Cyclic Peroxidic Carbon Dioxide Dimer Fuels Peroxyoxalate Chemiluminescence.

Peroxyoxalate chemiluminescence is used in self-contained light sources, such as glow sticks, where oxidation of aromatic oxalate esters produces a high-energy intermediate (HEI) that excites fluorescence dyes via electron transfer chemistry, mimicking bioluminescence for efficient chemical energy-to-light conversion. The identity of the HEI and reasons for the efficiency of the peroxyoxalate reaction remain elusive. We present here unequivocal proof that the HEI of the peroxyoxalate system is a cyclic peroxidic carbon dioxide dimer, namely, 1,2-dioxetanedione. Oxalic peracids bearing a substituted phenyl group were unable to directly excite fluorescent dyes; hence, they could be ruled out as the HEI. However, base-catalyzed cyclization of these species results in bright chemiluminescence, with decay rates and chemiexcitation quantum yields that are influenced by the electronic phenylic substituent properties. Hammett (ρ = +2.2 ± 0.1) and Brønsted (ß = -1.1 ± 0.1) constants for the cyclization step preceding chemiexcitation imply that the loss of the phenolate-leaving group and intramolecular nucleophilic attack of the percarboxylate anion occur in a concerted manner, generating 1,2-dioxetanedione as the unique outcome. The presence of better leaving groups influences the reaction mechanism, favoring the chemiluminescent reaction pathway over the nonemissive formation of aryl-1,2-dioxetanones.

Chemi- and Bioluminescence of Cyclic Peroxides.

Bioluminescence is a phenomenon that has fascinated mankind for centuries. Today the phenomenon and its sibling, chemiluminescence, have impacted society with a number of useful applications in fields like analytical chemistry and medicine, just to mention two. In this review, a molecular-orbital perspective is adopted to explain the chemistry behind chemiexcitation in both chemi- and bioluminescence. First, the uncatalyzed thermal dissociation of 1,2-dioxetane is presented and analyzed to explain, for example, the preference for triplet excited product states and increased yield with larger nonreactive substituents. The catalyzed fragmentation reaction and related details are then exemplified with substituted 1,2-dioxetanone species. In particular, the preference for singlet excited product states in that case is explained. The review also examines the diversity of specific solutions both in Nature and in artificial systems and the difficulties in identifying the emitting species and unraveling the color modulation process. The related subject of excited-state chemistry without light absorption is finally discussed. The content of this review should be an inspiration to human design of new molecular systems expressing unique light-emitting properties. An appendix describing the state-of-the-art experimental and theoretical methods used to study the phenomena serves as a complement.

Mechanism of activated chemiluminescence of cyclic peroxides: 1,2-dioxetanes and 1,2-dioxetanones.

Almost all chemiluminescent and bioluminescent reactions involve cyclic peroxides. The structure of the peroxide and reaction conditions determine the quantum efficiency of light emission. Oxidizable fluorophores, the so-called activators, react with 1,2-dioxetanones promoting the former to their first singlet excited state. This transformation is inefficient and does not occur with 1,2-dioxetanes; however, they have been used as models for the efficient firefly bioluminescence. In this work, we use the SA-CASSCF/CASPT2 method to investigate the activated chemiexcitation of the parent 1,2-dioxetane and 1,2-dioxetanone. Our findings suggest that ground state decomposition of the peroxide competes efficiently with the chemiexcitation pathway, in agreement with the available experimental data. The formation of non-emissive triplet excited species is proposed to explain the low emission efficiency of the activated decomposition of 1,2-dioxetanone. Chemiexcitation is rationalized considering a peroxide/activator supermolecule undergoing an electron-transfer reaction followed by internal conversion.

Unimolecular Decomposition Mechanism of 1,2-Dioxetanedione: Concerted or Biradical? That is the Question!

