-derived radicals (most likely C-radicals) that are generated during polymer degradation leads to fluorescent closed-shell alkoxy amines, which was used to obtain time-dependent degradation profiles to assess the relative stability of different polyesters towards weathering. The nitroxide probe couples excellent thermal stability and satisfactory photostability with high sensitivity and enables detection of free radical damage in polyesters under conditions that mimic exposure to the environment on a time scale of hours rather than months or years required by other testing methods. There are indications that the profluorescent nitroxide undergoes partial photo-degradation in the absence of polymer-derived radicals. Unexpectedly, it was also found that UV-induced fragmentation of the NO–C bond in closed-shell alkoxy amines leads to regeneration of the profluorescent nitroxide and the respective C-radical. The maximum fluorescence intensity that could be achieved with a given probe concentration is therefore not only determined by the amount of polyester radicals formed during accelerated weathering, but also by the light-driven side reactions of the profluorescent nitroxide and the corresponding alkoxy amine radical trapping products. Studies to determine the optimum probe concentration in the polymer matrix revealed that aggregation and re-absorption effects lowered the fluorescence intensity at higher concentrations of the profluorescent nitroxide, but too low probe concentrations, where these effects would be avoided, were not sufficient to trap the amount of polyester radicals formed upon weathering. The optimized experimental conditions were used to assess the impact of temperature and UV irradiance on polymer degradation during accelerated weathering.     

Thermal decomposition mechanisms of tert-alkyl peroxypivalates studied by the nitroxide radical trapping technique

The thermolysis of a series of tert-alkyl peroxypivalates 1 in cumene has been investigated by using the nitroxide radical-trapping technique. tert-Alkoxyl radicals generated from the thermolysis underwent the unimolecular reactions, beta-scission, and 1,5-H shift, competing with hydrogen abstraction from cumene. The absolute rate constants for beta-scission of tert-alkoxyl radicals, which vary over 4 orders of magnitude, indicate the vastly different behavior of alkoxyl radicals. However, the radical generation efficiencies of 1 varied only slightly, from 53 (R = Me) to 63% (R = Bu(t)()), supporting a mechanism involving concerted two-bond scission within the solvent cage to generate the tert-butyl radical, CO(2), and an alkoxyl radical. The thermolysis rate constants of tert-alkyl peroxypivalates 1 were influenced by both inductive and steric effects [Taft-Ingold equation, log(rel k(d)) = (0.97 +/- 0. 14)Sigmasigma - (0.31 +/- 0.04)SigmaE(s)(c), was obtained].     

Electron-transfer reaction of cinnamic acids and their methyl esters with the DPPH(*) radical in alcoholic solutions

The kinetic behavior of cinnamic acids, their methyl esters, and two catechols 1-10 (ArOH) in the reaction with DPPH(*) in methanol and ethanol is not compatible with a reaction mechanism that involves hydrogen atom abstraction from the hydroxyl group of 1-10 by DPPH(*). The rate of this reaction at 25 degrees C is, in fact, comparatively fast despite that the phenolic OH group of ArOH is hydrogen bonded to solvent molecules. The observed rate constants (k(1)) relative to DPPH(*) + ArOH are 3-5 times larger for the methyl esters than for the corresponding free acids and, for the latter, decrease as their concentration is increased according to the relation k(1) = B/[ArOH](0)(m), where k(1) is given in units of M(-1) s(-1), m is ca. 0.5, and B ranges from 0.02 (p-coumaric acid) to ca. 3.48 (caffeic acid) in methanol and from 0.04 (p-coumaric acid) to ca. 13 (sinapic acid) in ethanol. Apparently, the reaction mechanism of DPPH(*) + ArOH involves a fast electron-transfer process from the phenoxide anion of 1-10 to DPPH(*). Kinetic analysis of the reaction sequence for the free acids leads to an expression for the observed rate constant, k(1), proportional to [ArOH](0)(-1/2) in excellent agreement with the experimental behavior of these phenols. The experimental results are also interpreted in terms of the influence that adventitious acids or bases present in the solvent may have. These impurities dramatically influence the ionization equilibrium of phenols and cause a reduction or an enhancement, respectively, of the measured rate constants.     

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 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.     

