Die Entwicklung von Fluoreszenzsonden basierend auf natürlichen Sensorproteinen (H-NOX) zur in vivo Detektion von Sauerstoff und Stickstoffmonoxid in Signaltransduktionswegen
Zusammenfassung der Projektergebnisse
This research focuses on the development of fluorogenic probes for the selective intracellular detection, localization and quantification of nitric oxide (NO) and oxygen (O2). While O2 is a key factor in the development and growth of tumors as well as in their response to treatment, NO is an important signal molecule in the regulatory mechanism of many biological processes, ranging from communal behavior in bacteria to vasodilation and neurotransmission in mammals. Consequently, the development of methods to detect and measure intracellular O2 and NO are of broad scientific interest. Natural receptors for both gases are members from the recently discovered heme-protein family, the heme-nitric oxide/oxygen binding (H-NOX) domain. The main chemical component in the recognition and binding of the gas ligand is the cofactor (iron protoporphyrin IX, b-type heme), which is also one of the most abundant metalloporphyrins in nature. By itself, heme b is a non-fluorescent molecule. In order to achieve fluorescence, this research focused on electronic coupling of this cofactor with a fluorescence reporter, while at the same time maintaining structural compatibility with the H-NOX protein scaffold. The result of these considerations was a key intermediate, in which one methine position of the heme macrocycle was derivatized with a terminal alkyne. The chemical synthesis of alkynylated heme b, however, has not been reported. Therefore, a new method was developed for the direct functionalization of metalloporphyrins at the methine protons. The novelty was to use gold catalysis and hypervalent periodates to receive asymmetric alkynylated metalloporphyrins. With this single-step procedure, b-type heme was alkynylated and the product was incorporated into bacterial H- NOX gas sensor proteins. Hemoproteins with this type of engineered cofactor have several potential applications in labeling and imaging technologies. The terminal alkyne provided an attachment site for chemical tags in chemical biology experiments through copper(I)- catalyzed azide–alkyne cycloaddition (“click chemistry”) and other transition-metal-catalyzed reactions. In the next stage of this project, the terminal alkyne was coupled to selected fluorophores. In one heme-fluorophore conjugate the fluorescence of the dye moiety was completely suppressed. Binding of this cofactor surrogate to the heme-binding site of H-NOX gas sensor proteins and several mutants resulted in efficient restoration of fluorescence. The heme-fluorophore conjugates showed excellent unidirectional permeability into the cytosol of live mammalian cells. By imaging with confocal fluorescence microscopy of a fusion protein that carried a nuclear localization peptide sequence (H-NOX-mCherry-NLS) the in vivo formation of the holoprotein was demonstrated. Finally, the fluorogenic responses of the molecular assemblies towards signaling gases like NO and O2 demonstrated that fluorescence sensors can be engineered by specific cofactor-labeling of heme proteins.
Projektbezogene Publikationen (Auswahl)
- Angew. Chem. 2014, 126, 2649–2652 and Angew. Chem. Int. Ed. 2014, 53, 2611–2614
A. Nierth, M. A. Marletta
(Siehe online unter https://doi.org/10.1002/anie.201310145)
