Structure Elucidation and Synthesis of New Secondary ...

Structure Elucidation and Synthesis

of New Secondary Metabolites from Liverworts and

Microorganisms and Investigation of their Biogenesis

DISSERTATION

In Fulfillment of the Requirements for the Degree of Dr. rer. nat

at the Institute of Organic Chemistry, University of Hamburg

by

Stephan Heinrich von Reuß

Hamburg 2009

1. Gutachter: Prof. Dr. Dr. h.c. mult. W. Francke

Institut für Organische Chemie, Universität Hamburg

2. Gutachter: Prof. Dr. C. Meier

Institut für Organische Chemie, Universiät Hamburg

3. Gutachter: Prof. Dr. W. Boland

Department für Bioorganische Chemie,

Max Plank Institut für Chemische Ökologie, Jena

4. Gutachter: Prof. Dr. L. Wessjohann

Abteilung für Natur- und Wirkstoffchemie

Leibniz Institut für Pflanzenbiochemie, Halle

Tag der Disputation: 24 Juli 2009

The present work was performed from January 2004 to December 2008

under the supervision of Prof. Dr. Dr. h.c. mult. W. Francke in the laboratory of

the late Prof. Dr. W. A. König at the Institute of Organic Chemistry, as well as

in the laboratory of Dr. K. von Schwartzenberg at the Biozentrum Klein

Flottbek, University of Hamburg, Germany.

Dedicated to my parents

Acknowledgements

Acknowledgements

I like to express my deepest gratitude to the late Professor Dr. Wilfried A. König for his

support and inspiring enthusiasm. He initiated this investigation but passed away on Nov.19.

2004, after this investigation has just been started.

I am most grateful to Professor Dr. Dr. h.c. Wittko Francke for the opportunity to continue the

research under his supervision, his invaluable support, inspiring discussions and interesting

cooperations.

I am very grateful to Dr. Klaus von Schwartzenberg (Biozentrum Klein Flottbek, University

of Hamburg, Germany) for the opportunity to work under his guidance, his support in the

establishment of in vitro cultures, and the very pleasant atmosphere.

Support by the following people is gratefully acknowledged: Dr. Volker Sinnwell and his

team (Org. Chem.) for 1H and

13C NMR measurements, Dr. Erhard T. K. Haupt (Inorg.

Chem.) for 2D NMR measurements and helpful discussions, Dr. Stephan Franke, Gaby Graak,

Manfred Preuße and Annegret Meiners (Org. Chem.) for HREIMS, direct inlet EIMS and

FAB-MS measurements, Professor Dr. Paul Margaretha (Org. Chem.) for advice with the UV

isomerisations, Dr. Dietmar Keyser (Zoologisches Institut) for Scanning Electron Microscopy

(SEM), S. Bringe (Biozentrum Klein Flottbek) for technical assistance, and Dr. Hermann

Muhle (Abteilung Systematische Botanik und Ökologie, University Ulm, Germany) for

collection of requested liverworts and the joined fieldtrip to Portugal (March 2005), Professor

Dr. Yoshinori Asakawa (Tokushima, Bunri University, Japan), Professor Dr. Cecília Sérgio

(Herbário do Museu, Laboratório et Jardin Botânico de Lisboa, Portugal), Dr. Pavel Pribyl

(Culture Collection of Autotrophic Organisms, Trebon, Czech Republic), Dr. Tassilo Feuerer

(Herbarium Hamburgense, Hamburg, Germany) and Professor Dr. S. Robbert Gradstein (A-v-

H Institute, Göttingen, Germany) for providing plant material of Corsinia coriandrina and

Cronisia weddellii, respectively, Professor Dr. Wilhelm Boland (Max Planck Institute for

Chemical Ecology, Jena, Germany) for providing reference compounds of brown algal

pheromones, Dr. Alexey V. Tkachev (Novosibirsk Institute of Organic Chemistry,

Novosibirsk, Russia) for providing solid super acid, Dr. Detlef Hochmuth for providing the

MassFinder Software, and Dr. J. Rheinheimer (BASF AG, Ludwigshafen, Germany) for

biotests with synthetic Corsinia constituents..

Acknowledgements

Special thanks to all cooperative partners who significantly enriched my research:

Dr. Simon Jones (Department of Chemistry, University of Sheffield, Great Britain) for the

enantioselective synthesis of (R)-(–)-corsifuran A.

Professor Gerhard Gries and his group (Department of Biological Sciences, Simon Fraser

University, Burnaby, Canada) for providing (+)-spiroaxa-5,7-diene from Ulmus americana

infected with Ophiostoma novo-ulmi.

Professor Birgit Piechulla and Marco Kai (Institut für Biowissenschaften, University of

Rostock, Germany) for providing headspace samples from Serratia odorifera.

I also like to thank Professor Chia Li-Wu (Department of Organic Chemistry, Tamkang

University, Tan Shui, Taiwan), Professor Danuta Kalemba (Technical University of Lodz,

Institute of General Food Chemistry, Lodz, Poland), Dr. Claudia Höckelmann (Givaudan AG,

Dübendorf, Switzerland), and Dr. Florian Schiestl (Geobotanical Institute, ETH-Zürich,

Switzerland) for interesting cooperations.

Thanks to former members of W. A. König„s research group: Dr. Fernando Campos

Ziegenbein, Dr. Dennis Lass, Dr. Rita Richter, Dr. Heilemichael Tesso, Dr. Frank Werner,

and especially Dr. Adewale Martins Adio, Dr. Simla Basar, Dr. Thomas Hackl and Dr. Peter

Hansen; members of W. Francke‟s research group: Dr. Robert Twele and Armin Troeger; and

members of K. von Schwartzenberg‟s research group: Dr. Hanna Richter, Dr. Natalya

Yevdakova, Hanna Turcinov, and Marta Fernandez Nunez.

Financial Support by the „Deutsche Forschungsgemeinschaft” (DFG) and the „Deutsche

Akademische Austauschdienst” (DAAD) is gratefully acknowledged.

I also like to thank Sascha Ludolph for kindly providing technical equipment

and Mario Maiworm for computer programming.

I am most grateful to Marike J. Boenisch for participation, assistance, advice, and support.

Table of Contents

Table of Contents

1. Introduction ....................................................................................................................................... 1

2. Objective of Research ....................................................................................................................... 2

3. General Part ....................................................................................................................................... 3

3.1. Liverwort Biology ........................................................................................................................ 3

3.2. Secondary Metabolites of Liverworts .......................................................................................... 5

4. Special Part ........................................................................................................................................ 7

4.1. C11 Hydrocarbons from Fossombronia angulosa ......................................................................... 7

4.1.1. Fossombronia angulosa ............................................................................................................ 7

4.1.2. Volatile Constituents of Fossombronia angulosa ..................................................................... 8

4.1.3. Identification of Dictyopterene A (29) .................................................................................... 11

4.1.4. Identification of Dictyotene (32) ............................................................................................. 12

4.1.5. Identification of Ectocarpene (30) .......................................................................................... 13

4.1.6. Identification and Synthesis of n-Pentylbenzene (31) ............................................................ 14

4.1.7. Enantioselective GC Analysis of C11 Hydrocarbons ............................................................... 15

4.1.8. Discussion of C11 Hydrocarbons from Fossombronia angulosa ............................................. 16

4.2. Indole Alkaloids from Riccardia chamedryfolia ......................................................................... 17

4.2.1. Riccardia chamedryfolia ......................................................................................................... 17

4.2.2. Volatile Constituents of Riccardia chamedryfolia .................................................................. 17

4.2.3. Identification and Synthesis of 3-Chloro-6- and 7-prenylindoles (49, 50) ............................. 20

4.2.4. Identification of Chamedryfolian (51) .................................................................................... 23

4.2.5. Identification of 7-(3-Methylbutadienyl)indole (52)............................................................... 26

4.2.6. Discussion of Indole Alkaloids from Riccardia chamedryfolia.............................................. 27

4.3. Secondary Metabolites of Corsinia coriandrina ......................................................................... 28

4.3.1. Taxonomy of the Corsiniaceae ............................................................................................... 28

4.3.2. Volatile Constituents of Corsinia coriandrina ....................................................................... 29

4.3.3. Identification and Synthesis of 4-Methoxystyrenes from Corsinia coriandrina .............. 32

4.3.3.1. (Z/E)-Coriandrins (65) ..................................................................................................... 32

4.3.3.2. (Z/E)-O-Methyltridentatols B & A (72)........................................................................... 36

4.3.3.3. O-Methyltridentatol C (71) .............................................................................................. 37

4.3.3.4. (Z/E)-Corsinians (63) ....................................................................................................... 39

4.3.3.5. (Z/E)-Corsiandrens (67) ................................................................................................... 41

4.3.3.6. (Z/E)-Corsiandrenins (92) ................................................................................................ 43

4.3.3.7. (Z/E)-Tuberines (98) ........................................................................................................ 45

4.3.3.8. (Z/E)-Corsicillins (99) ..................................................................................................... 46

4.3.3.9. Discussion of 4-Methoxystyrenes from Corsinia coriandrina ........................................ 48

4.3.4. Identification and Synthesis of Stilbenoids from Corsinia coriandrina ............................ 51

4.3.4.1. (R)-(–)-Corsifuran A (73) ................................................................................................ 51

Table of Contents

4.3.4.2. Corsifuran B (114) ........................................................................................................... 55

4.3.4.3. Corsifuran C (74) ............................................................................................................. 56

4.3.4.4. (E/Z)-Corsistilbenes (68) ................................................................................................. 57

4.3.4.5. O,O-Dimethyllunularin (69) ............................................................................................ 60

4.3.4.6. 6-Hydroxy-O,O-dimethyllunularin (70) .......................................................................... 61

4.3.4.7. Discussion of Stilbenoids from Corsinia coriandrina ..................................................... 63

4.4. Synthesis of Deuterium Labelled Precursors ............................................................................. 67

4.4.1. Synthesis of (E)-[2-D]-Cinnamic Acids ([2-D]-89, [2-D]-93, [2-D]-152) ............................. 67

4.4.2. Synthesis of [3,3-D2]-3-Phenylpropanoic Acids (153 – 158) ................................................. 71

4.4.3. Synthesis of L-[CD3]-O-Methyltyrosine ([CD3]-155) ............................................................. 72

4.4.4. Synthesis of DL-[2-D]-Tyrosines ([2-D]-154, [2-D]-155) ...................................................... 73

4.4.5. Synthesis of DL-[2,3-threo-D2]-Tyrosine ([2,3-threo-D2]-154) .............................................. 73

4.4.6. Synthesis of [CD3]-O-Methyltyramine ([CD3]-168) ............................................................... 74

4.4.7. Synthesis of (±)-[2,3-threo-D2]-Phloretic Acid ((±)-[2,3-threo-D2]-157) .............................. 75

4.5. Axenic In Vitro Cultures of Corsinia coriandrina ...................................................................... 76

4.5.1. Collection of Corsinia coriandrina Spores ............................................................................. 76

4.5.2. Germination of Corsinia Spores and Propagation of Monoclonal Strains .............................. 77

4.5.3. Determination of Ploidy Levels .............................................................................................. 77

4.5.4. Optimization of Culture Media and Conditions ...................................................................... 78

4.5.5. Mixed Photo-Heterotrophic Growth ....................................................................................... 79

4.5.6. Aerated Liquid Submersion Cultures ...................................................................................... 80

4.5.7. Temporarily Immersed Cultures using RITA® ....................................................................... 82

4.5.8. Chemical Investigation of In Vitro Cultured Corsinia coriandrina ........................................ 82

4.6. Application Experiments ............................................................................................................. 84

4.6.1. Biosynthesis of Coriandrin in Corsinia coriandrina ........................................................... 86

4.6.1.1. The L-Tyrosine Origin of Coriandrin. ............................................................................. 87

4.6.1.2. Pulsed Application Experiments using RITA® ................................................................ 90

4.6.1.3. Tyrosine Decarboxylase Activity .................................................................................... 92

4.6.1.4. Tyrosine Ammonia Lyase Activity.................................................................................. 93

4.6.1.5. O-Methyl Transferase Activity ........................................................................................ 93

4.6.1.6. Stereospecific Elimination of the 3-Pro-S-Hydrogen of L-Tyrosine .............................. 95

4.6.1.7. The Glycine Origin of the O-Methyl Group of (Z)-Coriandrin ....................................... 96

4.6.1.8. Discussion of Coriandrin Biosynthesis in Corsinia coriandrina ..................................... 98

4.6.2. Biosynthesis of Corsifuran A in Corsinia coriandrina ..................................................... 102

4.6.2.1. Application Experiments using GC-EIMS Detection .................................................... 102

4.6.2.2. Application Experiments using 2D and

13C NMR Spectroscopy ................................... 103

4.6.2.3. The Phenylpropenoid Origin of Corsifuran A and Corsistilbene. ................................. 103

4.6.2.4. The Phenylpropenoid Origin of Aromatic Corsifuran C. .............................................. 106

4.6.2.5. Application of Dihydrocinnamic Acids ......................................................................... 109

4.6.2.6. Tyrosine Ammonia Lyase Activity................................................................................ 113

Table of Contents

4.6.2.7. Phenylalanine Transaminase Activity ........................................................................... 114

4.6.2.8. Application of D-3,3-Dideuterophenylalanine............................................................... 116

4.6.2.9. The Polymalonate Origin of Corsifuran A .................................................................... 118

4.6.2.10. -Carbonyl Group Reduction in Corsifuran A Biosynthesis ....................................... 120

4.6.2.11. Exclusion of Stilbenecarboxylic Acid Intermediates ................................................... 123

4.6.2.12. Discussion of Corsifuran A Biosynthesis in Corsinia coriandrina ............................. 126

4.6.3. Biosynthesis of Terpenoids in Corsinia coriandrina ......................................................... 130

4.6.3.1. Biosynthesis of (R)-(–)-(E)-Nerolidol ........................................................................... 130

4.6.3.2. Biosynthesis of (4S,6S)-( )- -Pinene ............................................................................ 132

4.6.3.3. Discussion of Terpenoid Biosynthesis in Corsinia coriandrina.................................... 136

4.7. (1S,2R)-(+)-Spiroaxa-5,7-diene associated with Dutch Elm Disease ...................................... 137

4.7.1. Durch Elm Disease (DED) .................................................................................................... 137

4.7.2. Identification of (1S,2R)-(+)-Spiroaxa-5,7-diene .................................................................. 139

4.7.3. Attempted Dehydration of (–)-Axenol .................................................................................. 141

4.7.4. Acid Catalyzed Rearrangement using Amberlyst ................................................................. 142

4.7.5. Solid Super Acid Catalyzed Rearrangement using TiO2/SO42–

............................................ 144

4.7.6. Discussion of (1S,2R)-(+)-Spiroaxa-5,7-diene from Ulmus americana ............................... 148

4.8. Octamethylbicyclo[3.2.1]octadienes from Serratia odorifera .................................................. 149

4.8.1. Serratia odorifera ................................................................................................................. 149

4.8.2. Identification of Odorifen ..................................................................................................... 149

4.8.3. Synthesis of Octamethylbicyclo[3.2.1]octadienes ................................................................ 152

4.8.4. Synthesis of Octamethylbicyclo[3.2.1]octa-2,6-diene and -2(10),6-diene ........................... 154

4.8.5. Biosynthesis of Odorifen ...................................................................................................... 156

4.8.6. Discussion of Octamethylbicyclo[3.2.1]octadienes from Serratia odorifera ....................... 157

5. Summary ........................................................................................................................................ 158

6. Zusammenfassung ......................................................................................................................... 160

7. Experimental Part ......................................................................................................................... 162

7.1. General Experimental Procedures ....................................................................................... 162

7.1.1. Nuclear Magnetic Resonance Spectroscopy (NMR) ........................................................ 162

7.1.2. Electron Impact Mass Spectrometry (EIMS) .................................................................... 162

7.1.3. High Resolution Electron Impact Mass Spectrometry (HREIMS) ................................... 162

7.1.4. Fast Atom Bombardment Mass Spectrometry (FAB-MS) ............................................... 162

7.1.5. Gas Chromatography (GC) ............................................................................................... 162

7.1.6. Enantioselective Gas Chromatography (eGC) .................................................................. 163

7.1.7. Preparative Gas Chromatography (PGC) ......................................................................... 163

7.1.8. Semi-preparative Gas Chromatography (SPGC) .............................................................. 163

7.1.9. Column Chromatography (CC) ........................................................................................ 163

7.1.10. Preparative Thin Layer Chromatography (TLC) ............................................................ 163

7.1.11. Polarimetry ..................................................................................................................... 164

7.1.12. Infrared Spectroscopy (IR) ............................................................................................. 164

Table of Contents

7.1.13. Ultraviolet Spectroscopy (UV) ....................................................................................... 164

7.1.14. UV Induced (E/Z)-Isomerisations ................................................................................... 164

7.1.15. Photography .................................................................................................................... 164

7.1.16. Microphotography .......................................................................................................... 164

7.1.17. Fluorescence Micrsocopy ............................................................................................... 164

7.1.18. Scanning Electron Microscopy (SEM) ........................................................................... 164

7.1.19. Quantum Mechanical Calculations ................................................................................. 164

7.2. Plant Materials & Extracts ................................................................................................... 165

7.2.1. Origin of Plant Materials .................................................................................................. 165

7.2.2. Deposition of Plant Material ............................................................................................. 165

7.2.3. Hydrodistillation ............................................................................................................... 165

7.2.4. Extraction of Air Dried Plant Material ............................................................................. 166

7.2.5. Extraction of Fresh Plant Material .................................................................................... 166

7.3. Axenic In Vitro Cultures of Corsinia coriandrina ............................................................... 166

7.3.1. Plant media ....................................................................................................................... 166

7.3.2. Establishment of Axenic In Vitro Cultures ....................................................................... 167

7.3.3. Flow Cytometry ................................................................................................................ 167

7.3.4. Application Experiments .................................................................................................. 167

7.4. Fossombronia angulosa .......................................................................................................... 168

7.4.1. Isolation of C11 Hydrocarbons from Fossombronia angulosa .......................................... 168

7.4.2. Synthesis of (±)-1-Phenylpentan-1-ol (41) ....................................................................... 168

7.4.3. Synthesis of (E)-1-Phenylpent-1-ene (42) ........................................................................ 169

7.4.4. Synthesis of n-Pentylbenzene (31) ................................................................................... 169

7.5. Riccardia chamedryfolia ......................................................................................................... 169

7.5.1. Isolation of Prenylindoles (14 and 15) .............................................................................. 169

7.5.2. Synthesis of 3-Chloro-7-prenylindole (49) ....................................................................... 170

7.5.3. Synthesis of 3-Chloro-6-prenylindole (50) ....................................................................... 170

7.5.4. Isolation of Chamedryfolian (51) ..................................................................................... 170

7.5.5. Chemical Correlation of Chamedryfolian (51) ................................................................. 171

7.5.6. Chemical Correlation of 7-(3-Methylbutadienyl)indole (52) ........................................... 171

7.6. Corsinia coriandrina ............................................................................................................... 171

7.6.1. Isolation of Secondary Metabolites ............................................................................... 171

7.6.1.1. Isolation of ( )- -Pinene (18) ....................................................................................... 171

7.6.1.2. Isolation of ( )-(E)-Nerolidol (64) ................................................................................ 172

7.6.1.3. Isolation of (E,E)- -Springene (66) ............................................................................... 172

7.6.1.4. Isolation of (Z)-Coriandrin ((Z)-65) ............................................................................... 172

7.6.1.5. Isolation of (Z)-O-Methyltridentatol B ((Z)-72) ............................................................ 172

7.6.1.6. Isolation of (R)-( -Corsifuran A (73) ........................................................................... 172

7.6.1.7. Isolation of Corsifuran C (74) ........................................................................................ 173

7.6.1.8. Isolation of (E)-Corsistilbene ((E)-68) .......................................................................... 173

Table of Contents

7.6.1.9. Isolation of 4-Methoxyphenylethanal (59) .................................................................... 173

7.6.2. Synthesis of Secondary metabolites from Corsinia coriandrina ................................. 173

7.6.2.1. Synthesis of 2-Bromo-1-(4-methoxyphenyl)ethanone (76) ........................................... 173

7.6.2.2. Synthesis of 2-Azido-1-(4-methoxyphenyl)ethanone (77) ............................................ 173

7.6.2.3. Synthesis of (±)-2-Amino-1-(4-methoxyphenyl)ethanol (78) ....................................... 174

7.6.2.4. Synthesis of (±)-2-Hydroxy-2-(4-methoxyphenyl)ethylammonium Chloride (79) ....... 174

7.6.2.5. Synthesis of (±)-2-Chloro-2-(4-methoxyphenyl)ethylammonium Chloride (80) .......... 174

7.6.2.6. Synthesis of (±)-2-Chloro-2-(4-methoxyphenyl)ethyl Isothiocyanate (81) ................... 175

7.6.2.7. Synthesis of (Z)- and (E)-Coriandrins (65) .................................................................... 175

7.6.2.8. Synthesis of (Z)- and (E)-O-Methyltridentatols (72) ..................................................... 176

7.6.2.9. Synthesis of (±)-5-(4-Methoxyphenyl)-2-S-methylthio-4,5-dihydrothiazole (82) ........ 176

7.6.2.10. Synthesis of O-Methyltridentatol C (71) ..................................................................... 177

7.6.2.11. Synthesis of (±)-2-Chloro-2-(4-methoxyphenyl)ethyl Isocyanate (87) ....................... 177

7.6.2.12. Synthesis of (Z)- and (E)-Corsinians (63).................................................................... 178

7.6.2.13. Synthesis of (E)-4-Methoxycinnamic acid (89) by Knoevenagel ................................ 178

7.6.2.14. Synthesis of (E)-3-(4-Methoxyphenyl)propenoyl Chloride (90) ................................. 178

7.6.2.15. Synthesis of (E)-3-(4-Methoxyphenyl)propenoyl Azide (91) ..................................... 178

7.6.2.16. Synthesis of (Z)- and (E)-Corsinians (63) by Curtius Rearrangement ........................ 179

7.6.2.17. Synthesis of (Z)- and (E)-Corsiandrens (67) ................................................................ 179

7.6.2.18. Synthesis of (Z)- and (E)-Corsiandrenins (92) ............................................................ 180

7.6.2.19. Synthesis of (E)-Cinnamic Acid (93) .......................................................................... 181

7.6.2.20. Synthesis of (E)-3-Phenylpropenoyl Chloride (94) ..................................................... 181

7.6.2.21. Synthesis of (E)-3-Phenylpropenoyl Azide (95) ......................................................... 181

7.6.2.22. Synthesis of (E)-2-Phenylethenyl Isocyanate (96) ...................................................... 181

7.6.2.23. Synthesis of Dehydroniranin A (97) ............................................................................ 182

7.6.2.24. Synthesis of (Z)- and (E)-Tuberines (98) ..................................................................... 182

7.6.2.25. Synthesis of (Z)- and (E)-Corsicillins (99) .................................................................. 183

7.6.2.26. Synthesis of (E)-4-Methoxycinnamonitrile (100) ........................................................ 183

7.6.2.27. Synthesis of (±)-Corsifuran B (114) ............................................................................ 183

7.6.2.28. Synthesis of (±)-Corsifuran A (73) .............................................................................. 184

7.6.2.29. Synthesis of (±)-[5-OCD3]-Corsifuran A ([5-OCD3]-73) ............................................ 184

7.6.2.30. Synthesis of Corsifuran C (74) .................................................................................... 185

7.6.2.31. Synthesis of 4-Methoxybenzyl chloride (126) ............................................................. 185

7.6.2.32. Synthesis of Diethyl-4-methoxybenzyl phosphonate (127) ......................................... 185

7.6.2.33. Synthesis of (E)-Corsistilbene ((E)-68) ....................................................................... 186

7.6.2.34. Isomerisation to (Z)-Corsistilbene ((Z)-68) ................................................................. 186

7.6.2.35 Synthesis of O,O-Dimethyllunularin (69) .................................................................... 187

7.6.2.36. Synthesis of 2-Hydroxy-5,4‟-dimethoxybibenzyl (70) ................................................ 187

7.6.3. Synthesis of Deuterium Labelled Precursors ................................................................... 187

7.6.3.1. Synthesis of (E)-Cinnamic Acid (93) by Perkin ............................................................ 187

Table of Contents

7.6.3.2. Synthesis of (E)-[2-D]-Cinnamic Acid ([2-D]-93) by Perkin........................................ 188

7.6.3.3. Synthesis of (E/Z)-Cinnamonitriles (149)...................................................................... 188

7.6.3.4. Synthesis of (E/Z)-[2-D]-Cinnamonitriles ([2-D]-149) ................................................. 188

7.6.3.5. Attempted Synthesis of (E)-[2-D]-Cinnamic Acid ([2-D]-93) ...................................... 189

7.6.3.6. Attempted 1H/

2D Exchange in Potassium (E)-Cinnamate ............................................. 189

7.6.3.7. Synthesis of (E)-[2-D]-Cinnamic Acid ([2-D]-93) by Hydrolysis with KOD ............... 189

7.6.3.8. Synthesis of (E)-[2-D]-Cinnamic Acid ([2-D]-93) by Hydrolysis with NaOD ............. 189

7.6.3.9. Synthesis of (E)-4-Methoxycinnamic Acid (89) by Hydrolysis .................................... 190

7.6.3.10. Synthesis of (E)-[2-D]-4-Methoxycinnamic acid ([2-D]-89) by Hydrolysis ............... 190

7.6.3.11. Synthesis of (E)-4-Hydroxycinnamonitrile (151) ........................................................ 190

7.6.3.12. Synthesis of (E)-4-Coumaric Acid (152) by Hydrolysis ............................................. 191

7.6.3.13. Synthesis of (E)-[2-D]-4-Coumaric Acid ([2-D]-152) by Hydrolysis ......................... 191

7.6.3.14. Synthesis of [3,5-D2]-4-Hydroxybenzaldehyde ([D2]-150) ......................................... 191

7.6.3.15. Synthesis of [D4]-Malonic Acid ([D4]-88) ................................................................... 192

7.6.3.16. Synthesis of (E)-[2,3‟,5‟-D3]-4-Coumaric Acid ([D3]-152) by Knoevenagel.............. 192

7.6.3.17. Synthesis of L- and D-[3,3-D2]-Phenylalanine (L-[3,3-D2]-153, D-[3,3-D2]-153) ...... 192

7.6.3.18. Synthesis of L-[3,3-D2]-Tyrosine (L-[3,3-D2]-154) ..................................................... 193

7.6.3.19. Synthesis of L-[3,3-D2]-O-Methyltyrosine (L-[3,3-D2]- 155) ..................................... 193

7.6.3.20. Synthesis of [3,3-D2]-Dihydrocinnamic Acid ([3,3-D2]-156) ..................................... 193

7.6.3.21. Synthesis of [2,2,3,3,3‟-D5]-Phloretic Acid ([D5]-157) ............................................... 194

7.6.3.22. Synthesis of [2,2,3‟-D3]-Tyramine ([2,2,3‟-D3]-158) .................................................. 194

7.6.3.23. Synthesis of L-N-Acetyltyrosine (159) ........................................................................ 195

7.6.3.24. Synthesis of [CD3]-Methyl L-N-Acetyl-[CD3]-O-methyltyrosinate ([CD3]-160) ....... 195

7.6.3.25. Synthesis of L-[CD3]-O-Methyltyrosine Hydrochloride ([CD3]-155) ......................... 195

7.6.3.26. Conversion to L-[CD3]-O-Methyltyrosine ([CD3]-155) .............................................. 196

7.6.3.27. Synthesis of DL-[2-D]-N,O-Diacetyltyrosine ([2-D]-161) .......................................... 196

7.6.3.28. Synthesis of DL-[2-D]-Tyrosine Hydrochloride ([2-D]-154) ...................................... 196

7.6.3.29. Synthesis of L-N-Acetyl-O-methyltyrosine (162) ....................................................... 197

7.6.3.30. Synthesis of DL-[2-D]-N-Acetyl-O-methyltyrosine ([2-D]-162) ................................ 197

7.6.3.31. Synthesis of DL-[2-D]-O-Methyltyrosine Hydrochloride ([2-D]-155) ........................ 197

7.6.3.32. Synthesis of (Z)-4-(4-Acetoxybenzylidene)-2-methyloxazol-5(4H)-one (164) .......... 198

7.6.3.33. Synthesis of (Z)-2-Acetamido-3-(4-acetoxyphenyl)propenoic Acid (165) ................. 198

7.6.3.34. Synthesis of DL-[2,3-threo-D2]-N,O-Diacetyltyrosine ([2,3-threo-D2]-161) .............. 198

7.6.3.35. Synthesis of DL-[threo-2,3-D2]-Tyrosine Hydrochloride ([2,3-threo-D2]-154) .......... 199

7.6.3.36. Synthesis of [CD3]-4-Methoxybenzaldehyde ([CD3]-58) ............................................ 199

7.6.3.37. Synthesis of [CD3]-4-Methoxy- -nitrostyrene ([CD3]-167) ........................................ 199

7.6.3.38. Synthesis of [CD3]-O-Methyltyramine ([CD3]-168) ................................................... 200

7.6.3.39. Synthesis of (±)-[2,3-threo-D2]-Phloretic Acid ([2,3-threo-D2]-157) ......................... 200

7.7. (1S,2R)-(+)-spiroaxa-5,7-diene from Ulmus americana ...................................................... 201

7.7.1. Isolation of (–)-Axenol (211) from Juniperus oxycedrus ................................................. 201

Table of Contents

7.7.2. Amberlyst Catalyzed Rearrangement of (+)-Aromadendrene ((+)-216) .......................... 201

7.7.3. Amberlyst Catalyzed Rearrangement of (+)-Ledene (218) .............................................. 201

7.7.4. Amberlyst Catalyzed Rearrangement of (–)-allo-Aromadendrene (217) ......................... 201

7.7.5. Solid Superacid Catalyzed Rearrangement of (+)-Aromadendrene ((+)-216) ................. 202

7.7.6. Solid Superacid Catalyzed Rearrangement of (+)-Ledene (218) ...................................... 202

7.7.7. Solid Superacid Catalyzed Rearrangement of (–)-allo-Aromadendrene (217) ................ 203

7.7.8. Isolation of (–)-ent-Aromadendrene ((–)-216) from Pellia epiphylla .............................. 203

7.7.9. Solid Superacid Catalyzed Rearrangement of (–)-ent-Aromadendrene ((–)-216) ............ 203

7.8. Serratia odorifera - Synthesis of Octamethylbicyclo[3.2.1]octadienes ............................... 203

7.8.1. Synthesis of 2,4-Dibromopentan-3-one (232) .................................................................. 203

7.8.2. 1,2,4,5,6,7,8-Heptamethylbicyclo[3.2.1]oct-6-en-3-ones (236 - 238) .............................. 203

7.8.3. bisaxial-1,2,4,5,6,7,8-Heptamethyl-3-methylenebicyclo[3.2.1]oct-6-ene (239) .............. 204

7.8.4. bisequatorial-1,2,4,5,6,7,8-Heptamethyl-3-methylenebicyclo[3.2.1]oct-6-ene (241) ..... 205

7.8.5. 1,2,3,4,5,6,7,8-Octamethylbicyclo[3.2.1]oct-6-en-3-ol (242) .......................................... 205

7.8.6. 1,2,3,4,5,6,7,8-Octamethylbicyclo[3.2.1]octa-2,6-diene (243) and .................................. 206

1,3,4,5,6,7,8-Heptamethyl-2-methylenebicyclo[3.2.1]oct-6-ene (244) ............................ 206

8. Hazardous chemicals ..................................................................................................................... 207

9. Colour Plates .................................................................................................................................. 210

10. References .................................................................................................................................... 218

Poster Presentations ...................................................................................................................... 240

Oral Presentations ......................................................................................................................... 241

Publications ................................................................................................................................... 242

CURRICULUM VITAE ............................................................................................................... 244

List of Abbreviations ..................................................................................................................... 245

1. Introduction

1

1. Introduction

»Organic Chemists have often been tempted to leave the security of their own proper

pastures and to graze, albeit speculatively, in the attractive fields of biochemistry.

It has seemed that, the structures of so many natural products having been established,

it should be possible to perceive some relations between them such as to suggest

schemes of biosynthesis.« Sir Robert Robinson, 1955

While primary metabolism refers to the anabolic and catabolic processes required for cell

maintenance and proliferation, secondary metabolism covers species specific natural products

that are not necessary for the individual cell, but thought to be required for the organisms‟

survival when interacting with its environment. The amazing structural diversity displayed by

natural products makes them a rewarding field for structure elucidation and synthesis. Apart

from the desire to isolate, identify and synthesize natural products in order to determine their

biological activity in an ecological or pharmacological context, our ever increasing

knowledge on their distribution among the different species allows for chemosystematic

classifications. Furthermore, since the availability of radioactive isotopes in the 1950‟s, the

discovery of new natural products has initiated the elucidation of precursor-product

relationships by application experiments with labelled compounds. Advances in NMR

spectroscopy then enabled application experiments using stable isotopes like 2D,

13C,

15N, and

18O (Schneider, 2007; Simpson, 1998; Vederas, 1987; Garson & Staunton, 1979). The

biosynthetic routes of many secondary pathways were outlined and provided the basis for the

enzymatic characterization of biosynthetic pathways in the 1970‟s and 1980‟s, followed by

the identification of the corresponding genes beginning in the late 1980‟s (Thomas, 2004;

Hartmann, 2007; Mahmud, 2007).

Liverworts (Hepaticae) have recently attracted attention, due to their phylogenetic position

basal to all other terrestrial plants (Qiu et al., 2006, 2007). Phytochemical studies on

liverworts are however hampered by the difficulties encountered with the acquisition of

sufficient quantities of pure plant material, unless axenic in vitro cultures are available

(Becker, 1990, 1994, 1995). It has been estimated that only 5 % of known liverwort species

have been cultured under in vitro conditions (Duckett et al., 2004; Hohe & Reski, 2005) and

less than 15 % have been chemically investigated so far (Asakawa, 1995, 2007, 2008).

2. Objective of Research

2

2. Objective of Research

The purpose of this study was the isolation, structure elucidation and synthesis of secondary

metabolites from liverworts and microorganisms, along with the investigation of their

biosynthesis. Research involves the acquisition of fresh liverwort plant material and its

botanical identification, along with the development of suitable extraction procedures

monitored by gaschromatography (GC). Unknown constituents are traced using coupled

gaschromatography electron impact mass spectrometry (GC-EIMS), in combination with a

mass spectral database (König & Hochmuth, 2004; König et al., 2004). Their isolation is

achieved by a combination of column chromatography (CC) and thin layer chromatography

(TLC) or preparative gaschromatography (PGC). Molecular formulas are determined by high

resolution mass spectrometry (HREIMS) and structures are deduced from mass spectral

fragmentation patterns and one- or two-dimensional nuclear magnetic resonance

spectroscopy, like 1H NMR,

13C {

1H} NMR,

13C PENDANT, COSY 90, HMQC, HMBC and

gp-NOESY. Structure assignments are then confirmed by chemical correlation and partial or

total synthesis. The absolute configuration of chiral compounds is finally determined by

enantioselective GC (eGC) using authentic reference samples and modified cyclodextrins as

stationary phase.

From more than 20 liverworts, screened for interesting compounds, 3 species were

investigated in more detail: Fossombronia angulosa, Fossombroniaceae (Chapter 4.1., page

7), Riccardia chamedryfolia, Aneuraceae (Chapter 4.2., page 17), and Corsinia coriandrina,

Corsiniaceae (Chapter 4.3., page 28). A variety of deuterium labelled precursors was prepared

(Chapter 4.4., page 67) and axenic in vitro cultures of Corsinia coriandrina were established

in cooperation with Dr. K. von Schwartzenberg at the Biozentrum Klein Flottbek, University

of Hamburg, Germany (Chapter 4.5., page 76). Application experiments were performed in

order to study the biosynthesis of 4-methoxystyrenes (Chapter 4.6.1., page 86), stilbenoids

(Chapter 4.6.2., page 102) and terpenoids (Chapter 4.6.3., page 130) in Corsinia coriandrina.

Research on microbial metabolites involves structure elucidation and synthesis of a

sesquiterpene hydrocarbon emitted by the American elm, Ulmus americana, upon infection

with the fungal pathogen Ophiostoma novo-ulmi (Chapter 4.7., page 137), performed in co-

operation with Prof. Gerhard Gries (Burnaby, Canada), and a new class of octamethyl-

tricyclo[3.2.1]octadienes emitted by the rhizobacterium Serratia odorifera (Chapter 4.8., page

149), which was performed in cooperation with Prof. Birgit Piechulla (Rostock, Germany).

3. General Part

3

3. General Part

»Man darf wohl sagen, daß der Chemie der ätherischen Öle auch bei den Kryptogamen

noch ein weites Arbeitsfeld offensteht, ein Gebiet, das dem bei den höheren Pflanzen an

Ausdehnung und Mannigfaltigkeit vielleicht nicht nachstehen wird.« Karl Müller, 1905

3.1. Liverwort Biology

The bryophytes are taxonomically placed between green algae (chlorophytes) and ferns

(pteridophtes) and regarded as the closest living relatives of the first land plants. It is

generally assumed that these organisms developed from Charophytean algae ancestors in

Ordovician times approximately 500 million years ago (Kenrick & Crane, 1997; Wellman et

al., 2003; Graham et al., 2004). Nevertheless, relationships among the three bryophyte classes

(liverworts, mosses, hornworts) and between them and other embryophytes have remained

unclear for several times (Beckert et al., 1999; Nickrent et al., 2000; Pruchner et al., 2001,

2002). Recent phylogenetic studies have placed the liverworts (Hepaticae, 6.000 – 8.000

species) as the most basal group of all terrestrial plants (Qiu et al., 2006, 2007). The life cycle

of liverworts is dominated by haploid gametophytes which form the liverwort plant and

develop gametangia for sexual reproduction. Male antheridia and female archegonia are either

located on separate thalli (dioecious) or present on the same thallus (monoecious). Diploid

sporophytes are only produced after water mediated fertilization and these short lived organs

are nourished by the gametophyte. After meiosis the resulting haploid spores are released to

germinate and develop new gametophytes. In addition, asexual propagation via gemma cups

is known from species like Lunularia cruciata or Marchantia polymorpha.

On the cellular level one of the most striking differences between the cells of the majority of

liverworts and those of all other terrestrial plants and algae is the presence of highly refractive

structures commonly known as oil bodies (see Colour Plates 1e, 3, and 5, pages 210 – 212). In

thalloid Marchantialean liverworts oil bodies are restricted to scattered specialized cells,

whereas in the leafy Jungermanniales they are generally present in all cells. Electron

microscope studies revealed that liverwort oil bodies are confined by a single lipid membrane

and contain lipid globules embedded in a carbohydrate matrix (Duckett & Ligrone, 1995).

3. General Part

4

The first extractions of liverworts performed by H. Lohmann (1903) and Karl Müller (1905)

afforded essential oils in up to 1.5 % of the dry weight, which were assumed to consist of

mono and sesquiterpenoids. Müller (1939) also concluded that these compounds were

contained in the oil body structures, because their number correlated with the amount of oil

obtained. Furthermore, the bluish oil bodies of several Calypogeia species were attributed to

1,4-dimethylazulene (1) as the major volatile constituent (Katoh & Takeda, 1990), and

3-methoxybibenzyl (8) has been identified as the main volatile constituent of isolated oil

bodies of Radula complanata (Flegel & Becker, 2000) (Figure 1, page 5). Histochemical

localization of enzymes related to terpenoid biosynthesis in oil body membranes of

Marchantia polymorpha have shown that these unique organelles are not merely storage

vesicles but constitute metabolically reactive compartments (Suire et al., 2000).

Liverworts are rich in carbohydrates but almost devoid of any mechanical protection against

herbivores, due to the imperfectly developed cuticula and the lack of lignins. Nevertheless,

they are only rarely affected by herbivory, which led to the assumption that they should

posses a kind of antifeedants. Antifungal, antibacterial, molluscicidal, and insecticidal, as well

as plant growth inhibiting or promoting activities of liverwort constituents suggested that

these compounds act as a chemical defence to become most efficient upon rupture of the plant

tissue (Becker & Wurzel, 1987; Zinsmeister & Mues, 1987; Wurzel et al., 1990; Zinsmeister

et al., 1991, 1994; Frahm, 2004; Asakawa, 2007, 2008).

In addition, liverworts form associations with proteobacteria and enterobacteria showing

antifungal and antibacterial activity (Opelt & Berg, 2004), as well as symbiotic and free-living

cyanobacteria (West & Adams 1997; Adams & Duggan, 2008; Rikkinen & Virtanen, 2008).

Epiphytic methylobacteria promote liverwort growth by releasing cytokinins and auxin

(Kutschera & Koopmann, 2005; Kutschera 2007). Furthermore, many liverworts form

mycorrhiza-like associations with fungal symbionts of the Glomeromycota (Read et al., 2000;

Russel & Bulman, 2005; Kottke & Nebel 2005; Ligrone et al., 2007; Kottke et al., 2008).

Fossilized fungal hyphae and spores from the Ordovician strongly resemble those of current

Glomus species, suggesting that fungal associations might have facilitated colonization of

terrestrial habitats (Redecker et al., 2000; Heckman et al., 2001). The extent of these

associations is best seen in the non-photosynthetic ghostwort, Cryptothallus mirabilis, which

forms mycorrhizal associations with Tulasnella to obtain photosynthate from other autotrophs

associated with the fungus (Bidartondo, et al., 2002, 2003).

3. General Part

5

3.2. Secondary Metabolites of Liverworts

Figure 1: Collection of secondary metabolites from liverworts (and hornworts (13)).

The liverworts are characterized by lunularic acid (5) an ubiquitous liverwort specific

phytohormone first isolated from Lunularia cruciata (Valio et al., 1969), and later shown to

be a precursor of liverwort bibenzyls (Pryce, 1972a) and bis(bibenzyls) (Friederich et al.,

1999a, 1999b). Lunularic acid (5) and the corresponding decarboxylation product lunularin

(6) (Pryce & Linton, 1974), as well as its labile precursor, prelunularic acid (7) (Ohta et al.,

1983, 1984; Abe & Ohta, 1984, 1985), have been detected in all liverworts examined, but not

in algae, mosses, hornworts, ferns, lichens, pteridophytes, or higher plants (Pryce, 1972b;

Gorham 1977a, 1977b). The single exception is the Garden Hortensia, Hydrangea macro-

phylla (Saxifragales), which contains lunularic acid (5) and hydrangenic acid (194, Figure

110, page 123), the corresponding stilbenecarboxylic acid (Gorham, 1977a).

3. General Part

6

In addition, flavonoids (10) of chalcone synthase (CHS) origin are abundant in liverworts

(Asakawa, 1982, 1995, 2004), whereas isoflavonoids are extremely rare (Anhut et al., 1984).

Furthermore, liverworts are known as rich sources of mono-, sesqui-, and diterpenoids

(Asakawa, 1982, 1995, 2004). Some sesquiterpenoids with unique skeletons like anastreptene

(2) (Andersen et al., 1978; von Reuß et al., 2004) and cyclomyltaylane (3) (Wu & Chang,

1992) have exclusively been reported from liverworts. In addition, many liverwort

sesquiterpenoids like (–)-longifolene (4) exhibit the opposite absolute configuration in

comparison to higher plant compounds (Huneck & Klein, 1967; Asakawa, 1982). These ent-

sesquiterpenoids and their derivatives from functional group transformations and

rearrangement reactions have become a valuable source of reference compounds required for

comparative enantioselective GC analysis using modified cyclodextrins as stationary phase

(Fricke et al., 1995; König et al., 1996; Bülow & König, 2000, König & Hochmuth, 2004).

