19F-Dehydrocoelenterazine as probe to investigate the active site of symplectin
Fluorinated dehydrocoelenterazines were synthesized to study molecular mechanisms of symplectin; a photoprotein of luminous squid S. oualaniensis L. The structure of model chromophores was determined by 19F NMR, Q-TOF-MS, and MS/MS analyses. Sequencing of chromopeptide of symplectin model was accomplished by MS/MS analysis.
Introduction
Many luminous lives possess photoprotein as a device for light emission. Tobiika, a luminous oceanic squid, is quite unique in the use of dehydrocoelenterazine (20) as an organic substrate of a photoprotein (symplectin: the photoprotein of a luminous squid Symplectoteuthis oualaniensis L.2), and for the existence of the photoprotein as adduct form of 20. Among many luminous lives, tobiika is so far the only one to use 20 as the organic substrate. Our studies on the bioluminescent mechanism of this squid have indicated to us the following bioluminescent mechanism: Michael addition of a sulfhydryl group of apo-symplectin to a dehydrocoelenterazine (DCT: 20) is the initial step in the symplectin bioluminescence to give a pseudo-reduced chromophore (1) (Scheme 1).3., 4. Structural changes of symplectin by mono-cations (Na+, K+)5 initiate the oxidation of this chromophore with oxygen to emit blue light (470 nm) and to give the oxidized form. Our interest is now focused on the active site of symplectin, especially on the cysteine residue to make a covalent bond with DCT. In this report, we describe the synthesis of fluorinated DCT (F-DCT) to make a covalent bond between a cysteine residue and DCT tighter to enable spectroscopic analysis (NMR and MS) of the chromopeptide under mild conditions. By studying a model bioluminescence prepared from F-DCT and thiols, we demonstrate that F-DCT is the best probe to investigate the active site of symplectin.
Section snippets
Stability of chromophore under neutral conditions
In our previous papers, we reported the preparation of a symplectin model (3) from 13C-labeled DCT analog (2) with thiol compounds such as dithiothreitol (DTT: 14) and glutathione (GSH: 15) under acidic conditions (pH 3) (asterisk indicates 13C-labeled position).6., 7. However, the symplectin model dissociated in equilibrium into DCT and DTT under neutral conditions (physiological condition) (Fig. 1).
This equilibrium between DCT and thiol adduct stops under acidic conditions, so that the thiol
Conclusions
Three fluorinated dehydrocoelenterazines (F-DCTs) were synthesized to study molecular mechanisms of symplectin. F-DCTs reacted with dithiothreitol (DTT) and glutathione (GSH) even under neutral-physiological condition to afford the stable chromopeptides as symplectin models. The structure of these chromopeptides was determined by 19F NMR, ESI-Q-TOF-MS, and MS/MS analyses. Sequencing of the chromopeptides was accomplished by using symplectin models prepared from three F-DCTs and GSH by
General
UV spectra were obtained on a JASCO U-best 50 spectrometer. Proton NMR spectra were recorded on a JEOL 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) as internal standard and coupling constants (J) in Hz. Carbon NMR were recorded on a JEOL GSX 270 for 67.8 MHz or a JEOL JNML-500 for 125.7 MHz or on a Bruker AMX-600 for 150.9 MHz. Chemical shifts are (δ) given in parts per
Acknowledgements
The authors are grateful for financial support from JSPS-RFTF 96L00504, The Naito Fundation, SUNBOR and Mitsubishi Chemical Corporation Fund for financial support.
References (13)
- et al.
Chem. Lett.
(1994)et al.BioMed. Chem. Lett.
(1993) - et al.
Tetrahedron
(2000)et al.BioMed. Chem. Lett.
(1998) - et al.
Tetrahedron
(1996)et al.Tetrahedron Lett.
(1995)et al.J. Chem. Soc., Chem. Commun.
(1986) - et al.
Nature
(2000)et al.J. Am. Chem. Soc.
(2001)et al.Biochemistry
(2001)et al.Tetrahedron Lett.
(2000)et al.Biochemistry
(1999)
Cited by (20)
Evidence for de novo Biosynthesis of the Luminous Substrate Coelenterazine in Ctenophores
2020, iScienceCitation Excerpt :Many marine organisms representing at least 9 phyla utilize an identical luciferin, coelenterazine: polycystine radiolaria in Retaria; Phaeodaria in Cercozoa; Ctenophora; Hexacorallia, Octocorallia, Scyphozoa, and Medusozoa in Cnidaria; Vampyroteuthid cephalopods and Pholas clams in Mollusca; Ostracoda, Copepoda, and Decapoda in Arthropoda; Ophiura in Echinodermata; Chaetognatha; Appendicularia, Myctophiformes, and Stomiformes in Chordata (Campbell and Herring, 1990; Haddock et al., 2010; Widder, 2010; Markova and Vysotski, 2015; Bessho-Uehara et al., 2020b). Some species modify coelenterazine before using it as luciferin: the firefly squid Watasenia scintillans uses coelenterazine disulfate (Inoue et al., 1976); the flying squid Sthenoteuthis oualaniensis uses a dehydrocoelenterazine as luciferin (Isobe et al., 2002). Other taxa modify luciferins for storage: the sea pansy Renilla reniformis possess luciferin enol-sulfate (Inoue et al., 1977a, 1977b); lanternfish Myctophidae possess an enol-ether form of coelenterazine bound with glucopyranosiduronic acid (Inoue et al., 1987).
Expression of recombinant apopholasin using a baculovirus–silkworm multigene expression system and activation via dehydrocoelenterazine
2020, Bioorganic and Medicinal Chemistry LettersCitation Excerpt :The task of developing a complete luminescent profile (including luminescent spectrum) for apopholasin–DCL is now underway as we are in the process of collecting protein starting material and cleaving the relevant His-and Flag®-tags using HRV 3C protease. Since Pholasin® is a glycoprotein,6a,20 apopholasinF and apopholasinM, as expressed in the baculovirus–silkworm multigene system, are also glycosylated. The precise structure of the sugar chain for both proteins is still unknown.
Chemistry around imidazopyrazine and ibuprofen: Discovery of novel fatty acid amide hydrolase (FAAH) inhibitors
2010, European Journal of Medicinal ChemistryCitation Excerpt :Classically, the synthesis of imidazo-[1,2-a]-pyrazin-3-(7H)-one (E) makes use of two building blocks, namely the α-ketoaldehyde (F) and the aminopyrazine (C) (Fig. 2). Their condensation is performed in aqueous acidic solutions [9,10]. In this article, we describe our persistent attempts to prepare the α-ketoaldehyde F derived from ibuprofen.
Pholasin luminescence is enhanced by addition of dehydrocoelenterazine
2008, Bioorganic and Medicinal Chemistry LettersLuminescence of imidazo[1,2-a]pyrazin-3(7H)-one compounds
2007, Bioorganic Chemistry