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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.

A convenient and environmentally friendly synthetic route from 2-vinylbenzimide to 3-hydroxy-isoindolinones through visible light-promoted transformations via iron/disulfide catalysis and molecular oxygen oxidation has been developed. A range of 3-hydroxy-isoindolinones was obtained in moderate to good yields, which exhibit excellent functional group compatibility and broad substrate scope. Further mechanistic investigations proved that dioxetane might be a key intermediate being involved in the reaction.

Protease chemiluminescent probes exhibit extremely high detection sensitivity for monitoring activity of various proteolytic enzymes. However, their synthesis, performed in solution, involves multiple synthetic and purification steps, thereby generating a major limitation for rapid preparation of such probes with diverse substrate scope. To overcome this limitation, we developed a general solid-phase-synthetic approach to prepare chemiluminescent protease probes, by peptide elongation, performed on an immobilized chemiluminescent enol-ether precursor. The enol-ether precursor is immobilized on a 2-chlorotrityl-chloride resin through an acrylic acid substituent by an acid-labile ester linkage. Next, a stepwise elongation of the peptide is performed using standard Fmoc solid-phase peptide synthesis. After cleavage of the peptide-enol-ether precursor from the resin, by hexafluoro-iso-propanol, a simple oxidation of the enol-ether yields the final chemiluminescent dioxetane protease probe. To validate the applicability of the methodology, two chemiluminescent probes were efficiently prepared by solid-phase synthesis with dipeptidyl substrates designed for activation by aminopeptidase and cathepsin-B proteases. A more complex example was demonstrated by the synthesis of a chemiluminescent probe for detection of PSA, which includes a peptidyl substrate of six amino acids. We anticipate that the described methodology would be useful for rapid preparation of chemiluminescent protease probes with vast and diverse peptidyl substrates.

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.

Natural rubber or synthetic rubber materials have been extensively used in our daily life and industrial production for more than 100 years due to their unique physical properties. However, a amount of rubber waste has been released into the environment, posing great challenges to the disposal of these polymers. In contrast to the disposal strategies such as combustion or stockpiling, enzyme catalyzed rubber degradation constitutes an environmentally friendly strategy. Latex clearing protein (Lcp) is an iron-heme containing endotype dioxygenase that catalyses the oxidative cleavage of the chemically inert poly( cis -1,4-isoprene) which is the main component of rubber, affording the products of aldehyde and ketone. It was found that active site E148 residue is key to the reactivity of Lcp based on the mutation experiments. However, the role of E148 and the overall mechanism are still controversial. It is generally accepted that both of dioxygen atoms are incorporated into the substrate. In previous studies, two possible mechanisms of Lcp catalyzed cleavage of poly( cis -1,4-isoprene) are proposed. In the first one, the distal oxygen atom of Fe(III)−O2− complex attacks onto the C=C double bond, leading to a three-membered epoxide intermediate. This is followed by the attack of Fe=O complex on the epoxide intermediate and subsequent C−C bond cleavage, affording the final ketone and aldehyde products. While in the second one, the proximal oxygen atom of Fe(III)−O2− complex attacks onto the C=C double bond of the substrate, which is followed by the attack of the distal oxygen atom onto the substrate radical to generate a four-membered cyclic dioxetane intermediate. The final O−O as and C−C bond cleavage in the dioxetane intermediate would afford the final products. In this study, based on the crystal structure of LcpK30, the combined molecular dynamic (MD) simulations and quantum mechanics/molecular mechanics (QM/MM) calculations are performed to elucidate the catalytic mechanism of Lcp catalysed cleavage of central double bond of poly( cis -1,4-isoprene). Our study shows E148 is protonated during the reaction and forms stable H bond interactions with Fe(III)−O2− species. Thus, the plausible role of E148 is related to the positioning of both Fe(III)−O2− species and the substrate. Moreover, it can be ruled out that the E148 acts as a base to abstract the proton from the substrate. The proposed mechanism of Lcp works as follows. Firstly, the distal oxygen of Fe(III)−O2− attacks onto the carbon−carbon double bond to generate the peroxide alky radical intermediate. This is followed by the rebound of the proximal oxygen to the carbon radical to generate the dioxetane intermediate, with Fe(III) being reduced to Fe(II). The following O−O bond cleavage occurs via the Fe(II)-mediated reductive cleavage of O−O bond of the dioxetane intermediate, leading to the vicinal diol radical anion intermediate. Finally, a facile C−C cleavage is undergone to yield the ketone and aldehyde products. In particular, our calculations show that the direct O−O cleavage of the dioxetane intermediate in the absence of Fe(II) catalysis requires a remarkably high barrier of 160 kJ/mol, indicating the Fe(II) catalysis is vital for the further transformation of the dioxetane intermediate. For comparison, we also investigated the previously proposed mechanism involving the three-membered epoxide intermediate. However, the attack of the epoxide by FeIV=O complex is found to be highly unfavourable thermodynamically and thus can be ruled out. As such, this study may not only expand our understanding of the catalytic mechanism of LcpK30, but also provide useful information for rational design or engineering of Lcp enzyme toward the degradation of synthetic rubber materials.

