Molecular Design of d-Luciferin-Based Bioluminescence and 1,2-Dioxetane-Based Chemiluminescence Substrates for Altered Output Wavelength and Detecting Various Molecules

Optical imaging including fluorescence and luminescence is the most popular method for the in vivo imaging in mice. Luminescence imaging is considered to be superior to fluorescence imaging due to the lack of both autofluorescence and the scattering of excitation light. To date, various luciferin analogs and bioluminescence probes have been developed for deep tissue and molecular imaging. Recently, chemiluminescence probes have been developed based on a 1,2-dioxetane scaffold. In this review, the accumulated findings of numerous studies and the design strategies of bioluminescence and chemiluminescence imaging reagents are summarized.     

Selected Least Studied but not Forgotten Bioluminescent Systems

Bioluminescence is a form of chemiluminescence generated by luminous organisms. Luminous taxa have currently been reported from about 800 genera and probably over 10 000 species in the world. On the other hand, their bioluminescent systems, including chemical structures of luciferins/chromophores and the genes encoding luciferases/photoproteins, have been elucidated from only a few taxonomic groups, for example beetles, bacteria, dinoflagellates, ostracods and some cnidarians. Research efforts to understand unknown bioluminescence systems are being conducted around the world, and recently, for example, novel luciferin structures of luminous enchytraeid potworms and fungi were identified by the authors. In this study, we review the current status and perspectives, in the context of postgenomic era, of most likely novel but less‐revealed bioluminescence systems of ten selected organisms: earthworm, parchment tubeworm, fireworm, scaleworm, limpet, millipede, brittle star, acorn worms, tunicate and shark, which indeed are the next focus of our international collaboration.     

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(-).     

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

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

QM/MM Study on the Light Emitters of Aequorin Chemiluminescence,Bioluminescence, and Fluorescence: A General Understanding of the Bioluminescence of Several Marine Organisms

Aequorea victoria is a type of jellyfish that is known by its famous protein, green fluorescent protein (GFP), which has been widely used as a probe in many fields. Aequorea has another important protein, aequorin, which is one of the members of the EF‐hand calcium‐binding protein family. Aequorin has been used for intracellular calcium measurements for three decades, but its bioluminescence mechanism remains largely unknown. One of the important reasons is the lack of clear and reliable knowledge about the light emitters, which are complex. Several neutral and anionic forms exist in chemiexcited, bioluminescent, and fluorescent states and are connected with the H‐bond network of the binding cavity in the protein. We first theoretically investigated aequorin chemiluminescence, bioluminescence, and fluorescence in real proteins by performing hybrid quantum mechanics and molecular mechanics methods combined with a molecular dynamics method. For the first time, this study reported the origin and clear differences in the chemiluminescence, bioluminescence and fluorescence of aequorin, which is important for understanding the bioluminescence not only of jellyfish, but also of many other marine organisms (that have the same coelenterazine caved in different coelenterazine‐type luciferases).     

Oxyluciferin Photoacidity: The Missing Element for Solving the Keto–Enol Mystery?

The oxyluciferin family of fluorophores has been receiving much attention from the research community and several systematic studies have been performed in order to gain more insight regarding their photophysical properties and photoprotolytic cycles. In this minireview, we summarize the knowledge obtained so far and define several possible lines for future research. More importantly, we analyze the impact of the discoveries on the firefly bioluminescence phenomenon made so far and explain how they re‐open again the discussion regarding the identity (keto or enol species) of the bioluminophore.     

发光分析

本文是《分析试验室》定期评述中“发光分析”课题的第四篇评述文章，它评述在１９９５年１月～１９９６年１２月间我国发光分析的发展概况，内容包括：荧光，磷光，化学发光和生物发光分析等方面，引用文献３５４篇。     

Chaetopterus variopedatus Bioluminescence: A Review of Light Emission within a Species Complex

Chaetopterus variopedatus has been studied for over a century in terms of its physiology, ecology and life history. One focus of research is on its intrinsic bioluminescent emissions, which can be observed as a blue light emitted from the extremities of individual body segments, or as a secreted mucus. Even though research shows that C. variopedatus is a species complex miscategorized as a single species, all of the variants of this polychaete produce light, which has been investigated in terms of both physiology and biochemistry. Despite decades of study, there are still many questions about the luminescence reaction, and, as of yet, no clear function for light emission exists. This review summarizes the current knowledge on C. variopedatus luminescence in addition to briefly describing its morphology, life cycle and ecology. Possible functions for luminescence were discussed using observations of specimens found in Brazil, along with a comparison of previous studies of other luminescent organisms. Further study will provide a better understanding of how and why C. variopedatus produces luminescence, and purifying the protein and luciferin involved could lead to new bioanalytical applications, as this reaction is unique among all known luminescent systems.     

