Conversion of pharmaceuticals and other drugs by fungal ... - IHI Zittau

2.5.8 IC-Method for organic acids ....................................................................... 48
2.6 Stoichiometrical analyses.................................................................................... 48
2.6.1 Sildenafil ..................................................................................................... 48
2.6.2 Safrole ......................................................................................................... 49
2.7 Enzyme kinetics .................................................................................................. 49
2.7.1 Pharmaceuticals........................................................................................... 49
2.7.2 Nitrobenzodioxole....................................................................................... 49
2.8 Experiments with 18 O-isotopes............................................................................ 50
2.8.1 Pharmaceuticals........................................................................................... 50
2.8.2 Nitrobenzodioxole....................................................................................... 50
2.9 Deuterium isotope effect experiment .................................................................. 50
2.10 Experiment with different peroxides and peroxide sources ................................ 51
2.11 In-vivo incubation experiment............................................................................. 51
2.12 Conversion of propranolol at environmentally relevant concentration............... 53
2.13 Long term experiments........................................................................................ 53
3 Results ......................................................................................................................... 55
3.1 Aromatic ring hydroxylation............................................................................... 55
3.1.1 Ring hydroxylation at pharmaceuticals....................................................... 55
3.1.2 Ring hydroxylation at drugs of abuse.......................................................... 58
3.2 Oxidation of alkyl side chains............................................................................. 59
3.3 O-Dealkylation .................................................................................................... 62
3.3.2 Ester cleavage.............................................................................................. 64
3.4 N-dealkylation ..................................................................................................... 64
3.5 O-Demethylenation ............................................................................................. 68
3.6 Oxidation of steroids ........................................................................................... 69
3.7 Stoichiometrical analyses.................................................................................... 71
3.7.1 Sildenafil ..................................................................................................... 71
3.7.2 Safrole ......................................................................................................... 72
3.8
18 O-Isotope incorporation.................................................................................... 72
3.9 Kinetics experiments ........................................................................................... 73
3.9.1 Michaelis-Menten kinetics .......................................................................... 73
3.9.2 Bisubstrate kinetics...................................................................................... 74
3.10 Deuterium isotope effect experiments................................................................. 75
3.11 Spectrophotometric nitrobenzodioxole assay...................................................... 75
3.12 Conversion of a set of human P450 substrates.................................................... 78
3.13 Difference spectroscopy...................................................................................... 81
3.14 Different peroxides and peroxide-generating systems ........................................ 82
3.15 In-vivo experiments ............................................................................................. 83
3.16 Tests with environmentally relevant concentrations of propranolol ................... 85
3.17 Long-term experiments ....................................................................................... 86
3.17.1 Pre-study with Vivaspin® (fed-batch method) ........................................... 86
3.17.2 Long-term experiment using a hollow fiber cartridge................................. 87
3.18 Scope of peroxygenase reactions concerning pharmaceuticals and drugs.......... 88
4 Discussion ................................................................................................................... 90
4.1 Preparation of human drug metabolites............................................................... 90
4.2 Mechanistic studies ............................................................................................. 99
4.2.1 Source of oxygen and stoichiometry........................................................... 99
4.2.2 Deuterium isotope effect ........................................................................... 100
4.2.3 Kinetics...................................................................................................... 100
4.2.4 Assays for human cytochrome P450 activities.......................................... 102
4.2.5 Peroxides and peroxides generation systems ............................................ 104
4.2.6 Difference binding spectra ........................................................................ 106
4.2.7 Phenoxyl radical scavenger....................................................................... 106
4.2.8 Stoichiometry and proposed mechanism of demethylenation................... 107
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