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.

Self-reporting polymers, which can indicate damage with perceptible optical signals in a tailored force range, are useful as stress-sensitive sensors. We demonstrate a simple approach to realize this function by embedding two distinct mechanophores — rhodamine (Rh) and bis(adamantyl)-1,2-dioxetane (Ad), in polyurethane/polylactic acid blends. The deformed blends generate red coloration and red chemiluminescence. Such a unique dual-responsive behavior was evaluated by solid-state UV-vis spectroscopy, macroscopic tensile tests with in situ RGB and light intensity analyses, which supported a stress-correlated occurrence of the ring-opening of Rh, the scission of Ad and the fluorescence resonance energy transfer process between the respective mechanochemical species. Complementarity stemming from the difference in properties and manifestations of the two mechanophores is essential. That is, the more labile Rh allows shifting the appreciable optical changes to a much lower force threshold; the transient nature and high dynamic range of mechanochemiluminescence from Ad map in real time where and when many of the covalently incorporated dioxetane bonds break; besides, the disrupted yet non-scissile structure of Rh acts as a fluorescent acceptor to effectively harvest chemiluminescence from ruptured Ad. The current strategy is thus empowering multi-functional mechano-responsive polymers with greatly improved sensitivity and resolution for multimodal stress reporting., Probing bond scission process in a broader force range was realized by embedding mechanochromic rhodamine and mechanochemiluminescent 1,2-dioxetane in polymer blends.

A computational study of 2,2-azobis(isobutyronitrile) (AIBN)-initiated aerobic oxidative cleavage of alkenes is carried out employing density functional theory (DFT) and high-level coupled-cluster methods, such as coupled-cluster singles and doubles with perturbative triples [CCSD(T)]. Our computations show that the barriers for the formation of dioxetane derivatives suggested by Xu and co-workers (J. Org. Chem., 2014, 79, 7220–7225) for the reaction mechanism of aerobic oxidative cleavage of alkenes are computed to be higher than 65 kcal mol−1. This barrier is relatively high under the reaction conditions. Our results for the Xu mechanism indicate that the reaction does not proceed via the formation of a dioxetane ring under the reaction conditions. Our results demonstrate that the reaction of aerobic oxidative cleavage of geminal alkenes in the presence of AIBN is initiated by the peroxyl radical 9, contrary to the isobutyronitrile radical 2. Our results show that the 2-(2-hydroxyl-1,1-diarylethoxy)-2-methylpropanenitrile radical (15) does not appear throughout the reaction scheme and the reaction progresses over the 2-(2-hydroxyl-2,2-diarylethoxy)-2-methylpropanenitrile radical (13) rather than the 2-(2-hydroxyl-1,1-diarylethoxy)-2-methylpropanenitrile radical (15). Our results are in agreement with the experimental results for the aerobic oxidative cleavage of the geminal disubstituted alkenes. Our results also demonstrate that the epoxide derivatives can be formed as an intermediate under the reaction conditions. This reaction is not applicable for pyridine derivatives due to the conversion of vinylpyridine derivatives to N-oxide derivatives.

