Chemically Initiated Electron Exchange Luminescence Research Articles

Water modulates the chemiexcitation efficiency of the peroxyoxalate reaction: Enhancement at low and decrease at higher concentrations

Chemiluminescence transformations are widely utilized in analytical and bioanalytical applications. Specifically, the peroxyoxalate system can be employed for sensitive quantification of a wide variety of analytes. Under anhydrous conditions, the peroxyoxalate reaction shows extremely high emission quantum yields, comparable to highly efficient bioluminescence systems. However, in aqueous media, important for bioanalytical applications, the emission efficiency suffers a significant decrease of several orders of magnitudes, although the reason for this loss in efficiency is unknown up to now.In the present work, we show the results of a detailed photophysical study on the effect of water on the chemiexcitation efficiency of the peroxyoxalate reaction using bis(2,4-dinitrophenyl) oxalate as reagent and imidazole as base catalyst in the presence of different chemiluminescence activators. It is shown that in 1,2-dimethoxyethane/water mixtures (DME:H2O), singlet quantum yields increase with increasing the water concentrations up to 0.7 mol/L, followed by a steady decrease at higher water concentrations. The decrease in quantum yields is not predominantly due to concurrent ester hydrolysis, as quantum yields could be corrected for this side reaction. This indicates that the quantum yield decrease is due to the diminished chemiexcitation efficiency in the presence of water. Using different activators, it is shown that chemiexcitation occurs via electron transfer and electron back-transfer steps between a high-energy intermediate and the activator. Additionally, its efficiency is governed by the energetics of the electron back-transfer step, in agreement with the Chemically Initiated Electron Exchange Luminescence (CIEEL) mechanism, also in partially aqueous environment. Lower excitation efficiency in aqueous media may be due to formation of solvent-separated radical ion pairs, inhibiting chemiexcitation.

Solvent polarity influence on chemiexcitation efficiency of inter and intramolecular electron-transfer catalyzed chemiluminescence

The parameters which determine the efficiency of chemiluminescence, and bioluminescence transformations are a continuous matter of experimental and theoretical studies. In this sense, we report here a study of the solvent polarity influence on four Chemically Initiated Electron Exchange Luminescence (CIEEL) systems. Two of them comprise intermolecular CIEEL systems with low efficiency – catalyzed decomposition of diphenoyl peroxide and spiroadamantyl-1,2-dioxetanone; the other two are highly efficient in producing excited states – the peroxyoxalate reaction through an intermolecular CIEEL, and the induced decomposition of a phenoxy-substituted 1,2-dioxetane derivative via intramolecular CIEEL. These four CIEEL systems are studied in binary solvent mixtures with similar viscosity but different polarity, as measured by the empirical solvent polarity parameter ET(30). Reaction rate constants for all the systems increase with increasing polarity, indicating the occurrence of electron or charge transfer in the rate-limiting step of these transformations. For the intermolecular systems, singlet quantum yields generally rise with medium polarity to a maximum value and decrease for higher polarities. This agrees with stabilization of the radical ion pair intermediate by solvation at an optimal medium polarity. In contrast, higher polarity promotes the separation of these charged species, thereby avoiding electron back-transfer between radical ion pairs, essential for chemiexcitation. The singlet quantum yields in the intramolecular CIEEL system show a steady increase with the medium polarity, reaching values close to the maximum possible quantum yield of 1.0 E mol−1, indicating the importance of biradical ion stabilization and the occurrence of the whole reaction sequence in an intramolecular manner.

Flower-like Gold Nanoparticles for In Situ Tailoring Luminescent Molecules for Synergistic Enhanced Chemiluminescence.

In recent years, gold nanoparticles (AuNPs) have attracted much attention due to their ease of surface modification, excellent biocompatibility, and extraordinary optoelectronic and catalytic activities. Herein, based on a AuNP-catalyzed reaction, a strategy for tailoring luminescent molecules in situ is proposed to trigger an ultrastrong chemiluminescence (CL). In the strategy, flower-like AuNPs are prepared using CL molecular probes (Probe-OH for NaClO/ONOO-) via one-pot synthesis and subsequently act as a tailor for Probe-OH to generate novel CL molecules, allowing a synergistic CL enhancement about 4 times that of initial Probe-OH. Furthermore, by modification with poly(vinylpyrrolidone) (PVP) on the surface, the CL signals (only for NaClO) are amplified by 100 times based on an intermolecular chemically initiated electron exchange luminescence (CIEEL) mechanism. Given the improved sensitivity and selectivity over Probe-OH, the thus-formed CIEEL nanoplatform (PVP-Au) is successfully developed for detecting NaClO in a wide range of 2.5-100 μM, and the detection limit is 10.68 nM. This work provides unprecedented perspectives for expanding this facile and effective strategy for CL amplification based on AuNP catalysis.