In contrast to vascular plants and aquatic algae, nitrogen- or sulfur-containing constituents are

virtually unknown from the liverworts (Asakawa, 2004, 1995). Considering the vast diversity,

distribution, and ecological importance of terrestrial plant alkaloids (Hesse, 2000), their

scarcity in lower plants is remarkable. Only 7- and 6-prenylindoles (14, 15) were described

from the Metzgerialean liverworts Riccardia chamedryfolia, R. incurvata, and R. multifida,

Aneuraceae (Benesova et al., 1969a, 1969b; Huneck et al., 1972; Nagashima et al., 1993),

whereas the alkaloid anthocerodiazonin (13) was reported from a hornwort, Anthoceros

agrestis (Trennheuser et al., 1994). Sulfur containing constituents are equally rare. The

isotachins A – C with -S-methylthio-(E)-acrylate structures (16) were obtained from the

Jungermannialean liverworts Isotachis japonica (Asakawa et al., 1985), Balantiopsis rosea

(Asakawa et al., 1986) and B. cancellata (Labbé et al., 2005). Some chlorinated bibenzyls (9)

and bis(bibenzyls) have recently been described from Lepidozia (Scher et al., 2003),

Plagiochila (Anton et al., 1997), Bazzania (Martini et al., 1998; Speicher et al., 2001),

Herbertus, Mastigophora (Hashimoto et al., 2000), and Riccardia species (Baeck et al., 2004;

Labbé et al., 2007).

4.1. C11 Hydrocarbons from Fossombronia angulosa

7

4. Special Part


4.1.1. Fossombronia angulosa

Fossombronia angulosa (Dicks.) Raddi is a member of the Fossombroniaceae, which form

thallose gametophytes with lamina divided into leaf-like lobes and a stem-like costa (Plate 1a,

page 210) carrying gametangia and sporophytes (Plate 1b). About 50 species have been

described of which 11 are represented in Europe, but some of these are difficult to distinguish

in the absence of gametangia and spores (Paton, 1999). Fossombronia angulosa forms thalli

up to 2.5 cm long and is restricted to lowland coastal regions with a mild climate. Samples

were collected on the Islands of Tenerife and Madeira in April 2003 and March 2007,

respectively. While former collection was lacking mature sporogons, the identity of the latter

specimen could be unambiguously established by inspection of spore morphology (Plate 1c),

which exhibited characteristic lamellae appearing on the spore margin as a continuous pale

wing (Plate 1d) as described by Paton (1999). Most thallus cells contain 6 – 10 oil bodies with

a diameter of 1 – 6 µm (Plate 1e).

Members of the Fossombroniaceae have previously been investigated for secondary

metabolites. Antibacterial diterpene dialdehydes were reported from in vitro cultured

Fossombronia pusilla (Sauerwein & Becker, 1990). Furthermore, biosynthesis of geosmin

(39) in F. pusilla has been shown to proceed via the mevalonate (MVA) pathway, in contrast

to its methylerythritol-4-phosphate (MEP) origin in Streptomces species (Spiteller et al.,

2002), and leucine origin in Myxobacteria (Dickschat et al., 2005). Hopane-type triterpenoids

and epi-neoverrucosane-type diterpenoids have been described from in vitro cultures of the

rare F. alaskana (Grammes et al., 1994, 1997) and their biosynthesis via the MVA or MEP

pathways, respectively, was established (Hertewich et al., 2001).


8

4.1.2. Volatile Constituents of Fossombronia angulosa

Figure 2: Gas chromatograms of hydrodistillation products of Fossombronia angulosa collected

on the Islands of Tenerife and Madeira (30 m, CpSil-5 CB, 50 °C, + 3 °C /min, to 250 °C).

Hydrodistillation of the carefully cleaned fresh plant material afforded an essential oil, which

was analyzed by GC (Figure 2) and GC-EIMS. Undecan-2-one (34) or tridecan-2-one (35)

was identified as major constituent by comparison of mass spectra and GC retention indices

with a spectral library established under identical experimental conditions (König et al., 2004;

König & Hochmuth, 2004). Small amounts of the corresponding alcohols (37 and 38) were

also detected, along with pentadecan-2-one (36). Monoterpene hydrocarbons like (–)- -

sabinene (20) and -phellandrene (26) were identified as major constituents of the specimen

from Tenerife only.

Madeira

Tenerife

17

18 19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

37

39 35

29

30

31

32

39 34

38 36

35

10 20 30 40 RT [min]

20 21

26

29 dictyopterene A 30 ectocarpene 31 pentylbenzene 32 dictyotene 33 terpinen-4-ol 34 undecan-2-one 35 tridecan-2-one 36 pentadecan-2-one 37 undecan-2-ol 38 tridecan-2-ol

39 geosmine

17 -thujene

18 -pinene 19 camphene

20 -sabinene

21 -pinene 22 myrcene

23 3-carene

24 -terpinene 25 p-cymene

26 -phellandrene

27 -terpinene

28 terpinolene


9

Three unidentified hydrocarbons with molecular ion signals at m/z 148 [M] (C11H16, 30) or

m/z 150 [M] (C11H18, 29 and 32) were isolated by a combination of column chromatography

on silica gel and semi-preparative GC. The identification of dictyopterene A (29), ectocarpene

(30), dictyotene (32), and n-pentylbenzene (31) is described in the following sections.

Figure 3: Volatile constituents identified in the hydrodistillates of Fossombronia angulosa

(* absolute configuration not determined).


10

Figure 4: Mass spectra (EI, 70 eV) of dictyopterene A (29), dictyotene (32),

ectocarpene (30), and n-pentylbenzene (31) from Fossombronia angulosa.

41

53

67

79

91

107121 150

40 60 80 100 120 140 160

20

40

60

80

100

41

53

67

79

93

107

121

150

40 60 80 100 120 140 160

20

40

60

80

100

39

5166

79

91

105

119

133148

40 60 80 100 120 140 160

20

40

60

80

100

4151 57

6578

91

105

119

148

40 60 80 100 120 140 160

20

40

60

80

100

C11H18

C11H18

C11H16

C11H16

C7H7


11

4.1.3. Identification of Dictyopterene A (29)

The mass spectrum of 29 (Figure 4) exhibited a molecular ion signal at m/z 150 [M]

indicating the molecular formula C11H18 with 3 units of unsaturation. The 1H NMR spectrum

(Figure 5) exhibited signals for one vinyl moiety at H 4.88 (1H, dd, 3JZ = 10.4 Hz,

2J = 1.6

Hz), 5.05 (1H, dd, 3JE = 17.0 Hz,

2J = 1.6 Hz) and 5.31 (1H, ddd,

3JE = 17.0 Hz,

3JZ = 10.4

Hz, 3J = 8.2 Hz), one trans-configured ethylene bond at H 4.97 (1H, dd,

3JE = 15.5 Hz,

3J =

7.6 Hz) and 5.46 (1H, dt, 3JE = 15.4 Hz,

3J = 6.6 Hz) adjacent to an allylic methylene group at

H 1.96 (2H, dt, 3J = 6.6 Hz), one overlapping signal at H 1.22 - 1.36

(6H, m), one methyl

group at H 0.86 (3H, t, 3J = 7.3 Hz), and a methylene group as part of a cyclopropyl moiety

as indicated by the chemical shift of H 0.67 (2H, t, 3J = 6.9 Hz).

Figure 5: 500 MHz 1H NMR spectrum of (+)-dictyopterene A (29, in C6D6) from F. angulosa.

Inspection of the 13

C PENDANT (Figure 6) and HMQC spectra confirmed the presence of a

vinyl moiety at C 112.0 (t) and 141.0 (d), one internal double bond at C 129.1 (d) and

132.1 (d), one methyl group at C 14.2 (q), one 1,2-disubstituted cyclopropyl-unit at C 14.9

(t), 23.9 (d), and 24.6 (d), and three methylene groups at C 22.6 (t), 32.2 (t), and 32.6 (t). The

connectivities between the partial structures were deduced from two dimensional COSY and

HMBC spectra, which indicated a 1-(hex-1-enyl)-2-vinylcyclopropane skeleton, previously

described as dictyopterene A (29) from marine brown algae (Pohnert & Boland, 2002). The

trans-configuration of the cyclopropyl moiety in 29 was assigned according to the gp-NOESY

spectrum, which exhibited NOE-correlations between 3-H and 6-H, as well as 2-H and 5-H.

This hypothesis was confirmed by comparison of the mass spectrum and GC retention index

H [ppm] 0.8 1.2 1.6 2.0 2.4 2.8 3.2 3.6 4.0 4.4 4.8 5.2

11-CH3

9,10-CH2

8-CH2 2-CH

3,5-CH

1-CHZ

1-CHE

6-CH

4-CH2

7-CH


12

with those of an authentic sample of (3R,5R)-(+)-dictyopterene A (29). The enantiomeric

composition of 29 was determined upon enantioselective GC (Section 4.1.7., page 15).

Figure 6: 100 MHz 13

C PENDANT spectrum of (+)-dictyopterene A (29, in C6D6) from F. angulosa.

4.1.4. Identification of Dictyotene (32)

The molecular ion signal at m/z 150 [M] in the mass spectrum of compound 32 (Figure 4,

page 10) suggested the molecular formula of C11H18 with 3 units of unsaturation. The 1H

NMR spectrum (Figure 7, page 13) indicated four partially overlapping signals for olefinic

protons at H 5.73 (1H, m) and 5.61 (3H, m), thus, suggesting a monocyclic structure.

Furthermore, one anisochoric methylene group at H 2.58 (1H, d.br, 2J = 19.5 Hz) and 2.86

(1H, d.br, 2J = 19.2 Hz), a chiral methine group at H 2.43 (1H, s.br.), one allylic methylene

group at H 2.13 (2H, m, 6-H), one propylene bridge at H 1.24 (6H, s.br), and one methyl

group at H 0.87 (3H, t, J = 7.3 Hz) were identified. Inspection of the COSY spectrum

allowed the assignment of a consecutive 7-butylcyclohepta-1,4-diene structure, previously

described as dictyotene (32) from marine brown algae (Pohnert & Boland, 2002). This

hypothesis was confirmed by comparison of the mass spectra and GC retention indices with

corresponding data of an authentic reference sample, whilst the enantiomeric composition of

dictyotene (32) was determined upon enantioselective GC (Section 4.1.7., page 15).

TMS

C [ppm]2030405060708090100110120130140

1-CH24-CH2

11-CH3

2-CH

7-CH

6-CH

9,8-CH2

10-CH2

3,5-CH

solvent

TMS

C [ppm]2030405060708090100110120130140

1-CH24-CH2

11-CH3

2-CH

7-CH

6-CH

9,8-CH2

10-CH2

3,5-CH

solvent


13

Figure 7: 500 MHz 1H NMR spectra of dictyotene (32) and ectocarpene (30, in C6D6)

from Fossombronia angulosa.

4.1.5. Identification of Ectocarpene (30)

The molecular ion signal at m/z 148 [M] in the mass spectrum of compound 30 suggested the

molecular formula of C11H16 with 4 units of unsaturation (Figure 4, page 10). The 1H NMR

spectrum of 30 was similar to that of dictyotene (32) (Figure 7), exhibiting signals for a

1.2 1.6 2.0 2.4 2.8 3.2 3.6 4.0 4.4 4.8 5.2 5.6

5-CH

1,4-CH

2-CH

8-CH

9-CH

3-CH

7-CH

3-CH’

6-CH2

10-CH2

11-CH3

[ppm] H

[ppm] 1.2 1.6 2.0 2.4 2.8 3.2 3.6 4.0 4.4 4.8 5.2 5.6

5-CH

3-CH’

H

3-CH

7-CH

8-CH2

1,2,4-CH

6-CH2

9,10-CH2

11-CH3


14

cyclohepta-1,4-diene-7-yl skeleton. Nevertheless, signals for the alkyl chain of dictyotene

(32) were replaced by those corresponding to a (Z)-configured internal double bond at H 5.48

(1H, dd, J = 10.7 Hz, JZ = 9.5 Hz) and 5.33 (1H, dt, JZ = 9.8, J = 7.3 Hz), adjacent to an

allylic methylene group at H 1.97 (2H, dq, J = 7.3 Hz, J = 7.6 Hz). The location of the double

bond in 30 was deduced from the chemical shift and triplet multiplicity of the methyl signal at

H 0.87 (3H, t, J = 7.6 Hz) and the fact that the chiral methine group at H 3.52 (1H, s.br) was

significantly shifted to lower field in comparison to the saturated dictyotene (32). The

structure of ectocarpene (30) was established by comparison of the mass spectra and GC

retention indices with an authentic sample while the enantiomeric composition was

determined upon enantioselective GC (Section 4.1.7., page 15).

4.1.6. Identification and Synthesis of n-Pentylbenzene (31)

GC-EIMS analysis of the hydrocarbon fraction of F. angulosa suggested the presence of trace

amounts (< 0.1 % of the total volatiles) of another C11H16 compound (31), as indicated by the

molecular ion signal at m/z 148 [M] (Figure 4, page 10). The base peak at m/z 91 (100)

[M – C4H9] for a tropylium ion (C7H7+) suggested a pentylbenzene structure. Considering the

co-occurring C11 hydrocarbons (29, 30 and 32) an unbranched alkyl residue was assumed.

This hypothesis was confirmed by comparison of the mass spectra and GC retention indices

with n-pentylbenzene (31) prepared as shown in figure 8. Racemic (±)-1-phenylpentanol (41)

was obtained in 79 % yield from benzaldehyde (40) upon reaction with n-butyl lithium. Acid

catalyzed dehydration was carried out using ion exchange resin Amberlyst® 15 in dichloro-

methane to give (E)-1-phenyl-1-pentene (42) in 66 % yield, which was hydrogenated using

palladium on charcoal to give n-pentylbenzene (31) in 90 % yield identical to the natural

product from Fossombronia angulosa.

Figure 8: Synthesis of n-pentylbenzene (31) from Fossombronia angulosa.


15

4.1.7. Enantioselective GC Analysis of C11 Hydrocarbons

The determination of absolute configurations and enantiomeric excess of dictyopterene A (29)

using gaschromatography with modified cyclodextrins as chiral selectors has previously been

described, but no suitable cyclodextrin derivative for the resolution of (±)-ectocarpene (30) or

(±)-dictyotene (32) was available at that time (Boland et al., 1989; Wirth et al., 1992). Of all

the different phases developed and tested till 2004 only heptakis(2,3-di-O-acetyl-6-O-tert-

butyldimethylsilyl)- -cyclodextrin (2,3-Ac-6-TBDMS- -CD) separated (±)-30 and (±)-32

(König, 2004). Using 2,3-Ac-6-TBDMS- -CD as the stationary phase at 60 °C isothermally,

the following -values were obtained; ectocarpene (30): (S)-(+)/(R)-(–) = 1.026, dictyotene (32):

(R)-(–)/(S)-(+) = 1.029, and dictyopterene A (29): (3S,5S)-(–)/(3R,5R)-(+) = 1.030 (Figure 9).

Figure 9: Enantioselective GC analysis of C11 hydrocarbons from Fossombronia angulosa

(29, 30 and 32) using 2,3-Ac-6-TBDMS- -CD at 60 °C isothermally.

(3R,5R)-(+)-dictyopterene A (29) from F. angulosa exhibited an enantiomeric excess of

ee = 72 and 74 %. The enantiomeric purities of (R)-(–)-dictyotene (32, ee = 87 %) and

(S)-(+)-ectocarpene (30, ee = 64 %) were high in samples from Tenerife, whereas almost

racemic mixtures were observed in samples from Madeira with (R)-(–)-dictyotene (32,

ee = 32 %) and (R)-(–)-ectocarpene (30, ee = 14 %) predominating.

RT [min]

20

18

16

(–)

(+)

(–)

(+)

(–)

(+)

(+)-30

( )-32

racemic standards

(–)-30

Tenerife Madeira

( )-32

(+)-29 (+)-29


16

4.1.8. Discussion of C11 Hydrocarbons from Fossombronia angulosa

Relying on GC-EIMS measurements and mass spectral databases Asakawa et al. have

recently reported the identification of dictyopterene (29), dictyotene (32), and (Z)-multifidene

(43) from F. angulosa (Ludwiczuk et al., 2008). The presence of 29 and 32 is now confirmed

by NMR spectroscopy. Because mass spectra and GC retention indices of (Z)-multifidene (43,

RI 1140) and (Z)-ectocarpene (30, RI 1136) are very similar (König et al., 2004) identification

of multifidene (43) might be in error as indicated by the characterization of ectocarpene (30)

using 1H NMR techniques. In addition, n-pentylbenzene (31) was identified as a trace

constituent by comparison with a synthetic sample. Furthermore, enantiomeric compositions

of ectocarpene (30) and dictyotene (32) are reported for the first time and shown to be highly

variable.

Figure 10: C11 hydrocarbons identified in Fossombronia angulosa (29– 32).

Compounds 29, 30 and 32 as well as related C11 hydrocarbons are well known from marine

brown algae (Phaeophyceae), which release enantiomeric mixtures as pheromones of female

gametes and in chemical defence (Pohnert & Boland, 2002). Ectocarpene (30) has also been

reported from Senecio isatideus, Asteraceae (Bohlmann et al., 1979). Dodecatrienoic acid was

recognized as its biogenetic precursor in this higher plant (Boland & Mertes, 1985; Neumann

& Boland, 1990), whereas C11-hydrocarbons of brown algae originate from eicosapentaenoic

acid (C11H16) or arachidonic acid (C11H18) (Stratmann et al., 1993; Pohnert & Boland, 2002).

While the biological function and biogenetic origin of C11 hydrocarbons in Fossombronia

angulosa remains to be clarified, the occurrence of arachidonic acid and eicosapentaenoic

acid in some liverworts (Asakawa, 1995) suggests a biogenetic pathway similar to those in

marine brown algae, although there are no close phylogenetical relationships between these

taxa.

4.2. Indole Alkaloids from Riccardia chamedryfolia

17


4.2.1. Riccardia chamedryfolia

The genus Riccardia S. Gray 1821 belongs to the family Aneuraceae, Metzgeriales. Riccardia

chamedryfolia (With.) Grolle 1969 forms dark green, 1 – 2 mm wide and 10 – 30 mm long

thalli consisting of 5 – 8 cell layers (Plate 2, page 211). Almost all epidermal cells contain

1 – 2 spherical, ellipsoidal, brownish oil bodies which range in size from 7 – 15 x 9 – 25 µm,

each composed of numerous small spherules (Plate 3, page 211).

As early as 1969 Šorm and co-workers reported on the isolation of 7-prenylindole (14) and

6-prenylindole (15) from Riccardia incurvata and R. sinuata (Hook.) Trev., a synonym for

R. chamedryfolia (Benesova et al., 1969a, 1969b). These results were confirmed by Huneck,

who reported local anesthetic properties of 15 and mentioned the presence of another

unidentified polar alkaloid in R. chamedryfolia (Huneck et al., 1972). 6- and 7-prenylindoles

(15 and 14) were also detected in R. multifida (L.) S. Gray subsp. decrescens (Steph.) Furuki,

(Asakawa et al., 1983; Nagashima et al., 1993). Furthermore, prenylindoles 14 and 15, as well

as the corresponding 3- and 5-isomers have been described from higher plants, such as

Annonaceae (Nwaji et al., 1972; Achenbach & Renner, 1985; Achenbach, 1986; Muhammad

et al., 1986; Nkunya et al., 2004; Boti et al., 2005) or Rutaceae (Delle Monache et al., 1989;

Kinoshita et al., 1989). Antifungal activities of 6-prenylindole (15), which has also been

described from Streptomyces sp. TP-A0595, Actinomycetes, suggested its role in chemical

defence (Sasaki et al., 2002).

4.2.2. Volatile Constituents of Riccardia chamedryfolia

Plant material of Riccardia chamedryfolia (With.) Grolle was collected on the Island of

La Palma in January 2006 and in Great Britain in 2007. The carefully cleaned fresh plant

material was frozen with liquid nitrogen, crushed and extracted with organic solvents using

sodium sulfate as drying reagent, or hydrodistilled, or air dried and solvent extracted.

Comparative GC-EIMS analysis indicated significant amounts of oxygenated artifacts in the

hydrodistillation products and diethyl ether extracts of air dried material, which were much

less pronounced in pentane extracts of fresh plants. GC and GC-EIMS investigations

indicated 7-prenylindole (14, 56 % of the total volatiles) and 6-prenylindole (15, 19 %) as

major constituents (Figure 11). In addition, sesquiterpene hydrocarbons like -maaliene

(44, 13 %), calarene (45, 1 %), -acoradiene (46, 1 %), bicyclogermacrene (47, 5 %), and


18

-cuprenene (48, 1 %) (Figure 12) were identified by comparison of mass spectra and GC

retention indices with a spectral library established under identical experimental conditions

(König et al., 2004). Extracts were fractionated by column chromatography on silica gel using

a hexane – diethyl ether gradient. Prenylindoles 14 and 15 were isolated upon repeated

column chromatography on silica gel 60 using a hexane – dichloromethane gradient.

Figure 11: TIC chromatogram of pentane / NaSO4 extract of fresh Riccardia chamedryfolia

from La Palma (30 m CpSil-5 CB, 80 °C, 2 min, + 10 °C/min, to 270 °C).

Figure 12: Sesquiterpene hydrocarbons (44 – 48) and prenylindoles (14, 15, 49 – 52)

from Riccardia chamedryfolia.

10 12 14 16 RT [min]

15

49

50 51

44

47

48

45

46

Sesquiterpenes Prenylindoles 50 TIC

[%]

14

10

20

30

40


19

Figure 13: Mass spectra (EI, 70 eV) of 7-prenylindole (14), 6-prenylindole (15),

3-chloro-7-prenylindole (49) and 3-chloro-6-prenylindole (50).

3951 63

77

83 89 103

117

130

143

155

170

185

40 60 80 100 120 140 160 180 200 220

20

40

60

80

100

39 51 63

77

84 89 103

117

130

143

155

170

185

40 60 80 100 120 140 160 180 200 220

20

40

60

80

100

41

51 6377

84101 115

128

141

154

164

169

188

204

219

40 60 80 100 120 140 160 180 200 220

20

40

60

80

100

41

55 63 6977 84

95102 115

128141

154

169

177

184

204

219

40 60 80 100 120 140 160 180 200 220

20

40

60

80

100

C13H15N●+

C13H15N●+

C9H8N+

C12H11N●+

C13H14NCl●+

C13H14NCl●+

C12H11NCl+

C12H11N●+

C12H11NCl+

C9H7NCl+

C12H12N●+

C12H12N●+


20

4.2.3. Identification and Synthesis of 3-Chloro-6- and 7-prenylindoles (49, 50)

Two chlorine containing indole alkaloids with molecular ion signals at m/z 219 [M] were

detected as minor (49) or trace constituents (50). The relative intensities of the [M + 2] signals

at m/z 221 indicated chlorine as a substituent (Figure 13). The molecular formula C13H14NCl

with seven units of unsaturation was established upon HREIMS. The mass spectrometric

fragmentation pattern of 49 was similar to those of 7-prenylindole (14) and exhibited

dominant chloroazaazulenium ion signals at m/z 204 [M – CH3] (100) for C12H11NCl+ and

m/z 164 [M – C4H7] (90) for C9H7NCl+, thus, indicating a prenylindole structure with an

aromatic chlorine substitution. The corresponding signals at m/z 204 [M – CH3] (60) and

m/z 164 [M – C4H7] (15) were less intense for the later eluting trace constituent 50, assumed

to represent the 6-prenyl derivative. Its mass spectrum was dominated by a base peak signal at

m/z 169 [M – CH3 – Cl] (100) for C12H11N●+

, which was significantly less pronounced for the

7-prenyl indole derivative (49) showing m/z 169 (50). Although rearranged chloroaza-

azulenium ions are almost identical for 6- and 7-prenylindoles, the observation that m/z 169

[M – CH3 – Cl] is predominating in the 6-prenyl isomer (50) only, suggested that different

charge distribution in the initial [M – CH3] fragment ion facilitates loss of the chlorine radical.

Quantum mechanical calculations (PM3, RHF) and Mulliken population analysis indicated

that the 6-butenylindol fragment ion [M – CH3] exhibits characteristic partial positive charges

located at 2-C, 3a-C, and 5-C of the indole nucleus, thus, suggesting the adjacent 3- (or 4-)

position for the chloro-substitution. This hypothesis was unambiguously established by

comparison with authentic samples of 7- and 6-prenyl-3-chloroindoles (49 and 50) obtained

by partial synthesis (Figure 14). Regioselective chlorination of indole (53) to 3-chloroindole

(54) has previously been described (De Rosa & Alonso, 1978). Reaction of 7- or 6-prenyl-

indole (14 and 15), isolated from Riccardia chamedryfolia, with N-chlorosuccinimide in

dichloromethane afforded the corresponding 3-chloro derivatives (49 and 50) in 85 and 80 %

yield, respectively.

Figure 14: Partial synthesis of 3-chloro prenylindoles (49 and 50).


21

Figure 15: 500 MHz 1H NMR spectra of 7-prenylindole (14) and 3-chloro-7-prenylindole (49).

The 3-chloro substitution was established by 1H NMR spectroscopy (Figures 15 and 16).

Comparison of the mass spectra and GC retention indices confirmed the identity of 49 and 50

with the natural products from Riccardia chamedryfolia.

2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 H [ppm]

12-CH3

11-CH3

8-CH2

9-CH

2-CH

NH

6-CH

5-CH

solvent

2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 H [ppm]

solvent

2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5

11-CH3

12-CH3

8-CH2

9-CH

3-CH

2-CH

6-CH

NH

5-CH

4-CH

X

4-CH


22

Figure 16: 500 MHz 1H NMR spectra of 6-prenylindole (15) and 3-chloro-6-prenylindole (50).

2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 H [ppm]

11-CH3

12-CH3

8-CH2

9-CH

2-CH

6-CH

NH

5-CH

4-CH

solvent

X

2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 H [ppm]

11-CH3

12-CH3

8-CH2

9-CH

3-CH

2-CH

7-CH

NH

5-CH

4-CH

solvent

X

X

X


23

4.2.4. Identification of Chamedryfolian (51)

One new oxazinoindole alkaloid (51) named chamedryfolian, with a molecular ion signal at

m/z 199 [M] (Figure 17) was isolated as a minor constituent by a combination of column

chromatography and thin layer chromatography on silica gel. The molecular formula

C13H13NO with eight units of unsaturation was established by GC-HREIMS. Keeping

structures of already identified indoles in mind and due to the base peak for an azaazulenium

ion C9H8N+

at m/z 130 [M – C4H5O] (100) the structure of a prenylindole carrying an oxygen

in the side chain was postulated.

Figure 17: Mass spectrum (EI, 70 eV) of chamedryfolian (51) from Riccardia chamedryfolia.

The 1H NMR and COSY spectra exhibited signals for a 7-substituted indole nucleus, along

with an oxygenated isopentenyl residue (Table 1, page 24), which, together with eight units of

unsaturation for the molecular formula C13H13NO implied a tricyclic indole alkaloid. 1H NMR

signals assigned to the sidechain pointed to one isopropenyl moiety and one anisochoric

benzylic methylene group at H 2.73 (1H, dd, 2J = 14.8 Hz,

3J = 3.8 Hz) and H 2.92 (1H, dd,

2J = 14.8 Hz,

3J = 7.9 Hz), adjacent to an oxygen linked methine group at H 4.38 (1H, dd,

3J = 7.9 Hz,

3J = 3.8 Hz). The large difference in vicinal coupling constants of the anisochoric

methylene hydrogens (3J = 7.9 Hz vs. 3.8 Hz) indicated their inclusion in a rigid structure and

suggested an oxacyclic bridge between the chiral methine group and the indole nitrogen

(Figure 18).

4151

6977 103

130

156171 184

199

40 60 80 100 120 140 160 180 200

20

40

60

80

100

[M] C13H13NO

C9H8N+


24

Figure 18: Important H,H-coupling constants, H,H-COSY and H,C-HMBC correlations

in chamedryfolian (51) from Riccardia chamedryfolia.

1H NMR 14 15 49 50 51

1-NH 7.25 1H s.br 6.77 1H s.br 6.91 1H s.br 6.38 1H s.br.

2 6.65 1H s.br 6.59 1H s.br 6.53 1H s.br 6.44 1H s.br. 6.72 1H s.br

3 6.56 1H s.br 6.49 1H s.br 6.55 1H s.br

4 7.63 1H

d 7.2

7.64 1H

d 8.2

7.71 1H

d 8.2

7.72 1H

d 7.9

7.64 1H

d 7.9

5 7.19 1H

dd 7.2 7.3

7.09 1H

d 7.9

7.12 1H

dd 7.9 7.3

7.03 1H

d 7.6

7.13 1H

dd 7.9 7.3

6 7.08 1H

d 7.3

7.01 1H

d 7.3

6.92 1H

d 7.3

7 7.00 1H s 6.83 1H s

8 3.32 2H

d 6.9

3.53 2H

d 7.3

3.18 2H

d 6.9

3.43 2H

d 7.3

2.73 1H

dd 14.8 3.8

2.92 1H

dd 14.8 7.9

9 5.34 1H

m

5.55 1H

m

5.24 1H

t 6.9

5.46 1H

t 7.2

4.38 1H

dd 7.9 3.8

11 1.57 3H s 1.67 3H s 1.53 3H s 1.63 3H s 4.79 1H s

4.82 1H s

12 1.61 3H s 1.72 3H s 1.61 3H s 1.70 3H s 1.58 3H s

Table 1: 1H NMR data of 7-prenylindole (14), 6-prenylindole (15), 3-chloro-7-prenylindole (49),

3-chloro-6-prenylindole (50), and chamedryfolian (51) from Riccardia chamedryfolia (chemical shift

H [ppm] in C6D6, integral, multiplicity, and coupling constants [Hz]; assignments derived from

1H NMR, COSY, HMQC, and HMBC spectra).


25

Although the isolated amount was too small to obtain 13

C {1H} NMR or

13C PENDANT

spectra, inverse detection of 13

C by a phase insensitive HSQC pulse sequence afforded the

chemical shifts of the hydrogen bond carbon atoms (Table 2). Inspection of the HMBC

spectrum confirmed the 7-prenylindole skeleton (Figure 18, page 24). Although the oxazino

indole moiety in 51 displayed only insignificant effects on the 1H and

13C NMR chemical

shifts of 2-and 3-methine groups in comparison to 7-prenylindole (14) (Table 1 and 2), the

9-methine group at C 90.1 ppm confirmed its inclusion in an oxacyclic ring. The 7-prenyl-

indole skeleton and the presence of a cleavable C-O-N bond were unambiguously established

by chemical correlation. Hydrogenation of 51 using Pd/C afforded the same 7-isopentyl-

indoline (56) as 7-prenylindole (14) (Figure 19). These results indicated the novel 2-(prop-1-

en-2-yl)-1,2-dihydro-[1,2]oxazino[4,3,2-hi]indole structure (51) which was termed chamedry-

folian.

13C NMR 14 15 49 50 51

2 123.8 d 123.6 d 120.8 d 120.3 d 123.8

3 103.3 d 102.6 d 107.0 s 106.2 s 102.8

3a 128.3 s 126.8 s 124.3 s 124.0 s nd

4 119.3 d 120.9 d 116.7 d 118.3 d 119.8

5 120.5 d 121.4 d 122.6 d 121.9 d 119.7

6 121.7 d 135.8 s 121.2 d 137.0 s 123.1

7 135.5 s 110.6 d 135.7 s 110.6 d nd

7a 135.6 s 136.8 s 134.3 s 135.6 s nd

8 30.7 t 35.2 t 30.0 t 34.7 t 34.5

9 122.8 d 125.1 d 122.8 d 124.3 d 90.1

10 132.8 s 131.5 s 133.1 s 131.8 s 143.8

11 17.8 q 17.8. q 17.7 q 17.7 q 113.2

12 25.6 q 25.9 q 25.6 q 25.8 q 17.7

Table 2: 13

C NMR data of 7-prenylindole (14), 6-prenylindole (15), 3-chloro-7-prenylindole (49),

3-chloro-6-prenylindole (50), and chamedryfolian (51) from Riccardia chamedryfolia (chemical shift

C [ppm] in C6D6 and multiplicity; assignments derived from 13

C {1H} NMR,

13C PENDANT, HMQC,

and HMBC spectra; 13

C NMR data of 51 derived from HSQC and HMBC spectra; nd = not detected).


26

4.2.5. Identification of 7-(3-Methylbutadienyl)indole (52)

A trace constituent with a molecular ion signal at m/z 183 [M] was enriched to 50 % purity by

column chromatography. The molecular formula C13H13N with eight units of unsaturation was

established by HREIMS. Although the available amount and its purity was too small to obtain

even a 1H NMR spectrum, the presence of 7-(3-methylbutadienyl)indole (52) was established

by chemical correlation with 7-prenylindole (14) (Figure 19). Hydrogenation of 52 and 14

with Pd/C afforded the same 7-isopentyl indole (55) and 7-isopentylindoline (56), as shown

by comparison of their mass spectra and GC retention indices. These results indicated a

7-(3-methylbutadienyl)indole (52) structure, but the stereochemistry of the internal double

bond remained unidentified. The corresponding (E)-6-(3-methylbutadienyl)indole has

previously been described from Monodora tenuifolia, Annonaceae (Nwaji et al., 1972; Ishii et

al., 1973; Ishii & Murakami, 1975; Onyiriuka & Nwaji, 1983; Somei, 1986).

Figure 19: Chemical correlation of 7-prenylindole (14) with chamedryfolian (51)

and 7-(3-methylbutadienyl)indole (52) (* absolute configuration not determined).


27

4.2.6. Discussion of Indole Alkaloids from Riccardia chamedryfolia

The reinvestigation of Riccardia chamedryfolia, one of the few liverworts known for the

presence of alkaloids, resulted in the identification of prenylindoles 14 and 15, as well as

3-chloro-7-prenylindole (49) and 3-chloro-6-prenylindole (50), along with a novel

oxazinoindole called chamedryfolian (51) (Figure 12, page 18). This tricyclic indole alkaloid

exhibits an unusual oxacyclic bridge between the 7-prenyl sidechain and the indole nitrogen.

N-Methoxyindole derivatives have previously been reported as phytoalexins from

Brassicaceae (Pedras et al., 2007). (Poly)-halogenated indoles are prominent in marine

organisms (Gribble, 1999) and 3-chloroindole (54) is known as the source of the disagreeable

odor of acorn worms, Ptychodera and Glossobalanus species. (Higa & Scheuer, 1975; Higa et

al., 1980), as well as the fungus Hygrophorus paupertinus, Agaricales (Wood et al., 2003). In

higher plants, however, organochlorine compounds are rare (Engvild, 1986; Monde et al.,

1998; Sugimoto et al., 2001). 4-Chloroindolacetic acid, 4-chlorotryptophan and related

derivatives were described from immature seeds of the tribe Vicieae, Fabaceae (Engvild,

1986). Chlorinated bibenzyls such as 9 (Figure 1, page 5) have recently been described from

the liverworts Riccardia marginata (Baeck, et al., 2004) and Riccardia polyclada (Labbé et

al., 2007), whereas chlorinated bis(bibenzyls) are known from Lepidozia incurvata (Scher et

al., 2003), Plagiochila deflexa (Anton et al., 1997), Bazzania trilobata (Martini et al., 1998;

Speicher et al., 2001), Herbertus sakuraii and Mastigophora diclados (Hashimoto et al.,

2000). Furthermore, the presence of haloperoxidase activity, capable of converting

bis(bibenzyls) to the corresponding chloro-derivatives, has been demonstrated in Bazzania

trilobata (Speicher et al., 2003). Consequently, conversion of prenylindoles (14 and 15) by in

vivo chloroperoxydase activity is proposed as the biosynthetic pathway to 3-chloroprenyl

indoles (49 and 50) from Riccardia chamedryfolia, similar to the partial synthesis (Figure 14,

page 20). Although GC-EIMS confirmed the presence of 14 (36 %) and 15 (16 %) in the

related Riccardia incurvata, 3-chloroprenylindoles (49 and 50) and chamedryfolian (51) were

exclusively present in R. chamedryfolia.

4.3. Secondary Metabolites from Corsinia coriandrina

28

4.3. Secondary Metabolites of Corsinia coriandrina

4.3.1. Taxonomy of the Corsiniaceae

The Marchantialean family Corsiniaceae Engler 1892, dedicated to the Italian botanist Tomas

Corsinii, comprise of two genera, Corsinia Raddi 1818 and Cronisia M.J. Berkeley 1857. The

monotypic genus Corsinia contains only a single species, Corsinia coriandrina (Sprengler)

Lindberg 1877 (Plate 4, page 212), whereas Cronisia consists of two species, Cronisia

weddellii (Mont.) Grolle 1977 (Plate 6, page 213) and Cronisia fimbriata (Nees.) Whittem. &

Bischl. 2001. The Corsiniaceae posses a peculiar combination of presumably primitive

features and are consequently regarded to fall nearest to a hypothetical ancestral type for the

Marchantiales (Bischler-Causse et al., 2005; Bischler & Whittemore, 2001).

Corsinia coriandrina forms light to yellow green dichotomously divided thalli, 3 – 6 mm

wide and up to 2 cm long (Plate 4, page 212). Antheridia developed at the thallus apex in a

groove and archegonia are initiated in a thallus crypt, shielded by a peltate scale. After

fertilization the calyptra proliferates into a sessile, warty structure which covers the

sporogonium (Plate 4). The 300 – 350 blackish-brown spores (90 – 130 µm in diam.) are only

released when the tissue around the sporophytes decays (Plate 7, page 213). Exceptional in

liverworts, sterile green cells, rather than elaters, are intermixed with the spores. Thalli

contain 1(2) layer(s) of large air chambers with chlorophyllose filaments on the ground (Plate

15, page 217) and a primitive air pore-like opening (Plate 5, page 212). Scattered oil cells are

widespread throughout the different tissues (Plate 15), each with a brownish oil-body ca.

25 x 30 µm in diameter (Plate 5). Because of its simple thallus, loosely aggregated

gametangia and absence of periodicity of gamete production, Corsinia is considered to be one

of the most primitive representatives of the order Marchantiales (Schuster, 1992).

Corsinia is distributed in warm-temperate climates, like Mediterranean- and Atlantic areas,

especially coastal portions of the Iberian Peninsula, and Macaronesia, in North America

within a limited area in Texas, and very rarely in the neotropics like Mexico, Brazil,

Argentina, and Chile. The disjunctive distribution (Schuster, 1984, 1992) and significant

genetic difference between Old and New World cytotypes (Boisselier-Dubayle & Bischler,

1998) has been interpreted as an indication for the development of the genus Corsinia in

Pangaean times prior to the separation of America from Europe due to plate tectonics. Recent

phylogenetic trees have placed Corsinia basal within the Marchantiales, thus, representing

one of the closest relative of the first terrestrial plants (Beckert et al., 1999; Pruchner et al.,

2001; Qiu, 2006). Three morphologically identical cytotypes have been described, which

4.3 Secondary Metabolites of Corsinia coriandrina

29

differ however in ploidy levels, habitat requirements, geographical-, and sex distribution.

Dioecious haploids (n = 8) are distributed in Atlantic areas, whereas two monoecious

polyploids (n = 16) of allopolyploid origin have been described from Mediterranean areas and

Texas, USA (Boisselier-Dubayle & Bischler, 1998).

Corsinia coriandrina was one of the first liverworts investigated for flavonoids in 1966 when

Reznik and Wiermann (1966) reported the presence of kaempferol (11) and quercetin (12)

(Figure 1, page 5), present mainly as 3-O-glycuronides (Markham, 1980; Müller & Mues,

1990) and Corsinia has remained a rare liverwort source for flavon-3-ols 11 and 12, which are

considered relatively advanced in the evolution of flavonoid metabolism (Stafford, 1991).

Furthermore, the liverwort specific phytohormone lunularic acid (5) and the corresponding

decarboxylation product lunularin (6) have been detected in C. coriandrina (Gorham, 1977a).

Comparative investigation of fatty acid composition indicated common saturated and

unsaturated compounds (Kohn et al., 1988).

4.3.2. Volatile Constituents of Corsinia coriandrina

Fresh plant material of dioecious Corsinia coriandrina (Sprengler) Lindberg 1877 from

Atlantic areas was collected by the late W.A. König on the Iberian Peninsula in spring 2003

and 2004. Additional samples were collected in the Guadiamar river basin (2005), a liverwort

hot spot (Guerra et al., 2002), as well as on the Islands of La Palma (2006) and Madeira

(2007). Bulk quantities were obtained from axenic in vitro cultures of monoclonal haploid

Corsinia coriandrina strain CC1 established from the spores of an Andalusian specimen

collected by the late W. A. König near St. Bartolomeo in April 2004 (Section 4.5, page 76).

In vitro cultured polyploid Corsinia coriandrina strain Lorbeer/33 was obtained from the

Culture Collection of Autotrophic Organisms (CCALA, Trebon, Czech Republic).

The carefully cleaned plant material was hydrodistilled or extracted with organic solvents.

Because some of the labile secondary metabolites were only detected in fresh plant material, a

procedure for solvent extraction of fresh thalli was developed, which uses sodium sulfate as a

drying reagent to bind tissue water and allows hydrophobic constituents to be extracted. Plant

material was frozen with liquid nitrogen, crushed in a mortar, mixed with sodium sulfate

while still frozen and the resulting mass extracted with organic solvents, usually diethyl ether.

Combined extracts were dried over sodium sulfate and concentrated in vacuum or a stream of

N2 to give green oils analyzed by GC, GC-EIMS (Figure 20) and GC-HREIMS.


30

Figure 20: TIC chromatogram of diethyl ether / Na2SO4 extract of fresh Corsinia coriandrina

from La Palma (30 m CpSil-5 CB, 80 °C, 2 min, + 10 °C/min, to 270 °C).

Several known compounds (Figure 21, page 31) were identified by comparison of the mass

spectra and GC retention indices with a spectral library established under identical

experimental conditions (König et al., 2004; König & Hochmuth, 2004). Monoterpene

hydrocarbons like -pinene (18) and myrcene (22) and sesquiterpenoids like (E)- -caryo-

phyllene (62) and (E)-nerolidol (64), as well as traces of sesquisabinene A and B,

-acoradiene, and -acoradiene were regularly detected, along with simple aliphatics like

decyl acetate (61) and trace amounts of aromatics like 4-methoxybenzaldehyde (58),

4-methoxyphenylethanal (59) and 4-methoxyphenylethanol (60). In addition, S,S-dimethyl-

trisulfide, S,S-dimethyltetrasulfide or S-methyl methanethiosulfonate (58) were identified as

minor constituents (Figure 21). The odd molecular ion signals of various unidentified

constituents ((Z/E)-63 at m/z 175 [M], (Z/E)-65 at m/z 191 [M], (Z/E)-67 at m/z 223 [M], 71 at

m/z 237 [M], and (Z/E)-72 at m/z 253 [M]) indicated nitrogen containing compounds, which

are virtually unknown in liverworts. Furthermore, various aromatic compounds with

molecular ion signals at m/z 240 [M] for (Z/E)-68, m/z 242 [M] for 69, m/z 258 [M] for 70,

m/z 256 [M] for 73, and m/z 254 [M] for 74 were detected. Crude diethyl ether extracts of

Corsinia coriandrina were fractionated by column chromatography on silica gel. Fractions

were analyzed by TLC, GC, GC-EIMS and enantioselective GC. Elution with hexane

afforded the terpenoid hydrocarbons (4S,6S)-(–)- -pinene (18, ee = 100 %), (–)-(E)- -caryo-

phyllene (62, ee = 100 %) and (E,E)- -springene (66), which were partially separated

60 61

(Z)-67

5 10 15 20

20

40

60

80

18

22 57

64

(Z)-65

(E)-65

73

74

(E)

68

66

(E)

72

(Z)

72

(Z)

68

69 71

(E)

67

(Z)-63

(E)-63

59 58

[%] TIC

RT [min]

70

62


31

according to their number of isoprene units (Figure 22). Structure elucidation of (E,E)- -

springene (66), previously described from glands of springbok, Antidorcas marsupialis

(Burger et al., 1978), caiman (Avery et al., 1993) and alligator species (Schulz et al., 2003),

male bumblebees (Bertsch et al., 2004), and the ectoparasitoid Habrobracon hebetor

(Hymenoptera) (Howard et al., 2003), was performed by 1H and

13C NMR techniques.

