Luciferin Analog Research Articles

An allylated firefly luciferin analogue with luciferase specific response in living cells.

An allylated firefly luciferin was successfully synthesized and its bioluminescence properties were evaluated. When applied to cellular imaging in combination with Eluc, which is one of the commercially available luciferases, this analogue displayed a luciferase-specific bioluminescence signal with prolonged emission (>100 min).

Quantum yield improvement of red-light-emitting firefly luciferin analogues for in vivo bioluminescence imaging

The dimethylamino group of AkaLumine ((4S)-2-[(1E,3E)-4-[4-(dimethylamino)phenyl]-1,3-butadien-1-yl]-4,5-dihydro-4-thiazolecarboxylic acid), a red-light-emitting firefly luciferin analogue, was replaced by cyclic amino groups (1-pyrrolidinyl, 1-piperidino, 1-azepanyl, and 4-morpholino) to give AkaLumine analogues exhibiting desirable bioluminescence with emission maxima in the red region (656–667 nm). In particular, a bioluminescence reaction of 1-pyrrolidinyl analogue with a recombinant Photinus pyralis luciferase showed a higher quantum yield than that with AkaLumine, giving an improved bioluminescence intensity. The 1-pyrrolidinyl analogue also showed the strongest luminescence in whole-body luciferase-expressing mice among the analogues, indicating that a quantum yield improvement of a luciferin analogue is effective to increase bioluminescence imaging intensity.

Parallel Screening for Rapid Identification of Orthogonal Bioluminescent Tools.

Bioluminescence imaging with luciferase enzymes and luciferin small molecules is a well-established technique for tracking cells and other biological features in rodent models. Despite its popularity, bioluminescence has long been hindered by a lack of distinguishable probes. Here we present a method to rapidly identify new substrate-selective luciferases for multicomponent imaging. Our strategy relies on parallel screening of luciferin analogues with panels of mutant enzymes. The compiled data set is then analyzed in silico to uncover mutually orthogonal sets. Using this approach, we screened 159 mutant enzymes with 12 luciferins. Thousands of orthogonal pairs were revealed with sufficient selectivity for use in biological environments. Over 100 pairs were validated in vitro, and three were applied in cell and animal models. The parallel screening method is both generalizable and scalable and will streamline the search for larger collections of orthogonal probes.

Luciferase Activity of Insect Fatty Acyl-CoA Synthetases with Synthetic Luciferins.

Long-chain fatty acyl-CoA synthetases (ACSLs) are homologues of firefly luciferase but are incapable of emitting light with firefly luciferin. Recently, we found that an ACSL from the fruit fly Drosophila melanogaster is a latent luciferase that will emit light with the synthetic luciferin CycLuc2. Here, we have profiled a panel of three insect ACSLs with a palette of >20 luciferin analogues. An ACSL from the nonluminescent beetle Agrypnus binodulus (AbLL) was found to be a second latent luciferase with distinct substrate specificity. Several rigid luciferins emit light with both ACSLs, but styryl luciferin analogues are light-emitting substrates only for AbLL. On the other hand, an ACSL from the luminescent beetle Pyrophorus angustus lacks luciferase activity with all tested analogues, despite its higher homology to beetle luciferases. Further study of ACSLs is expected to shed light on the features necessary for bioluminescence and substrate selectivity.

Rapid Access to a Broad Range of 6'-Substituted Firefly Luciferin Analogues Reveals Surprising Emitters and Inhibitors.

Light-emitting firefly luciferin analogues contain electron-donating groups in the 6'-position, but the scope of known 6'-substitution remains narrow. A two-step route to a broad range of 6'-substituted luciferin analogues was developed to fill this void and enable more extensive study of the 6'-functionality. This chemistry allowed direct access to "caged" amide and bright azetidine analogues, but also revealed thioether inhibitors and unexpectedly luminogenic aryl amine derivatives.

Electronic and Steric Effects of Cyclic Amino Substituents of Luciferin Analogues on a Firefly Luciferin–Luciferase Reaction

Bioluminescent and spectroscopic properties of luciferin analogues with cyclic amino substituents (1-pyrrolidinyl, piperidino, 1-azepanyl, and morpholino groups) at the 6′ position were investigated by comparing with those of 6′-dimethylaminoluciferin, to provide information on spatial and molecular environmental properties of the active site in Photinus pyralis luciferase.

