22-NBD-cholesterol as a novel fluorescent substrate for cholesterol-converting oxidoreductases

Docking simulations and experimental data indicate that 22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3β-ol (22-NBD-cholesterol), a common fluorescent sterol analog, binds into active sites of bovine cytochrome P450scc and microbial cholesterol dehydrogenases (CHDHs) and then undergoes regiospecific oxidations by these enzymes. The P450scc-dependent system was established to realize N-dealkylation activity toward 22-NBD-cholesterol, resulting in 7-nitrobenz[c][1,2,5]oxadiazole-4-amine (NBD-NH2) formation as a dominant fluorescent product. Basing on LC–MS data of the probes derivatized with hydroxylamine or cholesterol oxidase, both pregnenolone and 20-formyl-pregn-5-en-3β-ol were deduced to be steroidal co-products of NBD-NH2, indicating intricate character of the reaction. Products of CHDH-mediated conversions of 22-NBD-cholesterol were defined as 3-oxo-4-en and 3-oxo-5-en derivatives of the steroid. Moreover, the 3-oxo-4-en derivative was also found to be formed after 22-NBD-cholesterol incubation with pathogenic bacterium Pseudomonas aeruginosa, indicating a possible application of the reaction for a selective and sensitive detection of some microbes. The 3-keto-4-en derivative of 22-NBD-cholesterol may be also suitable as a new fluorescent probe for steroid hormone-binding enzymes or receptors.

Graphical abstractFigure optionsDownload full-size imageDownload as PowerPoint slideHighlights► 22-NBD-cholesterol is a fluorescent substrate for bovine cytochrome P450scc. ► P450scc catalyze 22-NBD-cholesterol N-dealkylation, resulting in NBD-NH2 formation. ► 22-NBD-cholesterol is a substrate for microbial cholesterol-dehydrogenases. ► Pathogen Pseudomonas aeruginosa converts 22-NBD-cholesterol into its 3-oxo-4-en derivative.