Figure 21: Known compounds identified in Corsinia coriandrina.

Figure 22: Alignment of TIC chromatograms from GC-EIMS measurements of five consecutive

column chromatographic fractions shows partial separation of terpenoid hydrocarbons like

(–)- -pinene (18), myrcene (22), (–)-(E)- -caryophyllene (62) and (E,E)- -springene (66).

5

66

62

22

18

10 15 RT [min]

20

40

60

80

TIC [%]

C10 C15 C20

CC (SiO2)


32

Stepwise increase of diethyl ether concentrations eluted the more polar compounds in the

following order: (Z/E)-coriandrins ((Z/E)-65), decyl acetate (61), (Z)-O-methyltridentatol B

((Z)-72), (E)-corsistilbene ((E)-68), O,O-dimethyllunularin (69), corsifuran C (74), (R)-(–)-

corsifuran A (73), (Z)-corsiandren ((Z)-67), (R)-(–)-(E)-nerolidol (64, ee = 75 – 85 %),

6-hydroxydimethyllunularin (70), as well as odd and even numbered fatty acids (C14 – C18).

For NMR investigation pure compounds were isolated from these enriched fractions by

repeated column chromatography, thin layer chromatography or preparative gas chromato-

graphy as described for the individual compounds.

4.3.3. Identification and Synthesis of 4-Methoxystyrenes from Corsinia coriandrina

4.3.3.1. (Z/E)-Coriandrins (65)

Figure 23: Mass spectrum (EI, 70 eV) of (Z/E)-coriandrins ((Z/E)-65) from C. coriandrina.

The major volatile constituent in Corsinia coriandrina extracts (50 – 70 % of the total

volatiles), a new isothiocyanate compound called (Z)-coriandrin ((Z)-65), was isolated by

column chromatography on silica gel, along with minor amounts of the corresponding

(E)-isomer ((E)-65) showing identical mass spectra (Figure 23). The dominating molecular

ion signal at m/z 191 [M] (100) indicated nitrogen containing compounds, whereas the

relative intensity of the [M + 2] signal (5) suggested the presence of sulfur in the molecule.

The molecular formula C10H9NOS with seven units of unsaturation was established by GC-

HREIMS.

3945

51

63

77

89

95

104

116

121

133

148

161

176

191

40 60 80 100 120 140 160 180 200 220 240 260

20

40

60

80

100

C10H9NOS

C9H6NOS

C8H6NS

C7H6N


33

Figure 24: 500 MHz 1H NMR spectrum of (Z)-coriandrin ((Z)-65 in C6D6) from C. coriandrina.

Figure 25: Section of the HMBC spectrum of (Z)-coriandrin ((Z)-65) showing long range

H,C-correlations of the (Z)-configured ethylene bridge to the isothiocyanate carbon.

3.84.24.65.05.45.86.26.67.07.4 H [ppm]

OCH3

solvent

2‘,6‘-CH 3‘,5‘-CH

2-CH

1-CH

3JZ = 8.2 Hz

3.84.24.65.05.45.86.26.67.07.4 H [ppm]

OCH3

solvent

2‘,6‘-CH 3‘,5‘-CH

2-CH

1-CH

3JZ = 8.2 Hz

5.6 5.4 5.2 5.0

144

136

128

120

112

104

1-H

2-H

1-CH

R-N=C=S

2-CH

1JH,C

3JH,C 4JH,C

3JH,C

2JH,C

3JH,C

2JH,C

3‘,5‘-CH

1‘-C

2-CH

[ppm]

(Z)-65

2‘,6‘-CH


34

Figure 26: GC-FTIR spectrum of (Z)-coriandrin ((Z)-65).

The 1H NMR spectrum of (Z)-coriandrin ((Z)-65) (Figure 24) exhibited one AA‟BB‟ spin

system for one para-disubstituted benzene unit, one AB spin system corresponding to a

(Z)-configured ethenyl bridge (3JZ = 8.2 Hz) and one singlet for a methoxy group, in

agreement with carbon signals for four types of methine groups, one oxygen-bound methyl

group, and two quaternary carbons in 13

C PENDANT and proton decoupled 13

C {1H} NMR

spectra. Inspection of two dimensional COSY, HMQC and HMBC spectra confirmed the

(Z)-4-methoxy- -styrene skeleton, which accounts for five of the seven units of unsaturation.

The remaining quaternary carbon at C 135.6 ppm was only detected by 3J- and

4J-H,C-

correlation signals in the HMBC spectrum (Figure 25). From its chemical shift as well as

intensive valence vibration bands at ν = 2096 cm–1

and 2058 cm–1

in the GC-FTIR spectrum

(Figure 26), the heterocumulene substituent was identified as an isothiocyanate group. As a

result, the target compound proved to be (Z)-2-(4-methoxyphenyl)ethenyl isothiocyanate

((Z)-65). Small amounts of the isomeric (E)-coriandrin ((E)-65) were also detected in the

diethyl ether extract of C. coriandrina.

The structure of the coriandrins ((Z/E)-65) was established unambiguously by synthesis as

shown in figure 27 (page 35). 1-(4-Methoxyphenyl)ethanone (75) was converted to the

2-bromo derivative (76). Nucleophilic substitution using NaN3 furnished 2-azido-

1-(4-methoxyphenyl)ethanone (77), which was reduced to (±)-2-hydroxy-2-(4-methoxy-

phenyl)ethylamine ((±)-78) using LiAlH4. After conversion to the hydrochloride (±)-79,

treatment with thionylchloride gave the corresponding (±)-2-chloro-2-(4-methoxyphenyl)-

ethylammonium chloride ((±)-80) in 48 % overall yield, which served as a precursor for the

synthesis of various Corsinia constituents.

70

80

90

100

4000 3600 3200 2800 2400 2000 1600 1200 800

Wavenumber (cm1)

1609

1512

1398

1177

1042

2058

2096

1260

[%]

C-O-C R-N=C=S

C=C

C=C


35

Thiophosgenation of (±)-80 under basic conditions afforded (±)-2-chloro-2-(4-methoxy-

phenyl)ethyl isothiocyanate ((±)-81) in moderate yield (Kniezo et al., 1978). Gas phase

dehydrohalogenation gave a 1:1 mixture of (Z)- and (E)-2-(4-methoxyphenyl)ethenyl

isothiocyanates ((Z/E)-65) identical to the coriandrins from Corsinia coriandrina (von Reuß

& König, 2005). (Z/E)-Coriandrins ((Z/E)-65) could also be detected in a herbarium specimen

of the related Cronisia weddellii, Corsiniaceae (Plate 6, page 213).

Figure 27: Synthesis of (Z/E)-coriandrins (65) and (Z/E)-O-methyltridentatols B & A (72).


36

4.3.3.2. (Z/E)-O-Methyltridentatols B & A (72)

Figure 28: Mass spectrum (EI, 70 eV) of O-methyltridentatols (72) from C. coriandrina.

Minor quantities of two isomeric S,S-dimethyl iminodithiocarbonate compounds ((Z/E)-72),

with a signal for the molecular ion at m/z 253 [M], were regularly observed in the diethyl

ether extract of C. coriandrina (< 3 % of the total volatiles). High-resolution mass

spectrometry afforded the molecular formula of C12H15NOS2 with six units of unsaturation.

The high sulfur content of the compounds was also indicated by a relatively intense signal at

[M + 2] (9 %). The mass spectra of (Z/E)-72 (Figure 28) were similar to those of the

coriandrins (Z/E)-65 (Figure 23, page 32) and exhibited a signal for a coriandrin radicalion

(C10H9NOS+) at m/z 191 [M – SCH3 – CH3], suggesting a similar carbon skeleton. A

(Z)-2-(4-methoxyphenyl)ethenyl structure was deduced from the 1H NMR spectrum of

isolated (Z)-72 in agreement with the long-range coupling correlations in the COSY spectrum.

Although predominantly present as the (Z)-isomer (Z)-72, the corresponding (E)-isomer

(E)-72 was also detected. The heteroatomic substituent of (Z/E)-72 could be deduced from the

mass spectrometric fragmentation, in combination with the HREIMS measurements. The

presence of a heterocyclic moiety was excluded as the signal at m/z 206 [M – SCH3] forms the

base peak, indicating an S,S-dimethyl iminodithiocarbonate moiety. This is in agreement with

the formation of 4-methoxyphenylethanal (59) and S,S-dimethyl dithiocarbonate upon

hydrolysis during hydrodistillation. The S,S-dimethyl (Z)- and (E)-2-(4-methoxyphenyl)-

ethenyl iminodithiocarbonate structures of (Z)-72 and (E)-72 were confirmed by comparison

of the mass spectra and GC retention indices with authentic samples prepared as shown in

figure 27. Addition of [sodium(18-crown-6)] methanethiolate complex in THF to coriandrins

((Z)-65 and (E)-65) and subsequent methylation afforded the corresponding S,S-dimethyl

iminodithiocarbonates (Z)-72 and (E)-72, identical to the natural products from Corsinia

coriandrina (von Reuß & König, 2005).

39 45 51

63 77

89

95 103 121

133

148

158

165

176

191

206

253

40 60 80 100 120 140 160 180 200 220 240 260

20

40

60

80

100

C10H9NOS

C9H6NOS

C11H12NOS

C12H15NOS2

C10H8NO


37

4.3.3.3. O-Methyltridentatol C (71)

Figure 29: Mass spectrum (EI, 70 eV) of O-methyltridentatol C (71) from C. coriandrina.

One aromatic compound (71) with a dominating molecular ion signal at m/z 237 [M] (Figure

29) was regularly detected as a trace constituent in Atlantic Corsinia collections (< 0.5 % of

the total volatiles). The molecular formula C11H11NOS2 with 7 degrees of unsaturation was

established by GC-HREIMS. The presence of two sulfur atoms was also indicated by a

relatively intense signal at [M + 2] (9 %) in agreement with a signal at m/z 204 [M – SH] for

the major ion fragment C11H10NOS+ by HREIMS. Additional signals of lower intensity

suggested the presence of an S-methylthio group due to C9H8NOS+ at m/z 178 [M – CSCH3],

possibly as part of an S-methylthiocarbimino substructure, as indicated by retro cleavage to

produce C9H8OS+ at m/z 164 [M – NCSCH3]. Furthermore, an aromatic methoxy group was

indicated by a signal at m/z 121 [M – NCSCH3 – COCH3] for C7H5S+. The observation of a

small signal at m/z 132 [M – NCS2CH3] for C9H8O+ consequently pointed to a 4- or 5-aryl-

2-methylthio-1,3-thiazole. Considering the carbon skeletons and the substitution patterns of

the co-occurring constituents (Z/E)-65 and (Z/E)-72 these results suggested 5-(4-methoxy-

phenyl)-2-methylthio-1,3-thiazole as the structure for 71.

This hypothesis was confirmed by a new synthesis as shown in figure 30. Treatment of

(±)-2-chloro-2-(4-methoxyphenyl)ethylammonium chloride ((±)-80) with carbon disulfide

and three equivalents of Hünig base, followed by methylation, furnished thiazolidine (±)-(82)

in excellent yield. Dehydrogenation of (±)-82 using 2,3-dichloro-5,6-dicyano-1,4-benzo-

quinone (DDQ) in 1,4-dioxane (Jayatilake & Baker, 1999) afforded 5-(4-methoxyphenyl)-2-

methylthio-1,3-thiazole (71), which was shown to be identical to the natural product from

Corsinia coriandrina by comparison of mass spectra and gas chromatographic retention

indices.

45 51 57 63 69

7789

121 132

149

164

178

189

204

222

237

40 60 80 100 120 140 160 180 200 220 240

20

40

60

80

100 C11H11NOS2

C11H10NOS

C9H8NOS

C9H8OS

C8H5OS

C7H5S

C9H8O


38

Figure 30: Synthesis of O-methyltridentatol C (71).

The free phenols ((E)-83, (Z)-83 and 84) and O-sulfates ((E)-85, (Z)-85 and 86) corresponding

to O-methyltridentatols A – C ((E)-72, (Z)-72 and 71) have previously been described to act

as defence compounds and sun protection agents of the marine hydroid Tridentata marginata

(Figure 31) (Lindquist et al., 1996; Stachowicz & Lindquist, 1997, 2000; Lindquist, 2002).

Due to potent antioxidant and UV-B absorbing properties the application of tridentatol A – C

((E/Z)-83, 84) and their O-methyl derivatives ((E/Z)-72, 71) in topical sunscreen and

antioxidant formulas has been patented (Lindquist, 1998; Lindquist & Loo, 1999; Johnson et

al., 1999).

Figure 31: Tridentatols A – C ((E/Z)-83, 84) and D – F ((E/Z)-85, 86) from the marine hydroid

Tridentata marginata and O-methyltridentatols A – C ((E/Z)-72, 71) from Corsinia coriandrina.


39

4.3.3.4. (Z/E)-Corsinians (63)

Figure 32: Mass spectrum (EI, 70 eV) of corsinians (63) from C. coriandrina.

Labile corsinians ((Z/E)-63), which represent the first isocyanate compounds of terrestrial

plant origin, were regularly detected as minor constituents of Corsinia coriandrina (< 5 % of

the total volatiles) but decomposed within hours after extraction of the fresh plant material

unless sodium sulfate was employed as a drying reagent during the extraction procedure. The

molecular ion signal at m/z 175 [M] was assigned a molecular formula of C10H9NO2 with 7

degrees of unsaturation by GC-HREIMS. An aromatic methoxy group was deduced from

fragment ion signals for C9H6NO2+ at m/z 160 [M – CH3] and C8H6NO

+ at m/z 132 [M –

COCH3]. The mass spectrometric fragmentation pattern (Figure 32) resembled those of

isothiocyanates (Z/E)-65 (Figure 23, page 32), indicating a similar structure, but the common

signal for C7H6N+ at m/z 104 [M – COCH3 – CO] suggested an isocyanate moiety in the case

of corsinians ((Z/E)-63). This hypothesis was established unambiguously by comparison with

synthetic (Z/E)-2-(4-methoxyphenyl)ethenyl isocyanates (63) prepared as shown in figure 33.

Figure 33: Synthesis of (Z/E)-corsinians (63) from Corsinia coriandrina.

39

51

63

77

91104

120

132

146

160

175

40 60 80 100 120 140 160 180 200 220 240 260 280

20

40

60

80

100

C10H9NO2

C8H6NO

C7H6N

C9H6NO2


40

(±)-2-Chloro-2-(4-methoxyphenyl)ethylammonium chloride ((±)-80) was available in 4 steps

from 4-methoxyphenylethanone (Figure 27, page 35). Phosgenation of (±)-80 with

triphosgene under basic conditions afforded the corresponding isocyanate (±)-(87) in

moderate yield, which could be isolated by column chromatography on silica gel. Gas phase

dehydrohalogenation of (±)-87 at 200 °C during GC-EIMS gave ca. 60 % of a 1:1 mixture of

(Z/E)-2-(4-methoxyphenyl)ethenyl isocyanates ((Z/E)-63) identical to the corsinians from

Corsinia coriandrina.

Attempted large scale conversion using a preparative GC equipped with a packed column of

polysiloxane SE-30 on chromosorb WHP was hampered by the high reactivity of the

isocyanates. Because isolated yields were unacceptably small (< 30 %), (E)-corsinian ((E)-63)

was prepared by Curtius rearrangement of (E)-4-methoxycinnamoyl azide (91) as shown in

figure 34 (Anzai, 1962; Massey & Harrison, 1977; Brettle & Mosedale, 1988). 4-Methoxy-

cinnamic acid (89) was obtained upon condensation of 4-methoxybenzaldehyde (58) with

malonic acid (88) and converted to the acid chloride (90) using thionylchloride. Nucleophilic

substitution with NaN3 furnished (E)-4-methoxycinnamoyl azide (91) which was rearranged

to the desired isocyanate (E)-63 upon reflux in toluene (Brettle & Mosedale, 1988). UV

irradiation at 350 nm afforded a 2:3 mixture of the (Z/E)-isomers (Z)-63 and (E)-63 that

proved to be identical to the natural products from C. coriandrina. Although (Z/E)-corsinians

((Z/E)-63) have previously been described as synthetic compounds (Anzai, 1962; Massey &

Harrison, 1977; Brettle & Mosedale, 1988), their detection in Corsinia coriandrina represents

the first example of isocyanates in terrestrial plants (Scheuer, 1992; Chang, 2000; Garson &

Simpson, 2004). (Z/E)-Corsinians ((Z/E)-63) could also be detected in a herbarium specimen

of the related Cronisia weddellii, Corsiniaceae (Plate 6, page 213).

Figure 34: Synthesis of (Z/E)-corsinians (63) by Curtius rearrangement.


41

4.3.3.5. (Z/E)-Corsiandrens (67)

Figure 35: Mass spectrum (EI, 70 eV) of corsiandrens (67) from C. coriandrina.

Two new isomeric styrene S-methyl thiocarbamates termed corsiandrens ((Z/E)-67) with a

molecular ion signal at m/z 223 [M] were occasionally observed as minor constituents of

Corsinia coriandrina (< 5 % of the total volatiles). The molecular formula C11H13NO2S with

6 degrees of unsaturation was established by GC-HREIMS. The mass spectra (Figure 35)

were very similar to those of the corsinians ((Z/E)-63) (Figure 32, page 39) and exhibited a

base peak signal at m/z 175 [M – HSCH3] for a corsinian radicalion (C10H9NO2+). The

presence of a methanethiol adduct was also indicated by a fragment ion signal at m/z 47 (19)

[SCH3]. A carbonyl-linked S-methylthio group was deduced from a characteristic signal for

C9H10NO+ at m/z 148 [M – COSCH3], which suggested an S-methyl thiocarbamate moiety for

the corsiandrens ((Z/E)-67).

The structure and relative configuration of the natural products was established by synthesis

as shown in figure 36. In contrast to the corresponding isothiocyanates (Z/E)-65 (Figure 27,

page 35), addition of [sodium(18-crown-6)] methanethiolate complex in THF to the

isocyanate (E)-63 afforded only 4-methoxyphenylacetaldehyde (59), most likely originating

from the hydrolysis of a presumed polymerisation product. Attempted addition of gaseous

methanethiol also failed. Treatment of (E)-corsinian ((E)-63) with a sodium methanethiolate

suspension in DMF afforded the desired S-methylthiocarbamate ((E)-67) in very small yield

(< 2 %), which was finally increased to ca. 50 % using a sodium methanethiolate solution in

DMSO as shown in figure 36.

39

47

63

77

91

104

120

132

148

160

175

223

40 60 80 100 120 140 160 180 200 220 240 260 280

20

40

60

80

100

C11H13NO2S

C10H9NO2

C9H10NO

C8H6NO

CH3S


42

Figure 36: Synthesis of (Z)- and (E)-Corsiandren ((Z/E)-67)

(E)-corsiandren ((E)-67) exhibited intensive UV absorption in the UV-B range ( max = 288

nm, = 25.700) and UV irradiation of (E)-67 at 350 nm afforded a 3:1 mixture of the (Z/E)-

corsiandrens ((Z/E)-67) identical to the natural products from C. coriandrina. Upon gas

chromatographic analysis, S-methylthiocarbamates (Z/E)-67 partially decomposed in the

injector port (200 °C) to afford the corresponding isocyanates (Z/E)-63. Nevertheless, the

genuine presence of (Z/E)-63 in C. coriandrina was confirmed by their detection in samples

devoid of (Z/E)-67.


43

4.3.3.6. (Z/E)-Corsiandrenins (92)

Figure 37: Mass spectrum (EI, 70 eV) of corsiandrenins (92) from polyploid C. coriandrina.

The GC-EIMS investigation of in vitro cultured polyploid Corsinia coriandrina strain

Lorbeer/33 obtained from CCALA (Section 4.5.3, page 77) indicated the presence of

coriandrins ((Z/E)-65), corsinians ((Z/E)-63), corsiandrens ((Z/E)-67) and O-methyl-

tridentatols A – C ((Z/E)-72 and 71), along with several yet unidentified nitrogen containing

compounds. One minor constituent that was termed corsiandrenin (92) exhibited a molecular

ion signal at m/z 265 (23) [M]. The molecular formula C13H15NO3S with 7 degrees of

unsaturation was established by HREIMS. The mass spectrum of 92 (Figure 37) was very

similar to those of the (Z/E)-corsiandrens (67) (Figure 35, page 41) and displayed fragment

ion signals at m/z 223 [M – CH2CO] for a corsiandren (67) radicalion (C11H13NO2S+),

m/z 175 [M – CH2CO – HSCH3] for a corsinian (63) radicalion (C10H9NO2+), and m/z 148

[M – CH2CO – COSCH3] for C9H10NO+. The loss of ketene and an intensive fragment ion

signal at m/z 43 (73) for CH3CO+ suggested an O- or N-acetyl group.

Because the S-methyl iminothiocarbonate O-acetic anhydride structure was considered

unstable (Galli et al., 1982) an N-acetyl S-methylthiocarbamate moiety was proposed,

although this functional group has never been described from a natural product before.

Because no synthesis of N-acetyl-N-ethenyl-S-methylthiocarbamates was found in the

literature, a new procedure had to be established. Attempted N-acetylation of (E)-corsiandren

((E)-67) under various conditions failed (acetic anhydride or acetyl chloride with pyridine,

Hünig base, DMAP, K2CO3 or pre-treatment with NaH). Upon reaction of isocyanate (E)-63

with sodium methanethiolate in DMSO, the corsiandren anion was obtained in situ, which

could subsequently be acetylated with equimolar amounts of acetic anhydride to give

(E)-corsiandrenin ((E)-92) in moderate yield (Figure 38).

43

5163

77

91 104121

132

148

160

175

223

265

40 60 80 100 120 140 160 180 200 220 240 260 280

20

40

60

80

100

C10H9NO2

C9H10NO

C2H3O

C13H15NO3S

C11H13NO2S


44

Figure 38: Synthesis of (Z/E)-corsiandrenins (92).

UV irradiation at 350 nm afforded a 3:1 mixture of (Z/E)-isomers ((Z/E)-92), of which the

(Z)-product (Z)-92 was identical to the natural product from polyploid Corsinia coriandrina

as shown by comparison of the mass spectra and GC retention times.

The new reaction procedure is also applicable to the synthesis of N-alkyl-N-ethenyl-S-methyl

thiocarbamates (Figure 39). Treatment of (E)-2-phenylethenyl isocyanate (96), obtained in

3 steps from (E)-cinnamic acid (93), with sodium methanethiolate in DMSO, followed by

dimethyl sulfate afforded (E)-2-phenylethenyl-N,S-dimethyl thiocarbamate (97). This

sequence represents the first total synthesis of antifungal dehydroniranin A (97) previously

described from Glycosmis cyanocarpa, Rutaceae (Greger et al., 1996).

Figure 39: Synthesis of dehydroniranin A (97) from Glycosmis cyanocarpa.


45

4.3.3.7. (Z/E)-Tuberines (98)

Figure 40: Mass spectrum (EI, 70 eV) of tuberines (98) from C. coriandrina.

Trace amounts of isomeric (Z/E)-tuberines ((Z/E)-98) were exclusively detected in cultured

plant material of haploid or polyploid Corsinia coriandrina (< 1 % of the total volatiles). The

mass spectrum exhibited a dominating molecular ion signal at m/z 177 [M] (Figure 40), which

was assigned the molecular formula of C10H11NO2 with 6 degrees of unsaturation by

HREIMS. Fragment ion signals at m/z 162 [M – CH3] and m/z 134 [M – COCH3] indicated

the presence of an aromatic methoxy group. The N-formamide moiety was deduced from

characteristic fragment ion signals at m/z 148 [M – CHO] for C9H10NO+, m/z 132

[M – H2NCHO] for a 4-methoxyphenylethin ion (C9H8O+), and m/z 121 [M – CHO – HCN]

for a methoxytropylium ion (C8H9O+), which suggested an 2-(4-methoxyphenyl)ethenyl

N-formamide structure known as tuberine (98) (Anzai, 1962; Nagatsu et al., 1963).

(E)-Tuberine ((E)-98) was prepared by reduction of (E)-corsinian ((E)-63) using lithium tris-

tert-butoxyaluminium hydride (Figure 41) (Massey & Harrison, 1977). UV irradiation of

(E)-98 afforded a 1:2 mixture of the (Z/E)-isomers (Z/E)-98, exhibiting identical mass spectra

and GC retention indices as the natural products from Corsinia coriandrina.

Figure 41: Synthesis of (Z/E)-tuberines (98).

39

51

63

77

89 104

121

134

148162

177

40 60 80 100 120 140 160 180

20

40

60

80

100

C10H11NO2

C8H8NO

C9H10NO

C9H8O

C8H9O

C9H8NO2


46

(E)-Tuberine ((E)-98) has previously been described from Streptomyces amakusaensis,

Actinomycetes (Anzai, 1962; Nagatsu et al., 1963), whereas the free phenol corresponding to

(Z)-tuberine ((Z)-98) has been known from Aspergillus fumigatus, Ascomycota (Umehara et

al., 1984).

4.3.3.8. (Z/E)-Corsicillins (99)

Two isomeric isonitriles called corsicillins ((Z/E)-99) with a molecular ion signal at m/z 159

(100) [M] were occasionally detected as trace constituents of in vitro cultured Corsinia

coriandrina only (< 1 % of the total volatiles). The molecular formula of C10H9NO with

7 degrees of unsaturation was established by HREIMS. Inspection of the mass spectral

fragmentation pattern (Figure 42) indicated an aromatic methoxy group due to signals at

m/z 144 [M – CH3] and m/z 116 [M – COCH3]. A nitrile (cyano; R-C≡N) or isonitrile

(isocyano; R-N+≡C ) moiety was deduced from a fragment ion signal at m/z 89

[M – COCH3 – HCN] for an ethinylcyclopentadienyl ion (C7H5+).

Figure 42: Mass spectra (EI, 70 eV) of (Z/E)-corsicillins ((Z/E)-99) from Corsinia coriandrina

and (E)-4-methoxycinnamonitrile ((E)-100).

39

51

63

75

89

102

116

129

144

159

40 60 80 100 120 140 160

20

40

60

80

100

(E)-100

39 51

63

75

89

102

116

129

144

159

40 60 80 100 120 140 160

20

40

60

80

100

C8H6N

C7H5

C9H6NO

C10H9NO


47

The mass spectra were almost identical to those of (E)-4-methoxycinnamonitrile (100) (Figure

42) obtained by condensation of 4-methoxybenzaldehyde (58) with acetonitrile (101) (Figure

43), but GC retention times were different. These results suggested 2-(4-methoxyphenyl)-

ethenyl isonitrile structures for (Z/E)-99. Attempted dehydration of (E)-tuberine ((E)-98)

using phosphoroxychloride in pyridine (Ugi & Meyr, 1960; Hagedorn et al., 1965) afforded

only traces of the desired isonitrile (E)-99 (< 1 %). Nevertheless, Wittig reaction of 58 with

diethylisocyanomethylene phosphonate (102) furnished a 1:1.7 mixture of (Z)- and (E)-

2-(4-methoxyphenyl)ethenyl isonitriles ((Z/E)-99) (Schöllkopf & Schröder, 1973; Hoppe &

Schöllkopf, 1984; Isshiki et al., 1987) identical to the natural products from Corsinia

coriandrina (Figure 43). Although isonitriles (Z)-99 and (E)-99 have previously been

described as synthetic compounds, their detection in Corsinia coriandrina represents the first

example for isocyanides in terrestrial plants, which have previously been described from

marine and microbial sources only (Scheuer, 1992; Chang, 2000; Garson & Simpson, 2004).

Figure 43: Synthesis of (E)-4-methoxycinnamonitrile ((E)-100) and (Z/E)-corsicillins ((Z/E)-99).


48

4.3.3.9. Discussion of 4-Methoxystyrenes from Corsinia coriandrina

Figure 44: 4-methoxystyrenes from Corsinia coriandrina and Cronisia weddellii (63, 65).

The 4-methoxystyrenes of Corsinia coriandrina are unique for liverworts and exhibit a

variety of rare functional groups like isocyanate (63), isocyanide (99), and N-formamide (98)

moieties, which have not been reported from terrestrial plants before (Figure 44). Results

from bioassays indicating insecticidal and fungicidal activities of (Z)-coriandrin ((Z)-65), the

major volatile constituent of Corsinia (40 – 70 % of the total volatiles), suggested their

importance in chemical defence against herbivore and fungal attacks. (Z/E)-Coriandrins

((Z/E)-65) and (Z/E)-corsinian ((Z/E)-63) were also detected in South American Cronisia

weddellii (Plate 6, page 213), the second of three species which constitute the family

Corsiniaceae (Bischler & Whittemore, 2001; Bischler-Causse et al., 2005). The presence of

characteristic 4-methoxystyrenes in European Corsinia coriandrina and South American

Cronisia weddellii, considered to have divided during the Ordovician period (490 – 440

million years ago) prior to the separation of land masses due to plate tectonics, indicated an

ancient biosynthetic pathway, dating back to the early beginning of terrestrial plant evolution.


49

Figure 45: Isothiocyanates and related compounds from terrestrial plants and marine organisms.

At least 120 glucosinolate derived isothiocyanates, commonly known as mustard oils (103

and 104) have been described from higher plants, clustered mainly among the Brassicaceae,

Capparaceae, and Caricaceae (Figure 45). Their origin from L-amino acids or chain elongated

homo-amino acids and the rearrangement of the corresponding glucosinolate intermediates to

the isothiocyanates (103 and 104, respectively) have been extensively studied (Halkier & Du,

1997; Fahey et al., 2001). In addition, S-methyl thiocarbamates ((Z/E)-67) and S,S-dimethyl

iminodithiocarbonates ((Z/E)-72) from Corsinia exhibit similarities to L-tryptophan derived

phytoalexins of the Cruciferae, like brassitin (105), brassenin (106), wasalexin (107), and

spirobrassenin (108) which are produced de novo via labile indol-3-ylmethyl isothiocyanate

(Monde et al., 1991, 1994, 1996; Pedras et al., 2000, 2004, 2007). Similar styrene derivatives

have also been reported from aquatic organisms. The free phenols (83 and 84) and O-sulfates

(85 and 86) corresponding to 71 and (Z/E)-72 from Corsinia have previously been described

as chemical defence and UV protection agents of the marine hydroid Tridentata marginata,

Sertulariidae (Figure 31, page 38) (Lindquist et al., 1996; Lindquist, 2002). Furthermore,

several sesqui- and diterpenoid isothiocyanates, isonitriles, N-formamides, and isocyanates

with various carbon skeletons like spiroaxane (227 – 230), cubebane, or cadinane structures,

have been described from marine invertebrates (Figure 133, page 148). Biosynthetic studies

using radioactive labelled compounds have shown that these compounds are derived from a

sesquiterpene carbocation and cyanide ions (CN ) (Garson & Simpson, 2004).


50

Figure 46: Isonitriles and N-formamides from microorganisms

Interestingly, the Corsinia styrenes exhibit their closest structural similarities with secondary

metabolites of microbial origin (Figure 46). Antibiotic xanthocillin X (109), a formal

1,1-dimer of (Z)-corsicillin ((Z)-99), was isolated as the first natural isocyanide compound

from Penicillium notatum by Rothe (1954). Its structure was determined by X-Ray crystal

structure analysis and proven by synthesis (Tatsuta & Yamaguchi, 2005). Numerous related

derivatives have been described from various ascomycotean fungi (Vesonder 1979; Itoh et al.,

1990; Tsunakawa et al., 1993; Morino et al., 1994; Zapf et al., 1995). Cordyformamide (110),

the corresponding N-formamide, has recently been described from Cordyceps brunnearubra

(Isaka et al., 2007). Furthermore, the corresponding monomer, antibiotic (E)-tuberine ((E)-98)

is known from the actinomycete Streptomyces amakusaensis (Anzai, 1962; Nagatsu et al.,

1963), while the (Z)-configured phenol WF-5239 ((Z)-111), a potent platelet aggregation

inhibitor, has been described from the ascomycotean fungi Aspergillus fumigatus (Umehara et

al., 1984). The biogenesis of these compounds has been subject of numerous investigations

(Herbert & Mann, 1983, 1984a, 1984b, Cable et al., 1987a, 1987b, 1987c, 1991). Due to the

significance of bacterial or fungal-type 4-methoxystyrenes in the primitive liverworts

Corsinia coriandrina and Cronisia weddellii it was decided to investigate the biosynthesis of

(Z)-coriandrin ((Z)-65) and related compounds in axenic in vitro cultures of C. coriandrina

using application experiments with stable isotope labelled precursors (Section 4.6.1., page

86).


51

4.3.4. Identification and Synthesis of Stilbenoids from Corsinia coriandrina

4.3.4.1. (R)-(–)-Corsifuran A (73)

Figure 47: Mass spectrum (EI, 70 eV) of corsifuran A (73) from Corsinia coriandrina.

A new 2-aryl-2,3-dihydrobenzo[b]furan named corsifuran A (73), with an unusual

4‟,5-dimethoxy substitution pattern was observed as a major component of diethyl ether

extracts of Corsinia coriandrina (1 – 10 % of the total volatiles) and could be isolated by a

combination of column chromatography and preparative thin-layer chromatography. The

mass spectrum (Figure 47) exhibited a strongly dominating molecular ion signal at m/z 256

[M] (100) and several fragment ion signals of low intensity, with those at m/z 241 [M – CH3],

m/z 225 [M – OCH3] and m/z 213 [M – COCH3] indicating a methoxy substituted aromatic

structure. By HREIMS the molecular formula of C16H16O3 with nine units of unsaturation

could be established.

The 1H NMR spectrum of corsifuran A (73) (Figure 48) exhibited an AA‟BB‟ spin system for

one 1,4-disubstituted benzene unit at H 7.20 (2H, d, 3J = 8.8 Hz) and H 6.75 (2H, d,

3J = 8.8

Hz), one 1,2,4-trisubstituted benzene unit at H 6.83 (1H, d, 3JAr = 8.5 Hz, 6-H), H 6.62 (1H,

dd 3JAr = 8.5 Hz,

4JAr = 2.8 Hz, 5-H) and H 6.70 (1H, d,

4JAr = 2.8 Hz, 3-H), two methoxy

groups at H 3.28 (3H, s) and H 3.38 (3H, s), and one ABM spin-system, corresponding to an

anisochoric methylene group at H 2.90 (1H, dd, 2J = 15.4 Hz,

3J = 8.5 Hz) and H 3.09 (1H,

dd, 2J = 15.8 Hz,

3J = 9.1 Hz), adjacent to a chiral methine group at H 5.48 (1H, dd,

3J =

3J =

8.6 Hz).

55 77 115121 141148 165 181 198 213 225241

256

40 60 80 100 120 140 160 180 200 220 240 260

20

40

60

80

100

C16H16O3


52

Figure 48: 500 MHz 1H NMR spectrum of corsifuran A (73, in C6D6) from Corsinia coriandrina.

Inspection of the COSY spectrum revealed a bibenzyl skeleton, whereas the chemical shift of

the stereogenic methine group at H 5.48 and the anisochrony of adjacent methylene protons

at H 2.90 and 3.09 indicated a 2-(4-methoxy-phenyl)-2,3-dihydrobenzofuran structure with a

5- (or 6-) methoxy substitution for corsifuran A (73).

The 2-(4-methoxyphenyl)-2,3-dihydrobenzofuran structure and the unusual 5-methoxy

substitution were proven unambiguously by synthesis as shown in figure 49 (page 53).

Cycloaddition between 4-methoxystyrene (112) and 1,4-benzoquinone (113) was effected

using ferrum(III)chloride in acetonitrile to give (±)-114 in moderate yield (Ohara et al., 2002).

Methylation of (±)-114 using methyl iodide furnished (±)-5-methoxy-2-(4-methyoxyphenyl)-

2,3-dihydrobenzo[b]furan ((±)-73) which was shown to be identical to corsifuran A from

Corsinia coriandrina by comparison of the EIMS and 1H NMR spectra (von Reuß & König,

2004). In addition, reaction of (±)-114 with [D3]-methyl iodide afforded (±)-[5-OCD3]-

corsifuran ([5-OCD3]-73), employed as internal standard for 2D NMR spectroscopy (Section

4.6.2.11., page 123).

H [ppm] 3.2 3.6 4.0 4.4 4.8 5.2 5.6 6.0 6.4 6.8 7.2

solvent

[ppm] 3.2 3.6 4.0 4.4 4.8 5.2 5.6 6.0 6.4 6.8 7.2

3-Pro-R-H

3-Pro-S-H

4‘-OCH3 5-OCH3

2-CH

6-CH

4-CH

7-CH

2‘,6‘-CH

3‘,5‘-CH


53

Figure 49: Synthesis of (±)-corsifuran B (114), (±)-corsifuran A (73), (±)-[5-OCD3]-corsifuran A

((±)-[5-OCD3]-73), and corsifuran C (74) from C. coriandrina.

Enantioselective GC analysis of racemic (±)-corsifuran A (73) using various modified

cyclodextrins indicated that the best enantiomer separation was achieved with 2,6-Me-3-Pe- -

CD as the stationary phase at 140 °C isothermally ( (R)-(–)/(S)-(+) = 1.030) (Figure 50).

Investigation of the enantiomeric composition of natural (–)-corsifuran A (73) isolated from

the various Corsinia samples indicated enantiopurities of ee = 75 – 95 %. (R)-(–)-73 could

also be detected in a herbarium specimen of Italian Corsinia coriandrina collected in 1887

and 1888, and was isolated by two dimensional thin-layer chromatography on silica gel.

Observed ee = 65 and 72 % indicated that racemisation of (R)-(–)-corsifuran A (73) in dry

plant material was insignificant. After several milligrams of 73 became available from in vitro

cultured Corsinia, the specific optical rotation of (–)-corsifuran A (73) D = – 11° (c = 0.1

in CDCl3) could be determined by polarimetry.

Figure 50: Enantioselective GC analysis of synthetic (±)-corsifuran A (73) and natural

(R)-(–)-corsifuran A (73) from C. coriandrina using 2,6-Me-3-Pe- -CD at 140 °C isothermally.

50

55

RT [min]

(R)-( )-corsifuran A (73) from Corsinia coriandrina

(±)-corsifuran A (73)


54

The absolute configuration of (R)-(–)-corsifuran A (73) from C. coriandrina was determined

in cooperation with Dr. Simon Jones at the University of Sheffield, UK, by enantioselective

synthesis as shown in figure 51 (Adams et al., 2008). 3-Methoxyphenylacetic acid (115) was

brominated (116), converted to the acid chloride (117), and reacted with methoxybenzene

(118) under Friedel Crafts conditions. The resulting 1,2-diarylethanone (119) was

enantioselectively reduced using 10 mol % of chiral oxazaborolidine catalyst (120). The

absolute configuration of the resulting alcohol (121) was determined by X-ray crystal

structure analysis. Palladium catalyzed cyclisation of the (R)-alcohol (121) afforded (R)-(–)-

corsifuran A (73), which was shown to be identical to the natural product from Corsinia by

comparative enantioselective GC analysis using 2,6-Me-3-Pe- -CD as the stationary phase.

Figure 51: Enantioselective synthesis of (R)-(–)-corsifuran A (73) (Adams et al., 2008).


55

Figure 52: Acid catalyzed racemisation of (R)-(–)-Corsifuran A (73) (R = 4-methoxyphenyl).

Treatment of synthetic (R)-(–)-73 (ee = 72 %) with acidic ion exchange resin Amberlyst® 15

in dichloromethane for 1 h resulted in complete racemisation to give (±)-corsifuran A (ee =

0 %) as shown by enantioselective GC. Racemisation of (R)-(–)-73 suggested reversible

cleavage of the benzofuran moiety as shown in figure 52, but only traces of the presumed

hydroxystilbene intermediate 124 could be detected by GC-EIMS. Treatment of (±)-73 with

modified [SO3D]-Amberlyst, obtained by 1H/

2D exchange of acidic protons with D2O,

resulted in the detection of deuterated [D1 – D3]-species, whereas 3,4‟-dimethoxybibenzyl

(69) was unaffected, thus, indicating reversible benzofuran ring cleavage and protonation-

deprotonation of intermediate hydroxystilbenes (124). These results suggested acid catalyzed

cyclisation of hydroxystilbenes (124) as a possible biosynthetic pathway to corsifuran A (73),

which was further examined by application experiments with deuterated precursors and

axenic in vitro cultures of Corsinia coriandrina (Section 4.6.2, page 102).

4.3.4.2. Corsifuran B (114)

Corsifuran B (114) was detected by GC-EIMS as a minor constituent (˂ 1.5 % of the total

volatiles) of the diethyl ether extract of Corsinia coriandrina collected near Tarifa, Spain. The

mass spectrum exhibited a dominating molecular ion signal at m/z 242 [M] (100), and the

molecular formula of C15H14O3 with nine units of unsaturation was established by HREIMS.

The mass spectrum was similar to that of corsifuran A (73) (Figure 47, page 51), suggesting

an O-desmethyl corsifuran structure. By comparison of the mass spectra and GC retention

indices with synthetic (±)-5-hydroxy-2-(4-methoxyphenyl)-2,3-dihydrobenzofuran (114)

(Figure 49, page 53) the identity with corsifuran B from Corsinia coriandrina could be

established. The absolute configuration was not determined, but is believed to correspond to

(R)-(–)-corsifuran A (73).


56

4.3.4.3. Corsifuran C (74)

Figure 53: Mass spectrum (EI, 70 eV) of corsifuran C (74) from Corsinia coriandrina.

Corsifuran C (74), didehydrocorsifuran A was isolated as a minor constituent of the diethyl

ether extract of Corsinia coriandrina collected at Palma (1.1 % of the total volatiles) using a

combination of column chromatography and preparative thin layer chromatography on silica

gel. The mass spectrum (Figure 53) exhibited a dominating molecular ion signal at m/z 254

[M] (100). By HREIMS the molecular formula of C16H14O3 with ten units of unsaturation

could be established. The presence of two methoxy groups attached to aromatic systems was

indicated by fragment ion signals at m/z 239 [M – CH3] and m/z 211 [M – COCH3], as well as

m/z 196 [M – COCH3 – CH3], and 168 [M – COCH3 – COCH3] (Parmar et al., 1985). The 1H

NMR spectrum was similar to those of corsifuran A (73) (Figure 48, page 52) but the signals

for the dihydrofuran moiety were replaced by a single broad signal at H 6.61 (d, 0.8 Hz),

indicating a 5-methoxy-2-(4-methoxyphenyl)-benzofuran structure, which was named

corsifuran C (74). This hypothesis was confirmed by comparison of the GC-EIMS and 1H

NMR data with those of an authentic sample obtained by dehydrogenation of (±)-corsifuran A

(73) using 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) in 1,4-dioxane (Figure 49, page

53) (von Reuß & König, 2004). Due to its intensive and broad UV absorption properties in the

UV-B range ( max = 316 nm and = 52.900) corsifuran C (74) and its derivatives have

previously been patented as sunscreen agents in topical applications (L‟Oreal, 1975).

63 106127

139 152168

183 196

211

224

239

254

40 60 80 100 120 140 160 180 200 220 240 260

20

40

60

80

100

C16H14O3

C15H11O3

C14H11O2

C12H8O2

C11H8O


57

4.3.4.4. (E/Z)-Corsistilbenes (68)

Figure 54: Mass spectrum (EI, 70 eV) of (E/Z)-corsistilbenes (68) from Corsinia coriandrina.

