AsLn2, a luciferin-related modified tripeptide from the bioluminescent earthworm Fridericia heliota

AsLn2, an unusual modified peptide, was isolated from the bioluminescent earthworm Fridericia heliota (Enchytraeidae). Its structure, elucidated by NMR and mass spectrometry, includes residues of tyrosine, CompX (a novel tyrosine modification product, reported in the accompanying paper), and N(omega)-acylated lysine. Chromatography, UV, and ¹H NMR data imply a close structural similarity of AsLn2 with F. heliota luciferin. AsLn2 appears to be an intermediate or by-product in F. heliota luciferin biosynthesis.     

Novel Peptide Chemistry in Terrestrial Animals: Natural Luciferin Analogues from the Bioluminescent Earthworm Fridericia heliota

We report isolation and structure elucidation of AsLn5, AsLn7, AsLn11 and AsLn12: novel luciferin analogs from the bioluminescent earthworm Fridericia heliota. They were found to be highly unusual modified peptides, comprising either of the two tyrosine‐derived chromophores, CompX or CompY and a set of amino acids, including threonine, gamma‐aminobutyric acid, homoarginine, and unsymmetrical N,N‐dimethylarginine. These natural compounds represent a unique peptide chemistry found in terrestrial animals and rise novel questions concerning their biosynthetic origin.     

Action spectrum and quantum yield for the photoinactivation of mnemiopsin, a bioluminescent photoprotein from the Ctenophore mnemiopsis SP.

Abstract— Ctenophores are bioluminescent marine invertebrates closely related to the coelenterates. The isolated bioluminescent systems of the ctenophores Mnemiopsis and Beroë and the hydrozoan jellyfish Aequorea are protein-luciferin complexes (photoproteins) which flash upon the addition of Ca²⁺ ions. The photoprotein mnemiopsin has an oxygen-independent quantum yield for photoinactivation of bioluminescence as high as 0.5, placing it among the most light-sensitive proteins known. We have measured the action spectrum for this photoinactivation at 107 narrow (3.4 nm) wavelength bands between 230 nm and 570 nm, covering a range of four decade units in the action. The action spectrum in the visible region is identical with the absorption spectrum of native photoprotein, implicating bound luciferin. The UV action spectrum implies that absorption by aromatic amino acid residues also leads to extremely efficient photoinactivation. Although photoinactivation is a rapid first-order reaction, destruction of the luciferin is a slower, multiple-order process. Therefore, protein-bound luciferin is not the ultimate target of the photoinactivation. Absorption of light results in the dissociation of “active oxygen” from the photoprotein. Therefore, the ctenophore photoprotein is a precharged enzyme already containing bound luciferin and oxygen.     

Luciferin Regeneration in Firefly Bioluminescence via Proton-Transfer-Facilitated Hydrolysis,Condensation and Chiral Inversion

Firefly bioluminescence is produced via luciferin enzymatic reactions in luciferase. Luciferin has to be unceasingly replenished to maintain bioluminescence. How is the luciferin reproduced after it has been exhausted? In the early 1970s, Okada proposed the hypothesis that the oxyluciferin produced by the previous bioluminescent reaction could be converted into new luciferin for the next bioluminescent reaction. To some extent, this hypothesis was evidenced by several detected intermediates. However, the detailed process and mechanism of luciferin regeneration remained largely unknown. For the first time, we investigated the entire process of luciferin regeneration in firefly bioluminescence by density functional theory calculations. This theoretical study suggests that luciferin regeneration consists of three sequential steps: the oxyluciferin produced from the last bioluminescent reaction generates 2-cyano-6-hydroxybenzothiazole (CHBT) in the luciferin regenerating enzyme (LRE) via a hydrolysis reaction; CHBT combines with L-cysteine in vivo to form L-luciferin via a condensation reaction; and L-luciferin inverts into D-luciferin in luciferase and thioesterase. The presently proposed mechanism not only supports the sporadic evidence from previous experiments but also clearly describes the complete process of luciferin regeneration. This work is of great significance for understanding the long-term flashing of fireflies without an in vitro energy supply.     