Rationalizing the role of electron/charge transfer in the intramolecular chemiexcitation of dioxetanone-based chemi-/bioluminescent systems

The thermolysis of dioxetanones is a key process in the intramolecular chemiexcitation step of several chemi- and bioluminescent reactions. This step is generally explained with mechanisms based on either electron transfer (ET), such as the Chemically Initiated Electron-Exchange Luminescence (CIEEL) mechanism, or charge transfer (CT), such as the Charge Transfer-Initiated Luminescence (CTIL) mechanism. Here, we have used a TD-DFT approach to characterize the thermolysis and chemiexcitation steps of model dioxetanones, to rationalize the role of ET/CT in those intramolecular processes. Our results showed that ET/CT can reduce the activation barrier of the thermolysis reaction, by reducing the repulsion between the reacting fragments (ketone and CO2 moieties) that originate during peroxide bond breaking. However, in terms of singlet chemiexcitation profiles, those of non-CIEEL/CTIL-based dioxetanones appear to be more efficient than of CIEEL/CTIL-based ones. Furthermore, the ground state to singlet excited state transitions were found to be local excitations, without CT between the peroxide ring and the electron-rich moiety. So, ET/CT appear to be responsible for tuning the activation barrier of the thermolysis reaction, without playing a role in efficient singlet chemiexcitation itself.

Use of coumarin derivatives as activators in the peroxyoxalate system in organic and aqueous media

The peroxyoxalate system – base catalyzed reaction of oxalic ester derivatives with hydrogen peroxide in the presence of a fluorescent activator – is one of the most efficient chemiluminescence transformations known and widely utilized in analytical and bioanalytical applications. Coumarin derivatives have found widespread application as fluorescent labels for the determination of enzymatic activity in biological fluids and in fluorescence immunoassays, among others. In this work, we show that coumarin derivatives can be utilized as activators in the peroxyoxalate reaction (with two oxalic esters, including a new, environmentally compatible derivative) in anhydrous and aqueous systems, leading to reproducible kinetic results and singlet quantum yields similar to that obtained with commonly utilized activators. Quantum yields are lower in aqueous than in anhydrous medium, however still considerably high to allow the application in analytical and bioanalytical assays. The chemiluminescence parameters (singlet quantum yields at infinite activator concentration and relative electron transfer rate constants) determined in the systems indicate the occurrence of electron or charge transfer steps according to the Chemically Initiated Electron Exchange Luminescence (CIEEL) mechanism.

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 onlysystem 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.

Emissive Enhancement of the Singlet Oxygen Chemiluminescence Probe after Binding to Bovine Serum Albumin.

A chemiluminescence probe for singlet oxygen 1O2 (SOCL) was investigated in phosphate buffer saline (PBS), either in the absence of proteins or containing bovine serum albumin (BSA). In the protein-free PBS, the reactivity of SOCL for methylene blue (MB)-photosensitized 1O2 was found to be moderate or low. The reaction yield increased with temperature and/or concentration of dissolved molecular oxygen. Unexpectedly, the presence of BSA boosted both the emissive nature and the thermal stability of the phenoxy-dioxetane intermediate formed in the chemiexcitation pathway. Isothermal titration calorimetry showed that SOCL has a moderate binding affinity for BSA and that entropy forces drive the formation of the SOCL-BSA complex. A model with two identical and independent binding sites was used to fit the binding isotherm data. Co-operative binding was observed when MB was present. Local viscosity factors and/or conformational restrictions of the BSA-bound SOCL phenoxy-dioxetane were proposed to contribute to the formation of the highly emissive benzoate ester during the chemically initiated electron exchange luminescence (CIEEL) process. These results led us to conclude that hydrophobic interactions of the SOCL with proteins can modify the emissive nature of its phenoxy-dioxetane, which should be taken into account when using SOCL or its cell-penetrating peptide derivative in living cells.