Two isomeric compounds (E/Z)-68 with identical mass spectra (Figure 54), characterized by a

dominating molecular ion signal at m/z 240 [M] (100), were detected as minor constituents of

Corsinia coriandrina (1 – 2 % and ˂ 0.5 % of the total volatiles, respectively). The molecular

formula C16H16O2 with nine units of unsaturation was established by HREIMS. The

dominating isomer (E)-68 was isolated from enriched fractions by preparative thin-layer

chromatography on silica gel using a hexane – ethyl acetate mixture.

The 1H NMR spectrum of (E)-68 (Figure 55) exhibited signals for two aromatic methoxy

groups at H 3.32 (3H, s) and 3.38 (3H, s), and one 1,4-substituted phenyl ring at H 6.79 (2H,

d, 3J = 8.8 Hz), and 7.29 (2H, d,

3J = 8.5 Hz). Although the signals of four of the six

remaining aromatic protons were considerably overlapping, the dd-multiplicity of one isolated

signal at H 6.74 and its vicinal (3J = 8.2 Hz) and allylic (

4J = 1.9 Hz) coupling constants

suggested a meta-substituted aryl unit. Furthermore, an (E)-configured ethenyl bridge was

deduced from the doublet multiplicity and large coupling constant of one isolated signal at

H 6.96 (1H, d, 3JE = 16.4 Hz), thus, suggesting a stilbenoid structure. Considering the co-

occurrence of lunularic acid (5) and lunularin (6) in C. coriandrina (Gorham, 1977a), these

results suggested (E)-3,4‟-dimethoxystilbene as the structure for (E)-68, which was named

corsistilbene.

4357

6369 76 83 97 112 120127 139

153

165

182 197 209225

240

40 60 80 100 120 140 160 180 200 220 240 260

20

40

60

80

100

C16H16O2


58

Figure 55: 500 MHz 1H NMR spectrum of (E)-corsistilbene ((E)-68, in C6D6) from C. coriandrina.

The structure of (E)-corsistilbene ((E)-68) was confirmed by comparison with a synthetic

sample obtained as shown in figure 56 (Huneck, 1976). 4-Methoxybenzylalcohol (125) was

converted to 4-methoxybenzyl chloride (126) upon treatment with thionylchloride. Michaelis

Abrusov reaction afforded the corresponding diethylphosphonate (127). Deprotonation with

sodium hydride gave the phosphoylide, which reacted with 3-methoxybenzaldehyde (128) via

a Wittig-Horner-Emmons reaction to furnish (E)-3,4‟-dimethoxystilbene ((E)-68) identical to

the natural product from C. coriandrina as shown by comparison of GC-EIMS and 1H NMR

spectra.

Figure 56: Synthesis of (E)-corsistilbene ((E)-68) from Corsinia coriandrina.

H [ppm]3.84.24.65.05.45.86.26.67.07.4

solvent

6.76.86.97.07.17.27.3

2‘,6‘-CH 3‘,5‘-CH

2-CH

7-CH8-CH

1H2H1H2H 4H

s

3JE = 16.3 Hz

H [ppm]3.84.24.65.05.45.86.26.67.07.4

solvent

6.76.86.97.07.17.27.3

2‘,6‘-CH 3‘,5‘-CH

2-CH

7-CH8-CH

1H2H1H2H 4H

s

3JE = 16.3 Hz

4-CH


59

Figure 57: UV spectrum of (E)-corsistilbene ((E)-68) in methanol

and UV induced (E/Z)-isomerisation.

(E)-Corsistilbene ((E)-68) exhibited intensive absorption properties in the UV-B range ( max =

320 nm, = 17.500) and was efficiently converted to the (Z)-isomer (Z)-68 upon UV

irradiation at 350 nm for 1 h (Figure 57). Although predominantly present as (E)-68 the

corresponding (Z)-isomer (Z)-68 was also detected in the diethyl ether extract of Corsinia

coriandrina (E/Z > 6).

Like other stilbenoid compounds, (E)-corsistilbene ((E)-68) exhibits characteristic bluish-

white fluorescence upon irradiation with UV-B light at 350 nm (Plate 16, page 217). Thin-

layer chromatography of methanol extracts indicated that only photosynthetic pigments, (E)-

corsistilbene ((E)-68) and corsifuran C (74) exhibit discernible fluorescence upon irradiation

with UV-B, and could thus be localized using UV-epifluorescence microscopy. Inspection of

Corsinia coriandrina thallus slides indicated that a bright fluorescence attributed to (E)-68

(and / or 74) was associated with selected oil bodies only, thus, suggesting their accumulation

within these compartments (Plate 16). Most surprisingly, fluorescence was restricted to

selected oil bodies only, indicating a high degree of specialization which has not been noticed

before. Biosynthetically related 3-methoxybibenzyl (8) has previously been identified as the

major volatile constituent in isolated oil bodies of Radula complanata (Flegel & Becker,

2000). Along with (E)-3,4-dihydroxy-3‟-methoxystilbene from Marchesina bongardiana,

Jungermanniales (Speicher & Schoeneborn, 1997) the (E/Z)-corsistilbenes ((E/Z)-68) from

Corsinia coriandrina represent rare examples for stilbene-type compounds in liverworts,

which are widely distributed in higher plants.


60

4.3.4.5. O,O-Dimethyllunularin (69)

Figure 58: Mass spectrum (EI, 70 eV) of O,O-dimethyllunularin (69) from Corsinia coriandrina.

One bibenzyl compound (69) with a molecular ion signal at m/z 242 [M] was regularly

detected as a trace constituent of Corsinia coriandrina extracts (< 0.1 % of the total volatiles).

The molecular formula C16H18O2 with eight units of unsaturation was established by

HREIMS. The mass spectrum was characterized by the presence of only a very few signals

(Figure 58). The highly dominating base peak at m/z 121 (100) [M – C8H9O+] was attributed

the molecular formula C8H9O+ by HREIMS, indicating symmetrical cleavage. No significant

ion of higher mass could be observed. The presence of one methoxy group in the major

fragment ion was indicated by signals for C7H7+ at m/z 91 [C8H9O – OCH2] and C6H6

+ at

m/z 78 [C8H9O – COCH3]. These results indicated a methoxytropylium ion for the base peak

signal and suggested a dimethoxybibenzyl skeleton with two methoxyphenyl units.

Considering the substitution patterns of the co-occurring corsistilbenes ((E/Z)-68) and the

bibenzlic lunularin (6) an O,O-dimethyllunularin structure (69) was assumed, which was

confirmed by comparison of the mass spectra and GC retention indices with an authentic

sample prepared by Pd/C catalyzed hydrogenation of (E)-corsistilbene ((E)-68) (Figure 59).

Figure 59: Synthesis of O,O-dimethyllunularin (69) by hydrogenation of (E)-corsistilbene ((E)-68).

44 78 91

121

242

40 60 80 100 120 140 160 180 200 220 240 260

20

40

60

80

100

C16H18O2

C8H9O


61

4.3.4.6. 6-Hydroxy-O,O-dimethyllunularin (70)

Figure 60: Mass spectrum (EI, 70 eV) of 2-hydroxy-4‟,5-dimethoxybibenzyl (70).

Fractionation of the diethyl ether extract of Corsinia coriandrina from La Palma by column

chromatography on silica gel and subsequent GC-EIMS analysis revealed the presence of one

polar minor constituent (70, < 1 % of the total volatiles) with a molecular ion signal at

m/z 258 [M]. The molecular formula C16H18O3 with eight units of unsaturation was

established by HREIMS. The mass spectrum (Figure 60) was similar to that of O,O-dimethyl-

lunularin (69) (Figure 58, page 60) and exhibited a dominating fragment ion signal at m/z 121

(100) [M – C8H9O2] for a methoxytropyliumion (C8H9O+), along with a small signal at

m/z 137 (5) [M – C8H9O] for a hydroxymethoxytropyliumion (C8H9O2+), thus, suggesting a

hydroxylated dimethyllunularin structure.

The large difference in relative intensities between these two fragment ions, which originate

from differential charge distribution during benzylic cleavage, indicated formation of the

C8H9O+ / C8H9O2

● couple to be favoured over the opposite combination. Cleavage of

bibenzyls affords benzylions and radicals as initial fragments, which subsequently rearrange

to the corresponding tropylium counterparts. Consequently, the substitution pattern of the

parent bibenzyl molecule ion [M+●

] has a great influence on charge distribution during

benzylic cleavage. Semi-empirical quantum mechanical calculations using the PM 3 potential

function and an unrestricted HF matrix (UHF) indicated that cleavage of dimethyllunularin

(69) to the 4-MeOBz+ / 3-MeOBz

● couple is energetically favoured by 11 kcal/mole in

agreement with previous studies on bibenzyl mass spectrometry, which have shown that the

dominating base peak in asymmetrically substituted bibenzyls is predominantly derived from

the para-substituted benzyl unit (McLafferty & Bursey, 1968).

77 91

121

137

258

40 60 80 100 120 140 160 180 200 220 240 260

20

40

60

80

100

C16H18O3

C8H9O

C8H9O2


62

Consequently, the base peak signal at m/z 121 (100) suggested a 4-methoxybenzyl moiety,

whereas the small signal at m/z 137 (5) was attributed to a meta-methoxy substituted benzyl

unit, which also keeps the additional hydroxyl unit. Although the presumed phenylpropanoid-

polymalonate origin of liverwort bibenzyls (Pryce, 1971; Friederich et al., 1999a) suggested a

3,5,4‟-substitution pattern, a 2,5,4‟-trioxygenated bibenzyl derivative was assumed, due to the

co-occurrence of 5,4‟-methoxy-2-arylbenzofurans like corsifuran A (73). Furthermore, the co-

occurrence of related bibenzyls like lunularin (6) (Gorham, 1977a) and O,O-dimethyl-

lunularin (69) indicated in vivo O-methyl transferase activity for para- and meta-hydroxy

groups, finally resulting in the proposal of a 2-hydroxy-4‟,5-dimethoxybibenzyl structure

(70). Semi-empirical quantum mechanical calculations (PM 3, UHF) confirmed that cleavage

of bibenzyl 70 to the 4-MeOBz+ / 2-HO-5-MeOBz

● couple is energetically favoured by

3 kcal/mole, in agreement with the dominating base peak signal at m/z 121 (100) in the mass

spectrum. This hypothesis was finally confirmed by comparison of the mass spectra and GC

retention indices with synthetic 2-hydroxy-4‟,5-dimethoxybibenzyl (70) prepared by

palladium catalyzed reductive ether cleavage of (±)-corsifuran A (73) (Figure 61).

Figure 61: Synthesis of 2-hydroxy-4‟,5-dimethoxybibenzyl (70) from (±)-corsifuran A (73).

Within the Marchantiales the free phenol, 2,5,4‟-trihydroxybibenzyl (144, Figure 63, page

65), and the corresponding 2-O- -D-glucopyranoside have previously been identified in

Ricciocarpos natans (Kunz & Becker, 1992, 1994), whereas the 2,5-di-O- -D-gluco-

pyranoside has been isolated as a water soluble constituent of Marchantia polymorpha (Qu, et

al., 2007). Within the Metzgeriales related bis(bibenzyls) like 14- or 14‟-hydroxy-perrottetine

have been reported from Pellia endiviifolia and P. epiphylla, respectively (Hashimoto et al.,

1991; Cullmann et al., 1993; Seoane, et al., 1996).


63

4.3.4.7. Discussion of Stilbenoids from Corsinia coriandrina

Several 2-arylbenzo[b]furans of phenylpropenoid-polymalonate origin have previously been

reported from higher plants and different biosynthetic pathways catalyzed by chalcone

synthase (CHS) or stilbene synthase (STS) were described (Figures 62 and 63) (Martin &

Dewick, 1979; Hano et al., 1989, 1994a, 1994b, 1994c). In the Fabaceae 2‟,4‟,6-trihydroxy-2-

arylbenzo[b]furans (133) co-occur with the corresponding isoflavenoids (132) (Veitch, 2007;

Erasto et al., 2004; Yenesew, et al., 2002; Demizu et al., 1988; Russell et al., 1984),

originating from the chalcone synthase (CHS) pathway via chalcone (129, 130) and flavanoid

(131) intermediates (Al-Ani & Dewick, 1984; Forkmann & Heller, 1999).

Figure 62: Biosynthetic key steps to phenylpropanoid-polymalonate derived 2-arylbenzo[b]furans of

CHS origin from higher plants of the Fabaceae (133 and 135) (phenylpropanoid- & methyl-groups of

acetate units in bold; CHS: chalcone synthase, FS: flavanoid synthase; IFS: isoflavonoid synthase).


64

Incorporation experiments with Vigna unguiculata indicated a phenylpropenoid derived 2-aryl

moiety and loss of the 3-methylene group during vignafuran (133) biosynthesis from

phenylalanine (Martin & Dewick, 1979; Al-Ani & Dewick, 1984), consistent with the

deformylation of a 2-hydroxyisoflavene intermediate (132) during ring contraction of the

coumestan (132) to the 2-arylbenzo[b]furan (133) skeleton (Kinoshita, 1997). Nevertheless,

the recent discovery of 3-formyl-2‟,4‟,6-trihydroxy-2-arylbenzo[b]furans (135) indicated that

an alternative pathway via co-occurring coumestrol (134) exists in some Fabacean species

(Macías et al., 1999; Halabalaki et al., 2000; Abdel-Kader, 2001; Kraft et al., 2001; Tanaka et

al., 2003; Belofsky et al., 2006; Salem & Werbovetz, 2006; Veitch, 2007).

Furthermore, numerous 3‟,5‟,6-trioxygenated 2-arylbenzo[b]furans related to moracin M

(138) a phytoalexin of mulberry (Morus sp.) are known to co-occur with the corresponding

stilbenoids, resveratrol (136) and oxyresveratrol (137) (Nomura, 1988; Takasugi et al., 1978),

originating from the stilbene synthase pathway (STS) (Figure 63) (Schröder, 1999). Formerly

believed to be restricted to the Moraceae (Nomura, 1988; Nomura & Hano, 1994; Nomura et

al., 1998), 3‟,5‟,6-trihydroxy-2-arylbenzofurans (138) and the corresponding 3‟,5‟,4-

trihydroxystilbenes (136) have recently been detected in some highly unrelated plant families,

like Fabaceae (Muhammad et al., 2001, 2003; Wanjala et al., 2002), Liliaceae (Zhou et al.,

1999; Kanchanapoom et al., 2002), Asparagaceae (Wiboonpun et al., 2004), Stemonaceae

(Pacher et al., 2002; Adams et al., 2005), and Gnetaceae (Li, et al., 1991; Huang et al., 2000;

Iliya et al., 2002; Yao et al., 2005). Application experiments using Morus alba cell cultures

indicated non-discriminatory incorporation of L-[1-13

C]-phenylalanine and L-[3-13

C]-tyrosine

into a dimeric moracin (138) derivative, and established the phenylpropanoid origin of the

benzo[b]furan moiety (Hano et al., 1994a, 1994b). Furthermore, the polymalonate origin of

the 3‟,5‟-dioxygenated aryl-moiety of resveratrols (136) and moracins (138) was established

by incorporation of 13

C labelled acetate (Hano et al., 1989, 1994c; Shimazaki et al., 2000).

Oxidative cyclisation of 2-hydroxystilbenes like oxyresveratrol (137) is known to afford the

corresponding 2-arylbenzofurans (138) (Nogami & Kurosawa, 1974; Pan et al., 2006),

whereas oxidative cyclisation of the related bibenzyls (139) (Schofield et al., 1971) was

recently proposed as a biosynthetic pathway to 2‟,3‟,5‟-trioxygenated-2,3-dihydro-

benzo[b]furans (140) in Bauhinia purpurea, Fabaceae (Boonphong et al., 2007). Two

different pathways leading to phenanthrens and 9,10-dihydrophenathrens via stilbenoid or

bibenzyl intermediates, respectively, have also been identified in Orchidaceae (Doux-Guyat et

al., 1983; Fritzemeier & Kindl, 1983; Reinecke & Kindl, 1994; Preisig-Müller, et al., 1995)

and Dioscoreaceae (Fritzemeier et al., 1984).


65

Figure 63: Biosynthetic key steps to phenylpropanoid-polymalonate derived 2-arylbenzo[b]furans of

STS origin from higher plants like Morus sp., Moraceae (138) or Bauhinia sp., Fabaceae (140), and

the liverwort Corsinia coriandrina (73 and 74 via steps C1 and C2) (phenylpropanoid- and methyl-

groups of acetate units in bold).


66

Figure 64: 2-arylbenzofurans from the moss Polytrichum pallidisetum (146) and yeast (147).

In addition, there are a few 2-arylbenzofurans of assumed phenylpropanoid-polymalonate

origin whose biosynthetic pathway remained obscure, like (E/Z)-pallidisetins ((E/Z)-146),

from the moss Polytrichum pallidisetum, Polytrichaceae (Zheng et al., 1994), or antioxidant

2‟,3‟,4‟,6‟-tetraoxygenated 2-arylbenzofuran (147) from yeast (McKittrick & Stevenson,

1984) (Figure 64). 4‟,5-Dioxygenated 2-arylbenzofurans like (R)-(–)-corsifuran A (73) from

the liverwort Corsinia coriandrina are virtually unknown in nature (Figure 63). Only racemic

bisphenol (±)-145 has been described as a metabolite of exogenic 4-coumaric acid (152) in

Bacillus megaterium, but radical coupling of intermediate 4-hydroxstyrene with a C6-unit like

1,4-benzoquinone (Figure 49, page 53) was proposed as a biosynthetic pathway (Torres y

Torres & Rosazza, 2001). Comparison of the substitution patterns suggested a similar STS

pathway to corsifurans (73, 74), corsistilbenes (68), lunularin (6) related bibenzyls (69, 70)

and lunularic acid (5) and indicated that the phenylpropanoid unit contributes to different

parts of the 2-aryl-benzofuran skeleton in higher plants and Corsinia (Figure 63, page 65).

Nevertheless, the relationship between stilbenoids (68), 2-arylbenzofurans (74) and the

corresponding dihydro derivatives (69 and 73) in Corsinia was completely unknown and at

least three different pathways had to be considered. Beside independent biosynthesis of

corsifuran A (73) and corsifuran C (74) by oxidative cyclisation of stilbenoid (step A1, 141

via 142) and bibenzyl (step B1, 6 via 144) intermediates, respectively, both corsifurans could

be metabolically connected by hydrogenation of corsifuran C (74, step A2) or dehydro-

genation of corsifuran A (73, step B2/C2), with the intermediates being derived from oxidative

cyclisation of stilbenoid (A1) or bibenzyl precursors (B1), or from acid catalyzed cyclisation

of stilbene intermediates (step C1) as observed upon acid catalyzed racemisation of (R)-(–)-

corsifuran A (73) via (E)-124 (page 55). Due to the significance of stilbenoid compounds for

liverworts it was decided to investigate the biosynthesis of (R)-(–)-corsifuran A (73) and

related compounds in Corsinia coriandrina by using application experiments with stable

isotope labelled precursors (Section 4.6.2., page 102).

4.4. Synthesis of Deuterium Labelled Precursors

67


For the incorporation experiments with in vitro cultures of Corsinia coriandrina stable

isotope labelled precursors were required. Deuterium (2D) was chosen as the isotope label for

the phenylpropanoid precursors due to its ability to provide regiochemical and stereochemical

information by the combination of 1H and

2D NMR techniques and to indicate subsequent

changes in the hybridisation state of adjacent sp3-carbons using multiply labelled compounds

or co-application experiments with different isotopomers. Furthermore, the increasing number

of known procedures for specific 1H/

2D-exchange in D2O, the cheapest isotope source

available, facilitated the synthesis of a large number of molecular probes.

4.4.1. Synthesis of (E)-[2-D]-Cinnamic Acids ([2-D]-89, [2-D]-93, [2-D]-152)

The condensation of benzaldehyde (40) and acetic anhydride (148) upon heating with

potassium acetate (Perkin reaction) afforded (E)-cinnamic acid (93) in up to 60 % yield

(Figure 65) (Rosen, 1991). When performed with [U-D6]-acetic anhydride ([U-D6]-148,

0.99 D), (E)-[2-D]-cinnamic acid ([2-D]-93) was obtained in 52 % yield, but deuterium

enrichment was low (> 0.74 D), due to significant participation of unlabelled acetate from

initial substitution reactions between [U-D6]-acetic anhydride ([U-D6]-148) and potassium

acetate (calc. 0.67 D for complete exchange).

Figure 65: Perkin synthesis of (E)-cinnamic acid (93) and (E)-[2-D]-cinnamic acid ([2-D]-93).

Therefore, a new two step approach via hydrolysis of [2-D]-cinnamonitrile ([2-D]-149)

obtained by condensation of benzaldehyde (40) and [D3]-acetonitrile ([D3]-101) was

investigated (Figure 66). Literature procedures were hampered by the large excess of

acetonitrile required but this problem was overcome by using THF as the solvent.

Condensation of 40 with 1 mol equivalent [D3]-acetonitrile ([D3]-101, > 0.98 D) using the

highly basic non-nucleophilic sodium hexamethyldisilazane (NaHMDS) in THF afforded

[2-D]-cinnamonitrile ([2-D]-149) in 12 % yield as a 5:1 mixture of the (E/Z)-isomers showing


68

[D]-enrichment of > 0.97 D. Nevertheless, attempted hydrolysis of the cyano group of (E/Z)-

[2-D]-cinnamonitrile ([2-D]-149) using 2 M potassium hydroxide solution resulted in the

complete loss of the [2-D]-label and furnished unlabelled (E)-cinnamic acid (93) (Figure 66).

This unexpected result suggested addition of hydroxide-ions to the -position of the double

bond, thus, leading to subsequent exchange of the [D]-label due to -CH acidity.

Nevertheless, Retro-Aldol-like cleavage was insignificant as indicated by the negligible

amounts of benzaldehyde (40) or benzoic acid detected. A similar 1H/

2D exchange in

cinnamonitrile (149) using sodium ethanolate in [D1]-ethanol has previously been described

(Zinn, et al., 1963). When (E)-cinnamic acid (93) was refluxed in alkaline D2O no exchange

of -H was observed, although 1H/

2D exchange has been reported for temperatures ≥ 190 °C

(Reed, et al., 1993) or acidic conditions (Noyce et al., 1962). Consequently, unlabelled (E/Z)-

cinnamonitrile (149) was prepared by condensation of benzaldehyde (40) and acetonitrile

(101) in 14 % yield and the [2-D]-label introduced upon base catalyzed hydrolysis in D2O to

afford (E)-[2-D]-cinnamic acid ([2-D]-93) in good yields (Figure 66).

Figure 66: Synthesis of (E)-[2-D]-cinnamic acids ([2-D]-93, [2-D]-89, and [2-D]-152)

by alkaline hydrolysis of cinnamonitriles (149, 100, and 151) in D2O.

Specific deuterium enrichment at the 2-position of (E)-[2-D]-cinnamic acid ([2-D]-93) was

unambiguously established upon 1H NMR spectroscopy (Figure 67, page 69), which revealed

an attenuated signal at H 6.47 (0.02H) for the residual 2-methine hydrogens of unlabelled

species. In addition, the signal at H 7.81 (1H) for 3-CH appeared as a broad singlet due to the

small 3JH,D coupling constant.


69

Figure 67: 500 MHz 1H NMR spectra of (E)-cinnamic acid (93) and (E)-[2-D]-cinnamic acid

([2-D]-93, > 0.97 D, in CDCl3) obtained by alkaline hydrolysis of cinnamonitrile (149) in D2O.

Observed [D]-enrichment was close to those calculated for complete 1H/

2D exchange and

reached > 0.97 D depending on the excess of D2O and synthetic details. The 1H/

2D exchange

upon hydrolysis of cinnamonitriles also proved useful for the synthesis of substituted

(E)-[2-D]-3-phenylpropenoic acids (Figure 66, page 68). (E)-[2-D]-4-methoxycinnamic acid

([2-D]-89) was prepared in 74 % yield from (E)-4-methoxycinnamonitrile (100), obtained in

22 % yield upon condensation of 4-methoxybenzaldehyde (58) and acetonitrile (101).

Attempted synthesis of the corresponding phenol (151) by condensation of 4-hydroxy-

benzaldehyde (150) and acetonitrile (101) failed, but treatment of (E)-4-methoxy-

cinnamonitrile (100) with boron tribromide (BBr3) in dichloromethane afforded

(E)-4-hydroxycinnamonitrile (151) in 20 % yield. Base catalyzed hydrolysis of 151 in D2O

furnished (E)-[2-D]-4-coumaric acid ([2-D]-152) in 68 % yield (Figure 66).

H [ppm]6.56.66.76.86.97.07.17.27.37.47.57.67.77.8

1H 1H2H 3H

CHCl3

3-H

2-H

2‘,6‘-H

3‘,4‘,5‘-H

H ppm]6.56.66.76.86.97.07.17.27.37.47.57.67.77.8

1H 0.02H2H 3H

CHCl33-H

residual 2-H

2‘,6‘-H

3‘,4‘,5‘-H

H [ppm]6.56.66.76.86.97.07.17.27.37.47.57.67.77.8

1H 1H2H 3H

CHCl3

3-H

2-H

2‘,6‘-H

3‘,4‘,5‘-H

H ppm]6.56.66.76.86.97.07.17.27.37.47.57.67.77.8

1H 0.02H2H 3H

CHCl33-H

residual 2-H

2‘,6‘-H

3‘,4‘,5‘-H


70

In contrast to (E)-[2-D]-cinnamic acid ([2-D]-93) the deuterium enrichment of (E)-[2-D]-

4-methoxycinnamic acid ([2-D]-89, > 0.84 D) and (E)-[2-D]-4-coumaric acid ([2-D]-152,

> 0.88 D) was significantly smaller than those calculated for complete 1H/

2D exchange.

Although not fully optimized yet, the new procedure provides an efficient route to highly

enriched [2-D]-3-phenylpropenoic acids. Its advantage over the Perkin and Knoevenagel

synthesis is the use of inexpensive D2O as the isotope source instead of deuterated organic

compounds like [U-D6]-acetic anhydride ([D6]-148) or [U-D4]-malonic acid ([D4]-88),

respectively (Figures 65 and 68). Although the overall yields from starting aldehydes are

much lower than those obtained by Perkin or Knoevenagel synthesis, improvement of

cinnamonitrile synthesis by Wittig reaction using cyanomethylene phosphonates (Appel et al.,

2009) or palladium catalyzed Heck reaction of aryl halides with acyrlonitrile (Bumagin et al.,

1989) might significantly add to the usefulness of the procedure.

Because the overall yield from the aldehyde (58) to (E)-[2-D]-4-coumaric acid ([2-D]-152)

was < 5 % (Figure 66) the corresponding (E)-[2,3‟,5‟-D3]-isotopomer ([2,3‟,5‟-D3]-152) was

preferably prepared in 56 % yield by Knoevenagel condensation (Figure 68). [U-D4]-Malonic

acid ([D4]-88, > 0.76 D by MS) was obtained from malonic acid (88) by repeated 1H/

2D-

exchange in D2O at 80°C. [3,5-D2]-4-hydroxybenzyldehyde ([3,5-D2]-150, > 0.65 D by 1H

NMR) was prepared from 4-hydroxybenzaldehyde (150) by repeated 1H/

2D-exchange in D2O

under alkaline conditions at 100 °C for 6 days using a Reactivial® (Al-Ani & Dewick, 1984).

Knoevenagel condensation of [3,5-D2]-4-hydroxybenzaldehyde ([3,5-D2]-150) and [U-D4]-

malonic acid ([D4]-88) afforded (E)-[2,3‟,5‟-D3]-4-coumaric acid ([2,3‟,5‟-D3]-152) with an

average enrichment of > 0.70 D as determined by mass spectrometry (ratio of 7 / 8 / 5 for

[D3]/[D2]/[D1] species). 1H NMR indicated specific enrichment of > 0.66 D for the aromatic

3‟,5‟-positions and > 0.78 D for the 2-position in [2,3‟,5‟-D3]-152 similar to the enrichment of

the reactants ([3,5-D2]-150 and [D4]-88).

Figure 68: Synthesis of (E)-[2,3‟,5‟-D3]-4-coumaric acid ([D3]-152) by Knoevenagel condensation.


71

4.4.2. Synthesis of [3,3-D2]-3-Phenylpropanoic Acids (153 – 158)

The synthesis of side-chain deuterated arylalkyl-type compounds by Pd/C catalyzed 1H/

2D

exchange in D2O has recently been described (Maegawa et al., 2005; Esaki et al., 2007).

1H/

2D exchange of benzylic hydrogens was affected by heating 0.5 mmol of the unlabelled

compound with 10 mg palladium on carbon (10 %, w/w) in 2 ml D2O (110.8 mmol) in a

sealed 10 ml flask filled with hydrogen gas at 110 °C for 5 h (Figure 69). Deuterium

enrichment and location was determined by 1H NMR spectroscopy, and the isotopomeric

composition was investigated by EIMS or FAB-MS.

Figure 69: Synthesis of benzyl-deuterated precursors

(153 – 158, see also Table 3, page 72).

The procedure provided highly enriched compounds in good yields (Table 3). In the case of

amino acids or -phenylethylamines attempted conversion of the corresponding hydro-

chlorides failed. Both L- and D-phenylalanine (L- and D-153) were converted into the

corresponding [3,3-D2] isotopomers (L-[3,3-D2]-153 and D-[3,3-D2]-153) in agreement with

FAB-MS molecular ion signals at m/z 168 [M + H]. Deuterium enrichments of ˂ 0.98 D and

˂ 0.92 D were determined for 3-Pro-R-H for 3-Pro-S-H of L-[3,3-D2]-phenylalanine

(L-[3,3-D2]-153) by 1H NMR. Comparison of the optical rotations indicated that racemisation

was insignificant under the reaction conditions. In comparison to [3,3-D2]-phenylalanines

([3,3-D2]-153) deuterium enrichment of L-[3,3-D2]-tyrosine (L-[3,3-D2]-154) was small and

partial exchange at the 3‟,5‟-positions was detected. For L-[3,3-D2]-O-methyltyrosine

(L-[3,3-D2]-155) 1H NMR spectroscopy indicated 0.95 D for 3-Pro-R and 0.94 D for 3-Pro-S

hydrogens, in agreement with a FAB-MS molecular ion signal at m/z 198 [M + H]. For

3-phenylpropanoic acids (156 and 157) significant 1H/

2D-exchange at the 2-methylene

position was observed. Conversion of 4-phloretic acid (157) afforded a [2,2,3,3,3‟-D5]-

isotopomer ([2,2,3,3,3‟-D5]-157), showing > 0.98 D for 3-methylene, > 0.92 D for 2-

methylene, and > 0.29 D for 3‟,5‟-methine positions. Similar 1H/

2D-exchange of

3‟,5‟-methine hydrogens was also observed with [2,2,3‟-D3]-tyramine ([2,2,3‟-D3]-158)

(Table 3).


72

Unlabelled Labelled R1

R2

R3

R4

Enrich.

[% D]

Yield

[%]

L-153 L-[3,3-D2]-153 COOH H (S) NH2 H > 95 87

D-153 D-[3,3-D2]-153 COOH H (R) NH2 H > 96 91

L-154

L-[3,3-D2]-154 I COOH H (S) NH2 OH > 25 57

L-155 L-[3,3-D2]-155 COOH H (S) NH2 OCH3 > 94 61

156 [3,3-D2]-156 COOH H/D II

H/D II

H > 89 82

157

[2,2,3,3,3‟-D5]-157 III

COOH H/D IV

H/D IV

OH > 98

92

158

[2,2,3‟-D3]-158 V H H NH2 OH > 90

67

Table 3: Isotope enrichment and yield of labelled precursors (153 – 158) prepared by Pd/C catalyzed

benzylic 1H/

2D exchange (I: 0.15 D for 3‟,5‟-H‟s; II: > 0.25 D for R

2 and R

3; III: > 0.29 D for

3‟,5‟-H‟s; IV: > 0.92 D for R2 and R

3; V: > 0.50 D for 3‟,5‟-H‟s by

1H NMR).

4.4.3. Synthesis of L-[CD3]-O-Methyltyrosine ([CD3]-155)

L-O-methyltyrosines (155) were prepared by O-methylation of N-protected tyrosine (159)

(Figure 70). L-Tyrosine (154) was converted to the L-N-acetyl derivative (159) upon treatment

with acetic anhydride in excess water at 90 °C (Behr & Clarke, 1932). O-Alkylation with

[D3]-methyl iodide or methyl iodide and K2CO3 in acetone gave methyl L-N-acetyl-

O-methyltyrosinates (160 or [CD3]-160). Hydrolysis in 3 M HCl afforded L-O-methyltyrosine

(155) or L-[CD3]-O-methyltyrosine ([CD3]-155) in agreement with FAB-MS signals at

m/z 196 [M + H] and 199 [M + H], respectively.

Figure 70: Synthesis of L-O-methyltyrosine (155) and L-[CD3]-O-methyltyrosine ([CD3]-155).


73

4.4.4. Synthesis of DL-[2-D]-Tyrosines ([2-D]-154, [2-D]-155)

-Deuterated tyrosine ([2-D]-154) and its O-methyl derivative ([2-D]-155) were prepared by

racemisation of N-acetyl derivatives (159, 162) in D2O as shown in figure 71 (Upson &

Hruby 1977; Fujihara & Schowen, 1984). L-amino acids (154, 155) were converted to their

L-N-acetyl derivatives (159, 162) upon reaction with acetic anhydride in water (Behr &

Clarke, 1932), and the -deuterium label was introduced by base catalyzed racemisation in

excess acetic anhydride – D2O to give DL-[2-D]-N,O-diacetyltyrosine ([2-D]-161) and

DL-[2-D]-N-acetyl-O-methyltyrosine ([2-D]-162). Hydrolysis of the N- (and O-) acetyl group

in 3 M hydrochloric acid finally afforded -deuterated DL-[2-D]-tyrosine ([2-D]-154) and

DL-[2-D]-O-methyltyrosine ([2-D]-155) with specific [2-D]-enrichment > 0.98 D as shown by

1H NMR.

Figure 71: Synthesis of DL-[2-D]-tyrosine ([2-D]-154) and DL-[2-D]-O-methyltyrosine ([2-D]-155).

4.4.5. Synthesis of DL-[2,3-threo-D2]-Tyrosine ([2,3-threo-D2]-154)

DL-[2,3-threo-D2]-tyrosine ([2,3-threo-D2]-154) was prepared as shown in figure 72 (Kirby &

Michael, 1973; Oba et al., 1995) Knoevenagel condensation of 4-hydroxybenzaldehyde (150)

with N-acetylglycine (163) and subsequent hydrolysis of the azlactone (164) furnished the

(Z)-2-acetamido-3-arylpropenoic acid (165) in ca. 50 % yield. Pd/C catalyzed syn-deuteration

in [D1]-ethanol afforded DL-[2,3-threo-D2]-N,O-diacetyl tyrosine (161) in 98 % yield. Acid

catalyzed hydrolysis in 3 M hydrochloric acid afforded DL-[2,3-threo-D2]-tyrosine ([2,3-

threo-D2]-154). By 1H NMR an specific enrichments of > 0.96 D for the 2,3-threo-positions

with a diastereomeric excess of de > 95 % were determined.


74

Figure 72: Synthesis of DL-[2,3-threo-D2]-tyrosine ([2,3-threo-D2]-154).

4.4.6. Synthesis of [CD3]-O-Methyltyramine ([CD3]-168)

[CD3]-O-Methyltyramine ([CD3]-168) was prepared using the classical -phenylethylamine

synthesis (Figure 73). Base catalyzed condensation of [CD3]-4-methoxybenzaldehyde ([CD3]-

58) prepared from 4-hydroxybenzyldehyde (150) and [CD3]-methyl iodide, with nitromethane

(166) afforded (E)-[CD3]-4-methoxy- -nitrostyrene ([CD3]-167), which was reduced with

LiAlH4 and isolated as [CD3]-O-methyltyramine hydrochloride ([CD3]-168) in agreement

with a FAB-MS signal at m/z 155 [M – Cl].

Figure 73: Synthesis of [CD3]-O-methyltyramine ([CD3]-168).


75

4.4.7. Synthesis of (±)-[2,3-threo-D2]-Phloretic Acid ((±)-[2,3-threo-D2]-157)

Racemic (±)-[2,3-threo-D2]-phloretic acid ([2,3-threo-D2]-157) was obtained from (E)-4-

coumaric acid (152) by [RhCl(PPh3)3] catalyzed syn-deuteration (Figure 74). The 2,3-threo-

configuration was deduced from the vicinal coupling constant of 3J2,3 = 6.3 Hz in the

1H NMR

recorded in CDCl3 according to (Dunham & Baird, 1975).

Figure 74: Synthesis of (±)-[2,3-threo-D2]-phloretic acid (192) (only one enantiomer is shown).

4.5. Axenic in vitro cultures of Corsinia coriandrina

76

4.5. Axenic In Vitro Cultures of Corsinia coriandrina

Axenic in vitro cultures of Corsinia coriandrina were established in cooperation with

Dr. Klaus von Schwartzenberg at the Biozentrum Klein-Flottbek, University of Hamburg.

Plant in vitro cultures consist of whole plants, plant tissues or cells, which grow under axenic

conditions (free from contaminating organisms) on artificial (defined) nutrient media under

controlled conditions (Dixon, 1985). Techniques for liverwort in vitro cultures have been

reviewed (Bopp & Knoop, 1984; Ohta et al., 1990; Duckett et al., 2004; Hohe & Reski, 2005).

Apart from various biological and molecular biological investigations, liverwort in vitro

cultures were previously used for isolation of secondary compounds (Takeda & Katoh, 1981;

Sauerwein & Becker, 1990; Morais & Becker 1991; Ono et al., 1992; Becker, 1990, 1994,

1995; Grammes et al., 1994, 1997; Geis et al., 1999), bioconversion of exogenous substrates

(Speicher & Roeser, 2002; Speicher et al., 2003), and application experiments to study

precursor-product relationships in terpenoid (Takeda & Katoh, 1983; Warmers & König,

2000; Adam et al., 1998; Nabeta et al., 1998; Tazaki et al., 1999; Hertewich et al., 2001;

Barlow, et al., 2001; Karunagoda et al., 2001; Spiteller et al., 2002; Itoh et al., 2003) or

bis(bibenzyl) (Friederich, 1999a, 1999b) biosynthesis.

4.5.1. Collection of Corsinia coriandrina Spores

Axenic in vitro cultures were established from the spores of Corsinia coriandrina, collected

by the late W. A. König near St. Bartolomeo, Andalusia, in April 2004. Male antheridia and

female archegonia were exclusively located on separate thalli, in agreement with the

dioecious, haploid cytotype described from Atlantic areas (Bousselier-Dubayle & Bischler,

1998). In contrast to most other liverworts sporogons of Corsinia coriandrina do not emit the

mature spores, which are only released upon decay of the surrounding plant tissue. Infertile

spores isolated from sporogons surrounded by a fleshy calyptra did not germinate. About 80

mature spores were obtained from a single sporogon pit located at the decaying thallus end

(Plate 7, page 213). Consequently, spores were considered highly contaminated with algae,

bacteria and fungi. To eliminate these concomitant organisms, spores were sterilized for 5 min

using 1 % calcium hypochlorite (Ca(OCl)2) solution, washed 5 times with sterile water and

centrifuged. Sterilized spores were stored in sterile water at 7 °C for up to one month. Spore

morphology was observed by scanning electron microscopy (SEM) (Plate 8, page 213) and

proved to be identical to literature descriptions of Corsinia coriandrina (Jovet-Ast, 1973).


77

4.5.2. Germination of Corsinia Spores and Propagation of Monoclonal Strains

From about sixty spores approximately fourtyfive germinated at 15 °C within 2 – 3 weeks

when placed on Knop agar supplemented with the antibiotics ampicilline, amphotecerin B,

and nystatine to suppress bacterial and fungal growth. The germ tube stadium was short and

plantlets differentiated to form rhizoids and calli (Plate 9, page 214). Within one month

twenty-nine calli could be transferred to fresh Knop agar before being overrun by resistant

bacteria, fungi or algae. Calli were subcultured every month using Knop agar supplemented

with abovementioned antibiotics. After 3 months, antibiotics were finally withdrawn and

axenic culture conditions were regularly tested by transfer of plant fragments and media to

liquid broth agar (LB) or potato dextrose agar (PDA). Cultures contaminated with bacteria or

fungi were discarded. Twenty-one calli grown under axenic conditions were employed for

vegetative propagation of the plant material, performed by using excised plant fragments as

new inoculums. To ensure the highest possible comparability of the application experiments,

three monoclonal strains (CC1 – 3) were raised from 3 selected plantlets of which CC1

exhibited fastest growth and proofed the most useful for vegetative propagation (Plate 10,

page 214).

4.5.3. Determination of Ploidy Levels

In addition to the monoclonal Corsinia coriandrina strains CC 1 – 3 derived from the spores

of a dioecious haploid specimen, in vitro cultured polyploid C. coriandrina strain Lorbeer/33

was obtained from the Culture Collection of Autotrophic Organisms (CCALA, Trebon, Czech

Republic). Originating from G. Lorbeers collection of liverworts, the specified strain was

cultivated at CCALA since the 1950‟s, but information on the collection site or date was

subsequently lost. Comparison of the relative chromosome number per nuclei using flow-

cytometry enabled different ploidy levels to be distinguished (Bousselier-Dubayle & Bischler,

1998). As shown in figure 75 the polyploid Lorbeer/33 strain contained twice the relative

amount of DNA per nuclei in comparison to the dioecious haploid CC1 strain from Andalusia.

The occurrence of additional 4-methoxystyrenes like (Z)-corsiandrenin ((Z)-92) (Figure 44,

page 48) in polyploid Corsinia strain Lorbeer/33 is in agreement with the proposed

allopolyploid origin (derived from a cross of two genetically distinct species) with the haploid

cytotype as one of the putative progenitors (Boisselier-Dubayle & Bischler, 1998).


78

Figure 75: Relative chromosome counts per nuclei for haploid Corsinia coriandrina strain CC1 and

polyploid Corsinia coriandrina strain Lorbeer/33 determined by flow-cytometry.

4.5.4. Optimization of Culture Media and Conditions

For propagation of Corsinia coriandrina chemically defined basal salt media like Knop,

Gamborg B5 (Dixon, 1985), and Murashige Skoog (MSK) (Murashige & Skoog, 1962) were

investigated. These high salt media were originally developed for in vitro cultures of higher

plants, but have been successfully applied for liverwort cultures as concentrated or diluted

media (Ohta, 1977; Katoh, 1980; Takeda & Katoh, 1981). Concentrations of macro nutritients

including optimized MSKP/5 medium are given in table 4. A 100 µM Fe(III)NaEDTA

supplement and trace elements listed in the Experimental Part (7.3.1, page 166) were identical

for all media.

[mM] NH4+

K+

Ca2+

Mg2+

Na+

NO3–

Cl–

PO43–

SO42–

Knop – 5.5 4 1 – 8 4 1.5 1

GB 2 25 1 1 1 25 2 1 2

MSK 41 20 3 1.5 – 60 3 1.25 1.5

MSKP/5 8 4.25 0.6 0.3 – 12 0.6 0.5 0.3

Table 4: Concentrations [mM] of macro nutritients in basal salt media for plant in vitro cultures.

