Wilhelm J. Baader

Studies on the Mechanism of the Excitation Step in Peroxyoxalate Chemiluminescence

Studies on the mechanism of excited state formation in the peroxyoxalate system have been performed, to corroborate the involvement of the well-known Chemically Initiated Electron Exchange Luminescence (CIEEL) mechanism in the chemi-excitation step of this complex sequence. The singlet quantum yields, extrapolated to infinite activator concentrations (ΦS∞), and relative rate constants (kCAT/kD) of the excitation step have been determined in the presence of several activators for two systems: (i) the complete peroxyoxalate reaction with bis(2,4,6-trichlorophenyl) oxalate; and (ii) the base-catalyzed reaction of 4-chlorophenyl O,O-hydrogen monoperoxyoxalate, an isolated key intermediate. For five activators commonly used in CIEEL studies (anthracene, 9,10-diphenylanthracene, 2,5-diphenyloxazole, perylene, and rubrene), a linear correlation of ln(kCAT/kD) with the voltammetric half-peak oxidation potential (Ep/2) of the activator was obtained for both systems. The values obtained with 9,10-dicyanoanthracene and 9,10-dimethoxyanthracene did not fit this correlation. A reasonable linear correlation between ln(ΦS∞) and Ep/2 was obtained for all activators. For the commonly used activators, this quantum yield (ΦS∞) dependence can be rationalized in terms of the free energy balance of the back electron transfer leading to the formation of the excited state of the activator. However, the ΦS∞ values obtained with 9,10-dimethoxyanthracene and 9,10-dicyanoanthracene cannot be explained on the basis of these considerations alone. Thus, although this work presents clear-cut evidence of the operation of the CIEEL mechanism in the peroxyoxalate reaction, the results obtained with less commonly used activators show that several mechanistic details of the CIEEL hypothesis remain to be elucidated.

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.

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.

Revision of Singlet Quantum Yields in the Catalyzed Decomposition of Cyclic Peroxides

The chemiluminescence of cyclic peroxides activated by oxidizable fluorescent dyes is an example of chemically initiated electron exchange luminescence (CIEEL), which has been used also to explain the efficient bioluminescence of fireflies. Diphenoyl peroxide and dimethyl-1,2-dioxetanone were used as model compounds for the development of this CIEEL mechanism. However, the chemiexcitation efficiency of diphenoyl peroxide was found to be much lower than originally described. In this work, we redetermine the chemiexcitation quantum efficiency of dimethyl-1,2-dioxetanone, a more adequate model for firefly bioluminescence, and found a singlet quantum yield (Φ(S)) of 0.1%, a value at least 2 orders of magnitude lower than previously reported. Furthermore, we synthesized two other 1,2-dioxetanone derivatives and confirm the low chemiexcitation efficiency (Φ(S) < 0.1%) of the intermolecular CIEEL-activated decomposition of this class of cyclic peroxides. These results are compared with other chemiluminescent reactions, supporting the general trend that intermolecular CIEEL systems are much less efficient in generating singlet excited states than analogous intramolecular processes (Φ(S) ≈ 50%), with the notable exception of the peroxyoxalate reaction (Φ(S) ≈ 60%).

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.

Studies on the Intramolecular Electron Transfer Catalyzed Thermolysis of 1,2-Dioxetanes

Abstract This work reports the synthesis and the chemiluminescence properties of the dioxetanes: 4-ethyl-4-methyl-3-(3-methoxyphenyl)-1,2-dioxetane (I), 4-ethyl-4-methyl-3-(3-tert-butyldimethylsilyloxyphenyl)-1,2-dioxetane (II), 4,4-dimethyl-3-(3-methoxybenzyl)-1,2-dioxetane (III) and 4,4-dimethyl-3-(3-tert-butyldimethylsilyloxybenzyl)-1,2-dioxetane (IV). While in the thermal decomposition of I–IV preferential formation of triplet excited states is observed, in the presence of fluoride ions the decomposition rate constants of II and IV increase drastically and singlet excited states are formed with high quantum yields. These results are discussed based on the CIEEL (‘Chemically Initiated Electron Exchange Luminescence’) mechanism where the decomposition of the dioxetanes should be initiated by an intramolecular electron transfer from the phenolate ion (generated by fluoride catalyzed deprotection of the silyloxy group), either directly bound to the peroxidic ring (II) or separated from it by a methylene bridge (IV).

Enols of aldehydes in the peroxidase/oxidase-promoted generation of excited triplet species.

General (acid and base) or specific (fluoride ion) catalysis generates the enol of isobutanal and propanal from the corresponding trimethylsilyl enol ethers. The enols are directly rapidly oxidized by peroxidase (acting as an oxidase) to triplet acetone or triplet acetaldehyde, respectively, and formic acid. Due to the faster rate of reaction and the absence of quenching by excess aldehyde, the excited carbonyl emits more strongly than when the aldehyde itself is the substrate. With both enols the emission is pure phosphorescence. Both triplet acetone and triplet acetaldehyde are generated within the enzyme, as shown by the different quenching by D- and L-tryptophan, and are somewhat protected from oxygen quenching, as attested by the very fact that phosphorescence is observed. The use of enol precursors as substrates opens wide possibilities for photochemical investigations in the absence of light over a much broader range of experimental conditions.

Fluoride-triggered decomposition of m-sililoxyphenyl -substituted dioxetanes by an intramolecular electron transfer (CIEEL) mechanism

Abstract The synthesis of the protected phenolate-substituted 1,2-dioxetanes 1 and 2, containing the substituent directly linked to the peroxidic ring or separated by a methylene group, is reported. The activation parameters and chemiluminescence quantum yields upon unimolecular and catalyzed decomposition of 1 and 2 are in agreement with the occurrence of an intramolecular CIEEL mechanism in the catalyzed decomposition of these dioxetanes.

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.

Facile chemiluminescent method for alkaline phosphatase determination

Abstract The determination of alkaline phosphatase activity is of wide applicability, both as a free enzyme or bound to antibodies (conjugates). Activity determinations employing chemiluminescent substrates have become increasingly important due to their high sensitivity, typically equivalent to or better than assays utilizing radioactive labels. We report here a new chemiluminescent methodology for the determination of alkaline phosphatase activity based on the hydrolysis of disodium 1-(2-methylpropenyl)phosphate, readily synthesized in three steps. The hydrolysis product, 2-methyl-1-propenol, is a known substrate of horseradish peroxidase, which catalytically oxidizes enols to a dioxetane intermediate, that in turn yields luminescence upon cleavage. The detection limit of this method was determined to be 1.5 femtomoles of free ALP per assay. This methodology was also tested with bound ALP conjugates showing excellent sensitivity. Since the horseradish peroxidase system consumes dissolved oxygen during oxidation of the enol, alkaline phosphatase quantification may also be performed by accompanying the oxygen uptake rate.