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In recent years, various methods for detecting exogenous and endogenous hypochlorite have been studied, considering its essential role as a biomolecule. However, the existing technologies still pose obstacles such as their invasiveness, high costs, and complicated operation. In the current study, we developed a glow-type chemiluminescent probe, hypochlorite chemiluminescence probe (HCCL)-1, based on the scaffold of Schaap's 1,2-dioxetane luminophores. To better explore the physiological and pathological functions of hypochlorite, we modified the luminophore scaffold of HCCL-1 to develop several probes, including HCCL-2, HCCL-3, and HCCL-4, which amplify the response signal of hypochlorite. By comparing the luminescent intensities of the four probes using the IVIS® system, we determined that HCCL-2 with a limit of detection of 0.166 μM has enhanced sensitivity and selectivity for tracking hypochlorite both in vitro and in vivo.

Here, we have developed a novel photoactivatable red chemiluminescent AIEgen probe (ACL), based on the combination of the red-emission AIEgen fluorophore (TPEDC) that shows excellent singlet oxygen (1O2)-generation ability and the precursor of Schaap's dioxetane (the linker connected to adamantane is the C═C bond) that can be modified to target various analytes, for in vitro and in vivo measurement of hydrazine. Prior to applying for sensing detection, the C═C bond connected to adamantane in ACL was first converted into dioxetane by irradiation to form the activated chemiluminescent AIEgen probe (ACLD). Then, the self-immolative reaction was triggered upon the deprotection of the acylated phenolic hydroxyl group in ACLD in the presence of hydrazine, resulting in the release of the high energy held in the 1,2-dioxetanes, and then, the chemiexcitation was triggered, thereby producing red chemiluminescence through the intramolecular chemiluminescence resonance energy transfer from Schaap's dioxetane to TPEDC. This chemiluminescent AIEgen probe was evaluated in a clean buffer environment as well as using living cells and mouse models.

Reacting with H2O2, tetrachloro-1,4-benzoquinone (TCBQ) produces chemiluminescence (CL), but chloranilic acid (CA), the dihydroxylation product of TCBQ, does not. However, an unprecedented strong CL generates from CA/H2O2 in the presence of Co(II). Why? We performed quantum chemical calculations on the entire reaction process of CA/H2O2 and CA/H2O2/Co(II) systems. The computational results indicate: for CA/H2O2 system, the reason leading to non-CL as: there is no free •OH produced by CA/H2O2, which prevents the subsequent reaction from taking place; for CA/H2O2/Co(II) system, the chemical process resulting in the CL as: First, a neutral dioxetane is formed via six sequential reactions. Then, the neutral dioxetane decomposes to generate a neutral excited-state (S1) product via a gradually reversible charge transfer initiated luminescence mechanism. A conical intersection of the ground and the S1-state potential energy surfaces facilitates the production of the S1-state product. Ultimately, the neutral S1-state product emits light as a practical light emitter. The key component for forming dioxetane and the following CL is the intrinsically generated •OH, which is roaming around at the region of C2 atoms of the CA moiety, instead of being free. The quantum chemical calculations supported the experimental observation and conclusion by providing the mechanistic explanation in detail.

Chemiluminescence imaging is imperative for diagnostics and imaging due to its intrinsically high sensitivity. To improve in vivo detection of biomarkers, chemiluminophores that simultaneously possess near-infrared (NIR) emission and modular structures amenable to construction of activatable probes are highly desired; however, these are rare. Herein, we report two chemiluminophores with record long NIR emission (>750 nm) via integration of dicyanomethylene-4H-benzothiopyran or dicyanomethylene-4H-benzoselenopyran with dioxetane unit. Caging of the chemiluminophores with different cleavable moieties produces NIR chemiluminescence probes (NCPs) that only produce signals upon reaction with reactive oxygen species or enzymes, for example, β-galactosidase, with a tissue-penetration depth of up to 2 cm. Thus, this study provides NIR chemiluminescence molecular scaffolds applicable for in vivo turn-on imaging of versatile biomarkers in deep tissues. Ministry of Education (MOE) Nanyang Technological University This work was supported by Nanyang Technological University (Start-up Grant: M4081627) and Singapore Ministry of Education Academic Research Fund Tier 1 (2019-T1-002-045,RG125/19) and Tier 2 (MOE2018-T2-2-042)

Compared with O2-dependent chemiluminescence (CL) and bioluminescence (BL), H2O2-dependent ones are far from being completely understood. A two-step mechanism for H2O2-dependent CL production from tetrachloro-1,4-benzoquinone (TCBQ) was proposed based on experimental evidence of detecting the formation of several intermediates and products. This mechanism is not yet supported by theoretical evidence, and its details remain unknown. In the present paper, we performed multireference and (time-dependent) density functional theory calculations on the complete reaction process of TCBQ with H2O2 to produce CL. The calculations reproduced the experimentally observed two-step CL. Although the reactants are different, the first and second CLs follow very similar reaction processes and mechanisms. First, an anionic dioxetane is formed via five sequential reactions. The intrinsically produced •OH is crucial for forming dioxetane. Subsequently, the anionic dioxetane decomposes to produce an anionic excited-state (S1) product. A conical interaction of the ground and the S1-state potential energy surfaces is responsible for producing the S1-state product. Finally, the S1-state anionic product changes to its neutral form, and the latter emits light as an actual light emitter. This mechanism could be extended to luminescent systems of all H2O2-dependent tetrahalogenated quinoids, including acorn worms, because TCBQ/H2O2 is a typical representative of these luminescent systems.

