Enzymatic Conversion of Cypridina Luciferyl Sulfate to Cypridina Luciferin with Coenzyme A as a Sulfate Acceptor in Cypridina (Vargula) hilgendorfii

In the luminous ostracod Cypridina (presently Vargula) hilgendorfii, Cypridina luciferyl sulfate (3‐enol sulfate of Cypridina luciferin) is converted to Cypridina luciferin by a sulfotransferase with 3′‐phosphoadenosine‐5′‐phosphate (PAP) as a sulfate acceptor. The resultant Cypridina luciferin is used for the luciferase–luciferin reaction of Cypridina to emit blue light. The luminescence stimulation with major organic cofactors was examined using the crude extracts of Cypridina specimens, and we found that the addition of coenzyme A (CoA) to the crude extracts significantly stimulated luminescence intensity. Further, the light‐emitting source in the crude extracts stimulated with CoA was identified as Cypridina luciferyl sulfate, and we demonstrated that CoA could act as a sulfate acceptor from Cypridina luciferyl sulfate. In addition, the sulfate group of Cypridina luciferyl sulfate was also transferred to adenosine 5′‐monophosphate (5′‐AMP) and adenosine 3′‐monophosphate (3′‐AMP) by a sulfotransferase. The sulfated products corresponding to CoA, 5′‐AMP and 3′‐AMP were identified using mass spectrometry. This is the first report that CoA can act as a sulfate acceptor in a sulfotransferase reaction.     

Syntheses and evaluation of the bioluminescent activity of (S)-Cypridina luciferin and its analogs

Cypridina luciferin is the substrate in the bioluminescence of a luminous ostracod Cypridina (Vargula) hilgendorfii. Cypridina luciferin contains a chiral center in the sec-butyl moiety. Here, we report a convenient method for the preparation of (S)-Cypridina luciferin by the condensation of (S)-1,1-diethoxy-3-methylpentan-2-one with ethioluciferin. The light yield of the synthesized (S)-luciferin in the presence of Cypridina luciferase was about 1.7 times as active as that of racemic form. Furthermore, several luciferin analogs prepared by the same condensation with different α-ketoacetal derivatives showed moderate light yield with Cypridina luciferase. These readily available Cypridina luciferin and analogs are applicable to the bioluminescent detection of Cypridina luciferase.     

Real‐time reaction monitoring by probe electrospray ionization mass spectrometry

Probe electrospray ionization (PESI) is a modified version of the electrospray ionization (ESI), where the capillary for sampling and spraying is replaced by a solid needle. High tolerance to salts and direct ambient sampling are major advantages of PESI compared with conventional ESI. In this study, PESI‐MS was used to monitor some biological and chemical reactions in real‐time, such as acid‐induced protein denaturation, hydrogen/deuterium exchange (HDX) of peptides, and Schiff base formation. By using PESI‐MS, time‐resolved mass spectra and ion chromatograms can be obtained reproducibly. Real‐time PESI‐MS monitoring can give direct and detailed information on each chemical species taking part in reactions, and this is valuable for a better understanding of the whole reaction process and for the optimization of reaction parameters. PESI‐MS can be considered as a potential tool for real‐time reaction monitoring due to its simplicity in instrumental setup, direct sampling with minimum sample preparation and low sample consumption. Copyright © 2010 John Wiley & Sons, Ltd.     

