Hydroxysteroid dehydrogenase transformations of 5B-scymnol and identification of oxoscymnol transformation products by liquid chromatography-tandem mass spectroscopy

A new and sensitive high performance liquid chromatography (HPLC) separation procedure coupled with tandem mass spectroscopy (MS and MS2) detection was developed to identify for the first time the oxidation products of 5ß-scymnol [(24R)-(+)-5ß-cholestan-3a,7a,12a,24,26,27-hexol] catalysed by bacterial hydroxysteroid dehydrogenase (HSD) reactions in vitro. The authentic scymnol (MW 468) standard yielded a protonated molecular ion [M+H]+ at m/z 469 Da, and higher mass adduct ions attributed to [M+NH4]+ (m/z 486), [M+H+CH3OH]+ (m/z 501) and [M+H+CH3COOH]+ (m/z 530). (24R)-(+)-5ß-Cholestan-3-one-7a,12a,24,26,27-pentol (3-oxoscymnol, m/z 467 Da, relative retention time (RRT) = 0.89) was identified as the principle molecular species of scymnol in the reaction with 3a-HSD pure enzyme. [S]0.5 for the reaction of 3a-HSD with scymnol as substrate was 0.7292 mM. (24R)-(+)-5ß-cholestan-7-one-3a,12a,24,26,27-pentol (7-oxoscymnol, m/z 467 Da, RRT = 0.79) and (24R)-(+)-5ß-cholestan-12-one-3a,7a,24,26,27-pentol (12-oxoscymnol, m/z 467 Da, RRT = 0.81) were similarly identified as principle molecular species in the respective 7a-HSD and 12a-HSD reactions. Polarity of the oxoscymnol species was established as 7-oxoscymnol > 12-oxoscymnol > 3-oxoscymnol > scymnol (in order from most polar to least polar). Confirmation that 5ß-scymnol is an oxidative substrate for steroid-metabolising enzymes was made possible by the use of sophisticated liquid chromatography-mass spectrometry (LC-MS) techniques that will likely provide the basis for further exploration of scymnol as a therapeutic compound