An interesting chemiluminescence (CL) phenomenon of cyclic peroxides originating from tetrahydrofuran hydrogen peroxide (THF-HPO) in the presence of BSA-stabilized Au NCs (Au@BSA NCs) was found for the first time. In this CL system, Au@BSA NCs can greatly accelerate the decomposition of THF-HPO, and then chemiluminescence resonance energy transfer (CRET) occurs between excited dioxetane derivatives and the Au@BSA NCs, yielding enhanced CL emission which can be further enhanced more than 10 times by the addition of copper ions. Based on this, a synergistic CL nanoprobe with a special signal amplification strategy was developed.

Hydrogen sulfide (H2S) is an important biomolecule and significant efforts have focused on developing chemical tools to aid different biological investigations. Of such tools, there are relatively few chemiluminescent or bioluminescent methods for H2S detection. Here we report two dioxetane-based chemiluminescent probes for H2S detection. With these probes, we directly compare the probe response to H2S-mediated azide reduction and nucleophilic displacement of 2,4-dinitrophenyl (DNP) motifs and demonstrate that the SNAr cleavage of the DNP group results in a larger response and greater stability in water.

The removal of ubiquitin (Ub) from a modified protein or Ub chain is a process that occurs regularly by the ubiquitin-proteasome system. This process is known to be mediated by various deubiquitinating enzymes (DUBs) in order to control the protein's half-life and its expression levels among many other signaling processes. Since the function of DUBs is also involved in numerous human diseases, such as cancer, there is an obvious need for an effective diagnostic probe that can monitor the activity of these enzymes. We have developed the first chemiluminescence probe for detection of DUBs activity. The probe was prepared by conjugation of the chemically synthesized C-terminally activated Ub(1-75) with a Gly-enolether precursor. Subsequent oxidation, under aqueous conditions, of the enolether conjuagate with singlet-oxygen furnished the dioxetane probe Ub-CL. This synthesis provides the first example of a dioxetane-luminophore protein conjugate. The probe's ability to detect deubiquitinating activity was successfully validated with three different DUBs. In order to demonstrate the advantage of our new probe, comparison measurements for detection of DUB UCH-L3 activity were performed between the chemiluminescent probe Ub-CL and the well-known Ub-AMC probe. The obtained data showed significantly higher S/N, for probe Ub-CL (>93-fold) in comparison to that observed for Ub-AMC (1.5-fold). We anticipate that the successful design and synthesis of the turn-ON protein-dioxetane conjugate probe, demonstrated in this work, will provide the insight and motivation for preparation of other relevant protein-dioxetane conjugates.

Quantification of biothiols in living systems is essential to understand their biological applications. Here, we developed two activatable chemiluminescence probes (SHCL and NCCL) and investigated their utility in the bioimaging of intracellular biothiols by directly tethering 2,4-dinitrobenzenesulfonyl to the hydroxyl group of phenoxy-dioxetane. The design of these two probes differed in substituents of phenol-dioxetane, i.e., SHCL contained the ortho chlorine, whereas NCCL had the para hydroxymethyl. Upon glutathione (GSH) cleavage, both probes emitted significantly “turn-on” chemiluminescent signals. However, the chemiluminescence intensity based on NCCL declined with increasing GSH level above 5 mM, while SHCL exhibited much higher chemiluminescent intensity and a wider concentration range (0.5 μM-50 mM), which was much more suitable for sensing endogenous biothiols. We further demonstrated that chlorine substitution in SHCL played an important role in bioimaging owing to the halogen effect, providing a lower pKa value and significant enhancement of the chemiluminescent emission. SHCL imaged the biothiols effectively in tumor cells and tumor-bearing mice. Additionally, this novel chemiluminescence probe can be easily used to evaluate the in vitro activity of acetylcholinesterase. Overall, we anticipate that SHCL may provide a facile and intuitive tool for studying the role of biothiols in diseases.

The oxidation of alkyl thiols to disulfides has been achieved under mild conditions using a chemiluminescent 1,2-dioxetane as a stoichiometric oxidant. Besides the mild and biocompatible reaction conditions, this approach offers the possibility to monitor the presence of thiols through oxidation and chemiluminescence of the remaining dioxetane.

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%.

Organic superbases represented by TBD (1,5,7-triazabicyclo[4.4.0]dec-5-ene) effectively induced the decomposition of hydroxyaryl-substituted dioxetanes in acetonitrile to give bright light. The color of the chemiluminescence from a dioxetane bearing a 4-(benzoxazol-2-yl)-3,5-dihydroxyphenyl moiety varied depending on the base used. In addition to this change in the color of emission, TBD increased the chemiluminescence efficiency 2- to 5-fold compared to the results with other base systems and accelerated decomposition of the dioxetane. These unique effects of TBD may be due to its “bifunctional” character, which is different from those of other organic superbases. Chemiluminescent decomposition of the dioxetane was effectively induced by superbases even in apolar p-xylene.

