Elsevier

Tetrahedron

Volume 58, Issue 11, 11 March 2002, Pages 2117-2126
Tetrahedron

19F-Dehydrocoelenterazine as probe to investigate the active site of symplectin

https://doi.org/10.1016/S0040-4020(02)00100-XGet rights and content

Abstract

Fluorinated dehydrocoelenterazines (F-DCTs) were synthesized to study molecular mechanisms of symplectin; a photoprotein of luminous squid Symplectoteuthis oualaniensis L. F-DCTs reacted with dithiothreitol and glutathione under neutral conditions to give the stable chromophores as symplectin model. Reconstructed symplectin was also obtained by addition of F-DCTs into apo-symplectin, and showed bioluminescence to emit 50–65% amount of light as natural symplectin. The structure of the chromophores was determined by 19F NMR, Q-TOF-MS, and MS/MS analyses. Sequencing of the chromopeptides of symplectin models prepared from F-DCTs and thiol compounds was accomplished by ESI-Q-TOF-MS/MS analysis.

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.

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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.

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