Fabrication of a new peroxyoxalate chemiluminescence system containing the Cy3 fluorophore and its application to carvedilol detection

Cy3 is a dye that has been widely applied in fluorescence microscopy and single molecule detection due to its low hydrophobicity and intense fluorescence emission. As a new application, this study proposes using Cy3 as the fluorophore of a peroxyoxalate chemiluminescence system. The system relies on the oxidative reaction of bis-(2,4,6-trichlorophenyl) oxalate (TCPO) with H2O2 in the presence of imidazole, as the catalyst, to produce dioxetane via a chemically initiated electron exchange luminescence (CIEEL) process, excite Cy3 by the excess energy of the dioxetane and permit Cy3 to emit yellow light. The chemiluminescence behavior of the system is investigated using different concentrations of TCPO, imidazole, the oxidant and Cy3 and by considering non-linear least squares curve fitting of the obtained intensity–time profiles into the pooled intermediate model. The evaluated kinetic parameters include experimental and theoretical maximum intensity, the rise and fall rate constants of the chemiluminescence process, experimental and theoretical time required to reach the maximum intensity and total emission yield. Finally, the developed system is adopted to detect a drug, i.e., carvedilol, in different pharmaceuticals. Under the optimal conditions, the system can provide a linear response for determination of 3 × 10− 7 to 3 × 10− 5 M carvedilol (r > 0.9869, n = 6) and a detection limit of 1.21 × 10− 7 M. The relative standard deviation of the detection results for six repetitive determinations is below 3.47% and the obtained recoveries are 99.6% and 101.9%.

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.

Peroxyoxalate chemiluminescence efficiency in polar medium is moderately enhanced by solvent viscosity

Abstract The peroxyoxalate reaction is one of the most efficient chemiluminescence transformations and the only system which is believed to occur by an intermolecular Chemically Initiated Electron Exchange Luminescence (CIEEL) mechanism with proven high efficiency. Although this reaction system is utilized in a variety of analytical applications, its exact reaction mechanism is still unknown, specifically with respect to the structure of the high-energy intermediate, the mechanism of its interaction with a chemiluminescence activator, as well as the reason for the extraordinarily high chemiexcitation efficiency. The solvent viscosity influence on the singlet quantum yields of the peroxyoxalate reaction is studied in this work, using the polar binary solvent mixture of ethyl acetate and dimethyl phthalate spanning a viscosity range of 0.42–14.4 cP. The viscosity increase of about forty times leads to a quantum yield increase of a factor of around five. Although the viscosity effect on the quantum yields is significant, it is considerably lower than that obtained before in less polar media. The quantum yield dependency on the viscosity can be described with both the collisional and the frictional model, with good correlation coefficients ( R 2 > 0.95). However, the α values obtained from the frictional model would be more compatible with an intramolecular transformation than with the intermolecular interaction of a high-energy intermediate with an external activator. This apparent contradiction is thought to be due to stabilization of the charge-transfer complex between the high-energy intermediate and the activator by the polar medium, avoiding solvent cavity escape and increasing chemiexcitation efficiency. The high stability of the charge-transfer complex in the peroxyoxalate reaction might be due to the high planarity of the possible high-energy intermediate in this transformation, 1,2-dioxetanedione, the peroxidic carbon dioxide dimer.

In vivo imaging of endogenous enzyme activities using luminescent 1,2-dioxetane compounds.

BackgroundHere we present a non-invasive imaging method for visualizing endogenous enzyme activities in living animals. This optical imaging method is based on an energy transfer principle termed chemically initiated electron exchange luminescence (CIEEL). The light energy is provided by enzymatic activation of metastable 1,2-dioxetane substrates, whose protective groups are removed by hydrolytic enzymes such as β-galactosidase and alkaline phosphatase. In the presence of a nearby fluorescent recipient, the chemical energy within the activated substrate is then transferred via formation of a charge-transfer complex with the fluorophore, a mechanism closely related to glow stick chemistry.ResultsEfficient CIEEL energy transfer requires close proximity between the trigger enzyme and the fluorescent recipient. Using cells stained with fluorescent dialkylcarbocyanines as the energy recipients, we demonstrated CIEEL imaging of cellular β-galactosidase or alkaline phosphatase activity. In living animals, we used a similar approach to non-invasively image alkaline phosphatase activity in the peritoneal cavity.ConclusionsIn this report, we provide proof-of-concept for CIEEL imaging of in vivo enzymatic activity. In addition, we demonstrate the use of CIEEL energy transfer for visualizing elevated alkaline phosphatase activity associated with tissue inflammation in living animals.

