- A NEW SYNTHESIS OF WATASENIA PRELUCIFERIN BY CYCLIZATION OF 2-AMINO-3-BENZYL-5-(p-HYDROXYPHENYL)PYRAZINE WITH p-HYDROXYPHENYLPYRUVIC ACID
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The reaction of 2-amino-3-benzyl-5-(p-hydroxyphenyl)pyrazine with p-hydroxyphenylpyruvic acid gave directly Watasenia preluciferin in a satisfactory yield without any reductive treatment.
- Inoue, Shoji,Okada, Kunisuke,Tanino, Hideo,Kakoi, Hisae
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- Enzymatic conversion of dehydrocoelenterazine to coelenterazine using FMN-bound flavin reductase of NAD(P)H:FMN oxidoreductase
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Coelenterazine (CTZ) is known as luciferin (a substrate) for the luminescence reaction with luciferase (an enzyme) in marine organisms and is unstable in aqueous solutions. The dehydrogenated form of CTZ (dehydrocoelenterazine, dCTZ) is stable and thought to be a storage form of CTZ and a recycling intermediate from the condensation reaction of coelenteramine and 4-hydroxyphenylpyruvic acid to CTZ. In this study, the enzymatic conversion of dCTZ to CTZ was successfully achieved using NAD(P)H:FMN oxidoreductase from the bioluminescent bacterium Vibrio fischeri ATCC 7744 (FRase) in the presence of NADH (the FRase–NADH reaction). CTZ reduced from dCTZ in the FRase–NADH reaction was identified by HPLC and LC/ESI-TOF-MS analyses. Thus, dCTZ can be enzymatically converted to CTZ in vitro. Furthermore, the concentration of dCTZ could be determined by the luminescence activity using the CTZ-utilizing luciferases (Gaussia luciferase or Renilla luciferase) coupled with the FRase–NADH reaction.
- Inouye, Satoshi,Nakamura, Mitsuhiro,Hosoya, Takamitsu
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- Formation of Coelenteramine from 2-Peroxycoelenterazine in the Ca2+-Binding Photoprotein Aequorin
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Aequorin consists of apoprotein (apoAequorin) and (S)-2-peroxycoelenterazine (CTZ-OOH) and is considered to be a transient-state complex of an enzyme (apoAequorin) and a substrate (coelenterazine and molecular oxygen) in the enzymatic reaction. The degradation process of CTZ-OOH in aequorin was characterized under various conditions of protein denaturation. By acid treatment, the major product from CTZ-OOH was coelenteramine (CTM), but not coelenteramide (CTMD), and no significant luminescence was observed. The counterparts of CTM from CTZ-OOH were identified as 4-hydroxyphenylpyruvic acid (4HPPA) and 4-hydroxyphenylacetic acid (4HPAA) by liquid chromatography/electrospray ionization–time-of-flight mass spectrometry (LC/ESI-TOF-MS). In the luminescence reaction of aequorin with Ca2+, similar amounts of 4HPPA and 4HPAA were detected, indicating that CTM is formed by two pathways from CTZ-OOH through dioxetanone anion and not by hydrolysis from CTMD.
- Hosoya, Takamitsu,Inouye, Satoshi,Nakamura, Mitsuhiro
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- ATP-INDEPENDENT BIOLUMINESCENT REPORTER VARIANTS TO IMPROVE IN VIVO IMAGING
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Provided herein are chemically modified luciferase substrates for spectrally shifted emission and enhanced water solubility. Provided herein are engineered luciferases. Moreover, provided herein are new ATP-independent bioluminescent reporters which have improved biochemical and photophysical properties and are expected to have broad applications. Finally, provided herein are spectral-resolved triple-color bioluminescent systems, suitable for flexible and convenient approaches to monitor multiple biological events in either qualitative or quantitative manners.
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Page/Page column 38; 45-46
(2020/09/12)
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- Gram-scale synthesis of luciferins derived from coelenterazine and original insights into their bioluminescence properties
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An original gram-scale synthesis of O-acetylated forms of coelenterazine, furimazine or hydroxy-bearing analogues of luciferins is described. The comparison over two hours of their bioluminescence, using the nanoKAZ/NanoLuc luciferase, provides remarkable insights useful for the selection of a substrate adapted for a given application.
- Coutant, Eloi P.,Goyard, Sophie,Hervin, Vincent,Gagnot, Glwadys,Baatallah, Racha,Jacob, Yves,Rose, Thierry,Janin, Yves L.
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p. 3709 - 3713
(2019/04/17)
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- Strategies for large-scale synthesis of coelenterazine for in vivo applications
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A new application of two Negishi-type coupling reactions for the synthesis of coelenterazine is reported. The synthesis of coelenterazine in high purity on a gram scale will enable numerous approaches to bioluminescence imaging and possibly photodynamic therapy of deep-seated tumors. Coelenterazine is the substrate for several luciferases, among them Gaussia luciferase (gLuc). This synthesis starts with pyrazin-2-amine and uses inexpensive starting materials and catalysts. Georg Thieme Verlag Stuttgart New York.
- Shrestha, Tej B.,Troyer, Deryl L.,Bossmann, Stefan H.