Influence of the nitroxide structure on the homolysis rate constant of alkoxyamines: a Taft-Ingold analysis

Alkoxyamines and persistent nitroxyl radicals are important regulators of living radical polymerizations. Because polymerization times decrease with the increasing rate of the homolytic C-O bond cleavage between the polymer chain and the nitroxide moiety, the factors influencing the homolysis rate are of considerable interest. Here, we present an analysis of the cleavage rate constants for 28 alkoxyamines carrying the styryl (PhEt) group as leaving alkyl radical in terms of polar inductive/field (sigmaL) and steric (Es) effects of the nitroxide substituents, using the Taft-Ingold equation, i.e., log(k/k0) = rhoLsigmaL + deltaEs. The rate constants are shown to increase with the increasing electron-donating capacities, the steric demand, and the intramolecular (hydrogen) bonding capabilities of the substituents. A good correlation, (R2 = 0.95, 23 data) log kd = -3.07sigmaL - 0.88Es - 5.88, is obtained, which should facilitate the design of new nitroxyl radicals and alkoxyamine regulators.     

Kinetics and mechanisms of the oxidation of phenols by a trans-dioxoruthenium(VI) complex

The kinetics of the oxidation of phenols by trans-[Ru(VI)(L)(O)(2)](2+) (L = 1,12-dimethyl-3,4:9,10-dibenzo-1,12-diaza-5,8-dioxacyclopentadecane) have been studied in aqueous acidic solutions and in acetonitrile. In H(2)O the oxidation of phenol produces the unstable 4,4'- biphenoquinone, as evidenced by a rapid increase and then a slow decrease in absorbance at 398 nm. The first step is first-order in both Ru(VI) and phenol, and rate constants are dependent on [H(+)] according to the relationship k(f) = k(x) + (k(y)K(a)/[H(+)]), where k(x) and k(y) are the rate constants for the oxidation of PhOH and PhO(-), respectively. At 298 K and I = 0.1 M, k(x) = 12.5 M(-1) s(-1) and k(y) = 8.0 x 10(8) M(-1) s(-1). At I = 0.1 M and pH = 2.98, the kinetic isotope effects are k(H(2)O)/k(D(2)O) = 4.8 and 0.74 for k(x) and k(y), respectively, and k(f)(C(6)H(5)OH)/k(f)(C(6)D(5)OH) = 1.1. It is proposed that the k(x) step occurs by a hydrogen atom abstraction mechanism, while the k(y) step occurs by an electron-transfer mechanism. In both steps the phenoxy radical is produced, which then undergoes two rapid concurrent reactions. The first is a further three-electron oxidation by Ru(VI) and Ru(V) to give p-benzoquinone and other organic products. The second is a coupling and oxidation process to give 4,4'-biphenoquinone, followed by the decay step, k(s). A similar mechanism is proposed for reactions in CH(3)CN. A plot of log k(x) vs O-H bond dissociation enthalpies (BDE) of the phenols separates those phenols with bulky tert-butyl substituents in the ortho positions from those with no 2,6-di-tert-butyl groups into two separate lines. This arises because there is steric crowding of the hydroxylic groups in 2,6-di-tert-butyl phenols, which react more slowly than phenols of similar O-H BDE but no 2,6-tert-butyl groups. This is as expected if hydrogen atom abstraction but not electron transfer is occurring.     

Nitroxide/substrate weak hydrogen bonding: attitude and dynamics of collisions in solution

The study of intermolecular collisions and bonding interactions in solutions is of critical importance in understanding and predicting solute/solvent properties. Previous work has established that stable paramagnetic nitroxide molecules are excellent probes of intermolecular interactions for hydrogen bonding in polar solvents. In this study, 1H, 2H, 13C, 15N NMR and liquid/liquid intermolecular transfer dynamic nuclear polarization (L2IT DNP) results are obtained for the paramagnetic probe molecule, TEMPO, interacting with the common aprotic and protic polar solvents, CH3CN and CH3CONH2, yielding a profile of both dipolar and scalar interactions. A significant scalar contact hyperfine is observed for the N-O...H-C interaction (13CH3 hyperfine, a/h=0.66 MHz) in the CH3CN/TEMPO system, whereas the N-O...H-C and N-O...H-N interactions for the TEMPO/CH3CONH2 system yield 13CH3 and 15N hyperfine couplings of a/h=0.16 and -0.50 MHz, respectively. The distance and attitude of the scalar interaction for the nitroxide hydrogen bonding at the methyl group in CH3CN and the amino group in CH3CONH2 are computed using density functional theory (DFT), yielding good agreement with the experimental results. These results show that the hyperfine coupling provides a sensitive probe of weak hydrogen-bonding interactions in solution.