Hydrogen sulfide (H2S), the third gaseous transmitter after CO and NO, is a double-edged sword in the human body. A specific concentration of H2S can attenuate myocardial ischemia–reperfusion injury by preserving mitochondrial function, in contrast, cause illness, including inflammation and stroke. There are already some probes for the real-time monitoring of the level of H2S in the biological environment. However, they have some disadvantages, such as phototoxicity, low sensitivity, and low quantum yield. In this research, by linking 4-dinitrophenyl-ether (DNP), a specific recognition group for H2S, with a chemiluminophore 1,2-dioxetane, we designed and synthesized the probe SCL-1. To tackle the barrier that the traditional chemiluminescent group has a short emission wavelength and is not easy to penetrate deep tissues, an acrylonitrile electron-withdrawing substituent was installed to the ortho-position of the 1,2-dioxanol hydroxy group. According to the same design strategy as SCL-1, the probe SCL-2 was designed with the modified chemiluminescent group. Studies have shown that SCL-2 with electron-withdrawing acrylonitrile has higher luminescence quantum yield and high sensitivity than SCL-1, realizing real-time detection of H2S in vitro and in vivo. The LOD of SCL-2 was 0.185 μM, which was the best among the currently available luminescent probes for detecting H2S. We envisage that SCL-2 may be a practical toolbox for studying the biological functions of H2S and H2S-related diseases.

Until recently, chemiluminescence cell images could only be obtained using luciferase-activated probes. Moreover, chemiluminescence microscopy cell-imaging has not been demonstrated for natively expressed enzymes like cathepsin B. Herein, we describe the design, synthesis, and evaluation of the first chemiluminescence probe for the detection and imaging of cathepsin B. The probe activation mechanism relies on the release of a dioxetane intermediate, which undergoes chemiexcitation to emit green light with high efficiency under physiological conditions. Using the probe, we obtained clear images of cancerous leukemia and colon cells. This is the first demonstration of chemiluminescence cell images obtained by a probe for a natively expressed endogenous enzyme. We anticipate that the concept presented in this study will be broadly used to develop analogous probes for other important proteases relevant to biomolecular processes.

Bringing together the concepts of self-immolative linkers and chemiluminogen dioxetane modules, a chemiluminescence-based sensor for fluoride with signal amplification is presented. Signal amplification is obtained by triggering two chemiluminescence events for each reacting fluoride ion that in turn releases two fluoride ions for each ion. As expected, the chemiluminescence signal starts to rise following an induction period. In addition to the analytical potential, this chemical system is also of interest as a demonstration of positive feedback loop character.

Chemiluminescence probes are considered to be among the most sensitive diagnostic tools that provide high signal-to-noise ratio for various applications such as DNA detection and immunoassays. We have developed a new molecular methodology to design and foresee light-emission properties of turn-ON chemiluminescence dioxetane probes suitable for use under physiological conditions. The methodology is based on incorporation of a substituent on the benzoate species obtained during the chemiexcitation pathway of Schaap’s adamantylidene–dioxetane probe. The substituent effect was initially evaluated on the fluorescence emission generated by the benzoate species and then on the chemiluminescence of the dioxetane luminophores. A striking substituent effect on the chemiluminescence efficiency of the probes was obtained when acrylate and acrylonitrile electron-withdrawing groups were installed. The chemiluminescence quantum yield of the best probe was more than 3 orders of magnitude higher than that of a standard, commercially available adamantylidene–dioxetane probe. These are the most powerful chemiluminescence dioxetane probes synthesized to date that are suitable for use under aqueous conditions. One of our probes was capable of providing high-quality chemiluminescence cell images based on endogenous activity of β-galactosidase. This is the first demonstration of cell imaging achieved by a non-luciferin small-molecule probe with direct chemiluminescence mode of emission. We anticipate that the strategy presented here will lead to development of efficient chemiluminescence probes for various applications in the field of sensing and imaging., A new molecular methodology to design and foresee light-emission properties of turn-ON chemiluminescence dioxetane probes is presented. The probes are suitable for use under physiological conditions and thus could provide exceptional live cell images.

We designed a chemiluminescent system using nanoparticles. We prepared mesoporous nanoparticles bearing naphthalene endoperoxide as a singlet oxygen generator in the pore and vinyl phenol derivative as an emission unit. The combination of these functional molecules gave highly efficient and bright chemiluminescence through the formation of a 1,2-dioxetane intermediate.

A pioneering chemiluminescent molecule reported by Schaap and co-workers, 3-(2′-spiroadamantane)-4-methoxy-4-(3″-hydroxy)phenyl-1,2-dioxetane (AMPD), does not require enzymatic activation but is unsuitable for use under physiological conditions. To overcome this limitation, we have developed a new AMPD derivative that contains an acetamido group at the ortho position of the hydroxy group as an intramolecular hydrogen-bonding site in order to lower the pKa value. This compound exhibits a superior chemiluminescence response to AMPD in the physiologically relevant pH range.

Chemiluminescence is being considered an effective imaging modality as it offers low background and high sensitivity. Recent discovery by our group has led to development of new phenoxy-dioxetane chemiluminescence luminophores, which are highly bright under physiological conditions. However, the current scope of probes based on these luminophores is limited, as they can only be turned on by phenol protecting group removal. Here we present a new chemiluminescence resonance energy transfer (CRET) system, Glow-CRET, in which light emission is triggered by proteolytic cleavage of a peptide substrate that links a dioxetane luminophore and a quencher. In order to compose such system, a new phenoxy-dioxetane luminophore, 7-HC-CL, was developed. This luminophore exhibits intense and persistent glow chemiluminescence; it undergoes very slow chemiexcitation, and it has the highest chemiluminescence quantum yield ever reported under physiological conditions. Based on 7-HC-CL, a Glow-CRET probe for matrix metalloproteinases, MMP-CL, was synthesized. Incubation of MMP-CL with its cognate protease resulted in 160-fold increase in chemiluminescence signal. MMP-CL was also able to detect matrix metalloproteinase activity in cancer cells with significantly higher signal-to-background ratio than an analogous fluorescence resonance energy transfer (FRET)-based probe. This work is expected to open new horizons in chemiluminescence imaging, as it enables to use the dioxetanes in ways that had not been possible. We anticipate that 7-HC-CL and future derivatives will be utilized not only for the construction of further Glow-CRET probes, but also for other applications, such as chemiluminescence tagging of proteins.