Time‐of‐flight mass spectrometry for time‐resolved measurements: Some developments and applications

In this paper, the time resolution for kinetic studies of reactions with mass spectrometric detection is characterized in detail, and it is shown how this allows faster kinetic processes to be determined. The time‐resolved technique used pulsed laser photolysis to initiate reaction and a time‐of‐flight mass spectrometer (TOFMS) to monitor progress, where the reactant gas was sampled by a sampling orifice and photoionized using pulsed, laser vacuum ultraviolet light before being analyzed by the TOFMS. Characterization of this setup has been carried out to identify the parameters that affect the time for “sampling,” which limits the fastest reactions that can be measured. A simple mathematical equation has been developed to correct for “sampling” delays (k_sampling～25, 000 s^?1), which extends the range of rate coefficients to be measured in a kinetic mass spectrometry reactor to k′ < 7000 s^?1. This method could be applied to any other kinetic mass spectrometry system where k_sampling can be measured; an important advantage since it allows the study of reactions over a wider range of conditions (e.g., larger concentrations of reagents/products can be used to minimize the contribution from wall losses). The system can produce reliable kinetic data whether monitoring reactant decay or product growth even when the reaction and sampling processes are occurring on a similar timescale (k′ < 7000 s^?1). Reproducible and reliable kinetic data have been obtained for the following reactions: SO + NO₂ → products (R1), ClSO + NO₂ → products (R2), where SO and ClSO were monitored under pseudo‐first‐order conditions, and HCO + O₂ → CO + HO₂ (R3), where CO was monitored by a [1+1] resonance enhanced ionization multiphoton ionization (REMPI) scheme with HCO reacting under pseudo–first‐order conditions. The limitations and potential developments of this setup are described. © 2011 Wiley Periodicals, Inc. Int J Chem Kinet 44: 532–545, 2012     

Marine Bioluminescence: Measurement by a Classical Light Sensor and Related Foraging Behavior of a Deep Diving Predator

Bioluminescence is produced by a broad range of organisms for defense, predation or communication purposes. Southern elephant seal (SES) vision is adapted to low‐intensity light with a peak sensitivity, matching the wavelength emitted by myctophid species, one of the main preys of female SES. A total of 11 satellite‐tracked female SESs were equipped with a time‐depth‐light 3D accelerometer (TDR10‐X) to assess whether bioluminescence could be used by SESs to locate their prey. Firstly, we demonstrated experimentally that the TDR10‐X light sensor was sensitive enough to detect natural bioluminescence; however, we highlighted a low‐distance detection of the sensor. Then, we linked the number of prey capture attempts (PCAs), assessed from accelerometer data, with the number of detected bioluminescence events. PCA was positively related to bioluminescence, which provides strong support that bioluminescence is involved in predator–prey interactions for these species. However, the limitations of the sensor did not allow us to discern whether bioluminescence (i) provided remote indication of the biological richness of the area to SES, (ii) was emitted as a mechanic reaction or (iii) was emitted as a defense mechanism in response to SES behavior.     

Luciferins, bioluminescent substances

The bioluminescence of the American firefly Photinus is due to the reaction of 2-(6-hydroxybenzothiazol-2-yl)-Δ²-1,3-thiazoline-4-carboxylic acid (“firefly luciferin”) with the enzyme luciferase in the presence of ATP and magnesium ion. In the crustacean Cypridina, on the other hand, the bioluminescence is due to the reaction of a luciferase with 8-(3-guanidinopropyl)-6-indol-3-yl-2-(1-methylpropyl)-3,7-dihydroimidazo[1,2-a]-pyrazin-3-one (“Cypridina luciferin”). The luciferin in Latia is 1,3,3-trimethyl-2-(4-formyloxy-3-methyl-3-butenyl)-1-cyclohexene and that in Renilla is a tryptamine derivative that has not yet been accurately identified; the luciferins of other luminescent organisms are not yet known. A review is given of the investigations which have been carried out on the above luciferins and the course of the luciferin-luciferase reaction is examined. Numerous spectral data obtained during the examination of these compounds are included in the text.     

The Chemical Basis of Fungal Bioluminescence

Many species of fungi naturally produce light, a phenomenon known as bioluminescence, however, the fungal substrates used in the chemical reactions that produce light have not been reported. We identified the fungal compound luciferin 3‐hydroxyhispidin, which is biosynthesized by oxidation of the precursor hispidin, a known fungal and plant secondary metabolite. The fungal luciferin does not share structural similarity with the other eight known luciferins. Furthermore, it was shown that 3‐hydroxyhispidin leads to bioluminescence in extracts from four diverse genera of luminous fungi, thus suggesting a common biochemical mechanism for fungal bioluminescence.     