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)

A computational study of N-hydroxyphthalimidecatalyzed aerobic oxidative cleavage of alkenes is carried out employing density functional theory and high-level coupled-cluster methods, such as coupled-cluster singles and doubles with perturbative triples [CCSD(T)]. Our results demonstrate that the reaction proceeds through the alkoxyl radicals, as opposed to the mechanism suggested by Jiao and co-workers (Org. Lett. 2012, 14, 4158-4161), in which the reaction proceeds via formation of the dioxetane ring. The barriers for the formation of dioxetane derivatives are computed to be higher than SO kcal mol(-1), while the barriers for the formation of alkoxyl radicals are as low as 13 kcal mol(-1). Our results also demonstrate that epoxide derivatives can be formed as intermediates or byproducts under the reaction conditions.

The b-type cytochrome LcpK30 is a latex clearing protein (Lcp), which acts as an endotype dioxygenase to catalyze the extracellular cleavage of the chemically inert aliphatic polymer poly(cis-1,4-isoprene), producing oligo-isoprenoids with different terminal carbonyl groups (aldehyde and ketone, -CH2-CHO and -CH2-COCH3). On the basis of the fact that the muteins of E148A, E148Q, and E148H have substantially reduced reactivity, and the E148-initiated reaction mechanism has been previously proposed, in which a cyclic dioxetane intermediate or an epoxide intermediate may be involved, however, open questions still remain. In this paper, on the basis of the crystal structure of LcpK30, the enzyme-substrate reactant model was constructed, and the cleavage mechanism of the central double bond of poly(cis-1,4-isoprene) was elucidated by performing quantum mechanics/molecular mechanics calculations. Our calculation results revealed that the oxidative cleavage reaction is triggered by the addition of the heme-bound dioxygen to the double bond of the polymer, and E148 does not act as the catalytic base to extract the allylic proton to assist the reaction as previously suggested. Of the two considered pathways, the pathway that involves the dioxetane intermediate was calculated to be more favorable. During the catalysis, the distal oxygen first adds to the double bond of the substrate to form a radical intermediate, and then the Fe-O1 (proximal oxygen) bond cleaves to generate the dioxetane intermediate, which can easily collapse affording the final ketone and aldehyde products. In general, the cleavage mechanism of double C-C bond catalyzed by LcpK30 is similar to those of indoleamine 2,3-dioxygenase, tryptophan 2,3-dioxygenase, and the nonheme stilbene cleavage oxygenase NOV1 that all depend on the iron-bound dioxygen to initiate the cleavage reaction.

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.

Mechanoresponsive polymers with intense red emission are highly desirable in view of the strong penetrability and high resolution of red luminescence. Herein we introduce the sensitized luminescence of rare-earth Eu(III) complexes into the mechanochemiluminescence from polymeric dioxetane in a covalent way. By controlling the energy-Transfer process from mechanically broken dioxetane to the Eu(III) center, bright red emission from transparent poly(methyl acrylate) films was achieved for the first time. Importantly, mechanoluminescence sensitivity is greatly improved, enabling the location and timing of bond scission with high resolution. A sensitive stretchable device with the polymer film as an emissive layer was fabricated by a facile surface-engineering method, which can emit patterned red light upon deformation. This sensitized mechanoluminescence design thus expands the useful space of luminescent stress probes.

A novel photocatalyzed radical addition/oxygen oxidation/cleavage of dioxetane/HAT domino process for cleavage of CC bonds has been described. This protocol used porous graphitic carbon nitrides (p-g-C3N4) as a metal-free recyclable photocatalyst for transformations of alkynes with diazonium salts to give two different aldehydes or esters, which enables the formation of multiple C–O bonds in a single reaction with operational simplicity. This reaction has an excellent substrate scope and gives the desired products in moderate to high yields. In addition, the heterogeneous semiconductor exhibits easy handling and excellent recyclability for at least 6 cycles without any apparent loss of activity. Furthermore, this reaction could be carried out under solar light irradiation and is applicable for the gram scale with satisfactory results.

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.

