“Biosynthesis of Morphine in Mammals” D i s s e r t a t i o n zur Erlangung des akademischen Grades Doctor rerum naturalium (Dr. rer. nat.) vorgelegt der Naturwissenschaftlichen Fakultät I Biowissenschaften der Martin-Luther-Universität Halle-Wittenberg von Frau Nadja Grobe geb. am 21.08.1981 in Querfurt Gutachter /in 1. 2. 3. Halle (Saale), Table of Contents I INTRODUCTION ........................................................................................................1 II MATERIAL & METHODS ........................................................................................ 10 1 Animal Tissue ....................................................................................................... 10 2 Chemicals and Enzymes ....................................................................................... 10 3 Bacteria and Vectors ............................................................................................ 10 4 Instruments ........................................................................................................... 11 5 Synthesis ................................................................................................................ 12 5.1 Preparation of DOPAL from Epinephrine (according to DUNCAN 1975) ................. 12 5.2 Synthesis of (R)-Norlaudanosoline*HBr ................................................................. 12 5.3 Synthesis of [7D]-Salutaridinol and [7D]-epi-Salutaridinol ..................................... 13 6 Application Experiments ...................................................................................... 15 6.1 Application to Papaver setigerum Seedlings ........................................................... 15 6.2 Injections into Mice and Collection of Urine .......................................................... 15 7 Preparation of Protein .......................................................................................... 15 7.1 Preparation of Crude Liver Protein ......................................................................... 15 7.2 Preparation of Microsomes from Liver ................................................................... 15 7.3 Heterologous Expression in E.coli .......................................................................... 16 7.3.1 Cloning of Amine N-Methyltransferase cDNA in Vector pET28a and Transformation into E. coli PlusKJ ......................................................................... 16 7.3.2 Isolation of Amine N-Methyltransferase His-tagged Protein from E. coli ................ 18 7.3.3 Thrombin Digestion of His-tagged Protein .............................................................. 19 8 Standard Test for Mammalian Liver Enzymes ................................................... 19 9 Solid Phase Extraction of Alkaloids ..................................................................... 20 9.1 Strata X-C (Phenomenex)-TCA method ................................................................. 20 9.2 Strata X-C (Phenomenex)-HClO4 method ............................................................... 20 9.3 Bond Elut Certify (Varian) ..................................................................................... 20 9.4 Sep Pak Plus C18 (Waters)-basic conditions ........................................................... 21 10 Chromatographic methods ................................................................................... 21 10.1 Thin Layer Chromatography (TLC) ........................................................................ 21 10.2 Analytical High Performance Liquid Chromatography ........................................... 22 10.3 Preparative High Performance Liquid Chromatography .......................................... 22 10.4 Liquid Chromatography-Mass Spectrometry (LC-MS) ........................................... 22 10.5 Liquid Chromatography-High Resolution Mass Spectrometry (HR-LC-MS) .......... 24 11 Protein Assay ........................................................................................................ 25 12 Radioactivity Measurements ................................................................................ 25 III RESULTS................................................................................................................. 26 PART A: MORPHINE ANALYTICS ......................................................................................... 26 1 Stability of morphine ............................................................................................ 26 2 Solid phase extraction with Strata X-C ............................................................... 29 3 Ion suppression ..................................................................................................... 31 4 MS-Instruments and Sensitivity ........................................................................... 35 I PART B: I.P. INJECTIONS INTO MICE AND THE MORPHINE PATHWAY IN MAMMALS .......... 38 1 The Biogenesis of Mammalian Morphinan Alkaloids ......................................... 38 1.1 Intraperitoneal Injection of (R,S)-Norlaudanosoline into Mice ................................ 41 1.1.1 Urinary Excretion of Tetrahydrobenzylisoquinoline Alkaloids after Application of (R,S)-Norlaudanosoline ...................................................................................... 44 1.1.2 Urinary Excretion of Tetrahydroprotoberberine Alkaloids after Application of (R,S)-Norlaudanosoline .......................................................................................... 45 1.1.3 Urinary Excretion of Aporphine and Morphinan Alkaloids after Application of (R,S)-Norlaudanosoline .......................................................................................... 46 1.2 Intraperitoneal Injection of (R)-Norlaudanosoline into Mice ................................... 47 1.3 Intraperitoneal Injection of (R)-[N-CD3]-Reticuline into Mice ................................ 49 2 Thebaine formation from Salutaridine in Mammals .......................................... 53 2.1 Intraperitoneal Injection of Salutaridine into Mice .................................................. 54 2.2 Intraperitoneal Injection of [7D]-Salutaridinol into Mice ........................................ 56 3 Morphine formation from Thebaine in Mammals .............................................. 59 3.1 Intraperitoneal Injection of [N-CD3]-Thebaine into Mice ........................................ 61 3.2 Intraperitoneal Injection of Oripavine into Mice ..................................................... 62 PART C: ENZYME STUDIES IN SELECTED MORPHINE BIOSYNTHETIC STEPS ...................... 66 1 Formation of 3,4-Dihydroxyphenylacetaldehyde (DOPAL) in Animals............. 67 2 Condensation to Norlaudanosoline ...................................................................... 71 3 N-Methylation in Mammalian Morphine Biosynthesis ....................................... 75 3.1 Heterologous Expression of two Human N-Methyltransferases ............................... 76 3.2 Activity of Recombinant Phenylethanolamine N-Methyltransferase and Amine N-Methyltransferase ............................................................................................... 78 3.3 Determination of Km, Vmax and the Catalytic Efficiency of NMT with (R)- configurated THBIQ Alkaloids as Substrates .......................................................... 80 4 The Oxidative C-C-Phenol-Coupling in Mammals .............................................. 84 4.1 The Phenol-Coupling Reaction in Mammals Catalyzed by Human CYP 2D6 and CYP 3A4 ................................................................................................................ 85 4.2 The Phenol-Coupling Reaction in Mammals Catalyzed by Rat CYP 2D2 ............... 89 4.3 Kinetic Analysis and Characteristics of Mammalian P450 Enzymes Catalyzing the Phenol Coupling Reaction................................................................................. 92 5 3-O-Demethylation of Thebaine and Codeine in Mammals Catalyzed by Human CYP 2D6 and Rat CYP 2D2 .................................................................... 97 6 6-O-Demethylation of Thebaine and Oripavine ................................................ 101 IV DISCUSSION .......................................................................................................... 103 V SUMMARY ............................................................................................................ 119 VI REFERENCES ........................................................................................................ 122 II List of Abbreviations APS ammonium persulfate cDNA complementary deoxyribonucleic acid cps counts per second CPR cytochrome P450 reductase CYP cytochrome P450 DLPC L-α-dilauroyl-sn-glycero-3-phosphocholine DNA deoxyribonucleic acid DOPAL 3,4-dihydroxyphenylacetaldehyde DOPA-pyruvate 3,4-dihydroxyphenylpyruvate DOPET 3,4-dihydroxyphenylethanol DW dry weight E. coli Escherichia coli EC-HPLC high perfomance liquid chromatography coupled to electrochemical detection EDTA ethylenediaminetetraacetic acid ELISA enzyme-linked immunosorbent assay EPI enhanced product ion EtOH ethanol FW fresh weight GC-MS gas chromatography-mass spectrometry GDP guanosine diphosphate GTP guanosine triphosphate HPLC high perfomance liquid chromatography HR-LC-MS liquid chromatography-high resolution mass spectrometry HR-MS high resolution mass spectrometry i.p. intraperitoneal LC-MS liquid chromatography-mass spectrometry