Fate of novel Quasi reverse steroidal substrates by Aspergillus tamarii KITA: bypass of lactonisation and an exclusive role for the minor hydroxylation pathway

The fungus Aspergillus tamarii transforms progesterone to testololactone in high yield through a flexible four-step enzymatic pathway. To date no studies have investigated the effect of transposition of steroidal functionality between ring-A and ring-D in order to determine the effect on steroidal metabolism. A series of novel quasi reverse steroids (7–9) were synthesised through Linz and Schäfer oxidation where 14-en-16-one functionality is generated on ring-D of the steroid. To retain parity with the normal series ring-D functionality was substituted onto ring-A of the analogues. All of the analogues (7–9) were handled through a minor 11β-hydroxylation pathway with no lactones being formed. In previous studies testololactone is produced within the first 12 h of metabolism. A time course experiment demonstrated that the transformation of these steroids initiated with the formation of a 3β-hydroxy group after which (48–96 h) hydroxylation through a minor pathway occurred, indicating that this hydroxylase was only then being induced. This is in contrast to the normal fungal metabolism of xenobiotic steroidal substrates where they are primarily hydroxylated. Furthermore, ring-D hydrogenation is reported for the first time as is reverse metabolism on this pathway. All metabolites were isolated by column chromatography and were identified by 1H and 13C NMR spectroscopy, DEPT analysis and other spectroscopic and crystallographic data.