Chemiluminescence offers advantages over fluorescence for bioimaging, since an external light source is unnecessary with chemiluminescent agents., Chemiluminescence offers advantages over fluorescence for bioimaging, since an external light source is unnecessary with chemiluminescent agents. This report demonstrates the first encapsulation of chemiluminescence phenoxy-adamantyl-1,2-dioxetane probes with trimethyl β-cyclodextrin. Clear proof for the formation of a 1 : 1 host–guest complex between the adamantyl-1,2-dioxetane probe and trimethyl β-cyclodextrin was provided by mass spectroscopy and NMR experiments. The calculated association constant of this host–guest system, 253 M–1, indicates the formation of a stable inclusion complex. The inclusion complex significantly amplified the light emission intensity relative to the noncomplexed probe under physiological conditions. Complexation of adamantyl-dioxetane with fluorogenic dye-tethered cyclodextrin resulted in light emission through energy transfer to a wavelength that corresponds to the fluorescent emission of the conjugated dye. Remarkably, the light emission intensity of this inclusion complex was approximately 1500-fold higher than that of the non-complexed adamantyl-dioxetane guest. We present the first demonstration of microscopic cell images obtained using a chemiluminescence supramolecular dioxetane probe and demonstrate the utility of these supramolecular complexes by imaging of enzymatic activity and bio-analytes in vitro and in vivo. We anticipate that the described chemiluminescence supramolecular dioxetane probes will find use in various biological applications.

Chemiluminescent luminophores are considered as one of the most sensitive families of probes for detection and imaging applications. Due to their high signal-to-noise ratios, luminophores with near-infrared (NIR) emission are particularly important for in vivo use. In addition, light with such long wavelength has significantly greater capability for penetration through organic tissue. So far, only a few reports have described the use of chemiluminescence systems for in vivo imaging. Such systems are always based on an energy-transfer process from a chemiluminescent precursor to a nearby emissive fluorescent dye. Here, we describe the development of the first chemiluminescent luminophores with a direct mode of NIR light emission that are suitable for use under physiological conditions. Our strategy is based on incorporation of a substituent with an extended π-electron system on the excited species obtained during the chemiexcitation pathway of Schaap's adamantylidene-dioxetane probe. In this manner, we designed and synthesized two new luminophores with direct light emission wavelength in the NIR region. Masking of the luminophores with analyte-responsive groups has resulted in turn-ON probes for detection and imaging of β-galactosidase and hydrogen peroxide. The probes' ability to image their corresponding analyte/enzyme was effectively demonstrated in vitro for β-galactosidase activity and in vivo in a mouse model of inflammation. We anticipate that our strategy for obtaining NIR luminophores will open new doors for further exploration of complex biomolecular systems using non-invasive intravital chemiluminescence imaging techniques.

International audience; Chemiluminescence is the emission of light as a result of a nonadiabatic chemical reaction. The present work is concerned with understanding the yield of chemiluminescence, in particular how it dramatically increases upon methylation of 1,2-dioxetane. Both ground-state and nonadiabatic dynamics (including singlet excited states) of the decomposition reaction of various methyl-substituted dioxetanes have been simulated. Methyl-substitution leads to a significant increase in the dissociation time scale. The rotation around the O-C-C-O dihedral angle is slowed; thus, the molecular system stays longer in the "entropic trap" region. A simple kinetic model is proposed to explain how this leads to a higher chemiluminescence yield. These results have important implications for the design of efficient chemiluminescent systems in medical, environmental, and industrial applications.

Singlet oxygen is among the reactive oxygen species (ROS) with the shortest life-times in aqueous media because of its extremely high reactivity. Therefore, designing sensors for detection of 1 O2 is perhaps one of the most challenging tasks in the field of molecular probes. Herein, we report a highly selective and sensitive chemiluminescence probe (SOCL-CPP) for the detection of 1 O2 in living cells. The probe reacts with 1 O2 to form a dioxetane that spontaneously decomposes under physiological conditions through a chemiexcitation pathway to emit green light with extraordinary intensity. SOCL-CPP demonstrated promising ability to detect and image intracellular 1 O2 produced by a photosensitizer in HeLa cells during photodynamic therapy (PDT) mode of action. Our findings make SOCL-CPP the most effective known chemiluminescence probe for the detection of 1 O2 . We anticipate that our chemiluminescence probe for 1 O2 imaging would be useful in PDT-related applications and for monitoring 1 O2 endogenously generated by cells in response to different stimuli.

WOS: 000388951300158, Chemiluminescent detection of H2O2 has been achieved by using self immolative dioxetane based probe which enables the signal amplification via disassembly of two chemiluminogenic modules at the same time in response to single analyte. Upon treatment of the probe with H2O2, boronate ester was deprotected subsequently to trigger the decomposition of 1,2-dioxetane ring via CIEEL mechanism which results in light emission as a selective sign of H2O2. (C) 2016 Elsevier B.V. All rights reserved.