NAPHTHYL- AND QUINOLYLLUCIFERIN: GREEN AND RED LIGHT EMITTING FIREFLY LUCIFERIN ANALOGUES

In the course of investigations on the possible involvement of the CIEEL (chemically initiated electron-exchange luminescence) mechanism in firefly bioluminescence, we have synthesized two novel firefly luciferin substrate analogues. D-Naphthylluciferin and D-quinolylluciferin were prepared by condensing D-cysteine with 2-cyano-6-hydroxynaphthalene and 2-cyano-6-hydroxyquinoline, respectively. These analogues are the first examples of bioluminescent substrates for firefly luciferase that do not contain a benzothiazole moiety. Firefly luciferase-catalyzed bioluminescence emission spectra revealed that compared to the normal yellow-green light of luciferin (lambda max = 559 nm), the emission from naphthylluciferin is significantly blue-shifted (lambda max = 524 nm); whereas quinolylluciferin emits orange-red light (lambda max = 608 nm). The fluorescence emission spectra, reaction pH optima, relative light yields, light emission kinetics and KM values of the analogues also were measured and compared to those of luciferin. Neither of the analogues produced the characteristic flash kinetics observed for the natural substrate. Instead, slower rise times to peak emission intensity were recorded. It appears that the formation of an intermediate from the analogue adenylates prior to the addition of oxygen is responsible for the slow rise times. The synthetic substrate analogues described here should be useful for future mechanistic studies.     

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.     

Synthesis of Latia luciferin benzoate analogues and their bioluminescent activity

The bioluminescent system of the univalve shell Latia neritoides exhibits a luciferin-luciferase reaction. We study the enol formate structure of Latia luciferin, which is expected to be important for luminescent activity. The Latia luciferin analogues with an enol substituted benzoate moiety were synthesized and their bioluminescent activity was measured. The Latia luciferin benzoate analogues delay emission for natural luciferin in bioluminescence, indicating that the Latia bioluminescent activity can be controlled by the design of the enol ester.     

BIOCHEMISTRY OF CENTIPEDE BIOLUMINESCENCE*

Abstract— The centipede (Orphaneous brevilabiatus) secretes a bioluminescent slime. The corrected emission spectrum of this luminescence was found to have maxima at about 510 and 480 nm. The reaction was found to require both a luciferin and luciferase and showed an unusually low pH optimum (4.6). Oxygen was required for the reaction, but oxygen could interact with one of the components allowing for anaerobic light emission.     

Two bioluminescent diptera: the North American Orfelia fultoni and the Australian Arachnocampa flava. Similar niche, different bioluminescence systems

Orfelia fultoni is the only bioluminescent dipteran (Mycetophilidae) found in North America. Its larvae live on stream banks in the Appalachian Mountains. Like their Australasian relative Arachnocampa spp., they build sticky webs to which their bioluminescence attracts flying prey. They bear two translucent lanterns at the extremities of the body, histologically distinct from the single caudal lantern of Arachnocampa spp., and emit the bluest bioluminescence recorded for luminescent insects (lambda(max) = 460 nm versus 484 nm from Arachnocampa). A preliminary characterization of these two bioluminescent systems indicates that they are markedly different. In Orfelia a luciferin-luciferase reaction was demonstrated by mixing a hot extract prepared with dithiothreitol (DTT) under argon with a crude cold extract. Bioluminescence is not activated by adenosine triphosphate (ATP) but is strongly stimulated by DTT and ascorbic acid. Using gel filtration, we isolated a luciferase fraction of approximately 140 kDa and an additional high molecular weight fraction (possibly a luciferin-binding protein) that activated bioluminescence in the presence of luciferase and DTT. The Arachnocampa luciferin-luciferase system involves a 36 kDa luciferase and a luciferin soluble in ethyl acetate under acidic conditions; the bioluminescence is activated by ATP but not by DTT. The present findings indicate that the bioluminescence of O. fultoni constitutes a novel bioluminescent system unrelated to that of Arachnocampa.     

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.     