Determination of the ground- and excited-state unimolecular decomposition mechanisms of 1,2-dioxetanedione gives a level of insight into bimolecular decomposition reactions of this kind for which some experimental results are reported. Although a few studies have put some effort to describe a biradical mechanism of this decomposition, there is still no systematic study that proves an existence of a biradical character. In the present study, state-of-the-art high-level multistate multiconfigurational reference second-order perturbation theory calculations are performed to describe the reaction mechanism of 1,2-dioxetanedione in detail. The calculations indicate that the decomposition of this four-membered ring peroxide containing two carbonyl carbon atoms occurs in concerted but not simultaneous fashion, so-called "merged", contrary to the case of unimolecular 1,2-dioxetane and 1,2-dioxetanone decompositions where biradical reaction pathways have been calculated. At the TS of the ground-state surface, the system enters an entropic trapping region, where four singlet and four triplet manifolds are degenerated, which can lead to the formation of triplet and singlet excited biradical species. However, these excited species have to overcome a second activation barrier for C-C bond cleavage for excited product formation, whereas the ground-state energy surface possesses only one TS. Thus our calculations indicate that the unimolecular decomposition of 1,2-dioxetanedione should not lead to efficient excited-state formation, in agreement with the lack of direct emission from the peroxyoxalate reaction.

Solvent viscosity influence on the chemiexcitation efficiency of inter and intramolecular chemiluminescence systems.

The effects of the medium viscosity on the chemiexcitation quantum yields of the induced decomposition of 1,2-dioxetanes (highly efficient intramolecular CIEEL system) and the catalyzed decomposition of diphenoyl peroxide and a 1,2-dioxetanone derivative (model systems for the intermolecular CIEEL mechanism, despite their low efficiency) are compared in this work. Quantum yields of the rubrene catalyzed decomposition of diphenoyl peroxide and spiro-adamantyl-1,2-dioxetanone as well as the fluoride induced decomposition of a phenoxy-substituted 1,2-dioxetane derivative are shown to depend on the composition of the binary solvent mixture toluene/diphenyl ether, which possess similar polarity parameters but different viscosities. Correlations of the quantum yield data with the medium viscosity using the diffusional and the frictional (free-volume) models indicate that the induced 1,2-dioxetane decomposition indeed occurs by an entirely intramolecular process and the low efficiency of the intermolecular chemiluminescence systems (catalyzed decomposition of diphenoyl peroxide and 1,2-dioxetanone derivative) is not primarily due to the cage escape of radical ion species.

Antiradical capacity assay for hydrophobic substances using hemin-catalyzed luminol chemiluminescence in cationic micelles.

Antioxidant substances which can diminish the steady-state concentration of free radicals in vivo are important in the human dietary to diminish the deleterious effects of oxidative stress. As the potential of certain substances as antioxidants is difficult to be verified in vivo, simple chemical in vitro assays which test the potential of substances as antioxidants are of great importance for the screening of new antioxidants. These assays measure the capacity of a substance to suppress free radicals. We describe here an antiradical capacity assay, based on luminol chemiluminescence, in cationic micellar medium, allowing the capacity determination of hydrophobic compounds. The antiradical capacity of antioxidants is determined using the Trolox standard by the measurement of the light emission inhibition area caused by the addition of different antiradical concentrations. The obtained results are compared to the values determined using the scavenging of stable free radicals be the substances and shown to be similar for compounds like uric acid, rutin, and quercetin. However, for vitamin E, the luminol assay results in a considerably higher antiradical capacity than the assay with a stable free radical, which is rationalized by the higher reactivity of the radical generated in the luminol assay and a specific localization of vitamin E in the micellar medium.

Solvent cage effects: basis of a general mechanism for efficient chemiluminescence.