During the oxidation of lipids in food or in vivo, fatty acids are initially converted to hydroperoxides, which undergo decomposition to various secondary decomposition products, including aldehydes and alkyl-furans. Aldehydes and alkyl-furans reduce the sensory quality of food by contributing to rancidity, and aldehydes reduce nutritional value by reacting with some essential nutrients such as lysine and thiamine. In vivo, reactions of aldehyde with proteins and DNA contribute to the pathogenesis of physiological disorders. Conversion of fatty acid hydroperoxides to aldehydes is generally believed to involve free radical reactions. However, it was recently hypothesized that lysine can catalyze the non-radical conversion of hydroperoxides to aldehydes. Thus the aim of the present study was to determine such lysine-catalysed conversion of linoleic acid hydroperoxides to aldehydic products. Linoleic acid hydroperoxides were prepared by the photosensitized oxidation of linoleic acid. The mixture of hydroperoxides was reacted with lysine, in the presence of a radical scavenger, and the organic fraction analysed by gas chromatography-mass spectrometry (GC-MS). Hexanal was detected as a major aldehydic product. The alkylfuran, 2-pentylfuran was also surprisingly detected under these conditions, and a pathway for its lysine-catalysed formation via the highly cytotoxic aldehyde, 4-hydroxy-2-nonenal proposed. The results of this study imply that, in the prevention of lipid oxidation-associated food deterioration and development of physiological disorders, more attention should be paid to pathways not involving free radical reactions, and which cannot be prevented by radical scavenging antioxidants.

Oxidation of olefins is initiated by addition of oxygen at the π-bond with formation of triplet biradical; therefore, dioxetane formation becomes dominant due to the Zeeman interaction stimulating triplet–singlet conversion through the T–S transition, with the contribution of the radical initiation being lower. In the case of saturated compounds, the Zeeman interaction results in the absence of the magnetic field effect on the rate of radical formation since the chain initiation is ruled by the hydrogen atom abstraction.

NOV1, a stilbene cleavage oxygenase, catalyzes the cleavage of the central double bond of stilbenes to two phenolic aldehydes, using a 4‐His Fe(II) center and dioxygen. Herein, we use in‐protein quantum mechanical/molecular mechanical (QM/MM) calculations to elucidate the reaction mechanism of the central double bond cleavage of phytoalexin resveratrol by NOV1. Our results showed that the oxygen molecule prefers to bind to the iron center in a side‐on fashion, as suggested from the experiment. The quintet Fe−O2 complex with the side‐on superoxo antiferromagnetic coupled to the resveratrol radical is identified as the reactive oxygen species. The QM/MM results support the dioxygenase mechanism involving a dioxetane intermediate with a rate‐limiting barrier of 10.0 kcal mol−1. The alternative pathway through an epoxide intermediate is ruled out due to a larger rate‐limiting barrier (26.8 kcal mol−1). These findings provide important insight into the catalytic mechanism of carotenoid cleavage oxygenases and also the dioxygen activation of non‐heme enzymes.

NOV1 is a stilbene cleavage oxygenase (SCO) responsible for the oxidative cleavage of the central double bond of stilbenes, forming two phenolic aldehydes. In the active site of NOV1, the iron cofactor is coordinated by four histidines and no negatively charged ligand, which is unusual for iron-containing enzymes. Here, on the basis of the recently obtained crystal structure of NOV1, we performed quantum mechanics/molecular mechanics (QM/MM) calculations to elucidate the reaction mechanism of NOV1. According to our calculations, two binding modes of O2 to Fe(II) (end-on and side-on modes) have been recognized, and the end-on mode is suitable for catalysis. The side-on mode, which has been obtained from the crystal, is an inactive form, which should first change to the end-on mode to initiate the catalytic reaction. For the reactant in end-on mode, the triplet and quintet states correspond to very similar energies. Of the three considered pathways, two pathways (path_a and path_b1, which differ only in the order of the first C–O bond formation) involving the dioxetane intermediate are calculated to be favorable, which can confirm the dioxygenase mechanism of NOV1. However, the pathway (path_b2) involving the epoxide intermediate corresponds to high energy barriers for the formation of the second C–O bond (27.49 and 31.56 kcal mol−1 on the triplet and quintet state surfaces) and can be ruled out. However, in path_b2, the formation of the epoxide intermediate is easy, which may react with the solvent water (non-enzymatic reaction) to form the by-product. In addition, the different protonation states of 4′-OH of the substrate and K134, as well as the coordination of additional water molecules with the iron, do not influence the reaction barrier.

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.