The decomposition of 3-oxyphenyl-3-methoxy-4-(2′-spiroadamantane)-1,2-dioxetane ( A ) and 5- tert -butyl-4,4-dimethyl-1-(3-oxyphenyl)bicyclo[3.2.0]heptane ( B ) in NaOH/H 2 O gives light in poor yield, which is several orders of magnitude lower than that in aprotic solvents. To understand the poor chemiluminescence efficiency in NaOH/H 2 O, we investigated the behaviors of the authentic emitters, methyl 3-oxidobenzoate ( C ) and 2,2,4,4-tetramethyl-3-oxopentyl 3-oxidobenzoate ( D ). We found that D was weakly fluorescent though hydrolyzed in NaOH/H 2 O, and estimated that the singlet-chemiexcitation efficiency Φ S was 6.1 × 10 −3 for the decomposition of B in NaOH/H 2 O. On the other hand, Φ S for A could not be estimated, since C was hydrolyzed too rapidly to observe its fluorescence.

Acridinium dimethylphenyl esters are widely used as chemiluminescent labels in automated immunoassays for clinical diagnostics in Siemens Healthcare Diagnostics' ADVIA Centaur® systems. Light emission from these labels and their conjugates is triggered with alkaline peroxide. Excited state acridone is believed to be the light emitting species that is formed from the initial peroxide adduct which subsequently undergoes a series of reactions leading to scission of the phenolic ester bond. Dioxetane and/or dioxetanone intermediates have been proposed as immediate precursors of excited state acridone. Despite the fact that acridinium esters have been widely used as chemiluminescent labels for decades, a substantive theoretical framework to guide acridinium ester design with improved properties over the basic structure is unavailable. We have relied on a more empirical approach to devise new acridinium esters with improved stability, higher light yield, fast light emission, low non-specific binding and improved immunoassay performance. In the current study, we have investigated the effect of branching in remote alkoxy substituents attached to C-2 and C-7 of the acridinium ring on light emission and chemiluminescence stability of two acridinium esters. We selected two, high light output acridinium dimethylphenyl esters that we described previously as a basis for these studies and report the synthesis of two new C-2 and C-7 alkoxy-substituted labels, compounds 5 and 10, with improved chemiluminescence stability and faster light emission respectively. Compound 5 exhibited better long term stability at both pH 6 and 7.4 (≥10%) compared to its unbranched counterpart compound 11 whereas compound 10 with branched hexa(ethylene) glycol substituents exhibited >4-fold faster light emission compared to its unbranched counterpart 12. Both parameters are important for immunoassay performance in automated instruments.

Fluoride plays an important role in our daily life as it associates with a variety of biological and pathological processes. Hence, an analytic method that can detect fluoride ions under physiological conditions with high sensitivity and specificity, and fast response, is needed. Herein, we introduced a dioxetane-based chemiluminescent probe, CL-F, specifically designed for the detection of fluoride ions in physiological environment. The probe CL-F emitted strong green chemiluminescent light within 1 min when incubating with fluoride ions specifically. The linear range for sensing fluoride ions and limit of detection were determined to be 0–30 μM and 0.91 μM, respectively. The probe CL-F was then successfully applied in quantifying fluoride ions in toothpaste and in vivo imaging in living mice, demonstrating CL-F as a promising tool for sensing fluoride ion in vitro and in vivo.

It was shown that acyclic intermediates play a role in the chemiexcitation of the chemiluminescence (CL) of lophine peroxides in addition to the dioxetane intermediates. Because the CL efficiencies of position-isomers (R)-9 and (R)-10, which theoretically give the common dioxetane intermediate, were different, the different CL efficiencies are attributable to the CL mechanism involving a partially cyclic transition structure at the chemiexcitation step.

Chemiluminescence is among the most sensitive methods for achieving a high signal-to-noise ratio in various chemical and biological applications. We have developed a modular practical synthetic route for preparation of turn-ON fluorophore-tethered dioxetane chemiluminescent probes. The chemiluminescent emission of the probes was significantly amplified through an energy-transfer mechanism under physiological conditions. Two probes were composed with green and near-infrared (NIR) fluorescent dyes tethered to Schaap's dioxetane. While both probes were able to provide chemiluminescence in vivo images following subcutaneous injection, only the NIR probe could provide a chemiluminescence image following intraperitoneal injection. These are the first in vivo images produced by Schaap's dioxetane chemiluminescence probes with no need of an enhancer. Previously, chemiluminescence cell images could only be obtained with a luciferin-based probe. Our NIR probe was able to image cells transfected with β-galactosidase gene by chemiluminescence microscopy. We also report, for the first time, the instability of dioxetane-fluorophore conjugates to ambient light. Our synthetic route effectively overcomes this limitation through a late-stage functionalization of the dioxetane intermediate. We anticipate that our practical synthetic methodology will be useful for preparation of various chemiluminescent probes for numerous applications.

In the present work the chemiluminescence characteristics of a peroxyoxalate system using berberine as a novel fluorophore has been investigated. Berberine can accept energy via the formation of a dioxetane and emits intense yellow light. The characteristics of the chemiluminescence for this system under various concentrations of reagents including bis (trichlorophenyl)oxalate, sodium salicylate (as catalyst), hydrogen peroxide and the fluorophore were also studied. These parameters including the rise and fall rate constants for the chemiluminescence emission, theoretical and experimental maximum intensity, theoretical and experimental time to reach maximum intensity and total light yield emission were evaluated from intensity–time profiles using the pooled intermediate model by a non-linear least-squares curve fitting program, KINFIT.