Poylploid strain Lorbeer / 33 (N = 3401 nuclei)

Haploid strain CC1 (N = 3712 nuclei)


79

To optimize the culture conditions for Corsinia coriandrina, plantlets were cultivated at 15 °C

or 25 °C on Knop, GB, or MSK basal salt agar for 2 months using a 16/8 day-night cycle

(10 µmol*m2*s

1). Morphological features like plant differentiation, increase in callus or

thallus size, and plant colour were compared in weekly intervals. Overall growth

characteristics observed at 15 °C were superior over those at 25 °C, in agreement with the

observation that Corsinia exhibits active growth during the moist winter season (Schuster,

1992). Growth of Corsinia on GB and MSK basal media was significantly better than Knop,

indicating preferred NH4+ uptake over NO3

– as previously reported for other liverworts like

Marchantia polymorpha (Katoh et al., 1980), Reboulia hemisphaerica (Morais & Becker,

1991), and Jungermannia subulata (Ohta et al., 1981). Nevertheless, after one month of

vigorous growth plantlets started to decay, indicating the requirements for further

optimization. To improve the concentration of macro-nutritients, concentrated and 1/2, 1/5,

and 1/10 diluted MSK basal salt agar were compared over a period of ˂6 months, indicating

the MSK/5 medium to be the most advantageous for constant growth and long durability in

comparison with higher concentrated media.

4.5.5. Mixed Photo-Heterotrophic Growth

Plant growth of in vitro cultures using basal salt media is restricted through the photosynthetic

capacity of the plants. Optimization of light intensity, composition and regime, as well as

increased CO2 levels have been reported to result in increased growth of plant cultures under

photoautotrophic conditions (Dixon, 1985). Cultures supplemented with suitable carbon

sources to be incorporated into the plants primary metabolism become independent from these

limitations, and increased growth is often observed under mixed photo-heterotrophic

conditions. In some cases, plant cultures even become independent from the photosynthetic

process itself and grow in complete darkness under chemoheterotrophic conditions (Takio et

al., 1990). A growth promoting effect of carbohydrates in liverwort cultures has been

reported, with D-glucose and D-sucrose (saccharose) being the most effective. In addition to

plant growth, carbohydrate supplements also affect secondary metabolism and either higher or

lower concentrations of secondary metabolites have been reported (Chopra & Sood 1973,

Morais & Becker 1991, Takeda & Katoh, 1981). For mixed photo-heterotrophic growth of

Corsinia coriandrina supplements of 25, 50, or 100 mM D-glucose, or 25 and 50 mM sucrose

were found to be effective leading to increased growth. Differentiation of thalli was only


80

observed with the lowest carbohydrate concentration of 25 mM D-glucose, whereas ≥ 50 mM

resulted in dedifferentiation and callus formation (Plate 11, page 215).

4.5.6. Aerated Liquid Submersion Cultures

Overall growth of Corsinia coriandrina on agar media even under optimized mixed photo-

heterotrophic conditions was painfully slow, thus, making propagation extremely time-

consuming. Consequently, liquid submersion cultures were tested using thallus or callus parts

placed in liquid media. Aerated liquid cultures were established in 250 or 500 ml glass vessels

by bubbling filter sterilized air into the media via a glass tube. Full strength Knop, GB, and

MSK, as well as diluted MSK/x media (dilution factor x = 2, 5, or 10) were tested using a

16/8 h day-night cycle (25 µmol*m2*s

1). Comparable to agar cultures the best results were

obtained with MSK/5 basal salt medium. Because phosphate has often been recognized as a

growth limiting factor, especially in diluted media (Katoh, et al., 1980), a phosphate enriched

media designated MSKP/5 with doubled phosphate concentrations was also tested and found

to be advantageous.

When using MSK media for liquid cultures of Corsinia coriandrina a severe drop from the

initial pH 5.7 to about pH 3.9 – 3.7 was observed during the first week, followed by a slow

decrease within the next three weeks, thus, indicating preferred and rapid NH4+/H

+ exchange.

With MSK/2 media pH values reached 3.0, and cultures decayed within 3 weeks. With the

diluted MSK/5 media pH values ranged at 3.4, and cultures decayed after 6 – 8 weeks. No

significant difference between MSK/5 and phosphate enriched MSKP/5 media was observed,

indicating that doubled phosphate levels were insufficient to buffer pH values. Comparable

acidification upon preferred ammonium uptake is well known in agriculture and has

previously been described for in vitro cultures of higher plants like Nicotiana tabacum,

Solanceaceae (Behrend & Mateles, 1976) and liverworts (Ohta et al., 1981, 1990). Organic

acids of the Citric-Acid cycle have been reported to efficiently buffer pH values during

ammonium uptake (Behrend & Mateles, 1976; Katoh et al., 1980; Takami & Takio, 1987;

Ohta et al., 1981, 1990).

Figure 76: Citric acid (169) and fumaric acid (170) supplemented to liquid media.


81

Consequently, potassium salts of citric acid (169) or fumaric (170) (Figure 76) at c = 2.5 mM

were tested for their ability to buffer pH values during ammonium uptake by Corsinia

coriandrina using MSK/5 agar. Fully differentiated monoclonal thalli (CC 1) were cut and

transferred to test media adjusted at pH 5.7. Cultures were inspected in weekly intervals and

pH values of the media were determined after 1 month. Plantlets cultivated with citrate (169)

supplement were of yellowish colour, exhibited significantly less growth in comparison to

control, and finally decayed within 3 weeks when the pH value ranged at 6.4, which is close

to the pKs3 of citric acid. Culture development on agar media supplemented with 2.5 mM

fumarate (170) was comparable to control, and pH values were efficiently buffered exhibiting

pH 4.9 versus pH 3.9 for control after 1 month.

Aerated liquid submersion cultures using MSK/5 medium supplemented with 2.5 mM

potassium citrate (169) decayed within 3 days, whereas 2.5 – 10 mM potassium fumarate

(170) was tolerated without any notable negative effect on plant growth and development.

Culture media were tested in weekly intervals, revealing a potent stabilization of pH values

for ˂ 4 weeks. After 8 weeks pH values finally increased to 7.0 – 8.0, indicating ammonium

(NH4+) depletion followed by subsequent nitrate (NO3

–) uptake. In conclusion, while citrate

(169) was found to have an undefined toxic effect on Corsinia coriandrina, 2.5 mM fumarate

(170) was shown to efficiently buffer pH values during NH4+/H

+ exchange without disturbing

plant development or ammonium uptake.

Aerated liquid cultures of monoclonal haploid Corsinia coriandrina strain CC1 were finally

established in 500 or 1000 ml glass vessels at 15 °C using MSKP/5 basal salt medium

supplemented with 25 mM D-glucose and 2.5 mM potassium fumarate. Under these

conditions plantlets failed to differentiate forming callus-like structures (Plate 12, left, page

215) exhibiting a doubling time of approximately 30 days. Nevertheless, when partly

differentiated thalli obtained with identical media under temporary immersion conditions with

RITA® (Section 4.5.7.) were retransferred to liquid culture conditions, no dedifferentiation

was observed (Plate 12, right, page 215). Microscopic investigation of differentiated thalli of

in vitro cultured Corsinia coriandrina indicated the presence of large air chambers, rhizoids,

ventral scales and oil bodies, comparable to the plant material from natural habitats (Plate 15,

page 217). Liquid cultures of undifferentiated calli and differentiated thalli of monoclonal

haploid Corsinia coriandrina strain CC1 (Plate 12, page 215) were maintained for

more than 2.5 years, produced almost the same constituents throughout, and were successfully

employed for the incorporation experiments with stable isotope labelled precursors (Sections

4.6., page 84).


82

4.5.7. Temporarily Immersed Cultures using RITA®

Temporary immersion systems (TIS), which combine unlimited ventilation of the plant tissue

and intermittent contact between its entire surface and the liquid medium, have recently been

described for in vitro cultures of higher plants (Etienne & Berthouly, 2002). Prevention of

hyperhydricity and more natural conditions are among the major advantages of the TIS

technique, which, however, requires optimization of the culture medium as well as immersion

frequency and duration (McAlister et al., 2005; Zhu et al., 2005). Although temporary

immersion systems appear highly suitable for reproducing the natural conditions of liverwort

habitats, this technique has not been applied to liverwort in vitro cultures before.

Temporary immersed cultures of Corsinia coriandrina strain CC1 were established using the

»Recipient for Automated Temporary Immersion« system (RITA®

) shown in Plate 14 (page

216) (Teisson & Alvard, 1995). The plant material is placed in the upper compartment on a

strainer, while the liquid medium can be pumped up from the lower compartment by applying

pressure, generated by a timer controlled aquarium-pump. Diluted MSKP/2, MSKP/5, and

MSKP/10 media were tested. Immersion times were synchronized with the light regime

(16/8 h day-night cycle, 25 µmol*m2*s

1), with immersion times for 3½ or 17½ minutes at

0, 6, 12 and 18 h (1.0 or 5.0 % of total immersion times of liquid cultures). By using

optimized diluted MSKP/5 medium supplemented with 25 mM D-glucose and 2.5 mM

potassium fumarate and applying immersion times of 3½ minutes every 6 hours a doubling

time of approximately 30 days was observed. Furthermore, spontaneous partial differentiation

of callus cultures occurred under these conditions (Plate 13, page 216). However, the factors

responsible for differentiation could not be identified. Resulting thalli could be transferred to

liquid culture conditions using identical media and propagated without dedifferentiation (Plate

12, right, page 215). In addition, TIS cultures of Corsinia coriandrina were used to limit

amino acid uptake in order to prevent excessive reversible transamination in application

experiments with L-tyrosine (154) (Section 4.6.1.2., page 90).

4.5.8. Chemical Investigation of In Vitro Cultured Corsinia coriandrina

GC and GC-EIMS investigation of in vitro cultured Corsinia coriandrina indicated the

presence of those compounds present in plant material from the natural habitat, although

quantitative differences were apparent (Figure 77). These results unambiguously established

the liverwort origin of Corsinia styrenes and stilbenoids. The predominance of (Z)-coriandrin

((Z)-65) in the in vitro cultured material indicated that (Z)-isomers constitute the initial


83

metabolites and are not derived from UV induced (E/Z)-isomerisation of (E)-isomers because

UV permeability of glass vessels and the RITA® compartment is low.

Figure 77: TIC chromatograms of diethyl ether / Na2SO4 extracts of Andalusian Corsinia coriandrina

and corresponding in vitro cultured plant material from liquid cultures and RITA® cultures.

in vivo

material

RITA®

in vitro culture

Liquid

in vitro culture

10 5 15

18

61

61

62

64

64

(E)-65

(E)-65

(Z)-72

RT [min]

RT [min]

RT [min]

TIC [%]

TIC [%]

66 (Z)-67

74

18

73

C16 acid

66

C16 acid

(Z)-65

(Z)-65

(Z)-65

73

(Z)-72

62

(Z)-67

73

50

100

100

50

100

50

TIC [%]

4.6. Application Experiments

84


Figure 78: Isotope labelled precursors applied to Corsinia coriandrina in vitro cultures.


85

Application experiments with deuterium or carbon-13 labelled precursors were performed

using aerated liquid submersion cultures of Corsinia coriandrina. Twenty molecular probes

representing 11 different precursors, like tyrosine (L-[U-D7]-154, DL-[2-D]-154, DL-

[2,3-threo-D2]-154), tyramine ([2,2,3‟-D3]-158), O-methyltyrosine (L-[3,3-D2]-155, DL-[2-D]-

155, L-[CD3]-155), O-methyl tyramine ([CD3]-168), L- and D-phenylalanine (L-[3,3-D2]-153,

D-[3,3-D2]-153), (E)-cinnamic acid ((E)-[2-D]-93), dihydrocinnamic acid ([3,3-D2]-156),

(E)-4-coumaric acid ((E)-[2-D]-152, (E)-[2,3‟,5‟-D3]-152), phloretic acid ([2,2,3,3,3‟-D5]-

157, [2,3-threo-D2]-157), acetate ([2-13

C]-189, [1,2-13

C]-189, [D3]-189) and glycine ([2-13

C]-

177) were applied (Figure 78, page 84).

Initial experiments with unlabelled phenylpropanoids indicated that concentrations c < 0.5

mM were readily tolerated by in vitro cultured Corsinia, whereas c > 0.8 mM proved

deleterious. (E)-Cinnamic acid (93, max = 268 nm at pH 5.7) or (E)-4-coumaric acid (152,

max = 285 nm at pH 5.7) at c = 0.2 mM were taken up within < 14 days as shown by UV

spectroscopy of the culture medium. For GC-EIMS analysis a total amount of 30 µmol of the

labelled precursor (Figure 78) was applied to 2 cm3 plant tissue at a concentration of c = 0.2

mM. Samples of ca. 0.5 cm3 (ca. 250 mg fresh weight) were collected after (7), 14, 21, or 28

days and their diethyl ether extracts analyzed by GC-EIMS with splitless injection. Partial

separation of deuterium labelled compounds eluting at the front of the GC peaks was

observed under gas chromatographic conditions (25 m CpSil-5, 80 °C for 2 min, +10 °C/min,

to 270 °C), enabling the convenient detection of enriched components by GC-EIMS. Isotope

enrichment was calculated from the differences of experimental ion intensities in comparison

to natural abundance compounds from control cultures by using the equation:

2DX or

13CX isotope enrichment [%] = [(M + X)L – (M + X)C / (M + n)L] * 100

(X = number of isotopes, n = 0, 1, 2, ...,; L = labelled, C = natural abundance from control)

Maximum isotope enrichment was observed after 14 – 21 days. L-Tyrosine was incorporated

into coriandrin (65) and related styrenes (Section 4.6.1., page 86), whereas L-phenylalanine

and the corresponding phenylpropanoids were incorporated into corsifuran A (73) and related

STS metabolites (Section 4.6.2., page 102).


86

4.6.1. Biosynthesis of Coriandrin in Corsinia coriandrina

Figure 79: Selected partial mass spectra (EI, 70 eV) of (Z)-coriandrin ((Z)-65) from control cultures of

Corsinia coriandrina; (Z)-[2,aryl-D5]-coriandrin ((Z)-[D5]-65) from application of L-[U-D7]-tyrosine

([U-D7]-154) to liquid cultures; (Z)-[1,2,aryl-D6]-coriandrin ((Z)-[D6]-65) from pulsed application of

L-[U-D7]-tyrosine ([U-D7]-154) to temporarily immersed cultures using RITA®; (Z)-[2-D]-coriandrin

((Z)-[D]-65) from application of L-[3,3-D2]-O-methyltyrosine ([3,3-D2]-155) to liquid cultures.

176

191

175 180 185 190 195 200

20

40

60

80

100

176

181

191

196

175 180 185 190 195 200

20

40

60

80

100

176

182

191

197

175 180 185 190 195 200

20

40

60

80

100

176

191

175 180 185 190 195 200

20

40

60

80

100 (Z)-[D]-65

(Z)-65

(Z)-[D6]-65

(Z)-[D5]-65 [M]

[M – CH3]

[D6]

[D6] [D1]

[D1]

[D5]

[D5]

C10H9NOS

C9H6NOS


87

4.6.1.1. The L-Tyrosine Origin of Coriandrin.

Application of uniformly labelled L-[U-D7]-tyrosine ([U-D7]-154; > 0.98 D) to aerated liquid

cultures of Corsinia coriandrina resulted in [D5]-enrichment of (Z)-coriandrin ((Z)-[D5]-65)

(Figure 79, page 86) and related compounds, thus, establishing their biogenetic origin from

L-tyrosine (154). Higher deuterium enrichment for (Z)-[D5]-65 over (E)-[D5]-65 suggested

that the predominating (Z)-configured styrenes are indeed the initial metabolites. Furthermore,

observed deuterium enrichments corresponded to isothiocyanates ((Z)-[D5]-65) and iso-

cyanates ((Z)-[D5]-63) being the precursors for S,S-dimethyl iminodithiocarbonates ((Z)-[D5]-

72) and S-methyl thiocarbamates ((Z)-[D5]-67), respectively (Figure 80).

Figure 80: Incorporation of L-[U-D7]-tyrosine ([U-D7]-154) into (Z)-[2,aryl-D5]-4-methoxystyrenes

([D5]-63, [D5]-65, [D5]-67, [D5]-72) and related compounds ([D5]-58, [D5]-59, [D5]- 60) by

in vitro cultured Corsinia coriandrina (enrichment after 21 days in atom % D).


88

Isotope enriched (Z)-[2,aryl-D5]-coriandrin ((Z)-[D5]-65) was isolated by preparative thin

layer chromatography on silica gel (hexane - EtOAc, 4:1, RF = 0.55, UV detection) and the

2-position of the side chain deuterium label was determined by alkaline hydrolysis using 1 M

aqueous KOH in THF (Figure 81). The resulting [aryl-D4]-4-methoxyphenylethanal ([aryl-

D4]-59) exhibited a molecular ion signal at m/z 154 [M] and a dominating fragment ion signal

at m/z 125 for [D4]-methoxytropylium ions, thus, indicating cleavage of a -CH-acidic

benzylic deuterium label due to keto-enol-tautomerism (171). These results suggested loss of

the -deuterium label of L-[U-D7]-tyrosine ([U-D7]-154) during (Z)-[2,aryl-D5]-coriandrin

((Z)-[D5]-65) biosynthesis under liquid culture conditions, in agreement with the inability of

DL-[2-D]-tyrosine (DL-[2-D]-154, ˂ 0.98 D) to label coriandrin (65) and related compounds.

Figure 81: Alkaline hydrolysis of (Z)-[2,aryl-D5]-coriandrin ((Z)-[D5]-65) to [aryl-D4]-4-

methoxyphenylethanal ([D4]-59).

Nevertheless, careful analysis of the isotopomer distribution of the molecular ion signal of

(Z)-[2,aryl-D5]-65 indicated that the relative intensity of the [M + 6] signal at m/z 197 (2.6)

exceeded what could be expected for natural abundance of [13

C,D5]-isotopomers (calc. 1.6),

thus, suggesting the presence of up to 1.0 % (Z)-[1,2,aryl-D6]-coriandrin ((Z)-[D6]-65) along

with 13.1 % of the [2,aryl-D5]-species ((Z)-[D5]-65). The predominance of [2,aryl-D5]-species

suggested that the -deuterium label of L-[U-D7]-tyrosine ([U-D7]-154) was possibly lost

during reversible transamination reactions via 4-hydroxypyruvate (172) as shown in figure 82

(page 89), catalyzed by tyrosine-amino-transferase activity (TAT, EC 2.6.1.5) (Wightman &

Forest, 1978; De-Eknamkul & Ellis, 1987). The assumption that alternative pathways for

L-tyrosine metabolism were operating in Corsinia was supported by the fact that L-[U-D7]-

tyrosine ([U-D7]-154) was also incorporated into [2,aryl-D5]-4-methoxyphenylethanal ([D5]-

59), [2,aryl-D5]-4-methoxyphenylethanol ([D5]-60), and [7,aryl-D5]-4-methoxybenzaldehyde

([D5]-58) (Figure 80), and their relative amounts increased.


89

Because [aryl-D4]-isotopomers were insignificant for [D5]-anisaldehyde ([D5]-58) and [D5]-2-

(4-methoxyphenyl)ethanol ([D5]-60) the presence of [aryl-D4]-4-methoxyphenylethanal ([D4]-

59) most likely resulted from the partial loss of the -CH acidic label after its biosynthesis or

during the isolation procedure. Similar metabolites were encountered upon application of L-

[3,3-D2]-phenylalanine (L-[3,3-D2]-153), which was incorporated into the STS metabolites,

but also afforded highly enriched [3,3-D2]-phenylethanal (186) and [3,3-D2]-2-phenylethanol

(187), unknown from C. coriandrina (Section 4.6.2.7., page 114).

Figure 82: Alternative biosynthetic pathways from L-tyrosine (154)

to 4-methoxyphenylethanal (59) and 4-methoxyphenylethanol (60).

Two different pathways from L-tyrosine (154) to 4-methoxyphenylethanal (59) have

previously been described (Figure 82). Transamination of L-tyrosine (154) by tyrosine amino

transferase (TAT, EC 2.6.1.5) affords 4-hydroxyphenylpyruvate (172) (Wightman & Forest,

1978; De-Eknamkul & Ellis, 1987), which is decarboxylated to furnish the aldehyde 175.

Alternatively, initial decarboxylation of L-tyrosine (154) with retention of the -hydrogen by

pyridoxal phosphate dependent tyrosine decarboxylase (TYDC, EC 4.1.1.25) affords tyramine

(158) (Battersby et al., 1980, Facchini et al., 2000), which can be oxidized by flavine (EC

1.4.3.4) or copper (EC 1.4.3.6) dependent amine-oxidases to afford aldehyde 175 upon

hydrolysis of the resulting aldimine 174 (Coleman et al., 1989; Scaman & Palcic, 1992).


90

Reduction by NADH dependent alcohol dehydrogenase gives rise to the alcohol 176, whereas

O-methylation by methyl transferase activity finally affords the corresponding methoxy

derivatives 59 and 60. The predominance of [2,aryl-D5]-4-methoxyphenylethanol ([2,aryl-

D5]-60) indicated that the -deuterium of L-[U-D7]-tyrosine (L-[U-D7]-154) is most likely lost

during TAT mediated transamination, while one benzylic deuterium label might be lost during

keto-enol tautomerism of the resulting 4-hydroxyphenylpyruvate intermediates 172 and 173.

Although this reaction is considered slow under physiological conditions, 4-hydroxy-

phenylpyruvate keto-enol tautomerase (EC 5.3.2.1) is known to catalyze the stereospecific

removal of the 3-Pro-R hydrogen of keto-acid 172 (Sciacovelli et al., 1976, Retey et al., 1977;

Leinberger et al., 1981; Krügel et al., 1985; Pirrung et al., 1993).

In conclusion it was speculated that the (partial) loss of the -deuterium label of L-[U-D7]-

tyrosine (L-[U-D7]-154) during (Z)-[2,aryl-D5]-coriandrin ((Z)-[D5]-65) biosynthesis resulted

from TAT mediated reversible transamination to afford L-[3,3,aryl-D6]-tyrosine (L-[3,3,aryl-

D6]-154). Furthermore, subsequent cleavage of the 3-Pro-R hydrogen of intermediate

[3,3,aryl-D6]-4-hydroxyphenylpyruvate ([3,3,aryl-D6]-172) by keto-enol tautomerase activity

(EC 5.3.2.1) (Retey et al., 1977; Leinberger et al., 1981, Krügel et al., 1985; Pirrung et al.,

1993) would result in L-[3-Pro-S,aryl-D5]-tyrosine (L-[3-Pro-S,aryl-D5]-154). If these

competitive reversible reactions are significantly faster than the first biosynthetic step in

coriandrin (65) biosynthesis, rapid amino acid uptake under liquid culture conditions could

result in almost the entire L-[U-D7]-tyrosine (L-[U-D7]-154) to be converted to L-[3,3,aryl-D6]-

or L-[3-Pro-S,aryl-D5]-tyrosine ([3,3,aryl-D6]-154 or [3-Pro-S,aryl-D5]-154) as possible

precursors to (Z)-[2,aryl-D5]-coriandrin ((Z)-[D5]-65) and [2,aryl-D5]-4-methoxyphenyl-

ethanol ([D5]-60).

4.6.1.2. Pulsed Application Experiments using RITA®

In order to limit amino acid uptake from the culture medium, pulse feeding experiments with

L-[U-D7]-tyrosine (L-[U-D7]-154, ˂ 0.98 D) were performed under temporary immersion

conditions using the RITA® system (Plate 14, page 216). Immersion times of 3½ minutes

every 6 hours were applied, corresponding to 1 % of the total immersion time of liquid

cultures. Samples were collected after (10), 20, 30, 40 (and 60) days, and their purified

extracts were investigated by GC-EIMS. Maximum deuterium enrichment observed after

30 - 40 days was smaller than those from liquid cultures, but the relative amount of

[1,2,aryl-D6]-coriandrins ([D6]-65) was clearly enhanced (Figure 79, page 86). Furthermore,


91

in the mass spectrum of (Z)-[D6]-coriandrin ([D6]-65) the [D6]-fragment ion signal at m/z 182

[M – CH3] for C9D6NOS from O-demethylation confirmed that all six deuterium labels were

exclusively attached to the L-[U-D7]-tyrosine (L-[U-D7]-154) derived carbon skeleton, thus,

establishing unambiguously that the biogenesis of (Z)-[1,2,aryl-D6]-coriandrin ((Z)-[D6]-65)

from L-[U-D7]-tyrosine (L-[U-D7]-154) proceeds with retention of the -hydrogen label.

Figure 83: Incorporation of L-[U-D7]-tyrosine (L-[U-D7]-154) into (Z)-[1,2,aryl-D6]-coriandrin

((Z)-[D6]-65), (E)-[1,2,aryl-D6]-coriandrin ((E)-[D6]-65), (Z)-[1,2,aryl-D6]-corsinian ((Z)-[D6]-63),

(Z)-[1,2,aryl-D6]-O-methyltridentatol B ((Z)-[D6]-72), (Z)-[1,2,aryl-D6]-corsiandren ((Z)-[D6]-67),

(Z)-[1,2,aryl-D6]-tuberin ((Z)-[D6]-98) and [7,aryl-D5]-4-methoxybenzaldehyde ([D5]-58) by in vitro

cultured C. coriandrina under TIS conditions using RITA®

(enrichment after 30 days in atom % D).


92

In addition to (Z)-[1,2,aryl-D6]-coriandrin ((Z)-[D6]-65), the corresponding (E)-isomer ((E)-

[D6]-65), (Z)-[1,2,aryl-D6]-corsinian ((Z)-[D6]-63), (Z)-[1,2,aryl-D6]-O-methyltridentatol B

((Z)-[D6]-72), (Z)-[1,2,aryl-D6]-corsiandren ((Z)-[D6]-67), (Z)-[1,2,aryl-D6]-tuberin ((Z)-[D6]-

98), and [7,aryl-D5]-methoxybenzaldehyde ([D5]-58) were also detected (Figure 83). Only

traces of 4-methoxyphenylethanal (59) or 4-methoxyphenylethanol (60) were observed, which

were too small to detect any possible enrichment.

The retention of the -deuterium of L-[U-D7]-tyrosine (L-[U-D7]-154) under TIS conditions

using the RITA® system supported the assumption that the loss of the -deuterium label under

liquid culture conditions resulted from competing reversible transamination catalyzed by TAT

activity. Moreover, retention of the -deuterium of L-[U-D7]-tyrosine (L-[U-D7]-154)

excluded TAT derived 4-hydroxyphenylpyruvate (172) (Figure 82, page 89) and the related

chain elongation step of the mustard oil pathway of higher plants to be involved in coriandrin

biosynthesis. Furthermore, decarboxylation of dehydrotyrosine intermediates (178, Figure

98), previously suggested as a pathway to primary enamides in microorganisms (Schmidt &

Lieberknecht, 1983), could also be excluded, because the introduction of the double bond

does not precede the decarboxylation step.

4.6.1.3. Tyrosine Decarboxylase Activity

The biosynthesis of higher plant alkaloids is often initiated by decarboxylation of L-tyrosine

(154) to tyramine (158) by tyrosine decarboxylase activity (TYDC, EC 4.1.1.25) (Battersby et

al., 1980; Facchini et al., 2000) (Figure 82, page 89). To identify whether decarboxylation

precedes dehydrogenation of L-tyrosine (154) in coriandrin (65) biosynthesis, [2,2,3‟-D3]-

tyramine ([D3]-158, ˂ 0.90 D for 2-D plus ˂ 0.50 D for 3‟,(5‟)-D) and [CD3]-O-methyl-

tyramine ([CD3]-168, ˂ 0.98 D) were applied. Complete uptake of tyramine (158) ( max = 275

nm at pH 5.7) within 14 d was deduced from UV spectroscopy of fumarate depleted media,

but no incorporation into coriandrin (65) and related compounds was detected. These results

excluded the TYDC metabolite tyramine (158) and its O-methyl derivative (168) as

biosynthetic intermediates in coriandrin (65) biosynthesis and indicated that decarboxylation

of L-tyrosine (154) is not the first biosynthetic step.


93

4.6.1.4. Tyrosine Ammonia Lyase Activity

After TAT and TYDC derived L-tyrosine metabolites were excluded as intermediates in

coriandrin biosynthesis, 4-coumaric acid (152), originating from tyrosine ammonia lyase

activity (TAL, EC 4.3.1.5) (Strange et al., 1972; Rösler et al., 1997; Watts et al., 2006) was

tested. (E)-[2,3‟,5‟-D3]-4-coumaric acid ((E)-[D3]-152; > 0.78 D for 2-D and 0.68 D for

3‟,5‟-D) was incorporated into the phenylpropanoid-polymalonate pathway to stilbenoids and

2-arylbenzofurans (4.6.2.4., page 106), but did not label coriandrin (65) and related

compounds. Furthermore, the observation that phenylpropanoid-polymalonate metabolites

were labelled by (E)-[2,3‟,5‟-D3]-4-coumaric acid ((E)-[D3]-152), but not by L-[U-D7]-

tyrosine (L-[U-D7]-154) indicated that there is no in vivo TAL activity in Corsinia

coriandrina.

4.6.1.5. O-Methyl Transferase Activity

Figure 84: Incorporation of L-[3,3-D2]-O-methyltyrosine (L-[3,3-D2]-155) into (Z)-[2-D]-coriandrin

((Z)-[2-D]-65), (E)-[2-D]-coriandrin ((E)-[2-D]-65), (Z)-[2-D]-corsinian ((Z)-[2-D]-63),

(Z)-[2-D]-O-methyltridentatol B ((Z)-[2-D]-72), and (Z)-[2-D]-corsiandren ((Z)-[2-D]-67)

by in vitro cultured Corsinia coriandrina (enrichment after 21 days in atom % D).


94

After TAT, TYDC and TAL metabolites of L-tyrosine (154) were ruled out as intermediates

in coriandrin biosynthesis by the previous experiments, O-methyltyrosine (155), derived from

L-tyrosine by O-methyl transferase activity, was tested as a biosynthetic intermediate.

Application of doubly labelled L-[3,3-D2]-O-methyltyrosine (L-[3,3-D2]-155, > 0.94 D)

resulted in deuterium enrichment of (Z)-[2-D]-coriandrin ((Z)-[2-D]-65, 13.9 % D) and related

compounds (Figure 84), as deduced from the significant increase of the [M + 1] signal (Figure

79, page 86). Single labelling of (Z)-[2-D]-coriandrin ((Z)-[2-D]-65) unambiguously

established that one of the enantiotopic 3-methylene protons of L-[3,3-D2]-O-methyltyrosine

(L-[3,3-D2]-155) is subsequently lost during introduction of the double bond.

Initially, these results let to the false assumption that O-methylation of L-tyrosine (154) could

be the first step in coriandrin biosynthesis. Nevertheless, L-[CD3]-O-methyltyrosine (L-[CD3]-

155) did not label coriandrin (65) or any other compound, which excluded O-methyltyrosine

(155) as a real biosynthetic intermediate in coriandrin biosynthesis (Figure 85). Moreover,

these results pointed to in vivo demethylation of L-[3,3-D2]-O-methyltyrosine (L-[3,3-D2]-155)

and subsequent incorporation of the resulting L-[3,3-D2]-tyrosine (L-[3,3-D2]-154) into [D1]-

coriandrin ((Z)-[2-D]-65) and related compounds.

Figure 85: In vivo O-demethylation of L-O-methyltyrosine (155) as deduced from application

experiments with L-[3,3-D2]-155, L-[CD3]-155, and DL-[2-D]-155 isotopomers.


95

The comparable enrichment of approximately 13 % D observed with L-[U-D7]-tyrosine

(L-[U-D7]-154) (Figure 80, page 87) and L-[3,3-D2]-O-methyltyrosine (L-[3,3-D2]-155)

(Figure 84, page 93) suggested that uptake and demethylation is not the limiting factor in

coriandrin (65) biosynthesis from exogenic O-methyltyrosine (155). In addition, the inability

of DL-[2-D]-O-methyltyrosine (DL-[2-D]-155, ˃ 0.98 D) to label coriandrin (65) and related

compounds not only confirmed the position of the deuterium label in (Z)-[2-D]-coriandrin

((Z)-[2-D]-65) obtained from L-[3,3-D2]-O-methyltyrosine (L-[3,3-D2]-155), but finally

demonstrated that the L-tyrosine skeleton derived upon demethylation becomes part of the

same precursor pool as did exogenic L-[U-D7]-tyrosine (L-[U-D7]-154), thus, resulting in

comparable dilution with endogenic L-tyrosine (154) and subsequent exchange of the

-hydrogen, due to reversible transamination by TAT activity (Figure 85, page 94).

4.6.1.6. Stereospecific Elimination of the 3-Pro-S-Hydrogen of L-Tyrosine

In order to determine whether the 3-Pro-S or 3-Pro-R hydrogen is lost upon assembly of the

(Z)-configured ethenyl moiety, racemic DL-[2,3-threo-D2]-tyrosine (DL-[2,3-threo-D2]-154,

> 0.97 D, de > 96 %) was applied to Corsinia under standard culture conditions, but no

isotope enrichment of methoxystyrenes whatsoever was detectable. Co-application of

60 µmol DL-[2,3-threo-D2]-tyrosine (DL-[2,3-threo-D2]-154) and L-[U-D7]-tyrosine (L-[U-D7]-

154) (20:1, w/w) afforded only [2,aryl-D5]-enriched species (Z)-[D5]-65 (0.9 % D5) (Figure

86). Although the -deuterium labels of L-[2,3-threo-D2]-154 and L-[U-D7]-154 are lost

during reversible transamination by TAT activity under liquid culture conditions, the

complete absence of single labelled [2-D1]-styrenes implied that neither the 3-Pro-S-deuteron

of L-[2,3-threo-D2]-tyrosine (L-[2,3-threo-D2]-154) nor the 3-Pro-R-deuteron of D-[2,3-threo-

D2]-tyrosine (D-[2,3-threo-D2]-154) are incorporated. That the unusual D-[2,3-threo-D2]-

tyrosine (D-[2,3-threo-D2]-154) did not label Corsinia styrenes might be due to different

reasons, like enantioselectivity of TAT for L-tyrosine (Wightman & Forest, 1978), or

stereospecific removal of the 3-Pro-R deuterium label of 4-hydroxyphenylpyruvate

intermediates [3-Pro-R-D]-172 and 173 by hydroxyphenylpyruvate keto-enol tautomerase

activity (EC 5.3.2.1) (Retey et al., 1977; Leinberger et al., 1981). Nevertheless, loss of the

benzylic deuterium label due to keto-enol tautomerism in [3-Pro-S-D]-172 was insignificant

during [2,aryl-D5]-coriandrin ((Z)-[D5]-65) biosynthesis from L-[U-D7]-tyrosine (L-[U-D7]-

154) as deduced from the [2,aryl-D5] / [aryl-D4]-ratio of > 8.


96

Consequently, the observation that the 3-Pro-S label of L-[2,3-threo-D2]-tyrosine (L-[2,3-

threo-D2]-154) was not incorporated in the co-application experiment can only be explained

by its stereospecific removal during formation of the (Z)-configured double bond. The

retention of 3-Pro-R-H and 2-H of L-tyrosine (154) during assembly of the (Z)-configured

ethenyl-bridge under TIS conditions using RITA®

and the specific loss of the 3-Pro-S-

hydrogen and the carboxyl unit indicated a concerted antiperiplanar decarboxylation-

dehydrogenation step as previously described from xanthocillin (109) biosynthesis in

Ascomycota (Herbert & Mann, 1984a, 1984b).

Figure 86: Stereospecific loss of the 3-Pro-S-hydrogen of L-tyrosine (154) during (Z)-coriandrin

((Z)-65) biosynthesis as deduced from co-application experiments with DL-[2,3-threo-D2]-tyrosine

(DL-[2,3-threo-D2]-154) and L-[U-D7]-tyrosine (L-[U-D7]-154) (20:1, w/w).

4.6.1.7. The Glycine Origin of the O-Methyl Group of (Z)-Coriandrin

After the L-tyrosine origin of (Z)-coriandrins ((Z)-65) and related compounds was established

by the previous experiments, the origin on the O-methyl group and the heterocumulene

carbon were investigated. Previous investigation on (E)-tuberin ((E)-98) biosynthesis in

Streptomyces amakusaensis indicated that both the O-methyl group and the N-formamide

carbon are derived from 2-C of glycine or 3-C of L-serine via tetrahydrofolate-linked

intermediates (Cable et al., 1987a, 1987b).


97

Application of 240 µmol [2-13

C]-glycine ([2-13

]-177) at c = 1.6 mM to liquid in vitro cultures

of Corsinia coriandrina for 1 month afforded only small enrichment of isolated (Z)-[13

C]-

coriandrin ((Z)-[13

CH3]-65; < 2 % 13

C) as deduced by GC/EIMS. Comparison of the relative

intensities of 13

C {1H} NMR signals of the isolated compound with those of unlabelled

control indicated specific 13

C enrichment of the O-methyl group (+165 % = +1.8 atom % 13

C;

Figure 87). Although the alternative S-adenosyl-methionine (SAM) mediated L-methionine

origin of the O-methyl group was not addressed (Roje, 2006), the incorporation of [2-13

C]-

glycine ([2-13

]-177) suggested that O-methylation in Corsinia is at least in part linked to the

tetrahydrofolate pathway.

Figure 87: Sections of 13

C {1H} NMR spectra (100 MHz, C6D6) of natural abundance (Z)-coriandrin

((Z)-65) and [13

CH3]-(Z)-coriandrin ([13

CH3]-65) from incorporation of [2-13

C]-glycine ([2-13

C]-177).

Because the isothiocyanate carbon could not be detected in the broadband decoupled 13

C {1H}

NMR or 13

C PENDANT spectra, intensities of H,C-correlation signals from two-dimensional

HMBC spectra were compared with those of natural abundance in (Z)-coriandrin (65). While

the specific 13

C-enrichment of the O-methyl group could be confirmed by comparing the

relative integrals of 1JH,C correlation signals with control spectra (+140 % peak space = +1.5

atom % 13

C), careful analysis of 3JH,C (1-H-C-NCS) and

4JH,C (2-H-C-C-NCS) correlation

signals failed to show 13

C-enrichment for the isothiocyanate carbon (Figure 25, page 33).

In conclusion, the results indicated a glycine origin of the O-methyl group of (Z)-coriandrin

((Z)-65) via the tetrahydrofolate pathway, whereas the origin of the isothiocyanate carbon

could not be elucidated.

60 70 80 90 100 110

60 70 80 90 100 110

(Z)-65

60 70

+165 % (Z)-[ 13CH3]-65 from application

of [2-13C]-glycine ([2-13C]-177)

x x

OCH3

3‘,5‘-CH

1-CH

C [ppm]


98

4.6.1.8. Discussion of Coriandrin Biosynthesis in Corsinia coriandrina

The L-tyrosine (154) origin of coriandrin (65) and related compounds was unambiguously

established by incorporation of L-[U-D7]-tyrosine (L-[U-D7]-154). While the loss of the

-label under liquid culture conditions was attributed to competing reversible transamination

by TAT, this could be (partly) suppressed by a pulsed application experiment under TIS

conditions using RITA®

to give (Z)-[1,2,aryl-D6]-coriandrin ((Z)-[D6]-65). A collection of

known L-tyrosine (154) metabolites like 4-coumaric acid (152) derived from tyrosine

ammonia lyase (TAL), 4-hydroxyphenylpyruvate (172) from tyrosine amino transferase

(TAT), tyramine (158) from tyrosine decarboxylase (TYDC), or O-methyltyrosine (155) from

O-methyl transferase activity, as well as dehydrotyrosine (178) (Schmidt & Lieberknecht,

1983) could be excluded as biosynthetic intermediates (Figure 88), suggesting that the first

biosynthetic step proceeds at the amino moiety (or the 3-Pro-S-hydrogen). Although the

origin of the isothiocyanate nitrogen has not been proven, the (partial) retention of the -

deuterium label of L-[U-D7]-tyrosine (L-[U-D7]-154) under TIS conditions strongly suggested

an L-tyrosine (154) origin.

Figure 88: Collection of known L-tyrosine (154) metabolites that were excluded

as intermediates in coriandrin biosynthesis.


99

Co-application experiments of DL-[2,3-threo-D2]-tyrosine (DL-[2,3-threo-D2]-154) and

L-[U-D7]-tyrosine (L-[U-D7]-154) indicated that it is the 3-Pro-S hydrogen which is stereo-

specifically lost during assembly of the (Z)-configured ethenyl-bridge of (Z)-coriandrin

((Z)-65). These results indicated a concerted antiperiplanar decarboxylative dehydrogenation

as a key step in (Z)-coriandrin ((Z)-65) biosynthesis in the liverwort Corsinia coriandrina

(Figure 89), similar to xanthocillin (109) biosynthesis in Dichotomomyces cejpii, Ascomycota

(Herbert & Mann, 1984a, 1984b; Cable et al., 1987c, 1991). Both pathways differ from

(E)-tuberine ((E)-98) biosynthesis in Streptomyces amakusaensis, Actinomycetes, which is

characterised by loss of the corresponding 3-Pro-R hydrogen (Herbert & Mann, 1983, 1984a,

1984b).

Figure 89: Proposed biosynthetic pathways from L-tyrosine (154) to (Z)-coriandrin (65),

(Z)-corsinian (63), and (Z)-corsicillin (99) in the ancient liverwort Corsinia coriandrina,

and 1,1‟-dimeric xanthocillin (109) in Dichotomomyces cejpii, Ascomycota, as well as

(E)-tuberine ((E)-98) in Streptomyces amakusaensis, Actinomycetes (ep = electron pair).


100

Furthermore, a tetrahydrofolate-linked glycine origin of N-formamide and O-methyl moieties

of (E)-tuberine ((E)-98) in Streptomyces amakusaensis has been reported, and isonitrile

intermediates were excluded by incorporation of [2,2-D2]-glycine ([2,2-D2]-176) (Cable et al.,

1987a, 1987b). Nevertheless, isonitrile hydratase capable of converting an isonitrile to the

N-formamide has been described from Pseudomonas putida (Goda, et al., 2001, 2002). In the

liverwort Corsinia, however, the isothiocyanate carbon of (Z)-coriandrin ((Z)-[13

CH3]-65) was

not labelled by [2-13

C]-glycine ([2-13

C]-177). While L-tyrosine (154) was identified as the

primary source for the isocyanide nitrogen of xanthocillins (109) the origin of the isonitrile

carbon could not be elucidated until recently (Herbert & Mann, 1984; Puar et al., 1985; Cable

et al., 1987c, 1991).

During the course of this work an isonitrile synthase gene cluster (isnA and isnB) was

identified from environmental DNA (eDNA) expressed in transformed E. coli to afford

(E)-indol-3-yl-ethenyl isonitrile ((E)-183) (Figure 90) (Brady & Clardy, 2005; Brady et al.,

2007; Clardy & Brady, 2007). The corresponding (Z)-indol-3-ylethenyl isonitrile ((Z)-183)

has been isolated from Pseudomonas NCIB 11237, -proteobacteria (Evans et al., 1976,

Hoppe & Schöllkopf 1984), and biosynthetically related hapalindoles, ambiguines,

fischerindoles and welwitindolinones are known from terrestrial cyanobacteria (Gademann &

Portmann, 2008; Raveh & Carmeli, 2007; Bornemann et al., 1988). The isonitrile synthase

operon encodes a protein capable of converting a -amino residue into an isonitrile group

(isnA) and a -ketoglutarate dependent oxygenase for decarboxylative dehydrogenation

(isnB). Similar sequences have been located in bacterial genomes and the free phenol

corresponding to the (E)-isomer of corsicillin ((E)-99) from Corsinia coriandrina was

detected in E. coli expressing the isonitrile synthase operon from Erwinia carotovora,

Enterobacteriales.

Figure 90: (E)-indol-3-yl-ethenyl isonitrile ((E)-183) biosynthesis by the isonitrile synthase operon

from eDNA expressed in E. coli and (Z)-indol-3-yl-ethenyl isonitrile ((Z)-183) from Pseudomonas sp.