Dioxetane intermediates readily decompose to chemiluminescent triplet carbonyls, giving rise to what has been paradoxically called photochemistry in the dark. In this issue of Photochemistry and Photobiology, Bechara et al. report on mechanistic advances in such a reaction. With the use of horseradish peroxidase for isobutyraldehyde-derived triplet acetone, light emission from acetone and singlet oxygen can be quenched. The experiments reveal that the reaction depends on oxygen and the amino acid. The analysis reveals that free tryptophan is a target of this form of "carbonyl stress," with the efficient formation of mono-, bi- and tricyclic compounds (N-formylkynurenine, indoline, 1λ2 -indole and 3H-indoles).

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.

We oxidized histidine residues in monoclonal antibody drugs of immunoglobulin gamma 1 (IgG1) using ultraviolet C irradiation (UVC: 200–280 nm), which is known to be potent for sterilization or disinfection. Among the reaction products, we identified asparagine and aspartic acid by mass spectrometry. In the photo-induced oxidation of histidine in angiotensin II, 18O atoms from H218O in the solvent were incorporated only into aspartic acid but not into asparagine. This suggests that UVC irradiation generates singlet oxygen and induces [2 + 2] cycloaddition to form a dioxetane involving the imidazole Cγ − Cδ2 bond of histidine, followed by ring-opening in the manner of further photo-induced retro [2 + 2] cycloaddition. This yields an equilibrium mixture of two keto-imines, which can be the precursors to aspartic acid and asparagine. The photo-oxidation appears to occur preferentially for histidine residues with lower pKa values in IgG1. We thus conclude that the damage due to UVC photo-oxidation of histidine residues can be avoided in acidic conditions where the imidazole ring is protonated.

Post-calculation analyses are often required to extract physical insights from ab initio molecular dynamics simulations. In the present work, we use different machine learning classifiers to take a new perspective on the decomposition reaction of dioxetane. Upon thermally activated decomposition, dioxetane can form products in an electronically excited state and can thus chemiluminesce. Simulated dynamics trajectories exhibit both successful and frustrated dissociations. As an exhaustive and systematic study of the decomposition mechanism “by hand” is beyond feasibility, machine learning models have been employed to study the relevant nuclear distortions governing molecular dissociation. According to all classifiers used in the study, the two sets of geometries differ by the in-phase planarisation of the two formaldehyde moieties. New insights are obtained from this analysis: if both moieties are not planar enough when the dissociation is attempted, it is frustrated and the molecule remains trapped. The postponing of the decomposition reaction by the so-called entropic trap enhances the chemiexcitation efficiency.

Two cholesterol secosterol aldehydes, namely, 3β-hydroxy-5-oxo-5,6-secocholestan-6-al (secosterol A) and its aldolization product 3β-hydroxy-5β-hydroxy-B-norcholestane-6β-carboxyaldehyde (secosterol B), are highly bioactive compounds which have been detected in human tissues and potentially contribute to the development of physiological dysfunctions such as atherosclerosis, Alzheimer’s disease, diabetes, and cancer. They were originally considered to be exclusive products of cholesterol ozonolysis and thus to be evidence for endogenous ozone formation. However, it was recently postulated that primary amines such as lysine may catalyse their formation from cholesterol-5α-hydroperoxide (Ch-5α-OOH), the main product of the oxidation of cholesterol with singlet oxygen. This involves cyclization of Ch-5α-OOH to an unstable dioxetane intermediate, which decomposes to form secosterol aldehydes with triplet carbonyl groups, whose return to the singlet state is at least partly coupled to the conversion of triplet molecular oxygen to singlet oxygen. Here, we subjected cholesterol to photosensitized oxidation, which predominantly produces Ch-5α-OOH and minor amounts of the 6α- and 6β-hydroperoxides, exposed the hydroperoxide mixture to lysine in the presence of the antioxidant 2,6-ditertiary-butyl-4-hydroxytoluene (BHT), and analysed the reaction mixture by liquid chromatography-electrospray ionization-mass spectrometry. Consistent with the postulated lysine-catalysed formation of secosterol aldehydes, we detected formation of the latter and several types of their lysine adducts, including carbinolamines, Schiff’s bases, and amide-type adducts. We propose that the amide type adducts, which are major biomarkers of lipid oxidation, are mainly formed by singlet oxygen-mediated oxidation of the carbinolamine adducts.

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.