Visualizing changes in brain-derived neurotrophic factor (BDNF) expression using bioluminescence imaging in living mice

Brain-derived neurotrophic factor (BDNF) plays a fundamental role in expressing various neural functions including memory consolidation. Alterations of BDNF levels in the brain are associated with neurodegenerative and neuropsychiatric disorders. Therefore, it is important to understand how levels of BDNF are controlled. Recently we generated a novel transgenic mouse strain, termed the Bdnf-Luciferase transgenic (Bdnf-Luc Tg) mouse, to monitor changes in Bdnf expression. In the present study, we detected the bioluminescence signal from living Bdnf-Luc Tg mice after intraperitoneal administration of d-luciferin. Despite high levels of Bdnf expression in the brain, it was difficult to detect a signal from the brain region, probably because of its poorly penetrable (short-wavelength) bioluminescence. However, we could detect the changes in the bioluminescence signal in the brain region using a luciferin analogue generating a near-infrared wavelength of bioluminescence. We also found a strong correlation between increases in body weight and bioluminescence signal in the abdominal region of Tg mice fed a high-fat diet. These results show that changes in Bdnf expression can be visualized using living mice, and that the Tg mouse could be a powerful tool for clarification of the role of Bdnf expression in pathophysiological and physiological conditions.

Synthetic analogue of Fridericia luciferin with improved spectral properties

New bioluminescent analogue of Fridericia luciferin was synthesized for the first time. Bioluminescence emission maximum of the compound demonstrates a 50-nm bathochromic shift compared to the luciferin. The obtained analogue may find use in the novel in vivo bioimaging applications.

Ultrasensitive chemiluminescence of tetracyclines in the presence of MCLA

In this article, five tetracyclines (TCs) showed ultrasensitive chemiluminescence (CL) based on the novel CL system of methoxylated Cypridina luciferin analogues (MCLA) and Ce(IV). It was found that the CL intensity of MCLA-Ce(IV) system was enhanced 30 times by adding 10 pmol chlortetracycline. Mechanisms of MCLA-Ce(IV)-TCs were investigated by CL spectra, radical scavengers and UV spectra. The results indicated that singlet oxygen, resulting from the reaction between Ce(IV) and TCs, was involved in the enhancement of CL phenomenon. This CL system coupling with flow injection analysis was developed for the determination of five common TCs. Linear ranged from 100fmol to 20pmol (r>0.999) with limit of detection of 20 to 50fmol (S/N=3). The sensitivity of the present method is comparable to that of UPLC-MS/MS detection for TCs.

Spectroscopic Properties of Amine-substituted Analogues of Firefly Luciferin and Oxyluciferin.

Spectroscopic and photophysical properties of firefly luciferin and oxyluciferin analogues with an amine substituent (NH2 , NHMe and NMe2 ) at the C6' position were studied based on absorption and fluorescence measurements. Their π-electronic properties were investigated by DFT and TD-DFT calculations. These compounds showed fluorescence solvatochromism with good quantum yields. An increase in the electron-donating strength of the substituent led to the bathochromic shift of the fluorescence maximum. The fluorescence maxima of the luciferin analogues and the corresponding oxyluciferin analogues in a solvent were well correlated with each other. Based on the obtained data, the polarity of a luciferase active site was explained. As a result, the maximum wavelength of bioluminescence for a luciferin analogue was readily predicted by measuring the photoluminescence of the luciferin analogue in place of that of the corresponding oxyluciferin analogue.

Determination of the activity of superoxide dismutase using a glassy carbon electrode modified with ferrocene imidazolium salts and hydroxy-functionalized graphene

A glassy carbon electrode was modified with a composite consisting of ferrocene imidazolium salts and hydroxy-functionalized graphene in a Nafion matrix. The electrode is shown to facilitate the reduction of oxygen to form the superoxide anion radical. Atomic force microscopy and electrochemical impedance spectroscopy were used to characterize the electrode. The in-situ production of superoxide anion was then studied with an analogue of methyl-Cypridina luciferin (also referred to as Vargulin). As a result, an electrochemiluminescence (ECL) based assay was developed for the improved determination of the activity of the enzyme superoxide dismutase (SOD). Under optimized conditions, the ECL based calibration plot is linear in the 0.5 to 6.5 U·mL−1 SOD activity range, with a 0.2 U·mL−1 detection limit (at a signal-to-noise ratio of 3).