Lophine derivatives as activators in peroxyoxalate chemiluminescence.

Lophine and four of its derivatives were used as activators (ACTs) of the chemiluminescent peroxyoxalate (PO) reaction of bis(2,4,6-trichlorophenyl)oxalate with H2O2, catalysed by imidazole. Kinetic emission assays have shown that with the tested compounds the reaction mechanism, regarding the formation of the high energy intermediate (HEI) of the PO reaction, occurs as previously seen for commonly used ACTs. A bimolecular interaction of the compounds with the HEI leads to chemiexcitation through the chemically initiated electron exchange luminescence (CIEEL) mechanism, as confirmed by a linear free-energy correlation between the relative catalytic rate constants and the oxidation potentials of the compounds. The yields of excited state formation and light emission, in the range of 10(-2)-10(-3) E mol(-1), are comparable to the ones seen with commonly used ACTs. A Hammett plot with ρ = -0.90 indicates the buildup of a partial positive charge on the transition step of the catalytic process, consistent with the formation of a radical cation of the ACT, being an additional validation of the CIEEL mechanism in this system.

Substituent effects on the decomposition of chemiluminescent tricyclic aromatic dioxetanes

Three tricyclic 1,2-dioxetane derivatives, 1a, 2a and 3a were synthesized from their corresponding 1,4-dioxin acenaphthylene compounds, 1, 2 and 3, by reaction with singlet-oxygen ((1) O2 ) in dichloromethane. Evidence for the formation of the dioxetanes 1a, 2a and 3a is provided by the chemiluminescence (CL) that corresponds to the emission from the electronically excited diesters 1b*, 2b* and 3b*, which are decomposed thermally from the dioxetanes 1a, 2a and 3a, respectively. The highly strained 1,2-dioxetane ring decomposes from a twisted geometry by simultaneous cleavages of the O-O and C-C bonds, producing the electronically excited diester that emits CL. It was observed that the CL from compound 2a is red-shifted relative to that of compounds 1a and 3a suggesting a higher degree of stabilization for the excited state by the electron-donating methoxy group. Also, a study of the solvent effect on fluorescence shows a significant red-shift in compound 2b, indicating a more polar excited state. The kinetics of the thermal decomposition of the 1,2-dioxetanes clearly demonstrate that the CL characteristics of compound 2a are quite different from those of compounds, 1a and 3a. These results are consistent with the proposed intramolecular chemically initiated electron exchange luminescence (CIEEL) mechanism which is triggered by the electron-donating group of compound 2a.

Role of background observed in aptasensor with chemiluminescence detection

One-step chemiluminescent aptasensor was developed using chemically initiated electron exchange luminescence (CIEEL) between high-energy intermediate formed from 1,1′-oxalyldiimidazole chemiluminescence (ODI-CL) reaction and G-quadruplex (ochratoxin A (OTA)-bound aptamer conjugated with TEX615) generated. The sensitivity of chemiluminescent aptasensor, optimized with various variables (e.g., property of microfibers fabricated with 3,4,9,10-perylenetetracarboxylic dimide, determination of fluorescent dye labeled with aptamer, physical properties of buffer solution), was dependent on the background (concentration of high-energy intermediate) generated in ODI-CL reaction. The limit of detection (LOD=background+3×standard deviation, 0.5nM) of ODI-CL aptasensor with lower background was lower than that (3.7nM) with 20 times higher background. Also, the ratio of signal to background (S/B) of ODI-CL aptasensor with low background was about 5-fold higher than that with high background. The sensitivities of ODI-CL aptasensors, with low as well as high background, capable of accurately and precisely quantifying OTA within 10min, were better than those of fluorescent aptasensors and as good as those of highly sensitive but time-consuming competitive enzyme-linked immune-sorbent assays (ELISAs) using expensive antibody produced with the sacrifice of small animals.

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%).