A Dual‐Color Far‐Red to Near‐Infrared Firefly Luciferin Analogue Designed for Multiparametric Bioluminescence Imaging

Red‐shifted bioluminescent emitters allow improved in vivo tissue penetration and signal quantification, and have led to the development of beetle luciferin analogues that elicit red‐shifted bioluminescence with firefly luciferase (Fluc). However, unlike natural luciferin, none have been shown to emit different colors with different luciferases. We have synthesized and tested the first dual‐color, far‐red to near‐infrared (nIR) emitting analogue of beetle luciferin, which, akin to natural luciferin, exhibits pH dependent fluorescence spectra and emits bioluminescence of different colors with different engineered Fluc enzymes. Our analogue produces different far‐red to nIR emission maxima up to λ_max=706 nm with different Fluc mutants. This emission is the most red‐shifted bioluminescence reported without using a resonance energy transfer acceptor. This improvement should allow tissues to be more effectively probed using multiparametric deep‐tissue bioluminescence imaging.     

Highly Sensitive Bioluminescent Probe for Thiol Detection in Living Cells

The sensitive detection of thiols including glutathione and cysteine is desirable owing to their roles as indispensable biomolecules in maintaining intracellular biological redox homeostasis. Herein, we report the design and synthesis of SEluc‐1 (s ulfinate e ster luc iferin), a chemoselective probe exhibiting a ratiometric and turn‐on response towards thiols selectively in fluorescence and bioluminescence, respectively. The probe, which was designed based on the “caged” luciferin strategy, displays excellent selectivity, high signal/noise ratio (>240 in the case of bioluminescence), and a biologically relevant limit of detection (LOD, 80 nm for cysteine), which are all desirable traits for a sensitive bioluminescent sensor. SEluc‐1 was further applied to fluorescence imaging of thiol activity in living human cervical cancer HeLa cell cultures, and was successfully able to detect fluctuations in thiol concentrations induced by oxidative stress in a bioluminescent assay utilizing African green monkey fibroblast COS‐7 cells and human breast adenocarcinoma MCF‐7 cells.     

Understanding the complete bioluminescence cycle from a multiscale computational perspective: A review

Bioluminescence (BL) is an amazing natural phenomenon whose visible light is produced by living organisms. BL phenomenon is quite pervasive and has been observed in 17 phyla of 4 kingdoms. This fascinating natural phenomenon has unceasingly attracted people’s curiosity from ancient era to today. For a very long time, we can only receive some sporadic and static information from experimental observations, the mechanism of most BL remains is unclear. How the chemical reaction of BL process is initiated? Where the energy for light emission comes from? How does the light emitter produce? What is the light emitter for a wild bioluminescent organism? How to regain luciferin for next bioluminescence when it is used up? The luciferin is utilized forthwith or stored and release for subsequent light emission? What factors affect the color and strength of a bioluminescence? How to artificially tune the bioluminescence for special application? Computational BL plays unreplaceable role in answering these mechanistic questions. In contrast with experimental BL, computational BL came very late. In the past two decades, computational BL has touched nearly all the bioluminescent systems with chemical bases via the method of multiscale simulation. In this review, the author firstly introduced the history, types and general chemical process of BL. Then, the computational scheme on BL was briefly epitomized. Using firefly BL as a paradigmatic case, the author summarized theoretical investigation on the six stages of general chemical process in a BL cycle: luciferin oxidation, peroxide thermolysis, light emission, luciferin regeneration, luciferin storage and luciferin release. At each stage, the available theoretical studies of other bioluminescent organisms are briefly introduced and compared with the firefly system. Basing on the mechanistic understanding, the author reviewed the up-to-date theoretical design on bioluminescent systems. Again, the firefly was mainly focused on, and the other possible systems were just briefly introduced. This review summarized the theoretical studies to date on BL and addressed the status, critical challenges and future prospects of computational BL.     

Chemiluminescence of Aldehydes in Pyrogallol–NaOH System Using Polyaniline/Cu(I) as Catalyst

Journal of Analytical Chemistry - In this research, chemiluminescence (CL) of several aldehydes (such as oxalaldehyde, N-isovaleryl-3-amino-propanal as the bioluminescent earthworm luciferin,...     

Bioluminescent Microcapsules: Applications in Activating a Photosensitizer

Bioluminescent microcapsules uploading D ‐luciferin have been fabricated by using the covalent assembly of firefly luciferase and alginate dialdehyde through a layer‐by‐layer technique. Such assembled microcapsules can produce visible light in the region of 520–680 nm, which can activate the photosensitizers rose bengal (RB) and hypocrellin B (HB) after adding ATP. The microcapsules uploading photosensitizers (RB or HB) have an obvious property to prevent the proliferation of tumor cells in the dark. The assembled bioluminescent microcapsules can be potentially used as photon donors for bioimaging, ATP detection, and photodynamic therapy.     