Semi‐heterogeneous Dual Nickel/Photocatalysis using Carbon Nitrides: Esterification of Carboxylic Acids with Aryl Halides

Cross‐coupling reactions mediated by dual nickel/photocatalysis are synthetically attractive but rely mainly on expensive, non‐recyclable noble‐metal complexes as photocatalysts. Heterogeneous semiconductors, which are commonly used for artificial photosynthesis and wastewater treatment, are a sustainable alternative. Graphitic carbon nitrides, a class of metal‐free polymers that can be easily prepared from bulk chemicals, are heterogeneous semiconductors with high potential for photocatalytic organic transformations. Here, we demonstrate that graphitic carbon nitrides in combination with nickel catalysis can induce selective C?O cross‐couplings of carboxylic acids with aryl halides, yielding the respective aryl esters in excellent yield and selectivity. The heterogeneous organic photocatalyst exhibits a broad substrate scope, is able to harvest green light, and can be recycled multiple times. In situ FTIR was used to track the reaction progress to study this transformation at different irradiation wavelengths and reaction scales.     

A Photodynamic System based on Endogenous Bioluminescence for in vitro Anticancer Studies

Photodynamic therapy (PDT) is becoming an important cancer treatment in recent years. However, at present, the therapeutic effect of PDT is limited due to insufficient penetration depth of light. In this study, a new photodynamic system (d ‐Lu)PCN‐224 is constructed by porphyrin‐based metal‐organic framework (MOF) PCN‐224 and bioluminescent molecule d ‐fluorescein (d ‐Lu). The bioluminescence (BL) spectrum of the reaction overlaps with the absorption spectrum of PCN‐224, so it is speculated that bioluminescence resonance energy transfer (BRET) between the MOF and d ‐Lu which indicates inner light can be gained and used for PDT. Confocal imaging analysis and cytotoxicity assays have demonstrated that (d ‐Lu)PCN‐224 can produce singlet oxygen and decrease the cell viability of SKOV‐3. This system provides a possibility of PDT for deep‐level organization without an external light source.     

Firefly Chemiluminescence and Bioluminescence: Efficient Generation of Excited States

Firefly luciferase catalyzes a light‐emitting reaction in which an excited‐state product is formed. Both experimental and theoretical methodologies are used to study this system, and the reactions catalyzed by luciferase are relatively well characterized. However, the mechanism by which an excited‐state product is formed is still unknown. This Minireview deals with the current understanding of firefly bioluminescence and chemiluminescence. Thermal decomposition of simple 1,2‐dioxetanes is also discussed, due to their role in formation of the excited‐state bioluminophore.     

Dynamic Diselenide Bonds: Exchange Reaction Induced by Visible Light without Catalysis

Dynamic covalent bonds are extensively employed in dynamic combinatorial chemistry. The metathesis reaction of disulfide bonds is widely used, but requires catalysis or irradiation with ultraviolet (UV) light. It was found that diselenide bonds are dynamic covalent bonds and undergo dynamic exchange reactions under mild conditions for diselenide metathesis. This reaction is induced by irradiation with visible light and stops in the dark. The exchange is assumed to proceed through a radical mechanism, and experiments with 2,2,6,6‐tetramethylpiperidin‐1‐yloxyl (TEMPO) support this assumption. Furthermore, the reaction can be conducted in different solvents, including protic solvents. Diselenide metathesis can also be used to synthesize diselenide‐containing asymmetric block copolymers. This work thus entails the use of diselenide bonds as dynamic covalent bonds, the development of a dynamic exchange reaction under mild conditions, and an extension of selenium‐related dynamic chemistry.     