Mechanistic Insight into the Chemiluminescent Decomposition of Firefly Dioxetanone

The peroxide decomposition that generates the excited-state carbonyl compound is the key step in most organic chemiluminescence, and chemically initiated electron exchange luminescence (CIEEL) has been widely accepted for decades as the general mechanism for this decomposition. The firefly dioxetanone, which is a peroxide, is the intermediate in firefly bioluminescence, and its decomposition is the most important step leading to the emission of visible light by a firefly. However, the firefly dioxetanone decomposition mechanism has never been explored at a reliable theoretical level, because the decomposition process includes biradical, charge-transfer (CT) and several nearly degenerate states. Herein, we have investigated the thermolysis of firefly dioxetanone in its neutral (FDOH) and anionic (FDO(-)) forms using second-order multiconfigurational perturbation theories in combination with the ground-state intrinsic reaction coordinate calculated via the combined hybrid functional with Coulomb attenuated exchange-correlation, and considered the solvent effect on the ground-state reaction path using the combined hybrid functional with Coulomb attenuated exchange-correlation. The calculated results indicate that the chemiluminescent decomposition of FDOH or FDO(-) does not take place via the CIEEL mechanism. An entropic trap was found to lead to an excited-state carbonyl compound for FDOH, and a gradually reversible CT initiated luminescence (GRCTIL) was proposed as a new mechanism for the decomposition of FDO(-).

Abstracts of the 17th International Symposium on Bioluminescence and Chemiluminescence ‐ (ISBC 2012)

WARNING : The light-emitting molecular structures responsible for the chemiluminescence and fluorescence phenomena are not necessarily the same!

Hypericin from St. John’s Wort (hypericum perforatum) as a novel natural fluorophore for chemiluminescence reaction of bis (2,4,6-trichlorophenyl) oxalate–H2O2–imidazole and quenching effect of some natural lipophilic hydrogen peroxide scavengers

Hypericin (HYP) molecule is a natural photoactive pigment, which plays a role as an effective photoreceptor in some plants of the Hypericum species (the most common of which is Saint John’s Wort) and some insect species. The present work deals with the first attempt to the study of peroxyoxalate chemiluminescence (POCL) system in the presense of HYP as a natural fluorophore. Reaction of bis (2,4,6-trichlorophenyl) oxalate(TCPO)–H2O2–imidazole can transfer energy to a HYP via formation of dioxetane through the chemically initiated electron exchange luminescence (CIEEL) mechanism and can emits a very intense red light. The effects of HYP, hydrogen peroxide, TCPO and imidazole concentrations on kinetic chemiluminescence parameters were also studied. These parameters including rise and fall rate constant for the chemiluminescence burst, theoretical and experimental maximum intensity, theoretical and experimental time to reach maximum intensity and total light yield emission were evaluated by using a pooled intermediate model for a non-linear least-squares curve fitting program, KINFIT. Moreover, quenching effect of two lipophilic natural antioxidant, Quercetin and β-carotene on it system was also investigated. The measurable concentration range of 7×10−6M to 7.5×10−5M of antioxidants were evaluated from the proper Stern–Volmer plots with satisfactory RSD% and corresponding detection limits of 2.2×10−6 and 3.7×10−6 for β-carotene and quercetin respectively.

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.

Spectroscopic study of the authentic emitter of AMPPD chemiluminescence in alkaline aqueous solution

To design more effective CIEEL (chemically initiated electron exchange luminescence) systems demands a complete picture of the dynamics of the chemiluminescence, which is often a challenge. In this work, photoluminescence of the methyl m-oxybenzoate anion - the authentic emitter of AMPPD (3-[2-spiroadamantane]-4-methoxy-4-[3-phosphoryloxy]-phenyl-1,2-dioxetane) in aqueous solvent has been studied. Combining the effect of solvent properties, e.g. pH value, and spectroscopic studies employing steady-state and ultrafast time-resolved emission and absorption and (1)H NMR techniques, a novel mechanism is proposed. We conclude that the deviation of emission peaks between chemiluminescence and photoluminescence of the authentic emitter of AMPPD i.e. the methyl m-oxybenzoate anion, in alkaline aqueous solvents is due to its hydrolysis, rather than the hydrogen-bonding effect as has been assumed so far. Besides, the hydrogen-bonding is suggested to play a key role in significantly decreasing the chemiluminescence yield of AMPPD in aqueous solution by shortening the lifetime of the excited authentic emitter to 10 ps order of magnitude - three orders of magnitude shorter than the previously reported value ( approximately 10 ns). These results shed light on the chemiluminescence dynamics of AMPPD and facilitate the design of more effective CIEEL systems.