Production of Drug Metabolites by Immobilised Cunninghamellaelegans: from Screening To

Production of drug metabolites by immobilised Cunninghamellaelegans: from screening to scale-up

Supplementary Information

Laura Quinn 1, Rita Dempsey 1, Eoin Casey 2, Ayla Kane 1and Cormac D. Murphy1*

1. School of Biomolecular and Biomedical Science, University College Dublin, Belfield, Dublin 4, Ireland
2. School of Chemical and Bioprocess Engineering, University College Dublin, Belfield, Dublin 4, Ireland

*Corresponding author,telephone: +353 (0)1 716 1311, email:

Analysis of drug metabolites

GC-MS was employed where possible to identify the drug metabolites, thus the presence of hydroxylated metabolites of diclofenac, flurbiprofen and ibuprofen, carboxyibuprofen and phenothiaizne sulphoxide (m/z=215)were confirmed. HPLC analysis of extracts from C. elegans incubated with flutamide, phenothiazine and flumequin are shown in Fig S1. The metabolites were identified by comparison of retention time data previously obtained in refs 1-3.

Identification of purified 4’-hydroxydiclofenac

The structure of 4’-hydroxydiclofenac was confirmed by GC-MS and 1H NMR. The NMR analysis showed that in the diclofenac standard there is a triplet present at 7.0 ppm which corresponds to the proton on the 4’ position. There is also a doublet present due to the splitting of two equivalent protons on the C3’ and C5’ positions. Both the triplet and doublet are replaced by a singlet in the NMR spectrum for the purified metabolite, which is consistent with the hydroxylation at the 4’ position (Fig S2).

Figure S1. HPLC analysis of supernatants from alginate immobilised and planktonic C. elegans after incubation with phenothiazine (A), flutamide (B), and flumequine (C).

Figure S2. 1H NMR spectra of the aromatic region of diclofenac (upper spectrum) and 4’-hydroxydiclofenac (lower spectrum).

References

1.Amadio J, Murphy CD (2011) Production of human metabolites of the anti-cancer drug flutamide via biotransformation in Cunninghamella species. BiotechnolLett 33:321-326.

2. Sutherland JB, Freeman JP, Heinze TM, Moody JD, Parshikov IA, Williams AJ, Zhang D (2001) Oxidation of phenothiazine and phenoxazine by Cunninghamellaelegans. Xenobiotica 31: 799-809

3. Williams AJ, Deck J, Freeman JP, Chiarelli MP, Adjei MD, Heinze TM, Sutherland JB (2007) Transformation of flumequine by the fungus Cunninghamellaelegans. Chemosphere 67:240-243.