Anionic synthesis and detection of fluorescence‐labeled polymers with a terminal anhydride group

To monitor polymer–polymer coupling reactions between two different monofunctional polymers in dilute polymer blends, fluorescence‐labeled anhydride‐functional polystyrene (PS) and poly(methyl methacrylate) (PMMA) were prepared by conventional anionic polymerization. Sequential trapping of lithiopolystyrene by 1‐(2‐anthryl)‐1‐phenylethylene (APE) and then di‐t‐butyl maleate (4) provided, after pyrolysis, anhydride‐functional fluorescent PS. Fluorescent PMMA anhydride (8) was synthesized with sec‐butyllithium/APE as an initiator for the anionic polymerization of methyl methacrylate, trapping by 4, and pyrolysis. These polymers could be reacted with amine‐functional polymers by melt blending, and the reaction progress could be monitored by gel permeation chromatography coupled with fluorescence detection. This technique not only allows monitoring of the coupling reaction with high sensitivity (ca. 100 times more sensitive than refractive index detection) but also permits selective detection because unlabeled polymers are invisible to fluorescence detection. This highly sensitive and selective detection methodology was also used to monitor the coupling reaction of 8 with PS‐NH₂ at a thin‐film interface, which was otherwise difficult to detect by conventional methods. © 2000 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 38: 2177–2185, 2000     

Exploring Bioluminescence Function of the Ca2+‐regulated Photoproteins with Site‐directed Mutagenesis

Site‐directed mutagenesis is a powerful tool to investigate the structure–function relationship of proteins and a function of certain amino acid residues in catalytic conversion of substrates during enzymatic reactions. Hence, it is not surprising that this approach was repeatedly applied to elucidate the role of certain amino acid residues in various aspects of photoprotein bioluminescence, mostly for aequorin and obelin, and to design mutant photoproteins with altered properties (modified calcium affinity, faster or slower bioluminescence kinetics, different emission color) which would either allow the development of novel bioluminescent assays or improvement of characteristics of the already existing ones. This information, however, is scattered over different articles. In this review, we systematize the findings that were made using site‐directed mutagenesis studies regarding the impact of various amino acid residues on bioluminescence of hydromedusan Ca²⁺‐regulated photoproteins. All key residues that have been identified are pinpointed, and their influence on different aspects of photoprotein functioning such as active photoprotein complex formation, bioluminescence reaction, calcium response and light emitter formation is discussed.     

Chemiluminescence of 6-aryl-2-methylimidazo[1,2-a]pyrazin-3(7H)-ones in DMSO/TMG and in diglyme/acetate buffer: support for the chemiexcitation process to generate the singlet-excited state of neutral oxyluciferin in a high quantum yield in the Cypridina (Vargula) bioluminescence mechanism

The chemiluminescence of 6-aryl-2-methylimidazo[1,2-a]pyrazin-3(7H)-ones (Cypridina luciferin analogues) in DMSO/1,1,3,3-tetramethylguanidine and in diglyme/acetate buffer was investigated. The results indicate that the reaction mechanism that produces a high chemiluminescence quantum yield involves a chemiexcitation process from a neutral dioxetanone intermediate possessing an electron-donating aryl group (σ_Ar <−0.6) to the singlet-excited state of neutral acetamidopyrazine. This result may be applied to the reaction mechanism for Cypridina (Vargula) bioluminescence.     

Photochemical Creation of Fluorescent Quantum Defects in Semiconducting Carbon Nanotube Hosts

Quantum defects are an emerging class of synthetic single‐photon emitters that hold vast potential for near‐infrared imaging, chemical sensing, materials engineering, and quantum information processing. Herein, we show that it is possible to optically direct the synthetic creation of molecularly tunable fluorescent quantum defects in semiconducting single‐walled carbon nanotube hosts through photochemical reactions. By exciting the host semiconductor with light that resonates with its electronic transition, we find that halide‐containing aryl groups can covalently bond to the sp² carbon lattice. The introduced quantum defects generate bright photoluminescence that allows tracking of the reaction progress in situ. We show that the reaction is independent of temperature but correlates strongly with the photon energy used to drive the reaction, suggesting a photochemical mechanism rather than photothermal effects. This type of photochemical reactions opens the possibility to control the synthesis of fluorescent quantum defects using light and may enable lithographic patterning of quantum emitters with electronic and molecular precision.     