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p. 646 - 652
(2014/03/21)
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- Development of luminescent coelenterazine derivatives activatable by β-galactosidase for monitoring dual gene expression
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Two bioluminogenic caged coelenterazine derivatives (bGalCoel and bGalNoCoel) were designed and synthesized to detect β-galactosidase activity and expression by means of bioluminescence imaging. Our approach addresses the instability of coelenterazine by introducing β-galactose caging groups to block the auto-oxidation of coelenterazine. Both probes contain β-galactosidase cleavable caging groups at the carbonyl group of the imidazo-pyrazinone moiety. One of the probes in particular, bGalNoCoel, displayed a fast cleavage profile, high stability, and high specificity for β-galactosidase over other glycoside hydrolases. bGalN-oCoel could detect β-galactosidase activity in living HEK-293T cell cultures that expressed a mutant Gaussia luciferase. It was determined that coelenterazine readily diffuses in and out of cells after uncaging by β-galactosidase. We showed that this new caged coelenterazine derivative, bGalNoCoel, could function as a dual-enzyme substrate and detect enzyme activity across two separate cell populations. Copyright
- Lindberg, Eric,Mizukami, Shin,Ibata, Keiji,Miyawaki, Atsushi,Kikuchi, Kazuya
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p. 14970 - 14976
(2013/11/06)
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- Regio- And chemoselective multiple functionalization of chloropyrazine derivatives. Application to the synthesis of coelenterazine
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Successive regio- and chemoselective metalations of chloropyrazines using TMPMgCl-LiCl and TMPZnCIIiCI furnish, after trapping with electrophiles, highly functionalized pyrazines in high yields. Application to a synthesis of coelenterazine, a bioluminescent natural product in jellyfish Aequorea victoria, in nine steps (9% overall yield) is reported.
- Mosrin, Marc,Bresser, Tomke,Knochel, Paul
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supporting information; experimental part
p. 3406 - 3409
(2009/12/01)
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- Synthesis of 3,7-dihydroimidazo[1,2a]pyrazine-3-ones and their chemiluminescent properties
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A series of 3,7-dihydroimidazo[1,2a]pyrazine-3-ones were prepared from 2-amino-3,5-dibromopyrazine. The concise synthesis of coelenterazine (5j), in three steps, 48% overall yield and >99% purity exemplifies the strategy. Further, the synthetic approach facilitated the regiospecific incorporation of carboxyalkyl linkers on the 3,7-dihydroimidazo[1,2a]pyrazine-3-one nucleus that are required for bioconjugation. Peroxymonocarbonate, an electrophilic oxidant, was used to initiate 'pseudo-flash' chemiluminescence from this class of molecules.
- Adamczyk, Maciej,Akireddy, Srinivasa Rao,Johnson, Donald D.,Mattingly, Phillip G.,Pan, You,Reddy, Rajarathnam E.
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p. 8129 - 8142
(2007/10/03)
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- Improved synthesis of Watasenia preluciferin
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On heating a mixture of 2-amino-3-benzyl-5-(p-hydroxyphenyl)pyrazine (2) and a large excess of p-hydroxyphenylpyruvic acid (4) at 130-140 °C in dioxane, without any reductive treatment, Watasenia preluciferin (1) was obtained directly in one batch process in 63% yield.
- Kakoi, Hisac,Inoue, Shoji
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p. 1669 - 1672
(2007/10/03)
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- Highly efficient and flexible total synthesis of coelenterazine
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A new total synthesis of the bioluminescent chromophore coelenterazine 1 is described.
- Keenan, Martine,Jones, Keith,Hibbert, Frank
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p. 323 - 324
(2007/10/03)
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- Chemi- and Bio-luminescence of Coelenterazine Analogues with Phenyl Homologues at the C-2 Position
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A series of phenyl homologues of coelenterazine substituted at the C-2 position were synthesized and their bio- and chemi-luminescence properties were investigated including the measurement of chemiluminescence spectra in various media.The light emitting species of each analogue was found to be a neutral form of a coelenteramide derivative in diethylene glycol dimethyl ether (DGM) containing a trace amount of acetate buffer (pH 5.60), while a monoanion was found only in dimethyl sulfoxide (DMSO) and a dianion was observed in DMSO containing a trace amount of aqueous sodium hydroxide.Based on pseudo first-order reaction kinetics, chemiluminescence rate constants were obtained in DGM containing a trace amount of acetate buffer.Each of the synthetic coelenterazine analogues was incorporated into recombinant apoaequorin to obtain a series of semisynthetic aequorins.Measurements of bioluminescence activities of the aequorins revealed that a benzyl group in the C-2 position was essential for efficient luminescence activity.A two-step incubation procedure was used to determine why some analogues gave less luminescence activity than the benzyl analogue and natural coelenterazine.
- Qi, Chen Feng,Gomi, Yasushiro,Hirano, Takashi,Ohashi, Mamoru,Ohmiya, Yoshihiro,Tsuji, Frederick I.
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p. 1607 - 1612
(2007/10/02)
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- Chemical Studies on Myctophina Fish Bioluminescence
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A new type of masked Watasenia preluciferin was isolated from the liver of myctophina fish and its structure was determined as Watasenia preluciferyl β-D-glucopyranosiduronic acid.
- Inoue, Shoji,Okada, Kunisuke,Tanino, Hideo,Kakoi, Hisae
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p. 417 - 418
(2007/10/02)
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