The induced decomposition of 1,2-dioxetanes results in the efficient formation of singlet-excited carbonyl compounds. This transformation has been assumed to involve two sequential electron-transfer steps, and the viscosity dependence of the chemiexcitation efficiency (solvent cage effect) has been considered as evidence for the occurrence of an intermolecular electron back-transfer, despite the very high chemiexcitation quantum yields observed. However, all other chemiluminescent reactions assumed to occur according to the entirely intermolecular mechanism, referred to as CIEEL, are inefficient, except for the peroxyoxalate system. Therefore, we have investigated the solvent cage effect on the singlet quantum yields in both the induced decomposition of 1,2-dioxetanes and the peroxyoxalate reaction. Analysis of the viscosity effect observed for both systems, using a collisional as well as a free-volume model, indicates a very distinct behavior, which was interpreted as the occurrence of intramolecular chemiexcitation in the induced 1,2-dioxetane decomposition. We propose a general mechanism for efficient chemiluminescence in which the required electron back-transfer and C-C bond cleavage are concerted and compete with conformational changes that compromise the chemiexcitation. This mechanism is in agreement with both experimental and theoretical data available on the induced 1,2-dioxetane decomposition as well as with the high quantum efficiency of this transformation.

An Update on General Chemiexcitation Mechanisms in Cyclic Organic Peroxide Decomposition and the Chemiluminescent Peroxyoxalate Reaction in Aqueous Media.

Four-membered ring peroxides are intimately linked to chemiluminescence and bioluminescence transformations, as high-energy intermediates responsible for electronically excited-state formation. The synthesis of 1,2-dioxetanes and 1,2-dioxetanones enabled mechanistic studies on their decomposition occurring with the formation of electronically excited carbonyl products in the singlet or triplet state. The third member of this family, 1,2-dioxetanedione, has been postulated as the intermediate in the peroxyoxalate reaction, recently confirmed by kinetic studies on peroxalic acid derivatives. Several general chemiexcitation mechanisms have been proposed as model systems for the chemiexcitation step in efficient bioluminescence and chemiluminescence transformations. In this review article, we discuss the validity and efficiency of the most important chemiexcitation mechanisms, extended to aqueous media, where the efficiency is known to be drastically reduced, specifically in the peroxyoxalate reaction, highly efficient in anhydrous environment, but much less efficient in aqueous media. Mechanistic studies of this reaction will be discussed in diverse aqueous environments, with special attention to the catalysis involved in the thermal reaction leading to the formation of the high-energy intermediate and to the chemiexcitation mechanism, as well as emission quantum yields. Finally, several recent analytical and bioanalytical applications of the peroxyoxalate reaction in aqueous media will be given.

The Molecular Basis of Organic Chemiluminescence.

Bioluminescence (BL) and chemiluminescence (CL) are interesting and intriguing phenomena that involve the emission of visible light as a consequence of chemical reactions. The mechanistic basis of BL and CL has been investigated in detail since the 1960s, when the synthesis of several models of cyclic peroxides enabled mechanistic studies on the CL transformations, which led to the formulation of general chemiexcitation mechanisms operating in BL and CL. This review describes these general chemiexcitation mechanisms-the unimolecular decomposition of cyclic peroxides and peroxide decomposition catalyzed by electron/charge transfer from an external (intermolecular) or an internal (intramolecular) electron donor-and discusses recent insights from experimental and theoretical investigation. Additionally, some recent representative examples of chemiluminescence assays are given.

Water Effect on Peroxyoxalate Kinetics and Mechanism for Oxalic Esters with Distinct Reactivities.

The peroxyoxalate reaction is being widely used for various analytical and bioanalytical applications, and however, few mechanistic studies are performed in aqueous media, important mainly for bioanalytical applications, where low chemiluminescence emission quantum yields are obtained. In this sense, we report here kinetic studies on the peroxyoxalate reaction, using two commercially available and widely utilized esters, bis(2,4-dinitrophenyl) oxalate (DNPO) and bis(2,4,6-trichlorophenyl) oxalate (TCPO), in 1,2-dimethoxyethane:water mixtures. The reaction of the much more reactive DNPO, in anhydrous and aqueous media, occurs by a direct nucleophilic attack of H2 O2 to the oxalic ester, not involving nucleophilic catalysis by imidazole. Contrary, in the reaction of the less reactive TCPO with H2 O2 , imidazole acts mainly as nucleophilic catalyst. For both esters, experimental conditions are established where precise kinetic data and emission quantum yields can be obtained. Interestingly, the quantum yields in 1,2-dimethoxyethane water mixtures increase up to a water concentration of 0.7 mol L-1 and decrease significantly with higher concentrations.

