RESUMEN
Identification of small-molecule ligands for a protein of interest can facilitate the analysis of the protein's functions in biological systems. Small-molecule microarrays have allowed for rapid detection of such ligand-protein interactions in a high-throughput manner, although a label on a protein is needed to observe these interactions. By combining SPR imaging technology with our recently developed photo-cross-linked small-molecule array platform, we developed a novel platform that allows in situ observation of interactions between photo-cross-linked small molecules on gold surfaces and nonlabeled proteins in solution. Interactions of estrogenic and androgenic substances with estrogen receptor alpha were observed using this platform.
Asunto(s)
Reactivos de Enlaces Cruzados/química , Oro/química , Análisis por Matrices de Proteínas/métodos , Proteínas/metabolismo , Resonancia por Plasmón de Superficie/métodos , Andrógenos/metabolismo , Sitios de Unión , Receptor alfa de Estrógeno/metabolismo , Estrógenos/metabolismo , Ligandos , Nanotecnología , Análisis por Matrices de Proteínas/instrumentación , Factores de TiempoRESUMEN
We have developed a unique photo-cross-linking approach for immobilizing a variety of small molecules in a functional-group-independent manner. Our approach depends on the reactivity of the carbene species generated from trifluoromethylaryldiazirine upon UV irradiation. It was demonstrated in model experiments that the photogenerated carbenes were able to react with every small molecule tested, and they produced multiple conjugates in most cases. It was also found in on-array immobilization experiments that various small molecules were immobilized, and the immobilized small molecules retained their ability to interact with their binding proteins. With this approach, photo-cross-linked microarrays of about 2000 natural products and drugs were constructed. This photo-cross-linked microarray format was found to be useful not merely for ligand screening but also to study the structure-activity relationship, that is, the relationship between the structural motif (or pharmacophore) found in small molecules and its binding affinity toward a protein, by taking advantage of the nonselective nature of the photo-cross-linking process.
Asunto(s)
Reactivos de Enlaces Cruzados/química , Genómica/métodos , Metano/análogos & derivados , Análisis por Matrices de Proteínas , Proteínas/química , Unión Competitiva , Vidrio , Hidrocarburos/química , Ligandos , Metano/química , Etiquetas de Fotoafinidad/química , Fotoquímica , Ensayo de Unión Radioligante , Relación Estructura-Actividad , Propiedades de Superficie , Rayos UltravioletaRESUMEN
The reactions of [60]fullerene with bis-o-quinodimethane precursors 3, 4, and 5 containing a dibenzo-18-crown-6, 24-crown-8 and 30-crown-10 moiety, respectively, were investigated. Both 3 and 4 provided trans-4 bisadducts 6a and 7a, respectively, as major product, though the selectivity in the latter was lower than that in the former. As minor product, 6b (cis-2) was produced from 3, while 7c (e) as well as 7b (cis-2) were produced from 4. Precursor 5 exclusively afforded e bisadduct 8 without any other regioisomers. These bisadducts showed different ionophoric properties from one another; for instance, 6a and 7b exhibited a high complexing ability toward the K+ ion, while 6b hardly showed complexation with any alkali metal ions. The selectivity coefficients (K(Na,K)(pot)) of 6a and 7b toward K+ over Na+ ion, determined with an ion-selective electrode, were much higher than that of dibenzo-18-crown-6.