101

Furthermore, inverse labelling by application of 12

C labelled carbohydrates to a fully 13

C

labelled culture background and the application of various E. coli knock-out mutants

transformed with the isonitrile synthase gene cluster for (E)-indol-3-ylethenyl isonitrile

((E)-183) synthesis allowed the identification of 2-C of D-ribose as the biogenetic precursor to

the isonitrile carbon (Brady & Clardy, 2005; Clardy & Brady, 2007).

The isonitrile synthase pathway might also account for (E)-tuberin ((E)-98) biosynthesis in

Streptomyces amakusaensis, Actinomycetes. Because only (E)-configured dehydroaminoacid

derivatives have been reported from isonitrile synthase operons so far, application

experiments with D-[2-13

C]-ribose and screening for comparable proteins or genetic sequences

will be required to clarify wheter isonitrile synthase related biosynthetic pathways are also

implicated with dimeric xanthocillins (109) from Ascomycota, as well as the corresponding

monomeric (Z)-corsicillins ((Z)-99) and related (Z)-coriandrins ((Z)-65) and (Z)-corsinians

((Z)-63) from the liverwort Corsinia coriandrina. The occurrence of characteristic

4-methoxystyrenes in two species of the family Corsiniaceae, European Corsinia coriandrina

and South American Cronisia weddellii, believed to have divided during the Ordovician

period (490 – 440 million years ago), indicates a relict biosynthetic pathway dating back to

the early beginning of terrestrial plant evolution. Considering the association of Corsinia

coriandrina and other liverworts with Glomeromycotean fungi (Ligrone et al., 2007),

believed to originate from the Ordovician period (Redecker et al., 2000; Heckman et al.,

2001), the presence of monomeric corsicillins (99) and the similarities in coriandrin (65) and

dimeric xanthocillin (109) biosynthesis in Corsinia and Ascomycota could hardly represent a

coincidence only. The recruitment of bacterial genes associated with secondary metabolism

during evolution of plant life has been discussed previously (Bode & Müller, 2003) and

horizontal gene transfer from associated fungi into mitochondrial introns of Marchantia

polymorpha was suggested (Ohyama & Takemura, 2008).


102

4.6.2. Biosynthesis of Corsifuran A in Corsinia coriandrina

4.6.2.1. Application Experiments using GC-EIMS Detection

Of the different phenylpropanoid precursors that have been applied to C. coriandrina (Figure

78, page 84), L-phenylalanine (L-153), (E)-cinnamic acid (93), (E)-4-coumaric acid (152), and

phloretic acid (157) were incorporated into (R)-(–)-corsifuran A (73) and related compounds

(Table 5).

Phenylpropanoid precursor [D]-73

[atom %]

[D]-74

[atom %]

[D]-69

[atom %]

[D]-68

[atom %]

L-[3,3-D2]-phenylalanine1 L-[D2]-153 11.8 D1

a < 0.5 9.2 D1

f 20.9 D1

D-[3,3-D2]-phenylalanine D-[D2]-153 < 0.5 < 0.5 40 D1 f < 0.5

(E)-[2-D]-cinnamic acid [2-D]-93 8.1 Db

< 0.5 nd 10.2 D

[3,3-D2]-dihydrocinnamic acid [D2]-156 < 0.5 < 0.5 < 1.0 < 0.5

(E)-[2-D]-4-coumaric acid [2-D]-152 10.4 Db < 0.5 nd 11.7 D

(E)-[2-D]-4-coumaric acid § [2-D]-152 7.2 D < 0.5 nd 5.7 D

(E)-[2,3‟,5‟-D3]-4-coumaric acid [D3]-152 11.0 D3c*

6.9 D2# 8.3 D3

g* 12.8 D3

*

[2,2,3,3,3‟-D5]-phloretic acid [D5]-157 13.9 D3d*

4.1 D1 10.8 D3h*

16.7 D3*

[2,3-threo-D2]-phloretic acid [D2]-157 12.1 D1e

< 0.5 4.3 D1 17.3 D1

Table 5: Isotope enrichment of phenylpropanoid-polymalonate metabolites after application of

30 µmol labelled precursor at c = 0.2 mM for 21 days (mean values from 2 GC-EIMS measurements

in comparison to unlabelled control, uncorrected for precursor enrichment), §: performed under

heterotrophic conditions with 100 mM D-glucose in absolute darkness; nd: not determined (see text);

*: [D3] + [D2]; #: [D2] + [D1]; a – e by 2D NMR, a: (R)-(–)-[2-D]-corsifuran A ([2-D]-73); b: (2R,3R)-

(–)-[3-D]-corsifuran A ([3-D]-73); c: (2R,3R)-(–)-[3,3‟,5‟-D3]-corsifuran A ([3,3‟,5‟-D3]-73);

d: (2R,3R)-(–)-[2,3,3‟-D3]-corsifuran A ([2,3,3‟-D3]-73); e: mixture of (2R)-(–)-[2-D]- and (2R,3R)-

(–)-[3-D]-corsifuran A ([2-D]-73 and [3-D]-73); f – h by EIMS, f: [7-D]-dimethyllunularin ([7-D]-69);

g: [3‟,5‟,8-D3]-dimethyllunularin ([3‟,5‟,8-D3]-69); h: [3‟,7,8-D3]-dimethyllunularin ([3‟,7,8-D3]-69);

1: [2,2-D2]-phenylethanal ([2,2-D2]-186, 22.2 % D2 & 27.4 % D1) and [2,2-D2]-2-phenylethanol

([2,2-D2]-187, 72.0 % D2 & 21.7 % D1) detected by EIMS.


103

4.6.2.2. Application Experiments using 2D and

13C NMR Spectroscopy

To unambiguously establish the specific incorporation of labelled phenylpropanoid precursors

into (R)-(–)-corsifuran A (73) the locations of deuterium atoms were determined by 2D NMR

spectroscopy at 61.4 MHz (Figures 92, 94, 98; pages 105, 107 and 111). A total amount of

120 µmol L-[3,3-D2]-phenylalanine (L-[3,3-D2]-153, ˂ 0.96 D), (E)-[2-D]-cinnamic acid

([2-D]-93, ˂ 0.97 D), (E)-[2,3‟,5‟-D3]-4-coumaric acid ([2,3‟,5‟-D3]-152, ˂ 0.78 D for 2-D,

˂ 0.66 D for 3‟,5‟-D), [2,2,3,3,3‟-D5]-phloretic acid ([2,2,3,3,3‟-D5]-157, ˂ 0.98 D for 3-D,

˂ 0.92 D for 2-D, ˂0.56 D for 3‟,(5‟)-D) or (±)-[2,3-threo-D2]-phloretic acid ((±)-[2,3-threo-

D2]-157) was applied to 20 cm3 Corsinia tissue at a concentration of c = 0.4 mM under

standard culture conditions. After 1 month 35 – 50 cm3 fresh plant material (20 – 30 g) was

collected and partially labelled (R)-(–)-corsifuran A (73) isolated by a combination of column

chromatography and thin-layer chromatography. Enantioselective gas chromatography

indicated enantiomeric excesses of ee > 85 – 95 %, suggesting that racemisation was

insignificant during the isolation procedure.

4.6.2.3. The Phenylpropenoid Origin of Corsifuran A and Corsistilbene.

Application of doubly labelled L-[3,3-D2]-phenylalanine (L-[3,3-D2]-153, ˃ 0.96 D) to liquid

in vitro cultures of Corsinia coriandrina afforded single labelled [2-D1]-corsifuran A ([2-D]-

73) in agreement with the initial loss of the 3-Pro-S deuterium label during conversion of

L-[3,3-D2]-153 to (E)-[3-D]-cinnamic acid ([3-D]-93) by phenylalanine ammonia-lyase

activity (PAL), considered as the starting point in phenylpropanoid-polymalonate metabolism

(Gorham, 1977). Consequently, the incorporation of isotopomeric (E)-[2-D]-cinnamic acid

([2-D]-93, ˂ 0.97 D) into [3-D]-corsifuran A ([3-D]-73) indicated that both - and -

hydrogens of the phenylpropenoid precursor are retained in corsifuran A biosynthesis (Figure

91 and Table 5, page 102). Because loss of the -hydrogen during isoflavenoid biosynthesis

via the CHS pathway has previously been demonstrated (Al-Ani & Dewick, 1984), these

results implied an STS pathway for corsifuran A (73) biosynthesis in Corsinia. Furthermore,

incorporation of (E)-[2-D]-4-coumaric acid ((E)-[2-D]-152, ˂ 0.88 D), biogenetically derived

from (E)-[2-D]-cinnamic acid ((E)-[2-D]-93) by cinnamic acid-4-hydroxylase activity (C4H)

(Pierrel et al., 1994), indicated that the para-substituted 2-arylring of corsifuran A (73) is

derived from the phenylpropanoid unit in contrast to its polymalonate origin in STS derived

2-arylbenzofurans from higher plants (Figure 63, page 65).


104

Figure 91: Incorporation of L-[3,3-D2]-phenylalanine ([D2]-153) via PAL derived (E)-[3-D]-cinnamic

acid ([3-D]-93) and (E)-[3-D]-4-coumaric acid ([3-D]-152) into [2-D]-corsifuran A ([2-D]-73) and

related compounds, and incorporation of isotopomeric [2-D]-cinnamic acid ([2-D]-93) and [2-D]-4-

coumaric acid ([2-D]-152) into [3-D]-corsifuran A ([3-D]-73) and (E)-[8-D]-corsistilbene ([8-D]-68)

(* absolute configuration not determined, enrichment of [8-D]-69 could not be detected).

The 2D NMR spectrum of (R)-( )-[2-D]-corsifuran A ([2-D]-73) from the incorporation of

L-[3,3-D2]-phenylalanine (L-[3,3-D2]-153) exhibited a single signal at D 5.4 ppm

corresponding to the 2-position (Figure 92, page 105), which unambiguously excluded

aromatic corsifuran C (74) as an intermediate in corsifuran A (73) biosynthesis (step A2 in

Figure 63, page 65). In addition, [3-D]-corsifuran A ([3-D]-73) from the application of (E)-

[2-D]-cinnamic acid ([2-D]-93) exhibited a signal at ca. D 3.0 ppm corresponding to one

hydrogen of the anisochoric 3-methylene group (data not shown). These results

unambiguously established regiospecific incorporation of - and -labels from the

phenylpropenoid precursors into corsifuran A (73) in agreement with an STS pathway (Figure

91). But in contrast to STS derived 2-arylbenzo[b]furans of higher plants, the 2-aryl moiety of

corsifuran A from Corsinia is derived from the phenylpropanoid unit (Figure 63, page 65).


105

Figure 92: Sections of the 61.4 MHz 2D NMR spectrum of (R)-(–)-[2-D]-corsifuran A ([2-D]-73,

in C6H6) from incorporation of L-[3,3-D2]-phenylalanine (L-[3,3-D2]-153) and

500 MHz 1H NMR spectrum of (R)-(–)-corsifuran A (73) (in C6D6).

In addition to corsifuran A, L-[3,3-D2]-phenylalanine (L-[3,3-D2]-153) or (E)-[2-D]-cinnamic

acid ([2-D]-93) and (E)-[2-D]-4-coumaric acid ([2-D]-152) were also incorporated into (E)-

[D1]-corsistilbenes ((E)-[7-D]-68 and (E)-[8-D]-68, respectively) (Figure 91 and Table 5,

page 102). Deuterium enrichment of (E)-[D1]-corsistilbenes ((E)-[7-D]-68 and (E)-[8-D]-68)

after 14 or 21 days was generally higher than those of the corresponding [D1]-corsifuran A

([2-D]-73 and [3-D]-73), supporting the proposal of stilbenoid intermediates in 2-aryl-

benzofuran biosynthesis (Table 5, page 102). Isomeric (Z)-corsistilbene ((Z)-68) was only

occasionally detected as a trace constituent, but its enrichment was always lower than those of

the corresponding (E)-isomer (E)-68, indicating that (E)-corsistilbene ((E)-68) is the initial

metabolite, in agreement with the stereochemistry of (E)-stilbenoids derived from in vitro

STS reactions (Schröder, 1999; Eckermann et al., 2003).

While molecular ion signals constitute the base peaks in the mass spectra of 2-arylbenzo-

furans (Figure 47 and 53, page 51 and 56) and stilbenoids (Figure 54, page 57), the

corresponding bibenzyls ([7-D]-69 and [8-D]-69) exhibited only a small relative intensity for

the molecular ion signal at m/z 242 [M] (17), which rendered the detection of single labelled

bibenzyls by GC-EIMS difficult (Figure 58, page 60). The base peak signal at m/z 121

[M – C7H7O] (100) for a methoxytropylium ion C7H7O+, which predominantly originates

from the para-substituted benzyl unit (Section 4.3.4.6, page 61), was employed to establish

incorporation of L-[3,3-D2]-phenylalanine (L-[3,3-D2]-153) into [7-D]-dimethyllunularin

([7-D]-69).

3.0 4.0 5.0 6.0 7.0

1H NMR

2-D

73

2D NMR

2-D

[ppm]


106

Because calculation of incorporation rates relied on the assumption that the observed

methoxytropylium ions are exclusively derived from the para-substituted methoxybenzyl

unit, their accuracy might be limited. Furthermore, due to the lack of [D]-methoxytropylium

ion signals in the mass spectra of [8-D]-dimethyllunularin ([8-D]-69) no enrichment resulting

from incorporation of (E)-[2-D]-3-phenylpropenoid acids [2-D]-93 and [2-D]-152 could be

determined (Table 5, page 102).

4.6.2.4. The Phenylpropenoid Origin of Aromatic Corsifuran C.

Figure 93: Incorporation of (E)-[2,3‟,3‟-D3]-4-coumaric acid ([2,3‟,3‟-D3]-152) into (E)-[3‟,5‟,8-D3]-

corsistilbene ([3‟,5‟,8-D3]-68), (2R,3R)-[3,3‟,5‟-D3]-corsifuran A ([3,3‟,5‟-D3]-73) and

[3‟,5‟-D2]-corsifuran C ([3‟,5‟-D2]-74).

In contrast to corsifuran A (73) the corresponding aromatic corsifuran C (74) was neither

labelled by L-[3,3-D2]-phenylalanine (L-[3,3-D2]-153) nor by [2-D]-3-phenylpropenoic acids

([2-D]-93 and (2-D]-152) (Table 5, page 102), excluding its possible role as an intermediate

in corsifuran A biosynthesis. To unambiguously establish the phenylpropenoid origin of

corsifuran C (74) triple labelled (E)-[2,3‟,5‟-D3]-4-coumaric acid ([2,3‟,5‟-D3]-152, ˂ 0.78 D

for 2-D, ˂ 0.66 D for 3‟,5‟-D) was applied to Corsinia cultures to afford [D3]-corsistilbene

([D3]-68), [D3]-corsifuran A ([D3]-73), and [D2]-corsifuran C ([D2]-74) (Figure 93). These

results indicated that aromatic corsifuran C (74) most likely originates from the

dehydrogenation of corsifuran A (73), which is characterized by the loss of both [D]-labels

originating from the - and - position of 4-coumaric acid (152) (step B2/C2 in Figure 63,

page 65). Deuterium enrichment after 21 days was in agreement with a biosynthetic pathway

from [D3]-4-coumaric acid ([D3]-152) via [D3]-corsistilbene ([D3]-68, 12.8 % atom % D) and

[D3]-corsifuran A ([D3]-73, 11.0 % D) to [D2]-corsifuran C ([D2]-74, 6.9 % D).


107

Incorporation of (E)-[2,3‟,5‟-D3]-4-coumaric acid ([D3]-152) into dimethyllunularin (69) was

deduced from the [D2]-methoxytropylium ion signal at m/z 123 [C7H5D2O], but deuterium

enrichment of 8.3 % D was lower than those of corsistilbene ([D3]-68) and corsifuran A ([D3]-

73), suggesting that bibenzyls do not serve as intermediates in corsifuran A biosynthesis

(Table 5, page 102).

Furthermore, the absolute configuration of the deuterium labelled 3-methylene group was

deduced in triple labelled (2R,3R)-(–)-[3,3‟,5‟-D3]-corsifuran A ([3,3‟,5‟-D3]-73), obtained

after incorporation of (E)-[2,3‟,5‟-D3]-4-coumaric acid ([2,3‟,5‟-D3]-152). The 2D NMR

spectrum (Figure 94) exhibited one signal at D 6.75 ppm for the aromatic 3‟,5‟-positions,

which unambiguously established the phenylpropanoid origin of the 2-aryl moiety.

Furthermore, this signal was employed as internal reference to determine the chemical shift of

the second smaller signal at D 3.05 ppm. The assignment of diasterotopic 3-methylene

protons to 1H NMR signals at = 2.95 and 3.05 ppm was finally derived from the

gp-NOESY spectrum (Figure 95). Both 3-methylene protons of 73 show NOE‟s with the

4-methine proton (Fig. 95; A & B), but only the syn-configured 3-Pro-S hydrogen at H 2.95

ppm exhibits an NOE with the 2‟,6‟-hydrogens of the 2-aryl moiety (C). These results

indicated a 3-Pro-R position of the deuterium label in (2R,3R)-[3,3‟,5‟-D3]-corsifuran A

([3,3‟,5‟-D3]-73), corresponding to a cis-configuration between the phenylpropanoid derived

hydrogens.

Figure 94: Sections of the 61.4 MHz 2D NMR spectrum of (2R,3R)-(–)-[3‟,5‟,3-D3]-corsifuran A

([3,3‟,5‟-D3]-73, in C6H6) from incorporation of (E)-[2,3‟,5‟-D3]-4-coumaric acid ([D3]-152) and

500 MHz 1H NMR spectrum of (R)-(–)-corsifuran A (73) (in C6D6).

3.0 4.0 5.0 6.0 7.0 [ppm]

1H NMR

2D NMR

3‘,5‘-D 3-Pro-R-D

73

Pro-S Pro-R


108

Figure 95: Section of the gp-NOESY spectrum and NOE interactions in (R)-(–)-corsifuran A (73)

Consequently, the observation that (E)-[2-D]-3-phenylpropenoic acids ([2-D]-93 and [2-D]-

152) were incorporated into (2R,3R)-[3-D]-corsifuran A ([3-D]-73) but did not label aromatic

corsifuran C (74), whereas (E)-[2,3‟,5‟-D3]-4-coumaric acid ([2,3‟,5‟-D3]-152) afforded

(2R,3R)-[3,3‟,5‟-D3]-corsifuran A ([3,3‟,5‟-D3]-73) and [3‟,5‟-D2]-corsifuran C ([D2]-74)

(Table 5, page 102) indicated that corsifuran C (74) is derived from corsifuran A (73) by syn-

dehydrogenation (Figure 96, page 109).

The specific labelling of the 3-Pro-R position in (2R,3R)-[3,3‟,5‟-D3]-corsifuran A

([3,3‟,5‟-D3]-73) indicated that one enzyme-derived hydrogen is stereo specifically introduced

at the 3-Pro-S position during biosynthesis of chiral (R)-(–)-corsifuran A (73) from achiral

(E)-4-coumaric acid (152), presumably upon H+ catalyzed cyclisation of the corresponding

stilbene intermediates. Consequently, the 2,3-cis-configuration of the phenylpropenoid

derived hydrogens in 73 implied a synfacial cyclisation of (E)-configured-, or an antarafacial

cyclisation of (Z)-configured stilbenoids as the key step to corsifurans. While concerted

synfacial cyclisation could be ruled out due to steric considerations, the antarafacial process

would require a previous (E/Z)-isomerisation step. To identify whether a light-inducible

(E/Z)-isomerisation of stilbenoid intermediates is crucial for the biosynthesis of corsifuran A

(73), (E)-[2-D]-4-coumaric acid ([2-D]-152) was applied to an aerated liquid Corsinia culture,

cultivated in absolute darkness under heterotrophic conditions using diluted MSKP/5 medium,

supplemented with 100 mM D-glucose. After 21 days, incorporation into [8-D]-corsistilbene

([8-D]-68) and [3-D]-corsifuran A ([3-D]-73) was detected by GC-EIMS (Table 5, page 102),

indicating that light is not a requirement for corsifuran A biosynthesis.

A B A B

C C

3-Pro-R 3-Pro-S


109

Although (Z)-stilbenoid intermediates could not be completely ruled out, these results

suggested a two step cyclisation mechanism from STS derived (E)-stilbenoids (124 or 142) to

2-aryl-2,3-dihydrobenzofurans in Corsinia (Figure 96). Electrophilic H+ catalyzed anti-

addition of water or some enzymatic hydroxyl group (184) and subsequent formation of the

furan ring upon bimolecular nucleophilic substitution, thus, resulting in inversion of the

absolute configuration at 2-C to give the cis-configuration of phenylpropenoid derived

hydrogens in corsifuran A (73).

Figure 96: Proposed cyclisation of stilbenoids to 2-aryl-dihydrobenzofurans.

4.6.2.5. Application of Dihydrocinnamic Acids

Although previous experiments suggested (R)-(–)-corsifuran A (73) to be derived from

(E)-4-coumaric acid (152) via stilbenoid intermediates, the identification of trace amounts of a

2,4‟,5-trioxygenated bibenzyl (70, Sect. 4.3.4.6., page 61), plausible intermediate in the

biosynthesis of bibenzyl derived 2-aryl-2,3-dihydrobenzo[b]furans, initiated further studies.

To clarify, whether the carbon skeleton of corsifuran A (73) is of stilbenoid- or bibenzyl-

origin, deuterium labelled 3-phenylpropanoic acids such as [3,3-D2]-dihydrocinnamic acid

([D2]-156, ˂ 0.89 D) and [2,2,3,3,3‟-D5]-4-phloretic acid ([D5]-157, ˂ 0.98 D for 2-D, ˂

0.92 D for 3-D, and ˂ 0.58 D for 3‟,(5‟)-D) were applied, designed as (selective) substrates

for the STS reaction to bibenzyls and related compounds (Fliegmann et al., 1992; Preissig-

Müller et al., 1995).


110

Figure 97: Observed isotopomers of corsifuran and related compounds from incorporation of

[D5]-phloretic acid ([D5]-157) and theoretically expected for biogenetic bibenzyl intermediates.

[3,3-D2]-Dihydrocinnamic acid ([D2]-156) was not incorporated into STS metabolites at all,

but multiple labelled [2,2,3,3,3‟-D5]-phloretic acid ([D5]-157) afforded [D3]-corsifuran A

([D3]-73) and [D1]-corsifuran C ([D2]-74), as well as [D3]-dimethyllunularin ([D3]-69) and

[D3]-corsistilbene ([D2]-68) (Figure 97 and Table 5, page 102). Although these results proved

the incorporation of 4-phloretic acid (157) into corsifuran A (73) and related compounds, the

numbers of incorporated deuterium atoms were not consistent with the expected [2,3,3,3‟-D4]-

corsifuran A ([D4]-73) and [3,3‟-D2]-corsifuran C ([D2]-73) isotopomers derived from [D5]-

phloretic acid ([D5]-157) via [7,7,8,8,3‟-D5]-bibenzyl intermediates ([D5]-69) (Figure 97). Of

the five deuterium labels present in [D5]-phloretic acid ([D5]-157), a maximum of three were

incorporated into corsifuran A and related compounds. Experimentally observed [D3]/[D2]-

ratios of [D3]-corsifuran A ([D3]-73), [D3]-dimethyllunularin ([D3]-69), and [D3]-corsistilbene

([D3]-68) were similar to the [D5]/[D4]-ratio of the [D5]-phloretic acid precursor ([D5]-157),

suggesting that unspecific loss of deuterium was insignificant. Because retention of [D]-labels

from the -, - and aromatic 3‟,5‟-positions of (E)-4-coumaric acid (152), the biogenetic

precursor to phloretic acid (157), had already been demonstrated for corsifuran A

biosynthesis, these results suggested that the second 3-methylene hydrogen in [2,3,3‟-D3]-

corsifuran A ([D3]-73) is not derived from [D5]-phloretic acid ([D5]-157), but introduced

during biosynthesis.


111

Figure 98: Sections of the 61.4 MHz 2D NMR spectrum of (2R,3R)-(–)-[2,3,3‟-D3]-corsifuran A

([2,3,3‟-D3]-73, in C6H6) from incorporation of [2,2,3,3,3‟-D5]-phloretic acid ([D5]-157)

and 500 MHz 1H NMR spectrum of (R)-(–)-corsifuran A (73) (in C6D6).

The 2D NMR spectrum of (2R,3R)-(–)-[2,3,3‟-D3]-corsifuran A ([2,3,3‟-D3]-73) from

incorporation of [2,2,3,3,3‟-D5]-phloretic acid ([D5]-157) exhibited three deuterium

resonances at D 6.75, 5.43, and 3.05 ppm in an approximate ratio of 0.7:1.0:0.7 (Figure 98),

corresponding to the aromatic 3‟-position, the 2-position, and the 3-Pro-R-position,

respectively, and, thus, unambiguously established a 2,3-cis-configuration of the

phenylpropanoid derived deuterium labels in (2R,3R)-(–)-[2,3,3‟-D3]-corsifuran A ([D3]-73).

In addition, specific [D]-labelling of the 3-Pro-R position confirmed that one enzyme derived

hydrogen is introduced stereo-specifically into the 3-Pro-S position during biosynthesis of

(2R,3R)-(–)-[2,3,3‟-D3]-corsifuran A ([D3]-73) from [2,2,3,3,3‟-D5]-phloretic acid ([D5]-157)

(Figure 97, page 110), comparable to the formation of (2R,3R)-[3,3‟,5‟-D3]-corsifuran A

([3,3‟,5‟-D3]-73) from (E)-[2,3‟,5‟-D3]-4-coumaric acid ([D3]-152) (Figure 93, page 106).

The position of the deuterium labels in [3‟,7,8-D3]-dimethyllunularin ([3‟,7,8-D3]-69) could

be deduced from the mass spectrum (Figure 99). While the molecular ion signal at m/z 245

indicated a [D3]-labelled species, the fragment ion signal at m/z 123 [M – C8H8DO] for a [D2]-

methoxytropylium ion (C8H7D2O+), which predominantly originated from the para-

substituted [3‟,7-D2]-4‟-methoxybenzyl-unit (Section 4.3.4.6, page 61), indicated a

[3‟,7,8-D3]-dimethyllunularin isotopomer ([3‟,7,8-D3]-69) of unknown stereochemistry. Small

signals at [M + 4] and m/z 124 were attributed to the [3‟,5‟,7,8-D4]-isotopomer and the

3.0 4.0 5.0 6.0 7.0

1H NMR

2D NMR

2-D 3‘,(5‘)-D

73

3-Pro-R-D

Pro-R

[ppm]

Pro-S


112

corresponding [D3]-methoxytropylium ions, respectively, originating from the small amount

of [2,2,3,3,3‟,5‟-D6]-phloretic acid ([D6]-157) present in the applied molecular probe.

These results indicated that [D5]-phloretic acid ([D5]-157) did not serve as a direct precursor

for the STS reaction to [D3]-corsifuran A ([D3]-73) and [D3]-bibenzyls ([D3]-69) in Corsinia.

The regiospecific loss of two deuterium labels due to hybridization changes at the - and -

position of [D5]-phloretic acid ([D5]-157) during biosynthesis of [D3]-dimethyllunularin

([D3]-69) pointed to the [2,3,3‟-D3]-isotopomer of (E)-4-coumaric acid ([2,3,3‟-D3]-152) as a

biosynthetic intermediate, which is also supported by the detection of (E)-[3‟,7,8-D3]-

corsistilbene ([3‟,7,8-D3]-68) (Figure 97, page 110).

Figure 99: Partial mass spectrum of [3‟,7,8-D3]-dimethyllunularin ([3‟,7,8-D3]-69)

from incorporation of [2,2,3,3,3‟-D5]-phloretic acid ([2,2,3,3,3‟-D5]-157).

The interconversion between phenylpropanoic acids and phenylpropenoic acids has

previously been described from phenylphenalenone biosynthesis in Anigozanthos preissii,

Haemodoraceae (Schmitt & Schneider, 1999, 2001) and mesembrine biosynthesis in

Sceletium strictum, Aizoaceae (Jeffs et al., 1978). In contrast to Anigozanthos species

phenylpropenoid reductase activity in Corsinia displays selectivity for 4-phloretic acid (157)

over dihydrocinnamic acid (156), which was not incorporated (Table 5, page 102).

To identify whether the dehydrogenation of [2,2,3,3,3‟-D5]-phloretic acid ([D5]-157) during

[D3]-corsifuran A ([D3]-73) biosynthesis exhibits syn- or anti-selectivity, racemic

(±)-[2,3-threo-D2]-phloretic acid ([2,3-threo-D2]-157) was applied to Corsinia to afford single

labelled [D1]-corsifuran A ([2-D]-73 and [3-D]-73) and [D1]-corsistilbene ([7-D]-68 and

[8-D]-68) with no evidence for the presence of doubly labelled species by GC-EIMS (Table 5,

page 102). The small [M + 2] signal detected was fully accounted for by natural abundance of

[2D,

13C]-isotopomers.

x 20 [M]

[D3] [D2]

x 20 [M]

[D2]

[M]


113

Incorporation rates of racemic (±)-[2,3-threo-D2]-phloretic acid ([2,3-threo-D2]-157) were

similar to those of the [D5]-isotopomer ([D5]-157) indicating that both enantiomers afforded

labelled metabolites. The 2D NMR spectrum of single labelled [D1]-corsifuran A from the

incorporation of (±)-[2,3-threo-D2]-phloretic acid ([2,3-threo-D2]-157) exhibited two signals

at D 5.43 (1.0 D) and 3.05 ppm (0.8 D) (data not shown), corresponding to a mixture of

(R)-[2-D]-corsifuran A ([2-D]-73) and (2R,3R)-[3-D]-corsifuran A ([3-D]-73), respectively.

These results suggested cleavage of one label from both enantiomers of (±)-[2,3-threo-D2]-

phloretic acid ([2,3-threo-D2]-157) during an anti-selective dehydrogenation, in agreement

with known anti-hydrogenation of cinnamic acid derivatives by Clostridium kluyveri

reductase (Bartl et al., 1977) and Baker‟s yeast (Kawai et al., 1996, 1998). The resulting

(E)-[2-D]- and (E)-[3-D]-4-coumaric acids ([2-D]-152 and [3-D]-152) serve as a substrate for

the STS reaction to afford single labelled [8-D]- and [7-D]-corsistilbene ([8-D]-68 and

[7-D]-68) as well as (2R,3R)-[3-D]- and (R)-[2-D]-corsifuran A ([3-D]-73 and [2-D]-73),

respectively (Figure 91, page 104).

4.6.2.6. Tyrosine Ammonia Lyase Activity

After L-phenylalanine (153) derived (E)-4-coumaric acid (152) was established as

intermediate in corsifuran biosynthesis, its possible alternative origin from L-tyrosine (154),

catalyzed by tyrosine ammonia-lyase activity (TAL, EC 4.3.1.5), was investigated. TAL

catalyzes the antiperiplanar elimination of the amino group and the 3-Pro-S-hydrogen of

L-tyrosine (154) to give (E)-4-coumaric acid (152) (Strange et al., 1972). TAL activity has

been reported from dicotyls mainly and the corresponding enzymes are closely related to PAL

(Rösler et al., 1997, Watts et al., 2006).

Application of L-[U-D7]-tyrosine (L-[U-D7]-154) afforded [D5]-labelled 4-methoxystyrenes

(Section 4.6.1, page 86), but no enrichment of phenylpropenoid-polymalonate metabolites

could be detected, indicating that L-tyrosine (154) is not converted into 4-coumaric acid (152)

due to a lack of in vivo TAL activity. Apparently, biosynthetic pathways to L-phenylalanine

(153) derived STS metabolites like (E/Z)-corsistilbenes ((E/Z)-68) and corsifurans A and C

(73 and 74) as well as L-tyrosine (154) derived coriandrin ((Z/E)-65) and related compounds

are already separated at the amino-acid stage, with no crosstalk between enzymes, due to the

lack of tyrosine-ammonia lyase (TAL) or phenylalanine-4-hydroxylase (P4H) activity.


114

4.6.2.7. Phenylalanine Transaminase Activity

Incorporation of L-[3,3-D2]-phenylalanine (L-[3,3-D2]-153) not only afforded labelled STS

metabolites (Section 4.6.2.3, page 103) but also furnished two highly enriched minor

constituents ([D2]-186 and [D2]-187) unknown from in vitro cultured Corsinia (Figure 100).

Upon application of unlabelled L-phenylalanine (153), phenylethanal (186, < 1 % of the total

volatiles) and phenylethanol (187, < 2 %) could be identified upon comparison of their mass

spectra and GC retention indices with those of authentic reference compounds. The benzylic

positions of deuterium labels in [2,2-D2]-phenylethanal ([D2]-186) and [2,2-D2]-2-phenyl-

ethanol ([D2]-187), obtained from L-[3,3-D2]-153, was deduced from [D2]-tropylium ion

signals at m/z 93 in the corresponding mass spectra (Figures 101 and 102, page 115).

Figure 100: Proposed biosynthetic pathway from L-phenylalanine (153) via phenylpyruvate (185) to

phenylethanal (186) and phenylethanol (187).

Because de novo synthesis of phenylethanol (187) is induced by addition of labelled

L-[3,3-D2]-phenylalanine (L-[D2]-153, 0.95 D) the extremely high enrichment of > 93 % D

[D2 + D1] observed for [2,2-D2]-2-phenylethanol ([D2]-187) suggested that the corresponding

precursor pool consisted almost exclusively of labelled species at the time of its biosynthesis,

and dilution with biogenic L-phenylalanine (153) was insignificant (Figure 100). The fact that

the [D2]-species largely predominates (72.0 % D2 vs. 21.7 % D) indicated that biosynthesis of

phenylethanol (187) from L-phenylalanine (153) proceeds without hybridisation changes at

the benzylic carbon. Biosynthetically related phenylethanal (186) and 2-phenylethanol (187)

most likely originate from L-phenylalanine (153) via phenylpyruvate (185) by phenylalanine

dehydrogenase (PHD, EC 1.4.1.20) activity. The observation that intermediate [2,2-D2]-

phenylethanal ([D2]-186) was only labelled to a lesser extent (22.2 % D2 and 27.4 % D) in

comparison to [D2]-187 might result from loss of the -CH acidic labels due to keto-enol

tautomerism.


115

Figure 101: Selected EIMS spectra of [2,2-D2]-phenylethanal ([D2]-186) and phenylethanal (186).

Figure 102: Selected EIMS spectra of [2,2-D2]-phenylethanol ([D2]-187) and phenylethanol (187).

39 51

65

77

91

103

122

40 60 80 100 120 140

20

40

60

80

100

C8H10O

187

51 66

93

124

40 60 80 100 120 140

20

40

60

80

100 C7H5D2

C8H8D2O

[D2]-187

20

40

60

80

100

40 60 80 100 120

39

51

65

91

120

C8H8O

C7H7

186

20

40

60

80

100

40 60 80 100 120

39 51

66

93

122

C8H6D2O

C7H5D2

[D2]-186

C7H7


116

4.6.2.8. Application of D-3,3-Dideuterophenylalanine

Because some of the labelled compounds used in previous experiments were applied in

racemic form only, the fate of unusual D-amino acids in Corsinia coriandrina was of interest.

It is known that D-amino acid uptake is similar to those of the L-forms (Aldag &Young, 1970;

Ladešić et al., 1971; Pokorny, 1974). Although racemic DL-amino acids have often been

applied, there is only one study on the incorporation of D-[14

C*]-phenylalanine (D-[14

C*]-153)

in buckwheat, Fagopyrum esculentum, Caryophyllales, which came to the conclusion that

high amounts were incorporated into flavonoids comparable to the L-isomer (Margna et al.,

1987). To test for the stereoselectivity of amino acid uptake and PAL in vivo, the unusual

D-[3,3-D2]-phenylalanine (D-[3,3-D2]-153, ˂ 0.96 D) was applied to Corsinia cultures. While

the corresponding L-[3,3-D2]-phenylalanine (L-[3,3-D2]-153) was a substrate for PAL and

entered the STS pathway to (E)-[7-D]-corsistilbene ([7-D]-68) and [2-D]-corsifuran A

([2-D]-73) (Figure 91, page 104), enantiomeric D-[3,3-D2]-phenylalanine (D-[3,3-D2]-153)

was not incorporated into these metabolites, indicating that D-[3,3-D2]-153 is not converted to

[3-D]-cinnamic acid ([3-D]-93), due to stereoselectivity of PAL for the L-form.

Nevertheless, D-[3,3-D2]-phenylalanine (D-[3,3-D2]-153) was a much better precursor for

single labelled [7-D]-dimethyllunularin ([7-D]-69) than the L-form (L-[3,3-D2]-153) or any

other precursor tested, resulting in the highest enrichment ever detected for the STS

metabolites in Corsinia (Figure 103 and Table 5, page 102). Application of D-[3,3-D2]-153

afforded [7-D]-dimethyllunularin ([7-D]-69, > 43 % D), along with traces of a second

bibenzyl, tentatively identified as [7-D]-4-O-methyl lunularin ([7-D]-188, > 54 % D), due to

the molecular ion signal at m/z 229 [M] and the base peak signal at m/z 122 [M – C7H7O] for

a [D]-methoxytropylium ion.

Figure 103: [7-D]-dimethyllunularin ([7-D]-69) and [7-D]-4-O-methyllunularin ([7-D]-188)

from the incorporation of D-[3,3-D2]-phenylalanine (D-[3,3-D2]-153.


117

The fact that only [D1]-species ([7-D]-69 and [7-D]-188) were observed after incorporation of

D-[3,3-D2]-phenylalanine (D-[3,3-D2]-153) indicated that biosynthesis of bibenzyls from

D-phenylalanine (D-153) proceeds with hybridization change at the benzylic position of the

phenylpropanoid precursor. Only traces of [2,2-D2]-phenylethanal ([D2]-186, < 0.1 % of the

total volatiles with 23 % D2 and 28 % D) were detected, suggesting that conversion of

D-phenylalanine (D-153) into phenylpyruvate (185) was insignificant in comparison to the

L-form (L-153). In conclusion, the biosynthetic intermediates of the transformation of

D-[3,3-D2]-phenylalanine (D-[3,3-D2]-153) into the bibenzyl compounds [7-D]-dimethyl-

lunularin ([7-D]-69) and [D]-4-O-methyl lunularin ([7-D]-188) remain unidentified. Because

D-phenylalanine (D-153) has not yet been identified in C. coriandrina, it is unknown whether

this pathway is of any biological significance under natural conditions. Nevertheless, the

results indicate the ability to convert D-[3,3-D2]-phenylalanine (D-[3,3-D2]-153) into

[D]-bibenzyls ([D]-69) only, with a complete lack of other labelled PAL derived metabolites

([D]-68 and [D]-73) (Table 5, page 102), which could not be readily explained. Nevertheless,

specific labelling of bibenzyls upon application of D-[3,3-D2]-phenylalanine (D-[3,3-D2]-153)

supported the conclusion that 2-arylbenzo[b]furans are not derived from bibenzyl

intermediates, but most likely originate from stilbenoid precursors.

Figure 104: Selected mass spectra (EI, 70 eV) of natural abundance dimethyllunularin (69) and

[7-D]-dimethyllunularin ([7-D]-69) from incorporation of D-[3,3-D2]-phenylalanine (D-[D2]-153).

20

40

60

80

100

40 60 80 100 120 140 160 180 200 220 240

4378

91

121

242

20

40

60

80

100

40 60 80 100 120 140 160 180 200 220 240

43 5578

91

122

243

C16H18O2

C16H17DO2

C8H9O

C8H8DO

[D]-69

69


118

4.6.2.9. The Polymalonate Origin of Corsifuran A

After participation of the phenylpropenoid pathway in the biosynthesis of (R)-(–)-corsifuran A

(73) was unambiguously established upon incorporation of [D]-labelled precursors and 2D

NMR spectroscopy, the assumed polymalonate origin of 4-CH, 5-C, 6-CH, 7-CH and 7a-C of

the benzo[b]furan unit was investigated using [13

C]-labelled acetate ([13

C]-189). Application

of 1.8 mmol sodium [2-13

C]-acetate ([2-13

C]-189) at c = 6 mM under standard culture

conditions for 1 month afforded 13

C-enriched corsifuran A ([13

C]-73) as shown by GC-EIMS

(+ 30 atom % 13

C over natural abundance). Comparison of the broadband decoupled

100 MHz 13

C {1H} NMR spectra of isolated [4,6,7a-

13C]-corsifuran A ([4,6,7a-

13C]-73) with

natural abundance corsifuran A (73) as control (Figure 106, page 119) indicated specific 13

C-

enrichment of 4-CH (+ 9.1 atom % 13

C over natural abundance), 6-CH (+ 10.3 % 13

C), and

7a-C (+ 10.0 % 13

C). Specific 13

C-enrichment of the corresponding 2-CH (+ 5.8 % 13

C), 4-CH

(+ 7.6 % 13

C), and 6-CH (+ 4.5 % 13

C) positions was also detected for (E)-[2,4,6-13

C]-

corsistilbene ([2,4,6-13

C]-68) isolated in small amounts (data not shown).

Figure 105: 13

C-enrichment of [4,6,7a-13

C]-corsifuran A ([4,6,7a-13

C]-73) and

(E)-[2,4,6-13

C]-corsistilbene ([2,4,6-13

C]-68) after incorporation of [2-13

C]-acetate ([2-13

C]-189)

(atom % 13

C over natural abundance, phenylpropanoid unit and C-2 of acetate drawn bold).

Specific labelling of both [4,6,7a-13

C]-corsifuran A ([4,6,7a-13

C]-73) and (E)-[2,4,6-13

C]-

corsistilbene ([2,4,6-13

C]-68) confirmed their phenylpropenoid-polymalonate origin via STS

catalyzed cyclisation of intermediate trisketo acid 190 (Figure 105), and also excluded

radical-coupling of 4-coumaric (152) acid derived 4-hydroxystyrene and a symmetrical C6-

unit such as hydroquinone or 1,4-benzoquinone (Figure 49, page 53), previously proposed as

metabolic pathway to racemic (±)-2-(4-hydroxyphenyl)-5-hydroxy-2,3-dihydrobenzo[b]furan

(145, Figure 63, page 65) in Bacillus megaterium (Torres y Torres & Rosazza, 2001).


119


C {1H} NMR spectra (CDCl3) of natural abundance corsifuran A (73),

[4,6,7a-13

C]-corsifuran A ([13

C]-73) from application of [2-13

C]-acetate ([2-13

C]-189), and

[4-13

C,5(6),7(7a)- 13

C2]-corsifuran A ([13

C2]-73) from [1,2-13

C2]-acetate ([1,2-13

C2]-189.

40 50 60 70 80 90 100 110 120 130 140 150 160

C [ppm] 50 60 70 80 90 100 110 120 130 140 150 160

●

□

40 50 60 70 80 90 100 110 120 130 140 150 160

solvent

solvent

solvent

4

6

7a

7a

40 50 60 70 80 90 100 110 120 130 140 150 160 40 50 60 70 80 90 100 110 120 130 140 150 160

50 60 70 80 90 100 110 120 130 140 150 160

■ ○

40 50 60 70 80 90 100 110 120 130 140 150 160

●

4

6 7

5

natural

abundance 73

5-OCH3

3 2 7

4

6

7a

5

4’

3a

4’-OCH3

1’

● ●

[13C2]-73 from [1,2-13C2]-189

[4,6,7a-13C]-73 from [2-13C]-189

x x

x

x

3’,5’ 2’,6’


120

4.6.2.10. -Carbonyl Group Reduction in Corsifuran A Biosynthesis

After the phenylpropenoid-polymalonate origin of corsistilbenes ((Z/E)-68) and corsifuran A

(73) was established by incorporation of [D]-phenylpropenoids and [2-13

C]-acetate ([2-13

C]-

189), the liverwort specific carbonyl group reduction and cyclisation of the intermediate

trisketo acid (190) were investigated. The majority of stilbenoids from higher plants are either

derived from 3,5-dioxygenated pinosylvine or 3,5,4‟-trioxygenated resveratrol (136, Figure

63, page 65), originating from (E)-cinnamic acid (93) or (E)-4-coumaric acid (152),

respectively. Although some cinnamic acid (93) derived 3,5-dioxygenated bibenzyls have

been described in liverworts, the 4-coumaric acid (152) derived 3,4‟-dioxygenated bibenzyls

and bis(bibenzyls) predominate (Asakawa, 2004), thus, indicating an unique intermediate

reduction step in the biosynthetic pathway to liverwort bibenzyls. Due to the co-occurrence of

prelunularic acid (7), lunularic acid (5), and lunularin (6) (Figure 1, page 5) selective

reduction of the -carbonyl group of the intermediate trisketo acid (190) has been postulated

but never proven unambiguously (Pryce, 1971; Ohta et al., 1983; Abe & Ohta, 1984;

Friederich et al., 1999a; Eckermann et al., 2003).