Mechanistic Study of the Peroxyoxalate System in Completely Aqueous Carbonate Buffer.

The peroxyoxalate reaction is one of the most efficient chemiluminescence transformations, with emission quantum yields of up to 50%; additionally, it is widely utilized in analytical and bioanalytical assays. Although the real reason for its extremely high efficiency is still not yet understood, the mechanism of this transformation has been well elucidated in anhydrous medium. Contrarily, only few mechanistic studies have been performed in aqueous media, which would be of great importance for its application in biological systems. We report here our experimental results of the peroxyoxalate reaction in completely aqueous carbonate buffer, using fluorescein as chemiluminescence activator. The kinetics are very fast in the used basic conditions (pH > 9); despite this, reproducible kinetic results were obtained. The reaction proceeds by specific base catalysis, with rate-limiting attack of hydrogen peroxide anion to the oxalic ester, in competition with ester hydrolysis by hydroxide ion. Emission quantum yields increase with the hydrogen peroxide concentration up to an optimal concentration of 10 mmol L-1 . The infinite singlet quantum yield of (5.8 ± 0.2) × 10-7 is much lower than in anhydrous medium; however, it is similar to quantum yields measured before in partially aqueous media.

Experimental evidence of the occurrence of intramolecular electron transfer in catalyzed 1,2-dioxetane decomposition.

The activation parameters for the thermal decomposition of 13 acridinium-substituted 1,2-dioxetanes, bearing an aromatic moiety, were determined and their chemiluminescence emission quantum yields estimated, utilizing in situ photosensitized 1,2-dioxetane generation and observation of its thermal decomposition kinetics, without isolation of these highly unstable cyclic peroxides. Decomposition rate constants show linear free-energy correlation for electron-withdrawing substituents, with a Hammett reaction constant of ρ = 1.3 ± 0.1, indicating the occurrence of an intramolecular electron transfer from the acridinium moiety to the 1,2-dioxetane ring, as postulated by the intramolecular chemically initiated electron exchange luminescence (CIEEL) mechanism. Emission quantum yield behavior can also be rationalized on the basis of the intramolecular CIEEL mechanism, additionally evidencing its occurrence in this transformation. Both relations constitute the first experimental evidence for the occurrence of the postulated intramolecular electron transfer in the catalyzed and induced decomposition of properly substituted 1,2-dioxetanes.

Mechanistic Studies on the Salicylate-Catalyzed Peroxyoxalate Chemiluminescence in Aqueous Medium.

The peroxyoxalate reaction is one of the most efficient chemiluminescence transformations known and the only system occurring by an intermolecular chemically initiated electron exchange luminescence (CIEEL) mechanism with confirmed high quantum yields. The peroxyoxalate chemiluminescence (PO-CL) is mainly studied in anhydrous organic medium; however, for bioanalytical application, it should be performed in aqueous media. In the present work, we study the peroxyoxalate system in a binary 1,2-dimethoxyethane/water mixture with bis(2,4,6-trichlorophenyl) oxalate (TCPO), bis(4-methylphenyl) oxalate (BMePO) and bis[2-(methoxycarbonyl)phenyl] oxalate (DMO), catalyzed by sodium salicylate, in the presence of rhodamine 6G as activator. Reproducible kinetic results are obtained for all systems; emission decay rate constants depend on the salicylate as well as hydrogen peroxide concentration, and the occurrence of a specific base catalysis is verified. Although singlet quantum yields determined are lower than in anhydrous media in comparable conditions, they are still considerably high and adequate for analytical applications. The highest singlet quantum yields are obtained for the "ecologically friendly" derivative DMO indicating that this derivative might be the most adequate substrate for the use of the peroxyoxalate system in bioanalytical applications.

Direct kinetic observation of the chemiexcitation step in peroxyoxalate chemiluminescence.