Progress in the Study of Bioluminescent Earthworms

Even though bioluminescent oligochaetes rarely catch people's eyes due to their secretive lifestyle, glowing earthworms sighting reports have come from different areas on all continents except Antarctica. A major breakthrough in the research of earthworm bioluminescence occurred in the 1960s with the studies of the North American Diplocardia longa. Comparative studies conducted on 13 earthworm species belonging to six genera showed that N‐isovaleryl‐3‐aminopropanal (Diplocardia luciferin) is the common substrate for bioluminescence in all examined species, while luciferases appeared to be responsible for the color of bioluminescence. The second momentous change in the situation has occurred with the discovery in Siberia (Russia) of two unknown luminous enchytraeids. The two bioluminescent systems belong to different types, have different spectral characteristics and localization, and different temperature and pH optima. They are unique, and this fact is confirmed by the negative results of all possible cross‐reactions. The bioluminescent system of Henlea sp. comprises four essential components: luciferase, luciferin, oxygen and calcium ion. For Friderica heliota, the luminescent reaction requires five components: luciferase, luciferin, ATP, magnesium ion and oxygen. Along with luciferin, more than a dozen analogues were isolated from worm biomass. These novel peptide‐like natural compounds represent an unprecedented chemistry found in terrestrial organisms.     

Cypridina bioluminescence—VII : The bioluminescence of Cypridina luciferin analogs

The relative rates of bioluminescence, as well as chemiluminescence, among Cypridina luciferin analogs, and the relative light yield between bioluminescence and chemiluminescence of each of the analogs have been measured with reference to Cypridina luciferin.     

Multicolor directional surface plasmon-coupled chemiluminescence

In reports over the past several years, we have demonstrated the efficient collection of optically excited fluorophore emission by its coupling to surface plasmons on thin metallic films, where the coupled luminescence was highly directional and polarized. This phenomenon is referred to as surface plasmon-coupled emission (SPCE). In this current study, we have extended this technique to include chemiluminescing species and subsequentially now report the observation of surface plasmon-coupled chemiluminescence (SPCC), where the luminescence from chemically induced electronic excited states couples to surface plasmons in thin continuous metal films. The SPCC is highly directional and predominantly p-polarized, strongly suggesting that the emission is from surface plasmons instead of the luminophores themselves. This indicates that surface plasmons can be directly excited from chemically induced electronic excited states and excludes the possibility that the plasmons are created by incident excitation light. This phenomenon has been observed for a variety of chemiluminescent species in the visible spectrum, ranging from blue to red, and also on a variety of metals, namely, aluminum, silver, and gold. Our findings suggest new chemiluminescence sensing strategies on the basis of localized, directional, and polarized chemiluminescence detection, especially given the wealth of assays that currently employ chemiluminescence-based detection.     

超氧负离子在生物发光中的作用

活性氧物种和超氧负离子是生物体内的重要物质,本文通过超氧负离子在生物发光反应中的作用,针对几种不同的典型生物发光体系,综述了超氧负离子参与发光反应的相关理论和实验研究进展.     

化学发光免疫分析技术在微生物检测中的应用

化学发光免疫分析技术凭借其化学发光的高灵敏性和免疫反应的高特异性在微生物快速检测中广泛应用。该文着重叙述了化学发光免疫分析技术核心检测体系在微生物检测中的研究进展,并对其涉及到的样品前处理和检测新方法进行了综述。化学发光免疫分析技术检测微生物用时短、成本低,但特异性识别方法和检测的灵敏度有待提升,新型的发光体系、发光放大方法、可替代抗体的识别分子以及相关的前处理技术是未来研究的重点。     

微流控免疫芯片检测方法的研究进展

微流控免疫芯片以其微型化、高通量、快速检测及低消耗等优点成为近年来分析领域的研究热点. 检测技术是微流控芯片的重要组成部分之一. 本文重点综述了近年来微流控免疫芯片的微系统研究及相应的检测方法和技术, 包括电化学检测及荧光检测、紫外-可见吸收光谱检测、化学发光和生物发光检测、表面增强拉曼散射检测、光纤检测、表面等离子体共振谱检测、热透镜显微镜检测和比色检测等光学检测及其它新型检测方面的进展, 并展望了其发展前景.     

Current Status of Research on Fungal Bioluminescence: Biochemistry and Prospects for Ecotoxicological Application

Over the last half decade the study of fungal bioluminescence has regained momentum since the involvement of enzymes has been confirmed after over 40 years of controversy. Since then our laboratory has worked mainly on further characterizing the substances involved in fungal bioluminescence and its mechanism, as well as the development of an ecotoxicological bioluminescent assay with fungi. Previously, we proved the involvement of a NAD(P)H‐dependent reductase and a membrane‐bound luciferase in a two‐step reaction triggered by addition of NAD(P)H and molecular oxygen to generate green light. The fungal luminescent system is also likely shared across all lineages of bioluminescent fungi based on cross‐reaction studies. Moreover, fungal bioluminescence is inhibited by the mycelium exposure to toxicants. The change in light emission under optimal and controlled conditions has been used as endpoint in the development of toxicological bioassays. These bioassays are useful to better understand the interactions and effects of hazardous compounds to terrestrial species and to assist the assessment of soil contaminations by biotic or abiotic sources. In this work, we present an overview of the current state of the study of fungal luminescence and the application of bioluminescent fungi as versatile tool in ecotoxicology.     

