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

Autor: Paul Wynne, Linda L. Glowacki, Nicolette Kalafatis, Paul F.A. Wright, Theodore A. Macrides, Lynn D. Hodges
Rok vydání: 2010
Předmět:
Zdroj: Steroids. 76(1-2)
ISSN: 1878-5867
Popis: A new and sensitive high performance liquid chromatography (HPLC) separation procedure coupled with tandem mass spectroscopy (MS and MS 2 ) detection was developed to identify for the first time the oxidation products of 5β-scymnol [(24R)-(+)-5β-cholestan-3α,7α,12α,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+NH 4 ] + ( m / z 486), [M+H+CH 3 OH] + ( m / z 501) and [M+H+CH 3 COOH] + ( m / z 530). (24R)-(+)-5β-Cholestan-3-one-7α,12α,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 3α-HSD pure enzyme. [S] 0.5 for the reaction of 3α-HSD with scymnol as substrate was 0.7292 mM. (24R)-(+)-5β-cholestan-7-one-3α,12α,24,26,27-pentol (7-oxoscymnol, m / z 467 Da, RRT = 0.79) and (24R)-(+)-5β-cholestan-12-one-3α,7α,24,26,27-pentol (12-oxoscymnol, m / z 467 Da, RRT = 0.81) were similarly identified as principle molecular species in the respective 7α-HSD and 12α-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 5β-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.
Databáze: OpenAIRE
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