A high-energy intermediate in the peroxyoxalate reaction can be accumulated at room temperature under specific reaction conditions and in the absence of any reducing agent in up to micromolar concentrations. Bimolecular interaction of this intermediate, accumulated in the reaction of oxalyl chloride with hydrogen peroxide, with an activator (highly fluorescent aromatic hydrocarbons with low oxidation potential) added in delay shows unequivocally that this intermediate is responsible for chemiexcitation of the activator. Activation parameters for the unimolecular decomposition of this intermediate (DeltaH(double dagger) = 11.2 kcal mol(-1); DeltaS(double dagger) = -23.2 cal mol(-1) K(-1)) and for its bimolecular reaction with 9,10-diphenylanthracene (DeltaH(double dagger) = 4.2 kcal mol(-1); DeltaS(double dagger) = -26.9 cal mol(-1) K(-1)) show that this intermediate is much less stable than typical 1,2-dioxetanes and 1,2-dioxetanones and demonstrate its highly favored interaction with the activator. Therefore, it can be inferred that structural characterization of the high-energy intermediate in the presence of an activator must be highly improbable. The observed linear free-energy correlation between the catalytic rate constants and the oxidation potentials of several activators definitely confirms the occurrence of the chemically initiated electron-exchange luminescence (CIEEL) mechanism in the chemiexcitation step of the peroxyoxalate system.

Revisiting the Mechanism of Hydrolysis of Betanin.

Betanin (betanidin 5-O-ß-D-glucoside) is a water-soluble plant pigment used as a color additive in food, drugs and cosmetic products. Despite its sensitivity to light and heat, betanin maintains appreciable tinctorial strength in low acidic and neutral conditions, where the color of other plant pigments, such as anthocyanins, quickly fades. However, betanin is an iminium natural product that experiences acid- and base-catalyzed hydrolysis to form the fairly stable betalamic acid and cyclo-DOPA-5-O-ß-D-glucoside. Here, we show that the decomposition of betanin in aqueous phosphate solution pH 2-11 is subject to general base catalysis by hydrogen phosphate ion and intramolecular general acid and base catalysis, providing new insights on the mechanism of betanin hydrolysis. UV/Vis absorption spectrophotometry, 1 H NMR spectroscopy and mass spectrometry were used to investigate product formation. Furthermore, theoretical calculations support the hypothesis that the nitrogen atom of the tetrahydropyridine ring of betanin is doubly protonated, as observed for structurally simpler amino dicarboxylic acids. Our results contribute to the study of betanin and other pigments belonging to the class of betalains and to deepen the knowledge on the chemical properties of imino acids as well as on iminium-catalyzed modifications of carbonyl compounds in water.

Mechanistic Studies on the Peroxyoxalate Chemiluminescence Using Sodium Salicylate as Base Catalyst.

Mechanistic studies on the peroxyoxalate chemiluminescence are often conducted with imidazole as base and nucleophilic catalyst. However, it is also known that this compound, at high concentrations, leads to a drastic reduction in the chemiluminescence quantum yields, apparently due to the destruction of the high-energy intermediate. Consequently, the search for new catalysts for this transformation is of importance for mechanistic studies as well as analytical application. Therefore, we report here a study on the mechanism of the peroxyoxalate reaction with sodium salicylate as base catalyst, which has already been utilized in this reaction; however, no detailed mechanistic investigation is known. In this work, a kinetic study on the sodium salicylate-catalyzed reaction of bis(2,4,6-trichlorophenyl) oxalate with hydrogen peroxide using 9,10-diphenylanthracene as activator is reported, where observed rate constants, singlet quantum yields and activation parameters are determined in different reaction conditions, leading to the formulation of a general mechanistic reaction scheme.

Chemiexcitation Efficiency of Intermolecular Electron-transfer Catalyzed Peroxide Decomposition Shows Low Sensitivity to Solvent-cavity Effects.