Figure 107: Different labelling patterns of corsifuran A (73) after incorporation of [1,2-13

C2]-acetate

([1,2-13

C2]-189) resulting from cyclisation of intermediate trisketo acids reduced at the - or -

carbonyl group (191 and 193), respectively (R = 4-hydroxy- or 4-methoxyphenyl) (●■, ○□, and

used to mark methyl- and carboxyl-atoms of acetate units, respectively)


121

Corsifuran A and C (73 and 74) as well as (E/Z)-corsistilbenes ((E/Z)-68) from Corsinia

coriandrina share the typical 3,4‟-substitution pattern of common liverwort bibenzyls, thus,

indicating a similar reduction step (Figure 63, page 65). To identify whether the - or -

carbonyl group is subsequently reduced in corsistilbene (68) and corsifuran A (73)

biosynthesis, the locations of the acetate units were determined by application of 1.2 mmol

sodium [1,2-13


C2]-189) at c = 4 mM under standard culture conditions for

one month. Depending on the reduction of one or the other of the carbonyl groups, two

different bond labelling patterns could be expected as shown in Figure 107 (page 120).

Inspection of the broadband decoupled 13

C {1H} NMR spectrum of isolated [4-

13C,5(6),7(7a)-

13C2]-corsifuran A ([

13C2]-73) indicated specific

13C-enrichment of the 4-C unit (s, +4.3 %

13C), while the signals for 5,6,7 and 7a-C were complemented by doublets due to

13C,

13C-

coupling in incorporated 13

C2-units (Figure 108). Comparison of the coupling constants

established incorporation of [1,2-13


C2]-189) into the 5,6-C2 (1J = 70.7 Hz)

and 7,7a-C2 (1J = 71.5 Hz) units. Additional

13C-coupling constants could be deduced from

multiple labelled corsifuran A, like 1J4,5 = 69.1 Hz and

2J4,6 = 4.0 Hz from the small dd-

signals corresponding to 4-C in [4,5,6-13

C3]-corsifuran A, or 1J6,7 = 60.2 Hz from small triplet

signals for 6- and 7-C in [5,6,7,7a-13

C4]-corsifuran A species.

Figure 108: Sections of the 13

C {1H} NMR spectrum of [4-

13C,5(6),7(7a)-

13C2]-corsifuran A

([13

C2]-73) from incorporation of [1,2-13


C2]-189).

109 110 111 112 113 114 153 154 155 109 110 111 112 113 114 153 154 155

5-13

C

7a-13

C

4-13

CH

7-13

CH

7,7a-13

C2 7a,7-

13C2

5,6-13

C2 6,5-13

C2

6-13

CH

1J 70.7 Hz

1J 70.7 Hz

1J 71.5 Hz 1

J 71.5 Hz

+ 4.3 % 13

C

13C4

13C3

●

□ ■ ○

3’,5’-13

CH

C [ppm]

13C4


122

Single labelling at the 4-position of [4-13

C,5(6),7(7a)-13


C2]-73) confirmed

that the adjacent 3a-C is not derived from [1,2-13


C2]-189) but originates

from the carboxyl group of the phenylpropenoid precursor (E)-4-coumaric acid (152), and

again excluded the coupling of 4-coumaric acid derived 4-hydroxystyrene and a C6-unit

(Figure 49, page 53), previously proposed for biosynthesis of corsifuran type compounds

(Torres y Torres & Rosazza, 2001). Furthermore, inspection of the 13

C {1H} NMR spectrum

of isolated (E)- [2-13

C,3(4),5(6)-13

C2]-corsistilbene ([13

C2]-68) (data not shown) indicated

significant 13

C2 enrichment of 3,4-C2 (d, 2J3,4 = 66.7 Hz) and 5,6-C2 (d,

2J5,6 = 58.8 Hz) units,

as well as a 13

C enriched 2-C unit (s, + 3.5 % 13

C) in the polymalonate derived 3-methoxy-

phenyl ring, corresponding to the labelling pattern in [4-13

C,5(6),7(7a)-13

C2]-corsifuran A

([13

C2]-73) (Figure 109).

Figure 109: 13

C {1H} NMR multiplicities and direct

1JC,C coupling constants of [4-

13C,5(6),7(7a)-

13C2]-corsifuran A ([

13C2]-73) and (E)-[2-

13C,3(4),5(6)-

13C2]-corsistilbene ([

13C2]-68) (●■, ○□, and

used to mark methyl- and carboxyl-atoms of acetate units, respectively).

Specific incorporation of [1,2-13

C]-acetate ([1,2-13

C]-189) unambiguously established the

2-position of (E)-[2-13

C,3(4),5(6)-13

C2]-corsistilbene ([13

C2]-68) and the corresponding

4-position of (R)-( )-[4-13

C,5(6),7(7a)-13


C2]-73) as former attachment

point of the subsequently cleaved carboxylic acid moiety (Figure 109). These results

established that it is the -carbonyl group of the intermediate trisketo acid (190), which is

subsequently reduced to 193 during (E)-corsistilbene (68) and corsifuran A (73) biosynthesis

in Corsinia (Figure 107, page 120), similar to the biosynthesis of hydrangenic acid (194,

R = hydroxyphenyl) in the Garden Hortensia, Hydrangea macrophylla, Saxifragales (Billek &

Kindl, 1962; Gorham, 1977a) or lunularic acid (5) in liverworts (Pryce, 1971; Friederich et

al., 1999a).


123

4.6.2.11. Exclusion of Stilbenecarboxylic Acid Intermediates

Although the possible implication of hydrangenic acid (194) as a biosynthetic intermediate in

corsistilbene (68) and corsifuran (73) biosynthesis is not supported by chemical analysis of

liverworts in general and Corsinia coriandrina in particular (Gorham, 1977a), [14

C*]-

hydrangenic acid ([14

C*]-194) is efficiently converted to [14

C*]-lunularic acid ([14

C*]-5) and

[14

C*]-lunularin ([14

C*]-6) by Lunularia cruciata (Pryce 1971). Nevertheless, stilbene-

carboxylic acids have recently been excluded as intermediates in the stilbene synthase

reaction (STS) due to mechanistic considerations (Austin et al., 2004) and the incorporation of

up to three deuterium labels from [2,2-D2]-malonyl-SCoA into (E)-[2,4,6-D3]-resveratrol

([2,4,6-D3]-136 , page 65) during the in vitro peanut STS reaction, thus, indicating

decarboxylation to occur prior to aromatisation of the polymalonate derived aryl unit

(Shibuya et al., 2002).

Figure 110: Hydrangenic acid (194) from Hydrangena macrophylla, Saxifragales,

and (E)-corsistilbene (68) and corsifuran A (73) from C. coriandrina.

To test for hydrangenic acid (194) as a possible intermediate in corsistilbene (68) and

corsifuran A (73) biosynthesis (Figure 110), sodium [D3]-acetate ([D3]-189) was applied to

Corsinia coriandrina. Initial experiments at 6 mM indicated that deuterium incorporation was

much smaller than those observed with [2-13

C]-acetate ([2-13

C]-189), presumably due to

extensive keto-enol tautomerism of [D3]-acetate derived [2,2-D2]-malonic acid ([2,2-D2]-88)

and various ketoacid intermediates (190 and 193) (Figures 105 and 107, page 118 and 120).

Nevertheless, consecutive application of 18 mmol [D3]-acetate ([D3]-189) over 3 months

afforded 120 g fresh plant material from which ca. 120 µmol (R)-(–)-[4,6-D]-corsifuran A

([4,6-D]-73) could be isolated, sufficiently enriched for 2D NMR analysis (ca. 12 % D by

EIMS and 13

C NMR).


124

After 0.5 mg synthetic (±)-[5-OCD3]-corsifuran A ([5-OCD3]-73) was added as internal

standard ( D 3.38 ppm) the dominating 2D NMR signal at D 6.6 ppm could be assigned to the

6-position. Furthermore, a partially overlapping smaller signal at D 6.7 ppm could be

assigned to the 4-position, while no evidence for labelling of the 7-methine group ( H 6.8

ppm) or any other position was obtained (Figure 111).

Figure 111: 1H NMR spectrum (500 MHz, C6D6) of corsifuran A (73) and

2D NMR spectrum

(61.4 MHz, C6H6) of (R)-(–)-[4,6-D]-corsifuran A ([4,6-D]-73) from incorporation of [D3]-acetate

([D3]-189) with (±)-[5-OCD3]-corsifuran A ([5-OCD3]-73) as internal standard.

Specific deuterium incorporation from [D3]-acetate ([D3]-189) into the 6- and 4- positions of

(R)-(–)-[4,6-D]-corsifuran A ([4,6-D]-73) was also supported by indirect detection of the

2D-labels using the -isotope shift on natural abundance

13C NMR signals. While the larger

-isotope shift (1

0.2 to 0.7 ppm) is difficult to detect in aromatics due to small signal

intensities resulting from the triplet multiplicity (1:1:1; 1JC,D ~ 25 Hz), poor relaxation and the

lack of signal enhancement by NOE, the -isotope shift (2

0.05 to 0.15 ppm) results in a

single sharp line due to the small values of the 2JC,D coupling constants (< 1.0 Hz) and

restores the possibility for signal enhancement in 2D

12C-

13C

1H species due to NOE acting on

hydrogen bound reporter nuclei (Dziembowska et al., 2004). First applied by Abell and

Staunton (1981) to determine the location of deuterium after incorporation of [D3,1-13

C]-

acetate, -isotope (2

) shifted natural abundance 13

C NMR signals were now observed in a

125 MHz 13

C {1H} NMR spectrum of [4,6-D]-corsifuran A ([4,6-D]-73) obtained by

accumulation of 15.000 scans using a relaxation delay time D1 = 4 sec (Figure 112, page 125).

3.0 4.0 5.0 6.0 7.0

(R)-(–)-[4,6-D]-73

4-D

6-D

7-H

5-OCD3

73

2D NMR

1H NMR

ppm


125

Figure 112: Sections of the 13

C {1H} NMR spectrum (125.8 MHz, CDCl3, NS 15.000, D1 = 4 sec) of

[4,6-D]-corsifuran A ([4,6-D]-73) from incorporation of [D3]-acetate ([D3]-189) showing -isotope

shifted signals for natural abundance 5-13

C, 3a-13

C and 7-13

CH; signals marked with 1JC,C originate

from natural abundance 13

C,13

C-coupling.

Deuterium substitution at the 6-position resulted in -isotope shifted 13

C NMR signals for

5-CO (2

= – 0.044 ppm, ca. 11 %) and 7-CH (2

= – 0.103 ppm, ca. 12 %). Furthermore,

retention of deuterium at the 4-position was indicated by additional -isotope shifted signals

for adjacent 5-CO (2

4 = – 0.088 ppm, ca. 2 %), and 3a-C (2

4 = – 0.110 ppm, ca. 2 %).

Observed -isotope shifts (2

) were in good agreement with values reported for [2-D]-

methoxybenzene and [2-D]-methylbenzene (Bell et al., 1972).

In conclusion, results from 2D NMR and

13C {

1H} NMR spectroscopy (Figures 111 and 112)

indicated incorporation of deuterium from [D3]-acetate ([D3]-189) into the 4- and the 6-

position of [4,6-D]-corsifuran A ([4,6-D]-73). Partial retention of deuterium at the 4-methine

position of corsifuran A indicated that decarboxylation at 2-C in the cyclised tetraketide

([2,4,6-D]-195) intermediate occurs prior to aromatization (Figure 113), and, thus, excluded

stilbenecarboxylic acids like hydrangenic acid (194) or lunularic acid (5) as intermediates in

corsifuran A (73) biosynthesis.

154.0 154.4 109.4 127.0 109.2 154.2 127.8 127.4 154.0 154.4 109.4 127.0 109.2 154.2 127.8 127.4

3a-13

C 5-13

C 7-13

CH

7-13

C,6-2D 3a-

13C,4-

2D

5-13

C,4-2D

5-13

C,6-2D

1JC,C

1JC,C

0.110 ppm

0.088 ppm

0.044 ppm

3’,5’-13

CH

∆2

∆2

∆2

∆2

C [ppm]

0.103 ppm


126

Figure 113: Proposed cyclisation of [D3]-acetate ([D3]-189) derived [2,4,6-D]-hydroxydiketo acid

([2,4,6-D]-193) via non-aromatic carboxylic acid [2,4,6-D]-195 to stilbenoids ([2,4,(6)-D-141) and

[4,6-D]-corsifuran A ([4,6-D]-73) (R = 4-hydroxy- or 4-methoxyphenyl).

4.6.2.12. Discussion of Corsifuran A Biosynthesis in Corsinia coriandrina

The biosynthetic origin of most atoms in corsifuran A (73) was deduced from incorporation

experiments with isotope labelled precursors and 2D and

13C NMR spectroscopy of isolated

corsifuran A (Figure 114). The phenylpropenoid origin of 3‟,5‟-H, 2-H, and 3-Pro-R-H, as

well as the polymalonate origin of 4-CH (), 5,6-CCH (□○), and 7,7a-C2 (■●) units was

unambiguously established. 7-H is attached to a carbon (■) originating from the carboxyl

moiety of acetate and is introduced during a liverwort specific reduction of the -carbonyl

group in the trisketo intermediate (190). Furthermore, the 3-Pro-S hydrogen is specifically

introduced, most likely during H+ catalyzed cyclisation of stilbene intermediates.

Figure 114: Biosynthetic STS origin of the corsifuran A (73) structure (phenylpropanoid unit bold;

●■, ○□, and used to mark methyl- and carboxyl-atoms of acetate units, respectively;

H introduced during biosynthesis upon reduction of -carbonyl group (Hred),

stilbene cyclisation (Hcycl) or keto enol tautomerism).


127

Figure 115: Proposed STS pathway from L-phenylalanine (153) to (E)-corsistilbene ((E)-68),

(R)-(–)-corsifuran A (73) and corsifuran C (74).

In conclusion, the results indicate an L-phenylalanine (153) origin and a phenylpropenoid-

polymalonate pathway to 2-arylbenzo[b]furans, stilbenoids and bibenzyls in Corsinia

coriandrina (Figure 115). L-Tyrosine (154) is a precursor of (Z/E)-coriandrins (65) and

related -amino styrenes (Figure 89, page 99), but does not participate in the

phenylpropenoid-polymalonate pathway, due to the lack of in vivo TAL activity. The

conversion of L-phenylalanine (153) to (E)-cinnamic acid (93) by phenylalanine ammonia

lyase (PAL) activity exhibits a strong preference for the L-enantiomer. Para-hydroxylation of

cinnamic acid (93) to 4-coumaric acid (152) is catalyzed by cinnamic acid 4-hydroxylase

(C4H) activity (Pierrel et al., 1994) and exhibits selectivity for cinnamic- over dihydro-

cinnamic acid. Although 4-coumaric acid (152) might be enzymatically hydrogenated to

afford phloretic acid (157) as the precursor for the STS reaction to lunularic acid (5) and

related bibenzyls (6) (Friederich et al., 1999), anti-dehydrogenation of phloretic acid (157) to

4-coumaric acid (152) was now observed in Corsinia. 4-Coumaric acid (152) is linked to


128

coenzyme A (HSCoA) by 4-coumaric acid ligase activity (4CL) and decarboxylative

condensation of 4-coumaroyl-SCoA with three acetate derived malonate-SCoA units by

stilbene synthase (STS) activity affords the trisketo acid (190). Reduction of the -carbonyl

group, previously proposed for lunularic acid biosynthesis (Ohta et al., 1983; Abe & Ohta,

1984; Friederich et al., 1999; Eckermann et al., 2003), has now been unambiguously

established for (E)-corsistilbene ((E)-68) and corsifuran A (73) biosynthesis using bond

labelling with [1,2-13


C2]-189). Cyclisation of the reduced ketoacid 193 to

furnish (E)-3,4‟-dihydroxystilbene (141) upon decarboxylative Knoevenagel condensation is

again catalyzed by STS activity. Possible involvement of the corresponding stilbene-2-

carboxylic acid, hydrangenic acid (194), as a biosynthetic intermediate was excluded because

of incorporation of [D3]-acetate ([D3]-189) into (–)-[4,6,D]-corsifuran A ([4,6-D]-73), which

indicated that the decarboxylation of sp3-hydridized 2-carboxylic acid intermediates (195)

occurs prior to aromatization of the polymalonate derived ring. The resulting (E)-3,4‟-

dihydroxystilbene (141) is converted to (E)-corsistilbene ((E)-68) by O-methyl transferase

activity. Para-hydroxylation of 3,4‟-dihydroxystilbene (141) to 2,5,4‟-trihydroxystilbene

(142) might be catalyzed by cytochrome P450‟s (Pierrel et al., 1994), and the corresponding

bibenzyl derivatives, O,O-dimethyllunularin (69) and 6-hydroxy-O,O-dimethyllunularin (70)

have been detected as minor constituents of Corsinia. The stereo specific labelling of the

(3-Pro-R)-position in (2R,3R)-(–)-[3-D]-corsifuran A ([3-D]-73) indicated that one enzyme

derived hydrogen (3-Pro-S) is introduced during H+ catalyzed cyclisation of the

corresponding stilbene intermediates. The 2,3-cis-configuration of the phenylpropanoid

derived deuterium labels in corsifuran A (73) implied an antarafacial cyclisation of

(Z)-configured stilbenoids as the key step to corsifurans, but no evidence for a light inducible

(E/Z)-isomerisation step was obtained and light was not a requirement for corsifuran A (73)

biosynthesis in Corsinia. Although (Z)-stilbenoid intermediates could not be ruled out

completely, these results suggested a two step cyclisation mechanism from STS derived

(E)-stilbenoids (142) to 2-aryl-2,3-dihydrobenzofurans (145) in Corsinia. Electrophilic H+

catalyzed anti-addition of water or some enzymatic hydroxyl group enables rotation of the

C7-C8 bond and subsequent formation of the furan ring during bimolecular nucleophilic

substitution in 184 to the 2-aryl-2,3-dihydrobenzofuran (145). Finally, syn-dehydrogenation

of (–)-corsifuran A (73) with loss of both phenylpropanoid derived - and - hydrogens

affords aromatic corsifuran C (74).


129

The presence of phenylpropanoid-polymalonate derived 2-arylbenzo[b]furans originating

from various biosynthetic pathways in highly unrelated plant families (Figure 62 and 63,

pages 63 and 65) is interesting from the point of evolutionary biology (Tropf et al., 1994).

While 2‟,4‟,6-trihydroxy-2-arylbenzo[b]furans of isoflavonoid (CHS) origin (133, 135) are

restricted to Fabaceae (Figure 62, page 63), 3‟,5‟,6-trihydroxy-2-arylbenzo[b]-furans of

stilbenoid (STS) origin (138, 140) are known from mono- and dicotyls, as well as

gymnosperms (Figure 63, page 65). In the liverwort Corsinia coriandrina a new biosynthetic

pathway to 4‟,5-dioxygenated 2-arylbenzofurans of STS origin (73 and 74) has now been

identified (C1/C2 in Figure 63, page 65). Both STS pathways share an ortho-hydroxylation

step of the intermediate stilbenoid (136 – 137 and 141 – 142) and subsequent cyclisation, but

the L-phenylalanine derived phenylpropanoid unit contributes to different parts of the 2-aryl-

benzofuran skeleton in liverworts and higher plants. While ortho-hydroxylation of

3,4‟-dihydroxystilbene (141) to 142 and its H+ catalyzed cyclisation to corsifurans (145)

might be facilitated by the para-position of 3- and 4‟-hydroxy groups, respectively, a different

hydroxylation mechanism from resveratrol (136) to oxyresveratrol (137) appears to operate in

higher plants. Furthermore, the requirement for the energetically less favoured

3‟,5‟-dihydroxy- -bibenzylcation in 2-arylbenzo[b]furan biosynthesis of higher plants

suggests intermediate formation of 2-hydroxystilbenepoxides, which, after cyclisation and

dehydration, would afford the aromatic 2-arylbenzo[b]furans (138) (Dupont & Cotelle, 2001;

Cotelle, 2005; Aslam et al., 2006). Furthermore, L-tyrosine (154) and L-phenylalanine (153)

participate equally in the biosynthesis of moracin M (138) and stilbenoids (136, 137) in

Morus alba (Hano et al., 1994), whereas L-phenylalanine (153) is the sole precursor to

corsifurans (73 and 74) in C. coriandrina, due to the lack of in vivo TAL activity.

While 2‟,4‟,6- and 3‟,5‟,6-trioxygenated 2-arylbenzofurans from higher plants (133, 135, 138)

exhibit antifungal activities, and some are known as phytoalexins (Ingham & Dewick, 1978;

Preston, et al., 1975; Nomura, 1988), 4‟,5-dioxygenated 2-arylbenzofurans like corsifuran C

(74) and the corresponding bis-phenol (143) were inactive against a range of phytopathogens

(Carter et al., 1978). Corsifurans A and C (73, 74), as well as (E)-corsistilbene ((E)-68) did

not exhibit fungicidal, herbicidal or insecticidal properties in in vitro and greenhouse tests

with the synthetic compounds. While the biological significance of (R)-(–)-corsifuran A (73)

as a major constituent in C. coriandrina is still unknown, aromatic corsifuran C (74, max =

316 nm, = 52.900) and (E)-corsistilbene ((E)-68, max = 320 nm, = 27.400) are potent

UV-B absorbents, thus, indicating sun screening properties, which were of great importance

for colonization of terrestrial habitats in ancient times (Rozema et al., 2002; Edreva, 2005).


130

4.6.3. Biosynthesis of Terpenoids in Corsinia coriandrina

The biosynthesis of terpenoids in Corsinia coriandrina was deduced from standard

application experiments with [2-13

C]-acetate ([2-13

C]-189) and [1,2-13


C2]-

189). Dominating compounds like (–)- -pinene (18), (–)-(E)-nerolidol (64), and (E,E)-

-springene (66) were chosen as representatives for mono-, sesqui-, and diterpenoids,

respectively, and isolated by column chromatography on silica gel. Inspection of their

13C {

1H} NMR spectra and comparison with those of the unlabelled compounds obtained

under identical experimental conditions indicated specific enhancement of 13

C NMR signals

in the case of (E)-nerolidol (64) (Figure 116, page 131) and -pinene (18) (Figure 118, page

133), whereas 13

C-enrichment of the diterpenoid -springene (66) was insignificant.

4.6.3.1. Biosynthesis of (R)-(–)-(E)-Nerolidol

Incorporation of [2-13

C]-acetate ([2-13

C2]-189) resulted in specific enhancement of 13

C NMR

signals corresponding to (E)-[2,4,6,8,10,12,13,14,15-13

C]-nerolidol ([13

C]-64) with an average

enrichment of +2.8 ±0.4 atom % 13

C over natural abundance per site (Figure 116, page 131).

Because the location of 13

C labels corresponds with positions 2-C, 4-C, and 5-C of each head-

tail fused isoprene unit ([2,4,5-13

C]-196), the incorporation of [2-13

C]-acetate ([2-13

C]-189)

indicated that (R)-( )-(E)-nerolidol (64) from C. coriandrina is derived via the classical

acetate-mevalonate (MVA) pathway (Figure 117, page 132). Upon application of [1,2-13

C2]-

acetate ([1,2-13

C2]-189) 13

C {1H} NMR signals for the 4-, 8-, and 12-position were

significantly enhanced (marked with ), whereas all other 13

C NMR signals were

complemented by doublets, due to direct 13

C,13

C-coupling in the incorporated 13

C2 units.

Although undiluted [1,2-13


C2]-189) at c = 4 mM was applied the 25 mM D-

glucose supplement for mixed photo heterotrophic growth was a sufficient source of 12

C2

acetate (189) to dilute the 13

C2 label, so that formation of multiple labelled nerolidol was

insignificant. Observed 13

C,13

C-coupling constants of 1Jcc = 40 – 45 Hz indicated

13C2

labelling of allylic bonds, and incorporation of intact 13

C2 units was established for [1,2-13

C2]

(1Jcc = 70.3 Hz), [3,15-

13C2] (

1Jcc = 39.4 Hz), [5,6-

13C2] (

1Jcc = 43.4 Hz), [7,14-

13C2] (

1Jcc =

42.2 Hz), [9,10-13

C2] (1Jcc = 44.6 Hz), and [11,13-

13C2] (

1Jcc = 42.6 Hz) units. Specific

13C2

incorporation into (E)-[13

C2]-nerolidol ([13

C2]-64) confirmed its origin from the acetate-

mevalonate pathway, whereas the specific labelling of (E)-[4,8,12-13

C]-nerolidol ([4,8,12-

13C]-64) by single labelled

13C units originating from 2-C of [1,2-

13C2]-acetate ([1,2-

13C2]-


131

189) indicates that subsequent conversion between IPP (196) and DMPP (197) by IPP

isomerase is highly stereospecific (Figure 117).


C {1H} NMR spectra (C6D6) of natural abundance (E)-nerolidol (64) from

C. coriandrina; (E)-[2,4,6,8,10,12,13,14,15-13

C]-nerolidol ([13

C]-64) from incorporation of

[2-13

C]-acetate ([2-13

C]-189); and (E)-[4,8,12-13

C,1(2),3(15),5(6),7(14),9(10),11(13)-13

C2]-nerolidol

([13



C2]-189) (●■, ○□, and used to mark

methyl- and carboxyl-atoms of acetate units, respectively).

2030405060708090100110120130140

2030405060708090100110120130140

C [ppm]30405060708090100110120130140

●■

□

●●

□

■

●

●●

●

●●

●

●●

□

○○

■○

x

2

7

11

6,10 1

3

48

15

1413

512

9solvent

x

solvent

solvent

4

815

12

1413

26,10

48 12

Application of [2-13C]-acetate

Application of [1,2-13C2]-acetate

Natural abundance

2030405060708090100110120130140

2030405060708090100110120130140

C [ppm]30405060708090100110120130140

●■

□

●●

□

■

●

●●

●

●●

●

●●

□

○○

■○

x

2

7

11

6,10 1

3

48

15

1413

512

9solvent

x

solvent

solvent

4

815

12

1413

26,10

48 12

Application of [2-13C]-acetate

Application of [1,2-13C2]-acetate

Natural abundance


132

Figure 117: 13

C-enrichment (atom % 13

C over natural abundance) of (E)-[2,4,6,8,10,12,13,14,15-13

C]-

nerolidol ([13

C]-64) from incorporation of [2-13

C]-acetate ([2-13

C]-189, c = 6 mM); and multiplicities

and 1JCC coupling constants (Hz) in (E)-[4,8,12-

13C,1(2),3(15),5(6),7(14),9(10),11(13)-

13C2]-nerolidol

([13



C2]-189, c = 4 mM) (●■, ○□, and used

to mark methyl- and carboxyl-atoms of acetate units, respectively).

4.6.3.2. Biosynthesis of (4S,6S)-( )- -Pinene

The biosynthetic pathway to the bicyclic monoterpene hydrocarbon (4S,6S)-( )- -pinene (18)

in Corsinia coriandrina was deduced from standard application experiments with

[13

C]-labelled acetate ([13

C]-189). [13

C]-enriched -pinene (18) was isolated by a combination

of column chromatography and preparative gas chromatography. Inspection of 100 MHz

broadband decoupled 13

C {1H} NMR spectra and comparison with those of natural abundance

( )- -pinene (18) recorded under identical experimental conditions indicated specific

13C incorporation from [2-

13C]-acetate ([2-

13C]-189) into [2,4,6,8,9,10-

13C]- -pinene

([13

C]-18) with an average enrichment of +9.3 ±1.1 atom % 13

C over natural abundance per

site (Figure 118, page 133). Specific 13

C incorporation into 2-C, 4-C, and 5-C of both head-

tail fused isoprene units ([2,4,5-13

C]-196) indicated that [2,4,6,8,9,10-13

C]- -pinene

([13

C]-18) from Corsinia is derived from the acetate-mevalonate (MEV) pathway (Figure 119,

page 134) and not via MEP/DXP, although in previous investigations MEP derived

monoterpenoids were described from the liverworts Ricciocarpus natans, Concocephalum

conicum (Adam et al., 1998), and Trichocolea tomentella (Barlow et al., 2001).


133

Figure 118: Broadband decoupled 13

C NMR spectra (100 MHz, C6D6) of natural abundance (4S,6S)-

( )- -pinene from C. coriandrina (18); [2,4,6,8,9,10-13

C]- -pinene ([13

C]-18) from incorporation of

[2-13

C]-acetate ([2-13

C]-189), and [2,9-13

C,1(10),3(4),5(6),7(8)-13

C2]- -pinene ([13

C2]-18) from

incorporation of [1,2-13


C2]-189) (●■, ○□, and used to mark methyl- and

carboxyl-atoms of acetate units, respectively).

20 30 40 50 60 70 80 90 100 110 120 130 140

30 40 50 60 70 80 90 100 110 120 130 140

□

○

□

■ ■

●

●

20 30 40 50 60 70 80 90 100 110 120 130 140

●

●

● ●

●

8

10 9

3,5

4

6

2

1

2

8

10 9

4 6

○

●

solvent

Natural abundance

20 30 40 50 60 70 80 90 100 110 120 130 140

30 40 50 60 70 80 90 100 110 120 130 140

20 30 40 50 60 70 80 90 100 110 120 130 140

1

2

10

4

[13

C]-18

(4S,6S)-(–)-18

8

9

10

2

[13

C2]-18

solvent

solvent

Application of 2-13

C -acetate

Application of 1,2-13

C2 -acetate

C [ppm]


134

Figure 119: 13

C-enrichment (atom % 13

C over natural abundance) of [2,4,6,8,9,10-13

C]- -pinene

([13

C]-18) from incorporation of [2-13

C]-acetate ([2-13

C]-189) and multiplicity and direct

1JCC coupling constants (Hz) of [2,9-

13C,1(10),3(4),5(6),7(8)-

13C2]- -pinene ([

13C2]-18) from

incorporation of [1,2-13


C2]-189) (nd: quaternary carbons not detected;

●■, ○□, and used to mark methyl- and carboxyl-atoms of acetate units, respectively)

Furthermore, the location of acetate units in the -pinene skeleton was derived from

application of [1,2-13

C]-acetate ([1,2-13

C]-189), resulting in the appearance of doublet signals

in the 13

C {1H} NMR spectrum of [

13C2]-18 due to

13C,

13C-coupling (Figure 118). By

comparison of the 1JC,C

coupling constants, the incorporation of intact

13C2 units was

unambiguously established for [3,4-13

C2] (1Jcc = 33.0 Hz), and [5,6-

13C2] (

1Jcc = 27.5 Hz)

(●■). In addition, coupling constants for [7,8-13

C2] (1Jcc = 37.6 Hz) and [1,10-

13C2]

(1Jcc = 44.0 Hz) were derived from doublet signals for the hydrogen bond 8-CH3 and 10-CH3

groups (○), whereas signal intensities for adjacent quaternary carbons 7-C and 1-C (□) were

too small to be detected. The 13

C NMR signal corresponding to 2-CH () appeared as

intensity enhanced singlet, marking the attachment point of IPP (196) at the tail of the DMPP

(197) unit and indicating that the interconversion of IPP (196) and DMPP (197) is highly

stereospecific, so that no randomisation of methyl bond 13

C2 label (○□ and ) occurs (Figure

119).


135

In the geminal dimethyl group of -pinene (18) the anti-configured 9-CH3 () at 26.5 ppm

appeared as intensity enhanced singlet, while the signal for syn-configured 8-CH3 (○) at 21.0

ppm was complemented by a doublet signal (2JCC = 37.6 Hz) indicating that C-9 () and C-8

(○) do not equilibrate, although an (S)-terpinen-7-carbenium ion (199) serves as biosynthetic

intermediate (Banthorpe & Le Patourel, 1972; Croteau et al., 1988, 1989) (Figure 120). The

assignment of the geminal dimethyl carbons was obtained from the combination of

gp-NOESY and HMQC spectra and indicated that cyclisation of GPP (198) via (S)-terpinen-

7-yl ion (199) to the pinane skeleton (18) is a synfacial process.

Figure 120: Stereochemical course of (4S,6S)-( )- -pinene (18) biosynthesis from GPP (198)

via (S)-terpenyl-7-ion (199) intermediates (●■, ○□, and used to mark methyl- and carboxyl-atoms

of acetate units, respectively)


136

4.6.3.3. Discussion of Terpenoid Biosynthesis in Corsinia coriandrina

Figure 121: Biosynthesis of (4S,6S)-( )- -pinene (18) and (R)-(–)-(E)-nerolidol (64) via the MVA

pathway in Corsinia coriandrina (●■, ○□, and used to mark methyl- and carboxyl-atoms of acetate

units, respectively).

Application experiments with [2-13

C]-labelled acetate indicated an acetate-mevalonate

(MVA) pathway to (4S,6S)-(–)- -pinene (18) and (R)-(–)-(E)-nerolidol (64) in Corsinia

coriandrina as shown in figure 121. The diterpenoid -springene (66) was not labelled and is

most likely of MEP origin (Adam et al., 1998; Rohmer, 2001, 2003: Dubey et al., 2003).

Furthermore, specific incorporation of [1,2-13

C]-acetate ([1,2-13

C]-189) indicated that

interconversion of IPP (196) and DMPP (197) is highly stereospecific and suggested a

synfacial cyclisation process from GPP (198) via a (S)-terpinen-7-yl ion (199) to the pinane

structure (18).

4.7. (1S,2R)-(+)-Spiroaxa-5,7-diene associated with DED

137

4.7. (1S,2R)-(+)-Spiroaxa-5,7-diene associated with Dutch Elm Disease

4.7.1. Durch Elm Disease (DED)

Fungal infections of American elms, Ulmus americana L., Ulmaceae, by Ophiostoma novo-

ulmi, Ascomycota, the decisive pathogen of Dutch Elm Disease, is transmitted by the native

elm bark beetle Hylurgopinus rufipes Eichhoff as the primary vector of the disease (Agrios,

2004). H. rufipes is attracted to a susceptible host by a blend of four elm-derived semio-

chemicals, (–)- -pinene (21), (–)- -cubebene (205), (+)- -cadinene (207) and the new

(+)-spiroaxa-5,7-diene (206) (Figure 122). All four compounds occur at low quantities in

healthy elms, and their production is up-regulated upon inoculation with O. novo-ulmi, thus,

enhancing the apparency of host trees to searching beetles and increasing the probability of

transportation of the pathogen to new hosts.

Figure 122: Semiochemicals of Ulmus americana infected with Ophiostoma novo-ulmi,

which are attractive to Hylurgopinus rufipes.

To isolate volatile semiochemicals of Ulmus americana, trunk sections infected with

Ophiostoma novo-ulmi were cut and ground to sawdust, which was placed in a glass aeration

chamber. Purified air was drawn through the chamber and a downstream Pyrex glass column

filled with Porapak Q for 96 h by a water driven pump. Trapped volatiles were eluted from

Porapak Q with pentane and aliquots were bioassayed in laboratory Y-tube olfactometer

experiments showing strong attraction of male and female Hylurgopinus rufipes to test

stimuli. Aliquots of Porapak Q extracts were analyzed by gas chromatography with

electroantennographic detection (GC-EAD) using antennae of H. rufipes (Figure 123). Four

compounds elicited consistent and significant antennal responses. By using combined gas

chromatography / mass spectrometry (GC/MS) including enantioselective GC, (–)- -pinene

(21), (–)- -cubebene (205) and (+)- -cadinene (207) could be identified upon comparison of

their mass spectra and GC retention indices with those of authentic samples (Figure 122).


138

One unidentified compound (206) occurred in trace quantities but elicited the strongest

antennal response. This was shown to be critically important in the 4 component blend

attractive to H. rufipes in laboratory assays and field tests (McLeod et al., 2005). The

identification and synthesis of this sesquiterpene hydrocarbon, (1S,2R)-(+)-spiroaxa-5,7-diene

((+)-206), is described in the following section.

Figure 123: GC-FID and GC-EAD chromatograms of volatiles emitted by Ulmus americana

upon infection with Ophiostoma novo-ulmi.

205 207 206 21


139

4.7.2. Identification of (1S,2R)-(+)-Spiroaxa-5,7-diene

The mass spectrum of 206 exhibited a molecular ion signal at m/z 204 (29) [M], indicating a

sesquiterpene hydrocarbon with the molecular formula of C15H24 (Figure 124). The

characteristic fragment ion signal at m/z 162 (81) [M – C3H6] suggested a 4-methyl-

cyclohexene moiety undergoing a Retro-Diels-Alder reaction. Dominant signals at m/z 147

(100) [M – C3H6 – CH3] and m/z 119 (56) [M – C3H6 – C3H7] corresponded to subsequent

loss of methyl or isopropyl radicals.

Figure 124: Mass spectrum (EI, 70 eV) and Retro-Diels Alder reaction of 5,7-spiroaxadienes (206).

A compound with identical mass spectra and GC retention indices was produced in up to 3 %

yield as a rearrangement product of (–)- -cubebene (205) upon impact of hydrogen on

palladium/BaSO4. HPLC and micro-preparative gas chromatography provided approximately

200 µg of 206 which exhibited identical biological activities as the natural product in both

GC-EAD experiments and field tests.

41

5565

77

91 105

119

133

147

162

175 189

204

40 60 80 100 120 140 160 180 200 220

20

40

60

80

100

C15H24

C12H18

C11H15

C9H11


140

Figure 125: 500 MHz 1H NMR spectrum of spiroaxa-5,7-diene (206, in C6D6).

The 1H NMR spectrum of 206 (Figure 125) displayed one secondary methyl group at H 0.90

(3H, d, J = 6.6 Hz), two isochoric secondary methyl groups at H 0.99 (6H, d, J = 6.9 Hz), one

olefinic methyl group at H 1.61 (3H, d, J = 1.0 Hz), and two signals for olefinic methine

protons at H 5.13 (1H, s) and 5.35 (1H, s), along with several overlapping signals. The

presence of two trisubstituted double bonds along with four degrees of unsaturation for the

molecular formula C15H24 implied a bicyclic carbon skeleton. In addition, the absence of any

high field shifted methine signal in the 1H NMR spectrum of 206 suggested cleavage of the

cyclopropyl ring during its formation from -cubebene (205). Furthermore, the isochrony of

two methyl groups and their doublet multiplicity (J = 6.9 Hz) indicated an olefinic isopropyl

group, in agreement with the chemical shift and septet multiplicity of the corresponding

allylic methine proton at H 2.13 (1H, sept, J = 6.9 Hz). These results excluded a cadinane

structure (208) and suggested cleavage of the C1-C6 or C5-C6 bond of (+)- -cubebene (205) to

form a guaiane (209) or spiroaxane (210) skeleton, respectively (Figure 126).

7-CH

15-CH3

14-CH3

11-CH

12-CH3 13-CH3

6-CH


141

Figure 126: Hypothetical cadinane (208), guaiane (209), and spiroaxane (210) skeletons originating

from C1-C5, C1-C6, or C5-C6 bond cleavage of the cyclopropyl unit of (–)- -cubebene (205)

(former bond attachment points expected to carry the newly formed double bond marked by dots).

Careful inspection of the COSY spectrum of 206 revealed only one additional methine group

at H 1.57 (1H, m) and four methylene groups, which, together with the remaining quaternary

carbon, most likely corresponded to a 5,7- or 5,8- spiroaxadiene structure (210) for compound

206, in agreement with the occurrence of the signal for a Retro-Diels-Alder fragment ion at

m/z 162 [M – C3H6] (81) in its mass spectrum (Figure 124, page 139).

4.7.3. Attempted Dehydration of (–)-Axenol

To test this hypothesis, the corresponding alcohol, (–)-axenol (211), previously described

from pine, juniper and cryptmeria trees (Kurvyakov et al., 1979; Khan et al., 1983; Barrero et

al., 1991), was isolated from Juniperus oxycedrus L., Cupressaceae, wood oil by a

combination of column chromatography and preparative gas chromatography using modified

cyclodextrins as stationary phases. Nevertheless, all attempts to obtain the corresponding

hydrocarbon (206) by dehydration of (–)-axenol (211) using a variety of reagents (amberlyst®

15 in hexane or dichloromethane; SOCl2 in pyridine; POCl3 in pyridine; mesylchloride in

pyridine; I2 / Ph3P / imidazol) furnished only (+)- -cadinene (207) and (–)-cadina-1(6),4-

diene (212), indicating that rearrangement is favoured over bimolecular anti-elimination

(Figure 127). Semi-empirical quantum mechanical modelling (PM3, RHF) of the spiroaxa-7-

en-6-carbocation (213) suggested that the geometrical proximity of the LUMO at 6-C and the

HOMO at 7-C and 8-C facilitates intramolecular cyclisation to the cubebane ion (214). This

ion is identical to those initially obtained upon acid catalyzed rearrangement of -cubebene

(205), which afforded the same (+)- -cadinene (207) and (–)-cadina-1(6),4-diene (212) via

cadina-4-en-1-carbeniumion (215) originating from cleavage of the C1-C5 bond.


142

Figure 127: Dehydration of (–)-axenol (211) from Juniperus oxycedrus.

4.7.4. Acid Catalyzed Rearrangement using Amberlyst

The synthesis of spiroaxadienes by acid catalyzed rearrangement of aromadendrene-type

hydrocarbons has recently been described (Polovinka et al., 2000). Treatment of (+)-aroma-

dendrene (216) in hexane with acidic ion exchange resin amberlyst®

15, a cross-linked

sulfonated polystyrene-divinylbenzene copolymer with a Hammet acidity factor of H0 = − 2.2,

afforded a complex mixture of sesquiterpenoids (> 46 compounds), but no traces of

spiroaxadienes. When using polar dichloromethane as the solvent, (1R,2R)-(–)-1-epi-spiroaxa-

5,7-diene (219), exhibiting almost identical mass spectra and GC retention indices as the

natural product (206), was formed in up to 10 % yield within 2 days, along with large

amounts of (+)-ledene (218) and numerous cadinane-type hydrocarbons. Under identical

conditions rearrangement of (+)-ledene (218) afforded the same 1-epi-spiroaxa-5,7-diene

(219) in up to 15 % yield (Figure 128), which was isolated by successive column

chromatography and semi-preparative gas chromatography using a DB-1701 thick-film

capillary column. By one- and two-dimensional NMR spectroscopic techniques the carbon

skeleton and the position of the double bonds could be established unambiguously as (1R,2R)-

(–)-1-epi-spiroaxa-5,7-diene (219).


143

Figure 128: Synthesis of (1R,2R)-(–)-1-epi-spiroaxa-5,7-diene (219) by rearrangement of

(+)-aromadendrene (216), (–)-allo-aromadendrene (217), or (+)-ledene (218) and attempted

rearrangement of (1R,4R,5R,7S)-(+)-guaia-10(14),11-diene (220) from Abies koreana.