The Sensitized Bioluminescence Mechanism of Bacterial Luciferase

After more than one‐half century of investigations, the mechanism of bioluminescence from the FMNH₂ assisted oxygen oxidation of an aliphatic aldehyde on bacterial luciferase continues to resist elucidation. There are many types of luciferase from species of bioluminescent bacteria originating from both marine and terrestrial habitats. The luciferases all have close sequence homology, and in vitro, a highly efficient light generation is obtained from these natural metabolites as substrates. Sufficient exothermicity equivalent to the energy of a blue photon is available in the chemical oxidation of the aldehyde to the corresponding carboxylic acid, and a luciferase‐bound FMNH‐OOH is a key player. A high energy species, the source of the exothermicity, is unknown except that it is not a luciferin cyclic peroxide, a dioxetanone, as identified in the pathway of the firefly and the marine bioluminescence systems. Besides these natural substrates, variable bioluminescence properties are found using other reactants such as flavin analogs or aldehydes, but results also depend on the luciferase type. Some rationalization of the mechanism has resulted from spatial structure determination, NMR of intermediates and dynamic optical spectroscopy. The overall light path appears to fall into the sensitized class of chemiluminescence mechanism, distinct from the dioxetanone types.     

Synthesis of Firefly Luciferin Analogues and Evaluation of the Luminescent Properties

Five new firefly luciferin ( 1 ) analogues were synthesized and their light emission properties were examined. Modifications of the thiazoline moiety in 1 were employed to produce analogues containing acyclic amino acid side chains ( 2 – 4 ) and heterocyclic rings derived from amino acids ( 5 and 6 ) linked to the benzothiazole moiety. Although methyl esters of all of the synthetic derivatives exhibited chemiluminescence activity, only carboluciferin ( 6 ), possessing a pyrroline‐substituted benzothiazole structure, had bioluminescence (BL) activity (λ_max=547 nm). Results of bioluminescence studies with AMP‐carboluciferin (AMP=adenosine monophosphate) and AMP‐firefly luciferin showed that the nature of the thiazoline mimicking moiety affected the adenylation step of the luciferin–luciferase reaction required for production of potent BL. In addition, BL of 6 in living mice differed from that of 1 in that its luminescence decay rate was slower.     

Simultaneous oxygen uptake and imaging chemiluminescence measurements

There are several studies that make a comparison between chemiluminescence and oxygen uptake. Although, there are several reasons to perform this comparison on the same sample at the same conditions, these studies were done by comparing results from separate oxygen uptake and chemiluminescence experiments.It is also postulated that the shape of the chemiluminescence and oxygen uptake curves is not related to the kinetics of the degradation chemistry but to the oxidizing area. To be able to measure the luminescence and the oxygen uptake at the same sample under exactly the same conditions an apparatus is developed in which both measurements can be done simultaneously. To be able to get information on the oxidizing area too, this apparatus is based on imaging chemiluminescence. The induction times determined with chemiluminescence and oxygen uptake are about comparable, although at lower temperatures the induction time according to oxygen uptake is shorter, which might be due to a decrease of the sensitivity of chemiluminescence with temperature.For stabilized and unstabilized polypropylene it was shown that the luminescence curve is related to the amount of material degrading and that the shape of the oxygen uptake and luminescence curves is not related to the kinetics of the degradation chemistry.     

Graphene materials-based chemiluminescence for sensing

Graphene has attracted considerable attention in multidisciplinary research fields and shown various promising applications due to its unique structure and extraordinary physicochemical properties. This review covers the latest advances in graphene materials-based chemiluminescence (CL) for sensing. Chemiluminescence resonance energy transfer and luminescence quenching of graphene materials are discussed. Graphene materials, such as graphene nanosheets, graphene quantum dots, graphene oxide, and reduced graphene oxide have been employed successfully in CL systems in recent years. Graphene materials can be utilized as catalysts, platforms, and energy acceptors to improve the performance of CL. Possible challenges and future perspective on this topic are also presented.     

Bioorganic studies on marine natural products--diverse chemical structures and bioactivities

The discovery of new molecules contributes to the development of basic scientific concepts, leads to valuable drug-oriented compounds, and suggests possible new pharmacological reagents. Newly discovered substances can even be responsible for the creation of new scientific fields. Due to the radically different habitats of marine organisms, several notable examples of secondary metabolites from marine organisms have been isolated. Two of the most remarkable properties of these compounds are their structural and physiological diversities. These bioactive compounds are candidates for drugs or biological probes for physiological studies. Palytoxin is a polyol compound that shows extreme acute toxicity. Halichondrins are remarkable antitumor macrolides from sponge. Pinnatoxins, potent shellfish poisons, cause food poisoning. This paper describes bioorganic studies on such newly discovered wonders of nature. Several bioactive marine alkaloids and important substances involved in dynamic ecological systems are also described.