Synthesis of 13C-Dehydrocoelenterazine and NMR Studies on the Bioluminescence of a Symplectoteuthis Model
Introduction
Molecular mechanisms of the bioluminescence and chemiluminescence have not been well-defined, although many studies on chemi- and bioluminescence have been undertaken in recent decades.2 We have studied a bioluminescence system of an oceanic luminous squid, Symplectoteuthis oualaniensis L. In 1981, Tsuji and Leisman reported that a homogenate of the luminous organ of this squid gave light in the presence of monocations such as Na+, K+ etc. and molecular oxygen at pH 7.8.3 This luminous system is potentially useful as a monitor of monocations in single cells. For monitoring dications, an aequorin system is especially famous for the determination of the Ca2+ concentration in living cells.4 In 1993, we succeeded in the extraction of the photoprotein, named symplectin, responsible for the bioluminescence in a 0.6 M KCl solution from the same squid S. oualaniensis collected in Okinawa, Japan. We have reported that this symplectin contains dehydrocoelenterazine (1a), a similar chromophore to coelenterazine (4), and that this chromophore may chemically be bound with the symplectin through a covalent bond such as thioether.5 The major evidence for this chromophore is that the mixing of dehydrocoelenterazine (1a) solution into apoprotein of this squid enhanced the bioluminescence in almost the same amount of light emission as from natural symplectin, but mixing with the yellow coelenterazine (4) solution did not show any bioluminescence. The reddish solution of 1a mixed with aposymplectin solution instantaneously changed into a yellowish color since the chromophore in 2 is the same as 4; this chemical change was supported by UV and Fluorescence spectra.4
The connecting position of this chromophore has been suggested from an acetone adduct (3) to a dehydrocoelenterazine (1a), which was isolated as an artifact from this photogenic organ.4A time course of fluorescence and absorption spectra were recorded during the symplectin bioluminescence.6 As a result of the spectra, the chromophore seemed to link with the cysteine residue of the symplectin through a thioether as illustrated in Scheme 1. As an evidence of this hypothesis, model studies on the bioluminescence were undertaken with DTT and GSH as a model for the symplectin. Firstly, the predicted carbon connecting to the sulfhydryl residue was labeled with 13C, and secondly, the chemical shift changing was measured by high-field NMR.
Section snippets
Result and Discussion
The retrosynthetic analysis of the 13C-labeled dehydrocoelenterazine (1a∗) is shown in Scheme 2. (In order to indicate and to distinguish 13C-labeled from natural abundance isotope compounds the asterisk is attached to the formula number such as N∗ in text.) Dehydrocoelenterazine (1a)7 is obtained from coelenterazine (4)8 by MnO2 oxidation, and 4 is synthesized from coelenteramine (5) and ketoaldehyde (6) by reported procedures.9 For the preparation of the 13C-labeled compound 6∗, it is
Experimental
All melting points were measured on Yanaco MP-S3 and uncorrected. UV spectra were taken on a JASCO U-best 50 spectrometer. IR spectra were determined on a JASCO FT/IR-7000S spectrophotometer. Proton NMR spectra were recorded on a JEOL EX 270 or GSX 270 for 270 MHz, a JEOL JNML-500 for 500 MHz or a Bruker AMX-600 for 600 MHz. Chemical shifts (δ) are given in parts per million relative to tetramethylsilane (δ 0.00) or CD3OD (δ 3.30) or DMSO-d6 (δ 2.49) as internal standard and coupling constants (J)
Acknowledgements
The authors are grateful for financial support from JSPS–RFTF 96L00504. They also thank Dr T. Kondo and Mr K. Koga for technical assistance in NMR measurements. M. Kuse thanks JSPS (Japan Society for the Promotion of Science) for granting a Research Fellowship for Young Scientists. We also express our thanks to Dr T. Franz for assistance in checking the manuscript.
References (13)
- et al.
BioMed. Chem. Lett.
(1993) - Preliminary communication about this paper was reported in the next literature. Isobe, M.; Kuse, M.; Yasuda, Y.;...
- (a) Isobe, M.; Fujii, T.; Swan, S.; Kuse, M.; Tsuboi, K.; Miyazaki, A.; Feng, M. C.; Li, J. Pure Appl. Chem. 1998, 70,...
- et al.
Proc. Natl. Acad. Sci. USA
(1981) - (a) Shimomura, O.; Johnson, F. H.; Saiga, Y. J. Cell. Comp. Physiol. 1962, 59, 223–240. (b) Shimomura, O.; Johnson, F....
- et al.
Chem. Lett.
(1994)
Cited by (32)
Expression of recombinant apopholasin using a baculovirus–silkworm multigene expression system and activation via dehydrocoelenterazine
2020, Bioorganic and Medicinal Chemistry LettersCitation Excerpt :Native ferric horseradish peroxidase is converted into Compound I by activation with H2O2; it is a two-equivalent oxidized intermediate. Compound II forms by one-electron reduction of Compound I.19 It postulated that Compound I and II oxidize Pholasin® to afford Oxypholasin (oxidized Pholasin®) with light emission.18 Thus, the use of a recombinant apopholasin and DCL was simply a reproduction of this biological system.
Suzuki-Miyaura coupling for general synthesis of dehydrocoelenterazine applicable for 6-position analogs directing toward bioluminescence studies
2011, TetrahedronCitation Excerpt :We have elucidated the chromophore structure as 2 but not 1 due to the fact that only 2 gives the bioluminescence with its apo-symplectin. The molecular mechanism of the bioluminescent system in symplectin has been solved in such a way that DCL or its 13C analog binds with an HS-residue of cysteine to form a conjugate adduct 3 as reported by Isobe’s group (Scheme 1).20–22 In 2008, the active center cysteine was determined to be 390-Cys in the 501 amino acids of symplectin.22
Chemistry around imidazopyrazine and ibuprofen: Discovery of novel fatty acid amide hydrolase (FAAH) inhibitors
2010, European Journal of Medicinal ChemistryCitation Excerpt :Aryl α-ketoaldehydes are usually obtained by oxidation of methylketones and diazoketones, or from α-(α′)-(di)haloketones [12–15]. Few methods are dedicated to benzyl α-ketoaldehydes [9,10,16] and none to the α-(methyl)benzyl analogs. In a first route (Scheme 1), we prepared the 1,3-dithianyl precursor 4a by substitution of the corresponding Weinreb amide 3a with 1,3-dithiane anion [17].
In vitro and in vivo studies of 6,8-(diaryl)imidazo[1,2-a]pyrazin-3(7H)-ones as new antioxidants
2009, Bioorganic and Medicinal ChemistryLuminescence of imidazo[1,2-a]pyrazin-3(7H)-one compounds
2007, Bioorganic Chemistry