Although both spiroaxa-5,7-dienes (206 and 219) from (–)- -cubebene (205) and (+)-aroma-

dendrene (216) exhibited almost identical mass spectra and gas chromatographic retention

indices, comparison of the 1H NMR spectra revealed small but significant differences (Table

6, page 147), indicating the presence of diastereoisomers. Nevertheless, rearrangement of

epimeric (+)-aromadendrene (216) and (–)-allo-aromadendrene (217) with amberlyst® 15

afforded the same epi-spiroaxa-5,7-diene (219) as shown by 1H NMR. Furthermore, the

common presence of large amounts of (+)-ledene (218) suggested that acid catalyzed

rearrangement of both (+)-aromadendrene (216) and (–)-allo-aromadendrene (217) using

amberlyst® 15 in dichloromethane proceeds via isomerisation to (+)-ledene (218), which

exclusively affords (1R,2R)-(–)-1-epi-spiroaxa-5,7-diene (219) (Figure 128).

The cyclopropyl unit appears to be critically important in the ring contraction step, because

rearrangement of (1R,4R,5R,7S)-(+)-guaia-10(14),11-diene (220), identified in cooperation

with Prof. Danuta Kalemba (Technical University of Lodz, Lodz, Poland) as a new compound

from Korean fir, Abies koreana (Baran et al., 2007), afforded no spiroaxadienes.


144

4.7.5. Solid Super Acid Catalyzed Rearrangement using TiO2/SO42–

Figure 129: Spiroaxadienes from the TiO2/SO42–

catalyzed rearrangement of (+)-aromadendrene (216)

and (–)-allo-aromadendrene (217) (224 – 226 detected by GC-EIMS only).

In order to obtain the desired spiroaxa-5,7-diene (206) the solid super acid TiO2/SO42–

with a

Hammett acidity factor H0 = 13.2 had to be employed as described in the original procedure

(Polovinka et al., 2000). TiO2/SO42–

was kindly provided by Professor Alexey V. Tkachev

(Novosibirsk Institute of Organic Chemistry, Novosibirsk, Russia). Treatment of (+)-aroma-

dendrene (216) with precalcinated TiO2/SO42–

in dichloromethane for 10 min afforded a

mixture of four spiroaxadiene isomers (206, 221 – 223, Figure 129), which were isolated by

successive column chromatography and semi-preparative gas chromatography using a

DB-1701 thick-film capillary column. The structures of spiroaxa-5,7-diene (206, 47 %),

spiroaxa-5,8-diene (221, 23 %), spiroaxa-4,7-diene (222, 8 %) and spiroaxa-4,8-diene (223,

6 %) were identified using one- and two-dimensional NMR spectroscopic techniques. By

comparison of the EIMS and 1H NMR data (Table 6, page 147), the carbon skeleton and the

position of the double bonds of compound 206 from -cubebene (205) could be established

unambiguously as spiroaxa-5,7-diene. The relative configuration was determined by

comparison with 2-epimeric spiroaxa-5,7-dienes, (1R,2S)-(–)-206 and (1R,2R)-(–)-219,

prepared by TiO2/SO42–

catalyzed rearrangement of (+)-aromadendrene (216) and (–)-allo-

aromadendrene (217), respectively, thus, indicating that the stereochemistry at 2-C of

epimeric spiroaxa-5,7-dienes (206 and 219) is determined by the absolute configuration at 1-C

in the aromadendrene precursors 216 and 217, respectively (Figure 129).


145

Relative configurations were deduced from their gp-NOESY spectra, which exhibited

significant NOE correlations between 3-H / 7-H and 2-H / 10-H for (1R*,2S*)-spiroaxa-5,7-

diene (206), and between 2-H / 7-H and 3-H‟ / 10-H for (1R*,2R*)-epi-spiroaxa-5,7-diene

(219) (Figure 130). These NOE interactions also indicated that the secondary methyl group at

2-C predominantly exhibits an equatorial conformation in both epimers in agreement with

semi-empirical quantum mechanical modelling (PM3, RHF). Comparison of the 1H NMR

spectra proved the relative configuration of 206 from -cubebene (205) to be different from

(1R*,2R*)-219, and identical to (1R*,2S*)-206 (Table 6, page 147).

2

3

710

2

3

7 10

Figure 130: Molecular models (PM3, RHF) and significant NOE interactions in (1R*,2S*)-spiroaxa-

5,7-diene (206), and (1R*,2R*)-epi-spiroaxa-5,7-diene (219) (20 hydrogens hidden for clarity).

The absolute configuration was finally established by comparative enantioselective GC

analysis with (1R,2S)-(–)-206 and (1S,2R)-(+)-206, obtained from (+)-aromadendrene

((+)-216) and (–)-ent-aromadendrene ((–)-216), respectively (Figure 131). The unusual

( )-ent-aromadendrene enantiomer ((–)-216) was isolated from the essential oil of the

liverwort Pellia epiphylla (L.) Corda, Metzgeriales, by a combination of column

chromatography and consecutive preparative gas chromatography using 6-TBDMS-2,3-Me- -

CD and 2,6-Me-3-Pe- -CD as the stationary phase.

206 219

NOE

NOE NOE

NOE


146

Figure 131: Synthesis of (1R,2S)-(–)-ent-spiroaxa-5,7-diene ((–)-206) and (1S,2R)-(+)-spiroaxa-5,7-

diene ((+)-206) by TiO2/SO42–

catalyzed rearrangement of (+)-aromadendrene ((+)-216)

and (–)-ent-aromadendrene ((–)-216) from the liverwort Pellia epiphylla.

Complete baseline separation of synthetic (±)-spiroaxa-5,7-diene ((±)-206) with an -value of

(+):(–) = 1.094 was observed using 2,6-Me-3-Pe- -CD as the stationary phase at 100 °C

(Figure 132). Consequently, the identity of (+)-206 from (–)- -cubebene (205) with (1S,2R)-

(+)-spiroaxa-5,7-diene ((+)-206) obtained by TiO2/SO42-

catalyzed rearrangement of (–)-ent-

aromadendrene ((–)-216) could finally be established by enantioselective GC. In comparative

GC-EAD analysis, synthetic (1R,2S)-(–)-ent-spiroaxa-5,7-diene ((–)-206) was less active than

its enantiomer (+)-206.

Figure 132: Enantioselective GC analysis of (1R,2S)-(–)-spiroaxa-5,7-diene ((–)-206) and (1S,2R)-

(+)-spiroaxa-5,7-diene ((+)-206) using 2,6-Me-3-Pe- -CD at 100 °C.

10

20

30

40

(+)-206 (+)-206 (–)-206

RT [min]

racemic standard natural product


147

1H NMR 5,7-diene 5,8-diene 4,7-diene 4,8-diene epi

206 221 222 223 219

2 1.57 m 1.57 m 1.64 m

1.62 m 1.60 m

3 1.58 m 1.57 m 1.75 m 1.76 br.d 1.39 m

3‟ 1.40 m 1.38 m 2.14 m 2.16 m

1.60 m

4 1.89 m 1.90 m 5.40 s.br 5.36 br.s 1.90 m

6 5.35 s 5.55 s 1.96 s.br 1.96 m 5.44 s.br.

7 5.11 s 2.37 m 5.32 s.br 2.19 m

1.84 d 17.0

5.10 s.br.

9 2.16 m

2.21 m

5.25 s.br 2.20 t 7.3 5.24 s.br 2.14 m

2.21 m

10 1.91 m 2.29

d.br 17.3

1.82 m 2.32

d.br 16.0

1.68 m

10‟ 1.81 m 1.97

d.br 17.3

1.64 m 2.13 m 1.94 m

11 2.13 qq 6.9 2.13 qq 6.6 2.14 m 2.10 m 2.13 qq 6.9

12 & 13 0.99 d 6.6 1.01 d 6.6 1.02 d 6.6 0.99 d 6.6 1.01 d 6.9

1.02 d 6.9

14 0.90 d 0.90 d 6.6 0.91 d 6.6 0.87 d 6.6 0.91d 6.9

15 1.61 s.br 1.63 s.br 1.67 s.br 1.65 s.br 1.66 s.br

Table 6: 1H NMR data (500 MHz) of spiroaxadienes prepared by acid catalyzed rearrangement of

aromadendrene-type compounds (chemical shift [ppm] in C6D6, multiplicities and coupling constants

[Hz], assignments from 1H NMR, COSY, NOESY and HMBC spectra).


148

4.7.6. Discussion of (1S,2R)-(+)-Spiroaxa-5,7-diene from Ulmus americana

(1S,2R)-(+)-spiroaxa-5,7-diene ((+)-206) from Ulmus americana represents the first

spiroaxane-type hydrocarbon identified as a natural product. (1S*,2R*)-spiroaxa-5,7-diene

(206) of unknown absolute configuration was also detected as a trace constituent of the

liverwort Tritomaria exsecta, Jungermanniaceae, collected in Austria. The corresponding

alcohol, known as (–)-gleenol or (–)-axenol (211) has previously been reported from various

sources, like the pine tree Picea glehnii (Kurvyakov et al., 1979), juniper and cryptmeria trees

(Khan et al., 1983, Barrero et al., 1991, Nagahama et al., 1996), and the marine brown algae

Taonia atomaria (de Rosa et al., 1994), whereas the enantiomeric (+)-axenol is known from a

marine sponge of the genus Eurypon (Barrow et al., 1988) (Figure 133). Furthermore, the

corresponding isonitrile, (+)-axisonitrile-3 (227) and the isothiocyanate, axisothiocyanate-3

(228) have been described from the marine sponge Axinella cannabina (Di Blasio et al., 1976)

whereas the corresponding isocyanate (229) has been detected in Phakellia ventilabrum

(Possner, 2005). Epimeric 2-epi-axisonitrile-3 (230) and related derivatives have also been

described from the marine sponge Geodia exigua (Uy et al., 2003). The spiroaxane skeleton

does not correspond with the isoprene rule of head-tail fused isoprene units and is most likely

derived biosynthetically upon Grob-like fragmentation of ( )- -cubebene (205) or related

compounds (de Rosa et al., 1994).

Figure 133: Spiroaxane type compounds as natural products.

4.8. Octamethylbicyclo[3.2.1]octadienes from Serratia odorifera

149


4.8.1. Serratia odorifera

The genus Serratia comprises Gram-negative, facultatively anaerobic Enterobacteriales

associated with plant roots. Serratia odorifera has recently attracted attention due to the

emission of volatile organic compounds capable of inhibiting growth of Arabidopsis thaliana

(Vespermann et al., 2007), as well as mycelial growth of the plant pathogen Rhizoctonia

solani (Kai et al., 2007). Rhizobaterial volatiles have been reported to influence plants, fungi

and other bacteria through largely unknown mechanisms (Ryu et al., 2005; Zhang et al., 2007;

Choudhary et al., 2008; Kai et al., 2009).

4.8.2. Identification of Odorifen

Figure 134: TIC chromatogram of VOCs from Serratia odorifera headspace (* structure proposals).

The scent of Serratia odorifera (Figure 134) was found to be dominated by a single

compound (239, ˂ 85 % of the total volatiles) which was termed odorifen. Its mass spectrum

(Figure 135) exhibited a molecular ion signal at m/z 218 [M] and an unusual set of highly

abundant fragments at m/z 136 (100), 135 (90), and 134 (80). Although an oxygenated

sesquiterpene was initially assumed, HREIMS indicated the molecular formula C16H26 with

four units of unsaturation. About 1 mg of the hydrocarbon (239) was collected from a 10 litre

culture of S. odorifera by adsorption on Super-Q and elution with organic solvents.

239 246* / 247*

245*

239

239


150

Figure 135: Mass spectrum (EI, 70 eV) of odorifen (239) from Serratia odorifera.

The 1H NMR spectrum in C6D6 exhibited signals for a symmetrically poly-methylated

compound (Figure 136), as suggested by the integral ratio of 1:2:3:6 along with the large

number of isochoric methyl groups. An exocyclic methylene group at H 4.63 (2H, s) and one

secondary methyl group at H 0.73 (3H, d, J = 6.9) connected to a methine group at H 2.04

(1H, q, J = 6.9) were identified. Furthermore, isochoric signals corresponding to two

secondary methyl groups at H 1.12 (6H, d, J = 7.3), each one connected to its own methine

group at H 2.08 (2H, q, J = 7.2) were identified, along with two quaternary methyl groups at

H 0.83 (6H, s), and two olefinic methyl groups at H 1.41 (6H, s).

Figure 136: 500 MHz 1H NMR spectrum of odorifen (239, in C6D6) from Serratia odorifera.

1.0 1.4 1.8 2.2 2.6 3.0 3.4 3.8 4.2 4.6

x x

x

11 - CH 2

14,15 - CH 3 9,13 - CH

3

10,12 - CH 3

16 - CH 3

2,4 - CH 8 - CH

2H

6H

3H 2H

6H

1H

6H

x

-

-

- - -

H [ppm]

40

55

67

83 91

105

121

136

148 162

189 203

218

40 60 80 100 120 140 160 180 200 220 240

20

40

60

80

100

C16H26

C10H16

239


151

The remaining 5 quaternary carbons required for C16H26 were observed in the 13

C PENDANT

spectrum (Figure 137) at C 51.5 (s), 134.2 (s), and 156.0 (s), in agreement with two isochoric

quaternary carbons, two isochoric olefinic carbons, and the remaining part of an exocyclic

methylene group at C 111.1 (t), respectively. Furthermore, two different types of methine

groups at C 39.9 (d) and 44.1 (d), as well as four types of methyl groups at C 9.6 (q), 11.0

(q), 17.7 (q), and 19.5 (q) were identified in agreement with the HMQC spectrum.


C PENDANT spectrum of odorifen (239, in C6D6) from Serratia odorifera.

The presence of one exocyclic- and one isochoric internal double bond, along with the

molecular formula indicated a bicyclic structure with Cs symmetry. Inspection of the COSY

and HMQC spectra confirmed the identification of five partial structures, which could be

connected according to the long range H,C-correlations from the HMBC spectrum to afford

the novel 1,2,4,5,6,7,8-heptamethyl-3-methylenebicyclo[3.2.1]oct-6-ene structure which was

termed odorifen (239). While the isochrony of the secondary methyl protons at H 1.12

(6H, d) already implied the presence of a meso-form, the stereochemistry was deduced from

the gp-NOESY spectrum. NOE interactions between the secondary methyl groups and the

bridging methine proton ( H 2.04, 1H, q) indicated an 8-anti-2,4-bisaxial (exo) configuration

(Figure 138).

C [ppm]

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160

x

x x

11 - CH 2

14,15 - CH 3

9,13 - CH 3

10,12 - CH 3

16 - CH 3 2,4 - CH

8 - CH

6,7 - C 3 - C

1,5-C

x

x

TMS

solvent

C [ppm]

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160

-

- 3

- 3

3

- 3 -

-

-


152

Figure 138: Important NOE interactions in odorifen (239) (17 hydrogens hidden for clarity)

4.8.3. Synthesis of Octamethylbicyclo[3.2.1]octadienes

The structure of odorifen (239) was unambiguously proven by synthesis as shown in figure

139. Access to the bicylo[3.2.1]octadiene skeleton via [3 + 4]-cycloaddition has previously

been described by H. M. R. Hoffmann (Hoffmann et al., 1972; Hoffmann & Iqbal, 1975;

Hoffmann & Vathke, 1980). Reaction of 2,4-dibromopentan-3-one (232), from the

bromination of pentan-3-one (231), with pentamethylcyclopentadiene (235) using the sodium

iodide / copper route (Hoffmann & Vathke, 1980; Ashcroft & Hoffmann, 1978) afforded

2 – 4:1 mixtures of 8-anti-configured 2,4-bisequatorial- (endo) and 2,4-bisaxial- (exo)

1,2,4,5,6,7,8-heptamethylbicyclo[3.2.1]oct-6-en-3-ones (238 and 236), respectively,

indicating that the intermediate 234 formed in situ via the diiodoketone 233 has an W-like

conformation. Trace amounts of the corresponding syn-configured 8-epimers were also

detected by GC-EIMS (< 0.5 %). Column chromatography using a pentane - diethyl ether

gradient afforded the bisequatorial-isomer (238) as colourless crystals, whereas the bisaxial-

isomer (236) was obtained as a colourless liquid. If contaminated with only traces of

2,4-diiodopentan-3-one (233), formed in situ from the bromo-derivative (232),

heptamethylbicyclo[3.2.1]oct-6-en-3-ones (236 and 238) readily decomposed unless kept

under nitrogen at temperatures below zero. For large scale synthesis of heptamethyl-

bicyclo[3.2.1]oct-6-en-3-ones (236 and 238), extraction with [Ag(NH3)2]+ complex had to be

employed to obtain a reaction mixture that could be distilled in vacuum.

NOEs

NOEs


153

The 2,4-bisaxial exo-isomer (236), required for the next step, was only obtained as the minor

product, and care had to be taken to prevent excessive epimerisation under acidic or alkaline

conditions and upon distillation resulting in the formation of mixed axial-equatorial

configured (±)-237. From the mixtures of epimers the undesired 2,4-bisequatorial isomer

(238) was removed by repeated crystallization from decreasing volumes of pentane - diethyl

ether at –20 °C to afford meso-8-anti-2,4-bisaxial-1,2,4,5,6,7,8-heptamethylbicyclo[3.2.1]oct-

6-en-3-one (236), which remained liquid under these conditions.

Figure 139: Synthesis of heptamethyl-3-methylenebicyclo[3.2.1]oct-6-enes (239 - 241).

Attempted Wittig reaction with bisaxial-236 let to epimerisation, whereas bisequatorial-238

was recovered unchanged. Olefination with Tebbe reagent (Cp2TiCH2AlCl(CH3)2) in THF

(Tebbe et al., 1978; Pine et al., 1985; Lamberth, 1994) afforded the corresponding

hydrocarbons (239 - 241) in up to 80 % yield. In contrast to Tebbe reagent, olefination under

Tebbe-type conditions using dibromomethane, magnesium and TiCl4 in THF (Hoffmann &

Vathke, 1980) led to significant epimerisation. Resulting hydrocarbons (239 - 241) were


154

isolated by a combination of column chromatography on silica gel using pentane and

preparative gas chromatography with polydimethylsiloxane SE 30 as stationary phase (120 °C

isothermally). Only meso-8-anti-2,4-bisaxial-1,2,4,5,6,8-heptamethyl-3-methylenebicyclo

[3.2.1]oct-6-ene (239) exhibited identical mass spectra and GC retention indices as well as

1H and

13C NMR spectra as the natural product from Serratia odorifera. The corresponding

(±)-2-epi-(240) and meso-2,4-di-epi (241) derivatives, as well as the corresponding 8-syn-

epimers were not detected in the headspace of Serratia odorifera.

4.8.4. Synthesis of Octamethylbicyclo[3.2.1]octa-2,6-diene and -2(10),6-diene

In addition to odorifen (239) co-occurring minor constituents exhibited related mass spectra

suggesting similar structures. Theoretical considerations indicated that there are 17 double

bond isomers with an octamethylbicyclo[3.2.1]octadiene skeleton. Depending on the structure

and symmetry of the compounds there are 8 – 32 possible stereoisomers for each double bond

isomer which sum up to a total number of 344 possible isomers including enantiomers.

Figure 140: Synthesis of octamethylbicyclo[3.2.1]octadienes (243 and 244).

Starting from the readily available 2,4-bisequatorial-heptamethylbicyclo[3.2.1]oct-6-en-3-one

(238), double bond isomers of odorifen were prepared by methylation using methyl lithium in

THF at 0 °C to afford the corresponding 2,4-bisequatorial-octamethylbicyclo[3.2.1]oct-6-en-

3-ols (242) as a 40:1 mixture of diastereoisomers (Figure 140). The dominating isomer was

isolated by column chromatography and preparative GC using a SE 30 column. Dehydration

of 242 with thionylchloride or phosphorylchloride in pyridine afforded predominantly

2,4-bisequatorial-3-methyleneheptamethylbicyclo[3.2.1]oct-6-ene (241), whereas treatment

with acidic ion exchange resin amberlyst® 15 in hexane furnished the corresponding double

bond isomers (±)-octamethylbicyclo[3.2.1]octa-2,6-diene (243) and (±)-heptamethyl-2-

methylenebicyclo[3.2.1]oct-6-ene (244), which were isolated by preparative GC using a

SE-30 column (120 °C to 130°C) along with trace amounts of 241 (Figure 140).


155

Figure 141: Mass spectra (EI, 70 eV) of octamethylbicyclo[3.2.1]octa-2,6-diene (243)

and heptamethyl-2-methylenebicyclo[3.2.1]oct-6-ene (244).

The mass spectrum of (±)-8-anti-4-equatorial-octamethylbicyclo[3.2.1]octa-2,6-diene (243)

(Figure 141) was identical to that of one of the minor volatiles of S. odorifera but retention

indices were different, thus, suggesting the presence of the corresponding 4-epimer (245) with

axial configuration (Figure 142). In addition, the mass spectrum of (±)-8-anti-4-equatorial-

heptamethyl-2-methylenebicyclo[3.2.1]oct-6-ene (244) was identical to those of two minor

constituents (Figure 141), but retention indices were again different (Figure 134, page 149).

While the stereochemistry at 3-C in synthetic 244 could not be deduced from the gp-NOESY

spectrum, the equatorial configuration at 4-C, originating from the ketone (238) and alcohol

(242) intermediates, could be confirmed, suggesting the corresponding 4-epimers (246 and

247) with axial configuration as natural products of S. odorifera (Figure 142), but attempted

synthesis was hampered by small yields and epimerization.

Figure 142: Proposed structures for three minor constituents of Serratia odorifera headspace.

41

55

69 77

83

91 97 105

121

136

147 163

173

189

203

218

40 60 80 100 120 140 160 180 200 220 240

20

40

60

80

100

244

41 55 77

91 105 119

135

147

162

203

218

40 60 80 100 120 140 160 180 200 220 240

20

40

60

80

100

243


156

4.8.5. Biosynthesis of Odorifen

The biosynthesis of odorifen (239) was investigated by application of sodium [2-13

C]-acetate

([2-13

C]-189) to label polyketide and mevalonate metabolites. GC-EIMS investigation of

collected volatiles indicated +11.8 atom % 13

C-enrichment of odorifen ([13

C]-239).

Comparison of the 13

C {1H} NMR spectrum of [

13C]-239 with data of natural abundance

odorifen (239) obtained under identical experimental conditions indicated specific enrichment

of 6/7-C (+2.0), 8-CH3 (+1.1), 8-CH (+1.1), 2/4-CH3 (+1.0), and 1/5-CH3 (+0.7), along with

small enrichment of 2/4-CH (+0.4) and 3-C (+0.4) (Figure 143). Although increase of 13

C

NMR signal intensities was small, calculated total 13

C-enrichment of 11.1 atom % 13

C over

natural abundance was similar to those deduced from GC-EIMS. Furthermore,

13C-enrichment of the pentamethylcyclopentene radicalion at m/z 136 (100) [C10H16] (Figure

135, page 150) was identical by EIMS and 13

C {1H} NMR (+7.9 %

13C). Due to the symmetry

of the meso-odorifen structure (239) six pairs of indistinguishable carbons are present, so that

it was impossible to distinguish between labelling of both isochoric carbons and twice

intensive single labelling of one carbon only. The labelling pattern in [13

C]-239 resulting from

incorporation of [2-13

C]-acetate ([2-13

C]-189) is difficult to interpret because the significant

13C-enrichment of adjacent 8-methyl and 8-methine carbons is neither consistent with a

polyketide nor a mevalonate origin. Considering the small degree of total incorporation and

the wide range of individual 13

C-enrichments (1:5 - 10), the results suggested that acetate may

not be a direct precursor to odorifen biosynthesis via polyketide or mevalonate pathways and

that 13

C from [2-13

C]-acetate ([2-13

C]-189) might enter the odorifen skeleton via different

intermediate pathways of primary metabolism.

Figure 143: 13

C-enrichment of [13

C]-odorifen ([13

C]-239)

from incorporation of [2-13

C]-acetate ([2-13

C]-189).


157

4.8.6. Discussion of Octamethylbicyclo[3.2.1]octadienes from Serratia odorifera

Odorifen (239) and related octamethylbicyclo[3.2.1]octadienes (245 – 247) from Serratia

odorifera represent an unprecedented class of novel natural products (Figure 144).

Polymethylated compounds like 1,2,2,3,4- and 1,2,3,4,4-pentamethylcyclopentanes (248 and

249) have previously been described from Carrion Beetles, Necrodes surinamensis (Roach et

al., 1990), and the obscure mealybug, Pseudococcus viburni (Millar et al., 2005), respectively.

While the isoprene origin of 248 and 249 via 2‟-3 / 4‟-2 and 2‟-2 / 3‟-4 linkages instead of the

normal 1‟-4 head to tail connections is obvious, no conclusive arrangement of isoprene units

could be identified in odorifen (239) and related compounds (245 – 247). Attempted 13

C-

labelling of polyketids and MVA derived terpenoids using [2-13

C]-acetate ([2-13

C]-189) gave

slight enrichment of [13

C]-odorifen ([13

C]-239), but results were inconclusive. Bioassays with

odorifen (239) did not show inhibitory effects on Arabidopsis thaliana, and, as a result, the

biological significance of the compound needs to be determined. On the other hand, the active

principle among the volatiles produced by Serratia odorifera causing inhibition of

Arabidopsis thaliana is still unknown.

Figure 144: Polymethyl compounds from Serratia odorifera (239, structure proposals 245 – 247),

Carrion Beetle (248), and the obscure mealybug (249).

5. Summary

158

5. Summary

Chemical investigation of Fossombronia angulosa, Fossombroniaceae, resulted in the

identification of C11 hydrocarbons, dictyopterene A (29), ectocarpene (30), and dictyotene

(32), known as brown algal pheromones, along with the new pentylbenzene (31). Absolute

configurations and enantiomeric compositions were assigned upon enantioselective GC using

2,3-Ac-6-TBDMS- -cyclodextrin.

In addition to the known 7-prenylindole (14) and 6-prenylindole (15), 3-chloro-7-prenylindole

(49) and 3-chloro-6-prenylindole (50) were identified from Riccardia chamedryfolia,

Aneuraceae, and their structures were confirmed by partial synthesis. Furthermore, the novel

oxazinoindole alkaloid chamedryfolian (51) was identified using NMR techniques and

chemical correlation.

Investigation of Corsinia coriandrina, Corsiniaceae, resulted in the identification of several

new 4-methoxystyrenes showing isothiocyanate (coriandrins, 65), isocyanate (corsinians, 63),

isocyanide (corsicillins, 99), N-formamide (tuberines, 98), S-methyl thiocarbamate

(corsiandrens, 67), N-acetyl-S-methyl thiocarbamate (corsiandrenins, 92), and S,S-dimethyl

iminodithiocarbonate (O-methyltridentatols, 72) moieties attached to a (Z)- or (E)-configured

2-(4-methoxyphenyl)ethenyl skeleton, as well as 5-(4-methoxyphenyl)-2-methylthio-1,3-

thiazol (O-methyl-tridentatol C, 71). Structures were deduced from NMR, FTIR or EIMS

investigations and confirmed by independent synthesis. Furthermore, the synthesis of

dehydroniranin A (97) from Glycosmis cyanocarpa was carried out for the first time.

In addition, corsifuran A (73), corsifuran B (114) and corsifuran C (74) with an unusual

2-(4-methoxyphenyl)-5-methoxy-benzo[b]furan skeleton, (E)- and (Z)-corsistilbenes (68),

dimethyllunularin (69) and its 6-hydroxy-derivative (70) were identified as new natural

products from Corsinia coriandrina by comparison with synthetic samples. The absolute

configuration of (R)-(–)-corsifuran A (73) was established by enantioselective synthesis in

cooperation with Dr. Simon Jones.

The authentic liverwort origin of 4-methoxystyrenes, corsifurans and corsistilbenes was

unambiguously established by their detection in plant material from axenic in vitro cultures of

Corsinia coriandrina established in cooperation with Dr. Klaus von Schwartzenberg. A

variety of deuterium labelled phenylpropanoid derivatives and aromatic amino acids were

synthesized and applied to liquid in vitro cultures of monoclonal haploid C. coriandrina.

Incorporation of labelled precursors was investigated by GC-EIMS, 2D NMR and

13C NMR

techniques. Application experiments established the L-tyrosine origin of coriandrin (65) and

5. Summary

159

related 4-methoxystyrenes in Corsinia coriandrina. A new pulsed application technique using

the temporarily immersion system RITA® was employed to minimize competing reversible

transamination mediated by TAT and unambiguously established that the -deuterium of

L-[U-D7]-tyrosine (L-[U-D7]-154) is retained in (Z)-[1,2,aryl-D6]-coriandrin ((Z)-[D6]-65)

biosynthesis. Co-application experiments with different isotopomers indicated that only the

3-pro-S-hydrogen and the carboxyl group are lost during assembly of the (Z)-configured

double bond, similar to the biosynthesis of xanthocillin in Ascomycota. The O-methyl group

originates from the methylene carbon glycine and is introduced at a later biosynthetic stage,

whereas the origin of the isothiocyanate carbon could not be identified.

Furthermore, the L-phenylalanine origin of corsifuran A (73) and corsistilbenes (68) via an

STS catalyzed phenylpropanoid-polymalonate pathway was unambiguously established. The

liverwort specific reduction of the -carbonyl group of the trisketo acid intermediate 190 was

confirmed for the first time and hydrangenic acid (194) and lunularic acid (5) were excluded

as biosynthetic intermediates. Biosynthetic relationships between stilbenoids (68), bibenzyls

(69) and the corresponding 2-arylbenzofurans (73 and 74) were investigated, indicating a two-

step cyclisation mechanism from (E)-stilbenoid precursors to corsifuran A (73), and

subsequent syn-dehydrogenation to corsifuran C (74).

Furthermore, the MVA pathway to -pinene (18) and (E)- -nerolidol (64) was established.

In cooperation with Prof. G. Gries a new sesquiterpene hydrocarbon emitted by Ulmus

americana upon infection with Ophiostoma novo-ulmi was identified as (1S,2R)-(+)-spiroaxa-

5,7-diene (206) by comparison with both enantiomers ((+)-206 and (–)-206) and both epimers

of four double bond isomers (206, 219, 221 – 226), obtained by solid super acid catalyzed

rearrangement of (+)- and (–)-ent-aromadendrene ((+)-216 and (–)-216) and (–)-allo-aroma-

dendrene (217) using the solid super acid TiO2/SO4. The unusual (–)-ent-aromadendrene

((–)-216) was isolated from the Liverwort Pellia epiphylla.

In cooperation with Prof. B. Piechulla a series of new natural products, with an

octamethylbicyclo[3.2.1]octadiene skeleton was identified from the rhizobacterium Serratia

odorifera. The structure and stereochemistry of odorifen (239), the major volatile constituent,

was deduced from 1H and

13C NMR in combination with 2D NMR spectra, and was

unambiguously established to be meso-8-anti-2,4-bisaxial-1,2,4,5,6,7,8-heptamethyl-3-

methylenebicyclo[3.2.1]oct-6-ene by comparison with synthetic compounds.

6. Zusammenfassung

160

6. Zusammenfassung

Chemische Untersuchungen von Fossombronia angulosa, Fossombroniaceae, führten zur

Identifizierung der C11-Kohlenwasserstoffe Dictyopteren A (29), Ectocarpen (30), und

Dictyoten (32), welche als Sex-Pheromone aus Braunalgen bekannt sind, sowie des neuen

Pentylbenzen (31). Die absoluten Konfigurationen und Enantiomerenreinheiten wurden

mittels enantioselektiver Gaschromatographie an 2,3-Ac-6-TBDMS- -CD untersucht.

Neben dem bereits bekannten 7-Prenylindol (14) und 6-Prenylindol (15) wurden in Riccardia

chamedryfolia, Aneuraceae, das neue 3-Chlor-7-prenylindol (49) und 3-Chlor-6-prenylindol

(50) nachgewiesen und die Strukturen mittels Partialsynthese bewiesen. Darüberhinaus wurde

ein Chamedryfolian (51) genanntes neuartiges Oxazinoindol Alkaloid mittels NMR

spektroskopischer Methoden und chemischer Korrelationen identifiziert.

Die Untersuchung von Corsinia coriandrina, Corsiniaceae, führte zur Identifizierung einiger

4-Methoxystyrene mit Isothiocyanat (Coriandrine, 65), Isocyanat (Corsiniane, 63), Isocyanid

(Corsicilline, 99), N-Formamid (Tuberine, 98), S-Methylthiocarbamat (Corsiandrene, 67), S-

Methyl-N-acetylthiocarbamat (Corsiandrenine, 92), und S,S-Dimethyliminodithiocarbonat (O-

Methyltridentatol A und B, 72) Gruppen an einem (Z)- oder (E)-konfigurierten 2-(4-Methoxy-

phenyl)ethenyl Gerüst, sowie 5-(4-Methoxyphenyl)-2-methylthio-1,3-thiazol (O-Methyl-

tridentatol C, 71). Die Strukturen wurden aus NMR oder EIMS Untersuchungen abgeleitet

und mittels unabhängiger Synthese bewiesen. Darüberhinaus wird die Synthese des

Dehydroniranin A (97) aus Glycosmis cyanocarpa erstmalig beschrieben.

Weiterhin wurden Corsifuran A (73), Corsifuran B (114) und Corsifuran C (74) mit einem

ungewöhnlichen 2-(4-methoxyphenyl)-5-methoxybenzo[b]furan Gerüst, sowie (E)- und (Z)-

Corsistilbene (68), O,O-Dimethyllunularin (69) und sein 6-Hydroxyderivat (70) durch

Vergleich mit synthetischen Proben als neue Naturstoffe aus Corsinia coriandrina

identifiziert. Die absolute Konfiguration des (R)-(–)-corsifuran A (73) wurde mittels

enantioselektiver Synthese in Kooperation mit Dr. Simon Jones aufgeklärt.

Die Bildung der 4-Methoxystyrene, Corsifurane und Corsistilbene durch das Lebermoos

wurde durch deren Detektion in sterilen in vitro Kulturen, angelegt in Kooperation mit Dr.

Klaus von Schwartzenberg, bestätigt. Eine Auswahl Deuterium markierter Phenylpropanoid

Derivate und aromatischer Aminosäuren wurden synthetisiert und an in vitro Flüssigkulturen

von C. coriandrina verabreicht. Einbau der markierten Vorstufen wurde mittels GC-EIMS,

2D-NMR und

13C-NMR Methoden untersucht. Einbauexperimente belegten den L-Tyrosin

Ursprung des (Z)-Coriandrins (65) und verwandter 4-Methoxystyrene aus Corsinia

6. Zusammenfassung

161

coriandrina. Eine neuartige gepulste Verabreichung unter Verwendung der TIS Technik

mittel RITA® wurde verwendet um die konkurrierende reversible Transaminierung durch

TAT zu unterdrücken und den Einbau des -Deuterium aus L-[U-D7]-Tyrosin (L-[U-D7]-154)

während der Biosynthese des (Z)-[1,2,aryl-D6]-Coriandrins ((Z)-[D6]-65) zu beweisen.

Koapplikations Experimente mit unterschiedlichen Isotopomeren belegten, dass während der

Bildung der (Z)-konfigurierten Doppelbindung nur der 3-Pro-S-Wasserstoff und die

Carboxyl- Gruppe abgespalten werden, vergleichbar mit der Biosynthese des Xanthocillins

(109) in Ascomycota. Die O-Methyl Gruppe entstammt dem 2-Methylenkohlenstoff von

Glycin und wird in einem späteren Biosyntheseschritt eingeführt, während die Herkunft des

Isothiocyanat Kohlenstoffs nicht aufgeklärt werden konnte.

Desweiteren wurde der L-Phenylalanin Ursprung der Corsifurane (73, 74) und Corsistilbene

(68) über einen STS katalysierten Phenylpropanoid-Polymalonat Weg nachgewiesen. Die

Lebermoos-spezifische Reduktion der -Carbonyl Funktion in der Trisketocarbonsäure

Zwischenstufe (190) wurde erstmalig bestätigt und Hydrangenasäure (194) und Lunularsäure

(5) als Zwischenstufen ausgeschlossen. Die biosynthetischen Zusammenhänge zwischen

Stilbenoiden (68), Bibenzylen (69) und den entsprechenden 2-Arylbenzofuranen (73 und 74)

wurden untersucht, wobei Hinweise für einen zweistufigen Cyclisierungsmechanismus,

ausgehend vom (E)-stilbenoiden Vorstufen zum Corsifuran A (73) und nachfolgende syn-

Dehydrogenierung zum Corsifuran C (74) erhalten wurden. Darüberhinaus wurde der MVA

Ursprung von -Pinen (18) und (E)- -Nerolidol (64) bewiesen.

In Zusammenarbeit mit Prof. G. Gries wurde ein neuer Sesquiterpen Kohlenwasserstoff,

welcher von Ulmus americana nach Infektion mit Ophiostoma novo-ulmi abgegeben wird, als

(1S,2R)-(+)-spiroaxa-5,7-diene (206) identifiziert. Zum Vergleich wurden beide Enantiomere

((+)-206 and (–)-206) und beide Epimere von vier Doppelbindungsisomeren (206, 219, 221 –

226) mittels Säure-katalysierter Umlagerung von (+)- und (–)-ent-Aromadendren (216) und

(–)-allo-Aromadendren (217) unter Verwendung der festen Super-Säure TiO2/SO4 hergestellt.

In Zusammenarbeit mit Prof. B. Piechulla wurde eine Klasse neuer Naturstoffe mit einem

Octamethylbicyclo[3.2.1]octadien Gerüst in dem Rhizobacterium Serratia odorifera

identifiziert. Die Struktur und Stereochemie der Odorifen (239) genannten Hauptkomponente

wurde aus 1H-,

13C-NMR und 2D-NMR Spektren abgeleitet und durch Vergleich mit

synthetischen Verbindungen eindeutig als meso-8-anti-2,4-bisaxial-1,2,4,5,6,7,8-

Heptamethyl-3-methylenbicyclo[3.2.1]oct-6-en bewiesen.

7. Experimental Part

162


7.1. General Experimental Procedures

7.1.1. Nuclear Magnetic Resonance Spectroscopy (NMR) 1H NMR measurements were carried out with a Bruker WM 400 instrument (400.1 MHz) or Bruker

WM 500 instrument (500.1 MHz). Broadband decoupled 13

C {1H} NMR and

13C PENDANT spectra

were recorded using Bruker WM 400 or Bruker AV 400 instruments (100.6 MHz). Two dimensional

NMR spectra (COSY, HMQC, HMBC and gp-NOESY) were recorded using Bruker WM 500

instrument (1H: 500.13 MHz,

13C: 125.8 MHz). Benzene-d6 (C6D6), chloroform-d1 (CDCl3), acetone-d6

(CD3COCD3), dimethylsulfoxide-d6 (CD3SOCD3), or water-d2 (D2O) were used as the solvent and

tetramethylsilane (TMS) served as internal standard ( = 0 ppm), except for DMSO-d6 ( H 2.50 ppm,

C 39.5 ppm) and water-d2 ( H 4.79 ppm), which were referenced to the solvent signal. 2D NMR

spectra of deuterium labelled corsifuran A ([D]-73) were measured with a Bruker AVANCE 400

instrument (61.4 MHz) using benzene (C6H6) as the solvent ( D 7.16 ppm) and cyclohexane-d12 ( D

1.40 ppm) or [5-OCD3]-corsifuran A ([5-OCD3]-73, D 3.38 ppm) as internal standard. 2D NMR

spectra of deuterium labelled precursors were measured with a Bruker AVANCE 400 instrument (61.4

MHz) using acetone as the solvent and internal reference ( D 2.05 ppm).

7.1.2. Electron Impact Mass Spectrometry (EIMS)

EIMS measurements were carried out on a HP 5890 gas chromatograph (Hewlett Packard) equipped

with a 25 m CPSil-5 CB (Chrompack) fused-silica capillary coupled to a VG Analytical 70-250S mass

spectrometer. Ionization energy of 70 eV, Temperature program from 80 °C to 270 °C at 10 °C/min.

Split- or splittless injection.

7.1.3. High Resolution Electron Impact Mass Spectrometry (HREIMS)

HREIMS measurements were carried out on a HP 5890 gas chromatograph (Hewlett Packard)

equipped with a 25 m CPSil-5 CB (Chrompack) fused-silica capillary coupled to a VG Analytical 70-

250S mass spectrometer.

7.1.4. Fast Atom Bombardment Mass Spectrometry (FAB-MS)

FAB-MS measurements were carried out on a VG Analytical 70-250S mass spectrometer.

7.1.5. Gas Chromatography (GC)

Gas chromatograms were run using a double column HRGC 5300 Mega instrument (Carlo Erba)

equipped with 25 m fused-silica capillary coated with polydimethylsiloxanes CPSil-5 CB or CPSil-19

CB (Chrompack). Hydrogen at 0.5 bar inlet pressure was used as the carrier gas. Split or splittless

injection at 200 °C (split ratio: 1:30); flame ionisation detector at 250 °C. Temperature program from

50 °C to 250 °C at 3 °C/min.


163

7.1.6. Enantioselective Gas Chromatography (eGC)

Enantioselective gas chromatography was performed using Fractovap 4160 instruments (Carlo Erba)

equipped with 25 m, 10 m, or 5 m fused-silica capillaries coated with:

heptakis(2,3-di-O-methyl-6-O-tert-butyldimethylsilyl)- -cyclodextrin in OV-1701 (1:1, w/w),

heptakis(2,3-di-O-acetyl-6-O-tert-butyldimethylsilyl)- -cyclodextrin in OV-1701 (1:1, w/w),

heptakis(2,6-di-O-methyl-3-O-pentyl)- -cyclodextrin in OV-1701 (1:1, w/w), or

octakis-(2,6-di-O-methyl-3-O-pentyl)- -cyclodextrin in OV-1701 (1:1, w/w).

Hydrogen at 0.5 bar inlet pressure was used as the carrier gas. Split injection at 200 °C (split ratio

approximately 1:30) or splitless injection; flame ionisation detector at 250 °C.

7.1.7. Preparative Gas Chromatography (PGC)

Preparative GC was carried out with a modified Varian 1400 gas chromatograph equipped with a

stainless steel column (1.85 m x 4.3 mm; Silcosteel, Amchro) packed with 10 % Polydimethylsiloxane

SE-30, 5 % octakis-(2,6-di-O-methyl-3-O-pentyl)- -cyclodextrin (2,6-Me-3-Pe- -CD) in OV-1701

(1:1; w/w), or 6.4 % heptakis-(6-O-tert-butyldimethylsilyl-2,3-di-O-methyl)- -cyclodextrin (2,3-Me-

6-TBDMS- -CD) in SE-52 (1:1; w/w) on Chromosorb W-HP. Helium was used as the carrier gas at a

flow rate of approximately 120 ml/min and 120 – 150 kPa column pressure. Injector and detector

(FID) temperatures were 200 °C and 250 °C, respectively. Eluting compounds were trapped in Teflon®

tubes cooled with liquid N2 (Hardt, 1994; Hardt & König, 1994).

7.1.8. Semi-preparative Gas Chromatography (SPGC)

Semi-preparative GC was carried out with a HP 6890 gas chromatograph (Hewlett Packard) equipped

with an auto sampler and a megabore thickfilm capillary column coated with polydimethylsiloxane

DB-1701 or DB-1705 (30 m, i.d. 0.53 mm, film thickness 5 µm) or FFAP (50 m, i.d. 0.53 mm, film

thickness 5 µm). Helium was used as carrier gas at constant flow of 2.5 ml/min. Injector and detector

temperatures were 200 °C and 250 °C, respectively. Eluting compounds were trapped at – 20 °C in

glass tubes using the automatic fraction collector PFC 1 from Gerstel and a cryostat.

7.1.9. