Electrochemical and enzymatic synthesis of oxidative drug metabolites for metabolism studies
Gül, Turan
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Publication date: 2017
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Gül, T. (2017). Electrochemical and enzymatic synthesis of oxidative drug metabolites for metabolism studies: Exploring selectivity and yield. Rijksuniversiteit Groningen.
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Electrochemical and Enzymatic
Synthesis of Oxidative Drug Metabolites
for Metabolism Studies
Exploring Selectivity and Yield
Turan Gül
University of Groningen Dutch Technology Foundation (STW)
Groningen University Institute for Drug Exploration
The research reported in this thesis was carried out at the University of Groningen in the Analytical Biochemistry group, member of the Groningen Research Institute of Pharmacy. The work was financially supported by the Dutch Technology Foundation STW (Grant 11957).
ISBN
978-90-367-9537-1 (printed version) 978-90-367-9536-4 (digital version)
Copyright content:
All rights reserved. No part of this book may be reproduced or transmitted in any form by any means without permission of the author.
Publisher: University of Groningen Paranymphs: Frank Klont & Andres Gil
Thesis Cover: EC-MS Method for Metabolism Studies
Visualized by: Piet van Rosmalen (Jongens van de Tekeningen) Printed by: Proefschriftmaken.nl, Vianen
Electrochemical and Enzymatic
Synthesis of Oxidative Drug
Metabolites for Metabolism Studies
Exploring Selectivity and Yield
PhD Thesis
to obtain the degree of PhD at the University of Groningen
on the authority of the Rector Magnificus prof. E. Sterken
and in accordance with the decision by the College of Deans. This thesis will be defended in public on
Friday 10 February 2017 at 16:15 hours
by
Turan Gül
born on 10 March 1987 in Iğdır, Turkey
Assessment Committee
Prof. G.J. Poelarends Prof. F.J. Dekker Prof. H. Oberacher
Dedicated to my beloved Grandfather whom I lost
recently and all the members of my big family…
Table of Contents
1. General introduction and thesis scope 1
1.1. Aim of the research 2
1.2. Phase I drug metabolizing enzymes 2
1.2.1. Cytochrome P450 (CYP) enzyme 2
1.2.2. Flavin-containing monooxygenase (FMO) enzyme 3 1.3. In vivo Phase I oxidation reactions 4
1.3.1. Heteroatom dealkylation 4
1.3.2. Heteroatom oxidation 5
1.3.3. Aliphatic/Aromatic hydroxylations 6 1.3.4. Dehydrogenation and Epoxidation 7 1.4. Electrochemical synthesis of drug metabolites 8
1.4.1. Direct electrochemistry 8
1.4.2. Indirect electrochemistry 9
1.5. Scope of the Thesis 11
1.6. References 12
2. Electrosynthesis methods and approaches for the preparative production of
metabolites from parent drugs 16
2.1. Introduction 17
2.2. Electrochemical Techniques Used in Drug Metabolism 19 2.3. Design of Electrochemical Cells for Drug Metabolite Synthesis 22
2.3.1.Electrode materials 22
2.3.2.Electrochemical Cell Types 24
2.3.2.1. Thin-layer Flow Cells 24 2.3.2.2. Porous Flow-through Cells 24 2.3.2.3. Batch Cell Reactors 25 2.4. Electrochemical Reaction Parameters 27
2.4.1. Solvents 27
2.4.2. Dissolved Gases 28
2.4.3. Solution pH 28
2.4.4. Supporting Electrolyte 29
2.4.5. Substrate Concentration 29
2.5. Conclusions and Future Trends 30
2.6. References 31
3. Optimization of reaction parameters for electrochemical oxidation of
lidocaine with a Design of Experiment approach 38
3.1. Introduction 39
3.2. Experimental Procedures 40
3.2.4. LC-MS Analysis 42
3.3. Results and Discussion 44
3.3.1. Optimization of EC parameters 44
3.3.2. pH effect on N-dealkylation 47
3.4. Conclusions 48
3.5. References 49
4. Mechanism of aromatic hydroxylation metabolites of lidocaine at a Pt
electrode under acidic conditions 52
4.1. Introduction 53
4.2. Materials and Methods 55
4.2.1. Reagents 55
4.2.2. Electrochemical Measurements 55
4.2.3. LC/MS analysis 56
4.3. Results and Discussions 57
4.3.1. Aromatic hydroxylation reactions of lidocaine under strong acidic conditions 57 4.3.2. Investigation of the oxygen source 57 4.3.3. Investigation of the aromatic hydroxylation mechanism 60
4.4. Conclusions 62
4.5. References 63
5. Microbial flavoprotein monooxygenases as mimics of mammalian
Flavin-containing monooxygenases for the enantioselective preparation of drug
metabolites 67
5.1. Introduction 68
5.2. Materials and Methods 69
5.2.1. Materials 69
5.2.2. Recombinant expression of BVMOs and preparation of cell extracts 70 5.2.3. Monooxygenase-catalyzed conversions 70
5.2.4. Chiral LC-MS/MS in the Selected Reaction Monitoring (SRM) mode 71
5.2.5. Circular Dichroism 72
5.3. Results 73
5.3.1. Conversion of the sulfide 3-methyl(thio)aniline 73 5.3.2. Conversion of the thioether drugs, albendazole and fenbendazole 76 5.3.3. Conversion of the amines, lidocaine and nicotine 78 5.3.4. Quantification of product yields with standard addition method 80 5.3.4.1. 3-methyl(thio)aniline 80 5.3.4.2. Albendazole sulfoxide 81
5.4. Discussion 84
