期刊
CHEMISTRY-A EUROPEAN JOURNAL
卷 26, 期 4, 页码 948-958出版社
WILEY-V C H VERLAG GMBH
DOI: 10.1002/chem.201904844
关键词
enzyme catalysis; heterocycles; luciferins; luminescence; natural products
资金
- Agence Nationale de la Recherche (ANR) [ANR-11-CRNT-0004]
- Global Care
- Universite Paris Descartes, Sorbonne Paris Cite
- AMGEN scholars fellowship
- association of the Institut Carnot Pasteur-Maladies Infectieuses
- association of the Institut Carnot Curie-Cancer
- association of the Institut Carnot Voir et Entendre
- association of the Institut Carnot Institut du Cerveau et de la moelle Epiniere
- association of the Institut Carnot Consortium pour l'Acceleration de l'Innovation et de son Transfert dans le domaine du Lymphome (CALYM)
- Agence Nationale de la Recherche (ANR) [ANR-11-CRNT-0004] Funding Source: Agence Nationale de la Recherche (ANR)
We describe here an extensive structure-bioluminescence relationship study of a chemical library of analogues of coelenterazine, using nanoKAZ/NanoLuc, a mutated luciferase originated from the catalytic subunit of the deep-sea shrimp Oplophorus gracilirostris. Out of the 135 O-acetylated precursors that were prepared by using our recently reported synthesis and following their hydrolysis to give solutions of the corresponding luciferins, notable bioluminescence improvements were achieved in comparison with furimazine, which is currently amongst the best substrates of nanoKAZ/NanoLuc. For instance, the rather more lipophilic analogue 8-(2,3-difluorobenzyl)-2-((5-methylfuran-2-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one provided a 1.5-fold improvement of the total light output over a 2 h period, a close to threefold increase of the initial signal intensity and a signal-to-background ratio five times greater than furimazine. The kinetic parameters for the enzymatic reaction were obtained for a selection of luciferin analogues and provided unexpected insights into the luciferase activity. Most prominently, along with a general substrate-dependent and irreversible inactivation of this enzyme, in the case of the optimized luciferin mentioned above, the consumption of 2664 molecules was found to be required for the detection of a single Relative Light Unit (RLU; a luminometer-dependent fraction of a photon).
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