Bicyclic dioxetanes, 1-(hydroxyaryl)-2-aza-6,7-dioxabicyclo[3.2.0]heptanes, bearing a syn-N-acyl or anti-N-acyl group underwent TBAF (tetrabutylammonium fluoride)-induced decomposition in DMSO or THF accompanied by the emission of bright light, the maximum wavelengths ( λ max CL ) of which were similar to each other. On the other hand, upon treatment with t-BuOK in THF, the dioxetanes bearing a syn-N-acyl group emitted light that showed a dramatic red-shift compared to that in a TBAF/THF system, while the dioxetanes bearing an anti-N-acyl group showed light with a blue-shift. For oxidoaryl-substituted dioxetanes bearing a syn-N-acyl group, t-BuOK most likely coordinated with both a syn-N-acyl and an oxygen of the dioxetane ring, so that two imide carbonyls in the produced emitter possessed a considerably regulated structure leading to a red-shift in the chemiluminescence. Such coordination of K+ was hardly expected for the case of the dioxetanes bearing an anti-N-acyl group, for which the strong contact of K+ to an oxidoaryl group presumably caused a blue-shift in chemiluminescence.

Bicyclic dioxetanes 3 and 4 tethering a fluorescein or 4-(benzothiazol-2-yl)-3-hydroxyphenyl moiety through a linker were synthesized by the use of dihydrofuran-intermediate 5 or its advanced intermediate 6. These dioxetanes underwent base-induced decomposition to effectively give light due to intramolecular energy-transfer from an excited oxidobenzoate to a tethered fluorophore. Although the chemiluminescence efficiency ΦCL values for 3 and 4 were only ca. 2-fold greater than that for parent 2 in a TBAF/acetonitrile system, these values were 30–550-fold greater than that for 2 in a NaOH/H2O system. Such marked increase of ΦCL was hardly observed by the simple addition of 25 or 26 as a model of a tethered fluorescer.

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 para- meters 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 b-carotene on it system was also investigated. The measurable concentra- tion range of 7 � 10 � 6 M to 7.5 � 10 � 5 M 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 b-carotene and quercetin respectively.

Hydroxyaryl-substituted dioxetanes 2-4 fused with a pyrrolidine ring were selectively synthesized by singlet oxygenation of the corresponding dihydropyrroles 5-7. These N-acylamino-substituted bicyclic dioxetanes were quite stable thermally, and 2a and 2b were estimated to possess half-lives of 32 and 34 y at 25 degrees C. When treated with TBAF (tetrabutylammonium fluoride) in DMSO, these dioxetanes underwent charge-transfer-induced decomposition (CTID) to emit yellow-orange light. The chemiluminescence efficiencies Phi(CL) for dioxetanes 2 ranged from 10(-6) to 10(-2). On the other hand, dioxetane 4 bearing a 4-(benzothiazol-2-yl)-3-hydroxyphenyl moiety showed chemiluminescent decomposition with high efficiency (Phi(CL) = 0.15) comparable to its oxy-analogue 26. Prominent characteristics for CTID of the present dioxetanes were that the N-acylamino-group influenced the color of chemiluminescence as well as the rate of decomposition k(CTID), and furthermore an N-acyl substituent could decisively affect the singlet-chemiexcitation efficiency, as observed for the case of 2b.

Organisms undergoing oxidative metabolism or stress chemically generate electronically excited species through dioxetane and tetroxide pathways. Consequently, luminescence is emitted which could be exploited for non-invasive label-free monitoring of oxidative stress. Spectral analysis of this endogenous chemiluminescence can serve as a tool for identification of the emitters as products of oxidation. Here we employ quantum mechanical calculations (Gaussian software package) and sensitive experimental photon counting methods to obtain deeper understanding of the emitter molecules both from theoretical and experimental perspective. We focused on the analysis of excited carbonyl groups of simple model species (formaldehyde, acetone, etc.) and carbonyls formed by fragmentation of linoleic acid since they are common product of oxidation and also potential emitters of endogenous chemiluminescence.

Bicyclic dioxetane 5 bearing a 3-hydroxynaphthalen-2-yl group and its analogs 14 and 15 decomposed to give light with efficiencies of only 0.002–0.005% in a tetrabutylammonium fluoride (TBAF)/THF system, which was as expected for dioxetanes with a so-called ‘ even ’ substitution pattern. However, the chemiluminescence efficiencies ( Φ CL ) markedly increased when these dioxetanes were decomposed with alkaline metal t- butoxide in THF. This enhancement of Φ CL by alkaline metal ion was most likely due to the highly ordered conformation of an aromatic ring by chelate formation of the metal ion with both an oxido anion and oxygen atom of a tetrahydrofuran ring in an intermediary dioxetane like 12 . Alkaline metal ion-enhanced chemiluminescence was similarly observed for dioxetane 6 bearing a 2-hydroxyphenyl group.

A bicyclic dioxetane 1 bearing a 4-(4-cyanophenyl)iminomethyl-3-hydroxyphenyl group was found to undergo base-induced decomposition with the accompanying emission of light, the colour of which changed depending on the base used and its concentration. When 1 was triggered with tetrabutylammonium fluoride (TBAF), 1 displayed an emission of glowing orange light. On the other hand, on treatment with a high concentration of potassium t-butoxide complexed with 18-crown-6 ether, 1 afforded a flash of blue light. The mechanistic study of this unprecedented phenomenon revealed that the emission of glowing orange light was due to the normal oxido anion of keto ester 11, whereas the emission of a flash of blue light was attributed to another species that was produced by addition of a nucleophile to an iminomethyl of unstable oxido anion of dioxetane 10. Copyright © 2008 John Wiley & Sons, Ltd.