A Chemo‐Enzymatic Cascade for the Smart Detection of Nitro‐ and Halogenated Phenols

The flavin‐dependent monooxygenase, HadA, catalyzes the dehalogenation and denitration of the toxicants, nitro‐ and halogenated phenols, to benzoquinone. The HadA reaction can be applied in one‐pot reactions towards the de novo synthesis of d ‐luciferin by coupling with d ‐Cys condensation. d ‐luciferin, a valuable chemical widely used in biomedical applications, can be used as a substrate for the reaction of firefly luciferase to generate bioluminescence. As nitro‐ and halogenated phenols are key indicators of human overexposure to pesticides and pesticide contamination, the technology provides a sensitive and convenient tool for improved biomedical and environmental detection at ppb sensitivity in biological samples without the requirement for any pre‐treatment. This dual‐pronged method combines the advantages of waste biodetoxification to produce a valuable chemical as well as a smart detection tool for environmental and biomedical detection.     

Fluorometric monitoring of organic reactions on solid phase

The direct monitoring of reaction progress on solid supports by fluorescence spectroscopy is described. An immobilized fluorescent tracer molecule (dansyl chloride) is used to monitor the reaction on OH resins (Argopore Wang, PS Wang, and Argogel Wang), both in batch and in parallel chemistry. Fluorescence measurements were obtained directly on solid phase. The method demonstrated to be a valuable tool for the quantitative determination of resin-bound hydroxyl groups, to study reaction kinetics and for continuously monitoring the progress of the conversion of the hydroxyl resins into the chlorinated ones. The procedure proposed is highly sensitive compared to the traditional ones. The system can be extended to monitor a variety of reactions on solid supports, and in conjunction with a well-established technique such as flow analysis, basic studies on solid-phase become possible.     

PHOTODYNAMIC EFFECTS INDUCED BY THE LUCIFERIN/LUCIFERASE SYSTEM

Abstract— Luciferin from Photinur pyralis is an effective sensitizer, when excited with UV light in the range of 310–390 nm. With histidine or dithiothreitol as substrate, a type II photooxidation occurs, as judged from the inhibitory effect of sodium azide. During the ATP-driven luciferin-luciferase reaction, the resulting bioluminescence does not induce photodynamic reactions, as there is no overlap between the bioluminescence spectrum and the excitation spectrum of luciferin. However, in the presence of a second sensitizer, excitable by the bioluminescent light, photodynamic reactions can take place in the absence of exogenous light. As a consequence several photosensitizers can thus provoke photodynamic inactivation of luciferase.     

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.     

Are the Bio‐ and Chemiluminescence States of the Firefly Oxyluciferin the Same as the Fluorescence State?

A usual strategy in both experimental and theoretical studies on bio‐ and chemiluminescence is to analyze the fluorescent properties of the bio‐ and chemiluminescence reaction product. Recent findings in a coelenteramide and Cypridina oxyluciferin model raise a concern on the validity of this procedure, showing that the light emitters in each of these luminescent processes might differ. Here, the thermal decomposition path of the firefly dioxetanone and the light emission states of the Firefly oxyluciferin responsible for the bio‐, chemiluminescence, and fluorescence of the molecule are characterized using ab initio quantum chemistry and hybrid quantum chemistry/molecular mechanics methods to determine if the scenario found in the coelenteramide and Cypridina oxyluciferin study does also apply to the Firefly bioluminescent systems. The results point out to a unique emission state in the bio‐, chemiluminescence, and fluorescence phenomena of the Firefly oxyluciferin and, therefore, using fluorescence properties of this system is reasonable.