Intermolecular chemically initiated electron exchange luminescence (CIEEL) systems are known to possess low chemiluminescence efficiency; whereas, the corresponding intramolecular transformations are highly efficient. As the reasons for this discrepancy are not known, we report in this work our studies of the solvent-cavity effect on the efficiency of two intermolecular CIEEL systems, the catalyzed decomposition of diphenoyl peroxide and of a relatively stable 1,2-dioxetanone derivative, spiro-adamantyl-1,2-dioxetanone. The results indicate a very low medium viscosity effect on the quantum yields of these systems, a priori not compatible with these bimolecular transformations, showing also that their low efficiency cannot be due to solvent-cavity escape of intermediate radical ion pairs. In addition, the solvent-cage effect on the CIEEL efficiency, after the occurrence of the initial electron transfer, proved also to be very low, indicating the intrinsic low viscosity effect on the chemiexcitation step. Therefore, it is concluded that the low efficiency of these systems is intrinsic to the chemiexcitation step and cannot be improved by medium viscosity effects, being possibly due to sterical hindrance on charge-transfer complex formation in the initial step of the CIEEL.

Evaluation of antiradical capacity by H2O2-hemin-induced luminol chemiluminescence.

This paper describes a screening method for antioxidant potential determination based on luminol/hemin/hydrogen peroxide chemiluminescence. The emission depletion, caused by an antiradical compound added during the chemiluminescence decay, is proportional to the number of reactive species trapped. Therefore, the difference between the areas of the emission decay curves, obtained in the absence and in the presence of the potential antioxidant, is a measure for the antiradical capacity of the sample. The technique has been applied to measure the antiradical capacity of pure compounds and complex mixtures from natural origin, providing reliable results that indicate the method's feasibility.

Efficiency of electron transfer initiated chemiluminescence.

Although the mechanisms of many chemiluminescence (CL) reactions have been intensively studied, no general model has been suggested to rationalize the efficiency of these transformations. To contribute to this task, we report here quantum yields for some well-characterized CL reactions, concentrating on recent reports of efficient transformations. Initially, a short review on the most important general CL mechanisms is given, including unimolecular peroxide decomposition, electrogenerated CL, as well as the intermolecular and intramolecular catalyzed decomposition of peroxides. Thereafter, quantum yield values for several CL transformations are compiled, including the unimolecular decomposition of 1,2-dioxetanes and 1,2-dioxetanones, the catalyzed decomposition of appropriate peroxides and the induced decomposition of properly substituted 1,2-dioxetane derivatives. Finally, some representative examples of quantum yields for complex CL transformations, like luminol oxidation and the peroxyoxalate reaction, in different experimental conditions are given. This quantum yield compilation indicates that CL transformations involving electron transfer steps can occur with high efficiency in general only if the electron transfer is of intramolecular nature, with the intermolecular processes being commonly inefficient. A notable exception to this general rule is the peroxyoxalate reaction which, also constituting an example of an intermolecular electron transfer system, possesses very high quantum yields.

1,3-diene probes for detection of triplet carbonyls in biological systems.

Electronically excited triplet carbonyls are formed during the oxidative degradation of polyunsaturated fatty acids, amino acids, and beta-dicarbonyl metabolites. Due to their long lifetime and high alkoxyl radical-like reactivity, triplet carbonyls may initiate deleterious reactions in biological systems. Here we study the quenching properties of conjugated dienes, specifically 2,4-hexadienoate (sorbate) and its alkyl ester, on triplet acetone generated chemically (thermolysis of tetramethyl-1,2-dioxetane) or enzymatically (horseradish peroxidase-catalyzed aerobic oxidation of isobutanal). Triplet acetone quenching rates were near diffusion control ( k q = 10 (8)-10 (9) M (-1) s (-1)) and accompanied by diene cis-trans isomerization. None of the dienes displays antioxidant activity in classical systems known to generate reactive oxygen species: superoxide anion radical, hydroxyl radical, alkoxyl and alkylperoxyl radicals, or singlet oxygen. Experiments with model systems used widely to study lipid peroxidation showed that sorbate can inhibit mitochondrial swelling induced by enzymically formed triplet benzophenone and quench the chemiluminescence of microsome preparations challenged with iron and ascorbate. Altogether, our data indicate that conjugated dienes can be used as specific quenchers of triplet carbonyls formed in biological systems during oxidative stress. Moreover, they suggest that the well-known food preservative properties of sorbate may be due to its triplet carbonyl quenching activity.