MECHANISTIC STUDIES ON DIAMINE OXIDASE A thesis presented in part fulfilm ent of the requirements for the Degree of Doctor of Philosophy by Stephen Scott Gavin Department of Organic Chemistry University of Glasgow July 1995 ProQuest Number: 13832535 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a com plete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest ProQuest 13832535 Published by ProQuest LLC(2019). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States C ode Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106- 1346 10ZW ) IE a Fx JtTI imun;; -;r,T j u s M r r f ( To m y wife Hazel and all m y family, One Love, One Blood. ACKNOWLEDGEMENTS I would like to offer my sincere thanks to Professor D. J. Robins for his guidance, both technically and personally, through the many challenges I have faced during my research. In particular for his help and patience in the writing of this thesis. I am most grateful to Dr. A. Cooper for his assistance with the biophysical work carried out as part of this thesis. I am also grateful for the help I received from the departmental technical staff during my studies. In particular, Miss I. Freer for the isolation of the crude pea enzyme and Mr. J. Tweedie for technical assistance in the laboratory. Also, Dr. D. S. Rycroft, Mr. J. Gall and Mr. J. McIver for NMR spectra; Mrs. K. Wilson for elemental analysis; Mr. A. Ritchie for mass spectra and Mr. G. McCulloch for IR spectra. The financial support from S.E.R.C. in the form of an Earmarked award is gratefully acknowledged. My deepest thanks go to my friends and colleagues, both past and present, in the unforgettable Henderson Lab. The memories of my time there w ill live with me forever. In particular, I must thank Greig, Paul, John and Henry for keeping me sane, and Gerry for his much needed help with the computers. Lastly, I dedicate this thesis to those in my heart, my family. To my mother Yolanda and father Patrick for their many years of love and support. Most of all to my wife Hazel, for her endless encouragement and without whom my achievements would be meaningless. ABBREVIATIONS BSA Bovine serum albumin BSAO Bovine serum amine oxidase CBDC Cyclobutanedicarboxylic acid CID Collision induced dissociation DACCP Diaminocyclohexylcarboxyphthalic acid Platinum (II) DAO Diamine oxidase DBU Diazabicyclo [5.4.0] undec-7-ene DEAE Diethylaminoethyl DEMO a-Difluoromethylornithine DMAB 3-(Dimethylamino)benzoic acid DMAP 4-Dimethylaminopyridine DMF D im ethylform am ide DMSO Dimethylsulphoxide DNA Deoxyribonucleic acid DNPH 2,4-Dinitrophenylhydrazine DOPA 3,4-Di hydroxy phenylalanine EDTA Ethylenediaminetetraacetic acid ee Enantiomeric excess EPR Electron paramagnetic resonance ESR Electron spin resonance FAD Flavine adenine dinucleotide FPLC Fast protein liquid chromatography HPLC High performance liquid chromatography IR Infrared LSIMS Liquid secondary ion mass spectrometry K m Michaelis Menten constant MBTH 3-Methyl-2-benzothiazolinone hydrazone MGBG Me thy lglyoxalbis (guany lhydrazone) NBT Nitroblue tetrazolium NMR Nuclear magnetic resonance ODC Ornithine decarboxylase PQCL Pyrroloquinoline quinone RNA Ribonucleic acid SDS Sodium dodecyl sulphate THF T etrahydrofuran TOPA Topaquinone UV Ultraviolet Vis V isible Vmax Maximum rate TABLE OF CONTENTS Page No. CHAPTER 1. In tro d u c tio n 1 1.1 The Growing Importance of Enzymes in Organic Synthesis 1 1.2 The Disadvantages of using Enzymes and Improvements using Immobilisation Techniques 3 1.3 Diamine Oxidase 4 1.4 Polyamine Metabolism and Cell Growth 5 1.5 Inhibitors of Polyamine Biosynthesis 7 1.6 Stereochemistry and Regiochemistry in Reactions Catalysed by Diamine Oxidase 8 1.7 A lka loid Biosynthesis 9 1.8 W ork Described In This Thesis 11 CHAPTER 2. A Review of Diamine Oxidase 12 2.1 Isolation and Purification of Diamine Oxidase 12 2.2 The Role of Copper with the DAO Enzyme 13 2.3 Early Proposals for Cofactors of Diamine Oxidase 14 2.4 The History of Pyrroloquinoline Quinone (PQQ) 15 2.5 Pyrroloquinoline Quinone as a Cofactor for Copper Amine Oxidases 16 2.6 Using Oxygen to Improve the “Hydrazine Method” 17 2.7 Alternative Methods for Detecting Enzyme Bound PQQ, 20 2.8 Naturally Occurring Forms of PQQ, 22 iv 2.9 The Mechanisms Involved with PQQ 23 2.10 A Further Insight Into the Chemistry of PQQ 28 2.11 Arguments Against PQQ.as a Cofactor for Amine Oxidases 32 2.12 Current Understanding of the Nature of the Cofactor 40 2.13 Interaction of the Prosthetic Groups in Diamine Oxidase 41 2.14 Assay Systems Used in the Determination