Determination and identification of estrogenic compounds generated with biosynthetic enzymes using hyphenated screening assays, high resolution mass spectrometry and off-line NMR

• Published in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Vol. 878; no. 7; pp. 667 - 674
• Main Authors: de Vlieger, Jon S.B., Kolkman, Ard J., Ampt, Kirsten A.M., Commandeur, Jan N.M., Vermeulen, Nico P.E., Kool, Jeroen, Wijmenga, Sybren S., Niessen, Wilfried M.A., Irth, Hubertus, Honing, Maarten
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 01.03.2010; Elsevier
• Subjects: Analysis; Analytical, structural and metabolic biochemistry; Bioaffinity; Biological and medical sciences; Bioreactors; Biosynthetic enzymes; Chromatography, Liquid - methods; Cytochrome P-450 Enzyme System - metabolism; Drug Discovery - methods; Estradiol - analogs & derivatives; Estradiol - chemistry; Estradiol - metabolism; Estrogen Receptor alpha - metabolism; Estrogen Receptor beta - metabolism; Estrogens - biosynthesis; Estrogens - chemistry; Estrogens - metabolism; Ethinyl Estradiol - analogs & derivatives; Ethinyl Estradiol - chemistry; Ethinyl Estradiol - metabolism; Fundamental and applied biological sciences. Psychology; General pharmacology; High resolution mass spectrometry; High resolution screening; Human estrogen receptor; Humans; Hyphenation; Liquid chromatography-mass spectrometry; Mass Spectrometry - methods; Medical sciences; Norethindrone - analogs & derivatives; Norethindrone - chemistry; Norethindrone - metabolism; Nuclear magnetic resonance spectroscopy; Nuclear Magnetic Resonance, Biomolecular - methods; Pharmacology. Drug treatments; Protein Binding; Protein Structure, Tertiary; Reproducibility of Results; Sensitivity and Specificity; Structure-Activity Relationship; HTS; CYPs; HRS; HLM; Bioaffinity; High resolution mass spectrometry; Hyphenation; Nuclear magnetic resonance spectroscopy; IT-TOF; RAD; hER; High resolution screening; Liquid chromatography–mass spectrometry; NET; Biosynthetic enzymes; Human estrogen receptor; Human; Estrogen receptor; High resolution; Enzyme; Coupled method; Liquid chromatography-mass spectrometry; Estrogen; HPLC chromatography; NMR spectrometry; Determination; Ovarian hormone; Screening; Qualitative analysis; Sex steroid hormone; Mass spectrometry; Quantitative analysis; Hormonal receptor
• ISSN: 1570-0232; 1873-376X; 1873-376X
• DOI: 10.1016/j.jchromb.2010.01.035

This paper describes the determination and identification of active and inactive estrogenic compounds produced by biosynthetic methods. A hyphenated screening assay towards the human estrogen receptor ligand binding domain (hER)α and hERβ integrating target–ligand interactions and liquid chromatography–high resolution mass spectrometry was used. With this approach, information on both biologic activity and structure identity of compounds produced by bacterial mutants of cytochrome P450s was obtained in parallel. Initial structure identification was achieved by high resolution MS/MS, while for full structure determination, P450 incubations were scaled up and the produced entities were purified using preparative liquid chromatography with automated fraction collection. NMR spectroscopy was performed on all fractions for 3D structure analysis; this included 1D- 1H, 2D-COSY, 2D-NOESY, and 1H- 13C-HSQC experiments. This multidimensional screening approach enabled the detection of low abundant biotransformation products which were not suitable for detection in either one of its single components. In total, the analytical scale biosynthesis produced over 85 compounds from 6 different starting templates. Inter- and intra-day variation of the biochemical signals in the dual receptor affinity detection system was less than 5%. The multi-target screening approach combined with full structure characterization based on high resolution MS(/MS) and NMR spectroscopy demonstrated in this paper can generally be applied to e.g. metabolism studies and compound-library screening.