Isolation and Structure Elucidation of Natural Products from Plants DISSERTATION In Fulfillment of the Requirements for the Doctorate Degree at the Institute of Organic Chemistry University of Hamburg By Hailemichael Tesso (from Alemaya University, Ethiopia) ___________________________________________________________________________ University of Hamburg Institute of Organic Chemistry Hamburg, March 2005 1. Gutachter: Prof. Dr. Dr. h.c. W. Francke Institut für Organische Chemie 2. Gutachter: Prof. Dr. J. Voss Institut für Organische Chemie 1. Prüfer: Prof. Dr. Dr. h.c. W. Francke Institut für Organische Chemie 2. Prüferin: Prof. Dr. E. Stahl-Biskup Institut für Pharmazie 3. Prüfer: Dr. S. Franke Institut für Organische Chemie The present work was carried out between April 2002 and January 2005 under the supervision of the late Prof. Dr. W.A. Koenig. The work was done at the Institute of Organic Chemistry, University of Hamburg. Part I: Table of contents Content Page Acknowledgements ....................................................................................................................5 List of Abbreviations ..................................................................................................................6 ABSTRACT...............................................................................................................................8 1. Introduction............................................................................................................................9 2. Terpenes ...............................................................................................................................10 2.1. Classifications of Terpenes................................................................................................11 2.1.1. Hemiterpenes..................................................................................................................11 2.1.2. Monoterpenes .................................................................................................................11 2.1.3. Sesquiterpenes ................................................................................................................12 2.1.4. Diterpenes.......................................................................................................................12 2.1.5. Triterpenes......................................................................................................................12 2.1.6. Tetraterpenes..................................................................................................................12 3. Terpenoid biosynthesis.........................................................................................................12 3.1. The Mevalonate pathway..................................................................................................12 3.2. The Deoxyxylulose Phosphate Pathway (Fig. 3) ..............................................................13 3.3 Isomerization of IPP to DMAPP ........................................................................................14 3.4. Prenyl transferases.............................................................................................................15 4. An Overview of the Analytical Methods..............................................................................16 4.1. Chromatographic Methods ................................................................................................16 4.1.1 Thin Layer Chromatography (TLC)................................................................................16 4.1.2 Column Chromatography (CC).......................................................................................17 4.1.3 High Pressure Liquid Chromatography (HPLC).............................................................17 4.1.4 Gel Permeation Chromatography....................................................................................18 4.1.5 Gas Chromatography (GC)..............................................................................................18 4.1.6. Chromatographic solvent "polarity"...............................................................................18 4.2. Extraction Techniques .......................................................................................................19 4.2.1 Solvent Extraction...........................................................................................................19 4.2.2 Hydrodistillation..............................................................................................................19 4.3. Spectroscopic Techniques.................................................................................................19 4.3.1. Nuclear Magnetic Resonance Spectroscopy (NMR)......................................................19 4.3.1.1. One dimensional NMR................................................................................................20 4.3.1.1.1. 1D-Proton NMR (1H-NMR).....................................................................................20 4.3.1.1.2. 1D-Carbon NMR (13C-NMR)...................................................................................20 4.3.1.2. Two dimensional NMR ...............................................................................................22 4.3.1.2.1 2D 1H,1H-COSY (COrrelated SpectroscopY)..........................................................22 4.3.1.2.2. 2D Nuclear Overhauser Enhancement SpectroscopY (NOESY).............................22 4.3.1.2.3. HMQC (Heteronuclear Multiple Quantum Correlation) ..........................................22 4.3.1.2.4. HMBC (Heteronuclear Multiple Bond Correlation) ................................................24 4.3.1.3. Other Spectoscopic methods .......................................................................................25 4.3.1.4. Gas Chromatography/Mass Spectrometry (GC/MS)...................................................25 4.3.1.4.1. Mass Spectrometery (MS)........................................................................................25 Ionizer.......................................................................................................................................25 Ion Analyzer .............................................................................................................................26 Detector ....................................................................................................................................27 4.3.1.4.2. Interpreting mass spectra..........................................................................................27 5. Plant Materials ......................................................................................................................29 6. Experimental Aspects...........................................................................................................29 6.1. Analysis of the Essential Oils ............................................................................................29 6.2. Analysis of the Solvent Extracts........................................................................................30 6.3. Identification and characterization of Isolated Unknowns ................................................30 7. Results and Discussion.........................................................................................................30 7.1. Otostegia integrifolia Benth. .............................................................................................30 7.1.1 Description of the plant and Literature Survey...............................................................30 7.1.2 Results and Discussion on O. integrifolia .......................................................................31 7.1.2.1. Essential oil of the leaves............................................................................................31 7.1.2.1.1 (+)-Axinyssene (1-methyl-4-(5,9-dimethyl-1-methylenedeca-4,8-dienyl)cyclohexene) ..................................................................................................................................................31 7.1.2.2. Chloroform extract of the leaves.................................................................................33 7.1.2.2.1 Otostegindiol (12)......................................................................................................33 7.1.2.2.2.Preotostegindiol (13).................................................................................................34 7.1.2.2.3.Pentatriacontane.......................................................................................................35 7.1.2.2.4. Stigmasterol..............................................................................................................36 7.2. Peucedanum tauricum .......................................................................................................36 7.2.1 Description of the plant and Literature Survey...............................................................36 7.2.2 Results and Discussion on P. tauricum...........................................................................36 7.2.2.1. Essential oil of the fruits..............................................................................................36 7.2.2.1.1 Guaia-1(10),11-diene (14) ........................................................................................38 7.2.2.1.2 Guaia-9,11-diene (15) ...............................................................................................38