of Diamine Oxidase Activity 43 2.15 Substrate Specificity and the Active Site 47 2.16 Diamine Analogues Containing Group VI Atoms (O, Se, Se) 51 2.17 Stereochemistry Involved in Reactions Catalysed by DAO 52 2.18 Regioselectivity and Stereoselectivity Involved with the DAO-Catalysed Reaction 55 2.19 The Necessity of Polyamines in Cell Growth and Replication 57 2.20 Inhibitors of Diamine Oxidase 58 2.21 Synthetic Applications of DAO 63 CHAPTER 3. Enzyme K in e tics 6 6 3.1 Introduction 6 6 3.2 Michaelis-Menten Kinetics 6 6 3.3 Determination of Vmax and Km by Experimental Methods 70 3.4 The Kinetics Involved with Inhibition 73 v CHAPTER 4. Mechanistic Studies on Diamine Oxidase 76 4.1 In tro d u ctio n 76 4.2 Synthesis of Deuterium Labelled Diamines 78 4.3 Results and Discussion 80 CHAPTER 5. Oxidation of Substituted Quinolines and Pyridines By Pea Seedling Diamine Oxidase 90 5.1 Extraction and Partial Purification of Diamine Oxidase from Pea Seedlings 90 5.2 The Determination of Protein Concentration 91 5.3 The Assay Procedure 91 5.4 Substituted Quinolines and Pyridines as Substrates for Pea Seedling DAO 93 5.4a Introduction 93 5.4b Synthesis of the Substituted Quinoline and Pyridine Derivatives 94 5.4c Results and Discussion 102 5.5 Inhibition of Diamine Oxidase by Quinoline and Pyridine Derivatives 105 5.5a Introduction 105 5.5b Results and Discussion 107 CHAPTER 6 . Oxidation of Substituted Thiophenes and Pyrroles By Pea Seedling Diamine Oxidase 111 6.1 In tro d u ctio n 111 v i 6 . 2 Synthesis of Substituted Thiophenes and Pyrroles 1 1 1 6 .2 a Synthesis of Substituted Thiophenes 1 1 1 6 .2 b Synthesis of Substituted Pyrroles 114 6.3 Results and Discussion 117 6.3a Substituted Thiophenes and Pyrroles as Substrates for Pea Seedling DAO 117 6.3b Substituted Thiophenes and Pyrroles as Inhibitors of Pea Seedling DAO 1 2 0 CHAPTER 7. Use of Diamines in the Synthesis of Cisplatin Analogues 1 2 2 7.1 Introduction 1 2 2 7.2 Mechanism of Action of Cisplatin 123 7.3 Interactions of Platinum Compounds with DNA 125 7.4 Synthesis of Cisplatin Analogues 127 7.4a Introduction 127 7.4b Synthesis of cis-l,4-Diaminobut-2-ene 129 7.4c Synthesis of Platinum Precursors and Cisplatin Analogues 130 CHAPTER 8 . Experimental 137 8.1 General Details 137 8.2 Extraction of Diamine Oxidase (EC 1.4.3. 6 ) from Pea Seedlings 138 8.3 Experimental for Chapter Four 145 8.4 Experimental for Chapter Five 156 v ii 8.5 Experimental for Chapter Six 181 8 . 6 Experimental for Chapter Seven 191 Appendix 200 References 203 via SUMMARY To investigate the enzyme pea seedling diamine oxidase our studies concentrated on three main areas: 1 . mechanistic studies of the oxidative deamination of primary diamines catalysed by diamine oxidase; 2 . oxidation of aromatic compounds with amine side chains by pea seedling diamine oxidase; and 3. inhibition of pea seedling diamine oxidase. To make use of diamines synthesised within the research group a fourth area was studied: 4. synthesis of cisplatin analogues. 1. Mechanistic Studies of the Diamine Oxidase-Catalvsed Deamination of Diamines Diamine oxidase catalyses the oxidative deamination of diamines to their corresponding aminoaldehydes (Scheme A). To test the hypothesis that the diamine oxidase-catalysed oxidation proceeds via an enamine intermediate (i) we prepared a number of a,co-diamines labelled with deuterium at the p-positions. nh2 (i) Diamine Oxidase CHO nh2 nh2 Scheme A ix The [2 H 4 ]-labelled diamines and the corresponding unlabelled diamines were incubated with pea seedling diamine oxidase and the products were trapped with benzoy lace tic acid in situ. (Scheme B). This gave substituted acetophenone products (ii) which were analysed by NMR and mass spectrometry. From a comparison of the spectroscopic data we were able to show that the enamine intermediate (i) is not involved in the enzymatic process. X NH2 Diamine Oxidase Benzoylacetic Acid NH X = H or D (“) n = 0 - 2 Scheme B 2. Oxidation of Aromatic Compounds with Amine Side Chains bv Pea Seedling Diamine Oxidase A range of quinoline, pyridine, thiophene and pyrrole derivatives with amine side chains were synthesised and tested as substrates of pea seedling diamine oxidase using a spectrophotometric assay which measures the hydrogen peroxide by-product of the enzymatic reaction. From this assay V m a x and K m values were obtained for the oxidation of each substrate using diamine oxidase. The V m a x is the maximal rate of oxidation and gives an indication of the oxidation rate for the various substrates.