Base-induced chemiluminescent decomposition of acylamino-substituted dioxetane 1b bearing a 3-hydroxyphenyl group proceeded according to dual phase kinetics due to syn – anti isomerism of the aryl group. For dioxetane 7 bearing a 6-hydroxynaphthalen-2-yl group, syn - and anti -rotamers were each isolated and their structures were determined by X-ray single crystallographic analysis. Both rotamers underwent base-induced decomposition accompanied by the emission of red light to exclusively give the same keto imide 8 . However, a marked difference in chemiluminescence efficiency was observed between anti - 7 and syn - 7 .

Dioxetanes bearing an aromatic electron donor decompose with an accompanying emission of light by an intramolecular charge-transfer-induced chemiluminescence (CTICL) mechanism. The color of the chemiluminescence from CTICL-active aryl-substituted dioxetanes is controlled by the design of the aromatic moiety and its substitution pattern, and the substituent on the carbon of the dioxetane ring. In addition to these color modulations, a change in the conformation of dioxetanes bearing a biaryl group was also found to cause a change in the color of CTICL in the coordination sphere of a crown ether complex. This new color modulation system was further developed for optically active dioxetanes bearing 2-hydroxy-1,1′-binaphthyl, the CTICL of which took place under the chiral recognition of optically active crown ether complexes. The spectra of the chemiluminescence from biaryl-type dioxetanes did not coincide with the fluorescence spectrum from the authentic emitter in the coordination sphere, but they did coincide with each other in a completely homogeneous system. This finding suggests that the emitter of CTICL possesses a transient structure, which retains the afterimage of the conformation of the dioxetane. © 2008 The Japan Chemical Journal Forum and Wiley Periodicals, Inc. Chem Rec 8: 213–228; 2008: Published online in Wiley InterScience (www.interscience.wiley.com) DOI 10.1002/tcr.20150

Four optical isomers of dioxetane 2 bearing a 2-hydroxy-1,1′-binaphthyl-4-yl moiety decomposed with accompanying emission of light on treatment with three base systems. The chemiluminescence efficiencies of all isomers were practically the same for tetrabutylammonium fluoride (TBAF) in DMSO system, whereas they split into two diastereomer-groups for 18-crown-6 ether complex of t -BuOK in benzene–THF system. For the base system using t -BuOK complex of optically active crown ether, ( Ra , Ra )- 7 or ( Sa , Sa )- 7 , the chemiluminescence efficiencies split further into four. The shape of chemiluminescence spectrum, maximum wavelength, and the rate of decomposition were also different between each of the four stereoisomers.

Mechanophores capable of releasing N-heterocyclic carbene (NHC), a strong base, are combined with triggerable chemiluminescent substrates to give a novel system for mechanically induced chemiluminescence. The mechanophores are palladium bis-NHC complexes, centrally incorporated in poly(tetrahydrofuran) (pTHF). Chemiluminescence is induced from two substrates, adamantyl phenol dioxetane (APD) and a coumaranone derivative, upon sonication of dilute solutions of the polymer complex and either APD or the coumaranone. Control experiments with a low molecular weight Pd complex showed no significant activation and the molecular weight dependence of the coumaranone emission supports the mechanical origin of the activation. The development of this system is a first step towards mechanoluminescence at lower force thresholds and catalytic mechanoluminescence.

Five pairs of diastereoisomeric dioxetanes, cis- and trans-2a–2e, were synthesized. These dioxetanes underwent intramolecular charge-transfer-induced decomposition with accompanying emission of orange light in TBAF in DMSO (system A) as a complete homogeneous system and in [K⊂(18C6)]+t-BuO− in PhH–THF (system B) as a sterically anisotropic environment. Maximum wavelength (λmaxCTICL) of chemiluminescence did not vary practically with the triggering system. The λmaxCTICL was little affected also by substituents on the upper-Nap of dioxetanes 2, nor by the difference in their stereochemistry, namely, cis- or trans-isomer. On the other hand, chemiluminescent efficiency was found to split up depending on stereochemistry of 2. Dioxetane 2b bearing a methoxycarbonyl group on the upper-Nap gave significantly weak light, while its free carboxylic acid analog 2c afforded light effectively.

The chemiluminescence (CL) mechanism of 3,4-bis(3-indolyl)-1H-pyrrole-2,5-dione (IPD) was investigated using liquid chromatography electrospray ionization mass spectrometry (LC–ESI-MS) of the products formed after the IPD CL reaction. We found that IPD produced strong CL via the decomposition of dioxetane formed after oxidation of the maleimide and indole moieties in the presence of CH3CN, H2O2 and NaOH. The IPD CL was used for evaluating the antioxidant effect on curcumin and epigallocatechin gallate.

Five bicyclic dioxetanes bearing a 2-hydroxynaphthyl group, 1aA – 1eA , were synthesized and their chemiluminescent decomposition was examined by the use of tetrabutylammonium fluoride (TBAF) as a base in DMSO. It was found that these dioxetanes hold completely the ‘ odd / even ’ relationship between the substitution pattern of hydroxy as a trigger on the naphthalene ring and their chemiluminescent efficiency, and that dioxetane 1aA exhibited chemiluminescence with the highest efficiency among those for the oxynaphthyl-substituted dioxetanes hitherto known. The significant change in chemiluminescent efficiency depending on the substitution pattern was clarified to be attributed to the marked change in singlet-chemiexcitation efficiency for charge-transfer-induced chemiluminescence (CTICL) of 1aA – 1eA . In respect of the rate of CTICL-decomposition, ‘ odd / even ’ relationship was observed for 1aA – 1dA .