Electrophilic aromatic substituted luciferins as bioluminescent probes for glutathione S-transferase assays

New highly sensitive latent bioluminescent luciferin substrates were designed and synthesized for monitoring mammalian glutathione S-transferase (GST) and Schistosoma japonicum enzyme activities.     

Novel dimeric cholesteryl-based A(LS)2 low-molecular-mass gelators with a benzene ring in the linker

Three novel dimeric cholesteryl-based A(LS)(2) low-molecular-mass organic gelators (LMOGs) with phthaloyl, isophthaloyl, or terephthaloyl moieties in the linkers were designed and prepared. According to the linker structures, the compounds are denoted as 1 (o-), 2 (m-), and 3 (p-), respectively. Gelation tests revealed that the difference of relative positions of two cholesterol moieties in the benzene ring can produce a dramatic change in the gelation behaviors of the compounds. Importantly, 2 and 3 are more efficient gelators than 1, and their self-assembly behaviors are also very different from each other as revealed by scanning electron microscopy (SEM) measurements. Very interestingly, 2 gels xylene spontaneously at room temperature, and the sol-gel phase transition of the system is mechanically controllable. FTIR and (1)H NMR spectroscopy studies revealed that hydrogen bonding and pi-pi interactions between the molecules of the gelators play an important role in the formation and maintenance of the gels. The X-ray diffraction (XRD) analysis revealed that in the gel of 2/benzene, 2 aggregated into a layered structure with an interlayer distance of 3.54 nm, which is just the length of 2.     

Fluorescence excitation spectroscopy for the measurement of epidermal proliferation.

Fluorescence excitation spectroscopy was used to assess cellular turnover in human skin by monitoring changes of endogenous fluorescence. Epidermal proliferation was induced with alpha-hydroxy acids. Commercially available glycolic acid creams (8 and 4% wt/wt concentration) and a vehicle cream (placebo) were applied in a randomized double blinded fashion on subjects' forearms, twice daily for 21 days. Excitation spectra were recorded (excitation 250-360 nm, emission 380 nm) at days 0, 1, 3, 7, 10, 11, 14, 17 and 21. The 295 nm excitation band (assigned to tryptophan moieties) was used in this study as a marker for cellular proliferation. To further reduce the day-to-day variability of the skin fluorescence the intensity of the 295 nm band was normalized to the 334 nm band (assigned to collagen crosslinks). The fluorescence emission intensity from placebo-treated skin remained practically unchanged over the period of the measurements while the fluorescence intensity measured from the glycolic acid-treated skin increased monotonically with treatment. The rate of increase of the excitation intensity with treatment was found to be dose dependent. The epidermal 295 nm band may be used as a quantitative marker to monitor the rate of proliferation of epidermal keratinocytes noninvasively.     

Predictions of the electronic absorption and emission spectra of luciferin and oxyluciferins including solvation effects

The ground and excited state properties of luciferin (LH(2)) and oxyluciferin (OxyLH(2)), the bioluminescent chemical in the firefly, have been characterized using the configuration interaction singles (CIS) and time dependent density functional (TDDFT) methods. The effects of solvation on the electronic absorption and emission spectra of luciferin and oxyluciferin are predicted with a self-consistent isodensity polarized continuum model of the solvent using both the configuration interaction singles model and time dependent density functional theory. The S(0)-->S(1) vertical excitation energies in the gas phase and in water are obtained with both methods. Optimizations of the excited state geometries permit the first predictions of the fluorescence spectra for these biologically important molecules. Shifts in both the absorption and emission spectra on proceeding from the gas phase to aqueous solution also are predicted.     

Structure of cypridina biluciferyl,a dimer of cypridina luciferyl radical having bioluminescent activity

Structure of $\text{Cypridina}$ biluciferyl (luciferyl radical dimer), which is produced by chemical oxidation of $\text{C}$. luciferin with such as ferricyanide, was determined to be the symmetric 5,5′-dimer of $\text{C}$. luciferin. It gives light in the presence of $\text{C}$. luciferase, although the bioluminescent rate is very low. We suggest that the biluciferyl is an intermediate in the oxidation of the luciferin to $\text{C}$. luciferinol.