Development of a luminescence-controllable firefly luciferin analogue using selective enzymatic cyclization

In this study, a new firefly luciferin analog that can switch firefly bioluminescence (BL) activity from ‘off’ to ‘on’ state was designed and synthesized. BL inactive N-Ac-γ-glutamate luciferin 3 contains an acyclic precursor of the thiazoline moiety. Enzymatic treatment of 3 with aminoacylase resulted in a smooth removal of the acyl protecting group and concomitant cyclization to provide BL active carboluciferin 2.

Carbonylated proteins exposed to UVA and to blue light generate reactive oxygen species through a type I photosensitizing reaction

BackgroundCarbonylated proteins (CPs) are generated by the reaction of basic amino acid residues in proteins with aldehyde compounds produced during lipid peroxidation. CPs in the stratum corneum (SC) impact skin conditions such as skin moisture functions including water content and transepidermal water loss (TEWL). In addition, CPs can be frequently seen in the SC from sun-exposed sites compared with sun-protected sites. ObjectiveThe aim of this study was to reveal whether CPs could be a generation source of reactive oxygen species (ROS) in the SC following exposure to ultraviolet (UV) radiation and to identify the type of ROS and its generation mechanism. MethodsROS generation was detected using a methyl cypridina luciferin analog (MCLA) chemiluminescence system and an ESR spin-trapping method. CPs in porcine SC, in a keratin film and in bovine serum albumin (BSA) were prepared by reaction with acrolein. Levels of protein carbonylation were quantified by detecting aldehyde residues. ResultsCP levels in the SC were increased in a UVA energy-dependent manner. That result suggested that a source of ROS generation existed in the SC initiated and produced the carbonylation of SC proteins. Carbonylated BSA and carbonylated porcine SC sheets exhibited fluorescence spectra at an excitation wavelength of 430nm and an emission wavelength of 520nm. Irradiation of the SC with UVA increased protein carbonylation and the amount of autofluorescence in the SC. ROS generation in the SC caused by UVA and by short-wavelength visible light (blue light, 400–470nm) was detected by the MCLA chemiluminescence system. Artificially carbonylated porcine SCs and keratin films had increases of chemiluminescence intensity after exposure to both light sources as well. The addition of superoxide dismutase to the MCLA system completely abolished the incremental chemiluminescence intensity after both UVA and blue light exposure of the SC. In addition, acrolein-treated BSA gave ESR signals like hydroxyl radicals (OH) converted from superoxide anion radicals (O2−) during irradiation with a xenon arc lamp containing UVA and visible light. From the sum of these results, we consider that CPs are produced from O2− initially generated from exposure to UVA and blue light. ConclusionCPs are excited by absorbing sunlight, particularly UVA and blue light, and result in the generation of O2− through a CPs progress new protein carbonylation in stratum corneum through ROS generation. photosensitizing reaction. Further, the results suggest that the O2− produces CPs in the SC through lipid peroxidation in the sebum, and finally affects skin conditions including color and moisture functions.

Innovation of New Luciferin Analog for In Vivo Imaging

【Introduction】 We are innovating new imaging technologies by using organic chemistry. For making advanced medicinal technology, in vivo imaging is needed. So, we have been developing compounds suitable for deep in vivo imaging. 【Background】 It is very difficult for deep in vivo imaging to be absorbed and scattered light in living tissue. But, it is well known that the light in the near infrared region (NIR) has high permeability for the living tissue. It is a challenge to visualize in vivo deep imaging by developing an imaging material that has NIR emission. We have got structure-activity relationship data on the firefly luciferin luciferase reaction. According to the structure-activity relationship data on luciferin analogues, we have designed and marked NIR probe “AkalumineⓇ, (WAKO pure chemical LTD.). AkalumineⓇ gives a higher brightness compared with the natural firefly luciferin because of high permeability AkalumineⓇ in tissue. 【Purpose】 We try to rise concentration of the firefly luciferin for high dose. But we faced difficulty that AkalumineⓇ has low water-soluble. For various applications, “high dose” is key technology on in vivo imaging. Based on the AkalumineⓇstructure, new luciferin analogues have designed and synthesized three types. 【Result and conclusion】 We made the substances of three new luciferin analogues. One of these emits in the NIR and another has water-solubility increased by 15 fold for AkalumineⓇ. We will apply new probe to the animal for conforming practical ability and discuss these new luciferin analogues for in vivo imaging technology.