A flow-injection chemiluminescence method for the determination of tryptophan was proposed, which was based on an intense chemiluminescence of tryptophan in hydrogen peroxide–nitrite–sulfuric acid medium. The chemiluminescence reaction was attributed to peroxidation and epoxidation of tryptophan by peroxynitrous acid, and subsequent decomposition of the formed dioxetane. The chemiluminescence intensity was linear with tryptophan in the range of 6.0 × 10−7 to 3.0 × 10−5 mol l−1 and the limit of detection (S/N = 3) was 1.8 × 10−7 mol l−1. The proposed method was applied to the analysis of tryptophan in pharmaceutical preparations and human serum.

A class of high-energy molecules, the 1,2-dioxetanes, have received a great deal of attention because of their unique ability to decompose thermally into electronically excited carbonyl products. Their chemistry originates from studies on molecular mechanisms of bioluminescence and has a history of over 30 years. However, the luminescent efficiency of the dioxetanes could hardly be compared with that for bioluminescence until a dioxetane bearing an easily oxidized aryl group was found to afford efficient chemiluminescence by the intramolecular chemically initiated electron exchange luminescence (CIEEL) mechanism. Nowadays, the CIEEL-type dioxetanes are being applied to modern biochemical and biomedical analyses. In this review, the advanced chemistry of the CIEEL-type dioxetanes as highly efficient chemiluminescent substrates is described, focusing on their molecular design and synthesis. Singlet chemiexcitation processes for the intramolecular CIEEL decay of dioxetanes and triplet chemiexcitation processes for the thermolysis of dioxetanes are also discussed.

A chemiluminescence assay for detection of coliforms was developed based on the reaction of .-galactosidase enzyme from Escherichia coli with a phenylgalactosidase-substituted dioxetane substrate, leading to the generation of light at 530 nm. Polymixin B was added to increase cell membrane permeability. Light emitted from the reaction was measured in a luminometer, and data were correlated with counts of E. coli enumerated on MacConkey agar plates. A comparison of light intensity from cultures grown in nutrient broth and lactose broth (LB) showed LB to be a better choice as growth medium. A randomized block design analysis comprising a two-way treatment structure with six dilution levels and 25, 50, or 55 .L of E. coli culture indicated maximum sensitivity of the assay at 55 .L of E. coli with 500 .L of the dioxetane substrate. An exponential increase in the intensity of light emitted was observed with an exponential increase in numbers of E. coli. The detection limit for the assay was 102 to 103 CFU in 60 min. The assay was able to detect the presence of coliforms in milk, sprouts, and water samples tested, as confirmed by plating the samples on MacConkey agar. With further testing, this enzyme-based chemiluminescence assay may provide a simple and rapid method for detection of E. coli contamination in food and water.

The experimentally observed reduction of chemiluminescence in the spiro-substituted dioxetane with increasing diffusion is analyzed in terms of the diffusional theory of spin-dependent recombination and separation of radical ions produced by chemically initiated decomposition of the source reactant. The partitioning between singlet and triplet products of recombination crucially depends on the spin conversion in the radical ion pair and the relative rates of the competing reactions of backward transfer to the excited singlet (light emitting) or triplet state of the recombination products.

The chemiluminescence (CL) intensities of various indole derivatives substituted with a glyoxylyl group at the 3-position and a hydroxyl group at the 5-position of the indole ring were compared upon the addition of H2O2 in alkaline media. The CL intensities of 3-indoleglyoxylyl chloride, 3-indoleglyoxylic acid, 5-hydroxyindole and 5-benzyloxyindole in CH3CN were 5.9-, 48-, 5.9- and 3.3-fold stronger than that of 3-methylindole. A lasting CL of 3-indoleglyoxylyl chloride was found. Under appropriate conditions, the CL emission reached a maximum within 10 min after the addition of H2O2 in the presence of NaOH, and the intensity was retained for 25 min. One of the final products via the CL reaction of 3-indoleglyoxylyl chloride was indole-3-carboxylic acid. 3-Indoleglyoxylyl chloride emitted light by decompositions via both dioxetane and dioxetanedione. An enhancement effect of beta-cyclodextrin and bovine serum albumin on the CL of 3-indoleglyoxylyl chloride was also found.

Bicyclic dioxetanes bearing a 3-hydroxy-4-isoxazolylphenyl moiety ( 3a–c ) were synthesized. All these dioxetanes ( 3a–c ) underwent base-induced CIEEL-decay to afford light with high efficiency in an NaOH/H2O system as well as in a TBAF (tetrabutylammonium fluoride)/acetonitrile system. Among them, a dioxetane bearing a 3-hydroxy-4-[5-(trifluoromethyl)isoxazol-3-yl]phenyl moiety ( 3a ) emitted light in the aqueous system with the highest efficiency which parallels that attained in an aprotic solvent system. Fluorescence study and the AM1 calculations for 3a and the parent CIEEL-active dioxetanes ( 1 ) suggested that one important factor affecting chemiluminescence efficiency in an aqueous system should be the hydrogen-bonding at the carbonyl oxygen site of an oxyanion of hydroxyarenecarboxylate as the emitter produced by the CIEEL-decay.