Resveratrol, piperine and apigenin differ in their NADPH-oxidase inhibitory and reactive oxygen species-scavenging properties

BackgroundMany plant-derived chemicals have been studied for their potential benefits in ailments including inflammation, cancer, neurodegeneration, and cardiovascular disease. The health benefits of phytochemicals are often attributed to the targeting of reactive oxygen species (ROS). However, it is not always clear whether these agents act directly as antioxidants to remove ROS, or whether they act indirectly by blocking ROS production by enzymes such as NADPH oxidase (NOX) enzymes, or by influencing the expression of cellular pro- and anti- oxidants. Hypothesis/PurposeHere we evaluate the pro- and anti-oxidant and NOX-inhibiting qualities of four phytochemicals: celastrol, resveratrol, apigenin, and piperine. Study DesignThis work was done using the H661 cell line expressing little or no NOX, modified H661 cells expressing NOX1 and its subunits, and an EBV-transformed B-lymphoblastoid cell line expressing endogenous NOX2. ROS were measured using Amplex Red and nitroblue tetrazolium assays. In addition, direct ROS scavenging of hydrogen peroxide or superoxide generated were measured using Amplex Red and methyl cypridina luciferin analog (MCLA). ResultsOf the four plant-derived compounds evaluated, only celastrol displayed NOX inhibitory activities, while celastrol and resveratrol both displayed ROS scavenging activity. Very little impact on ROS was observed with apigenin, or piperine. ConclusionThe results of this study reveal the differences that exist between cell-free and intracellular pro-oxidant and antioxidant activities of several plant-derived compounds.

Multicolor Bioluminescence Obtained Using Firefly Luciferin.

Firefly bioluminescence is widely used in life science research as a useful analysis tool. For example, the adenosine-5`-triphosphate (ATP)-dependent enzymatic firefly bioluminescence reaction has long been utilized as a microbial monitoring tool. Rapid and sensitive firefly luciferin-luciferase combinations are used not only to measure cell viability but also for reporter-gene assays. Recently, bioluminescence was utilized as a noninvasive, real-time imaging tool for living subjects to monitor cells and biological events. However, the number of commercialized luciferase genes is limited and tissue-permeable near-infrared (NIR) region emitting light is required for in vivo imaging. In this review, recent studies describing synthetic luciferin analogues predicted to have red-shifted bioluminescence are summarized. Luciferase substrates emitting red, green, and blue light that were designed and developed in our laboratory are presented. The longest emission wavelength of the synthesized luciferin analogues was recorded at 675 nm, which is within the NIR region. This compound is now commercially available as "Aka Lumine®".

Molecular Origin of Color Variation in Firefly (Beetle) Bioluminescence: A Chemical Basis for Biological Imaging.

Firefly shows bioluminescence by "luciferin-luciferase" (L-L) reaction using luciferin, luciferase, ATP and O2. The chemical photon generation by an enzymatic reaction is widely utilized for analytical methods including biological imaging in the life science fields. To expand photondetecting analyses with firefly bioluminescence, it is important for users to understand the chemical basis of the L-L reaction. In particular, the emission color variation of the L-L reaction is one of the distinguishing characteristics for multicolor luciferase assay and in vivo imaging. From the viewpoint of fundamental chemistry, this review explains the recent progress in the studies on the molecular mechanism of emission color variation after showing the outline of the reaction mechanism of the whole L-L reaction. On the basis of the mechanism, the progresses in organic synthesis of luciferin analogs modulating their emission colors are also presented to support further developments of red/near infrared in vivo biological imaging utility of firefly bioluminescence.

Luciferase gets deep and sensitive

A luciferin analog enables highly sensitive bioluminescent imaging from deep within biological tissue samples.

A luciferin analogue generating near-infrared bioluminescence achieves highly sensitive deep-tissue imaging.

In preclinical cancer research, bioluminescence imaging with firefly luciferase and D-luciferin has become a standard to monitor biological processes both in vitro and in vivo. However, the emission maximum (λmax) of bioluminescence produced by D-luciferin is 562 nm where light is not highly penetrable in biological tissues. This emphasizes a need for developing a red-shifted bioluminescence imaging system to improve detection sensitivity of targets in deep tissue. Here we characterize the bioluminescent properties of the newly synthesized luciferin analogue, AkaLumine-HCl. The bioluminescence produced by AkaLumine-HCl in reactions with native firefly luciferase is in the near-infrared wavelength ranges (λmax=677 nm), and yields significantly increased target-detection sensitivity from deep tissues with maximal signals attained at very low concentrations, as compared with D-luciferin and emerging synthetic luciferin CycLuc1. These characteristics offer a more sensitive and accurate method for non-invasive bioluminescence imaging with native firefly luciferase in various animal models.

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 (λmax =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.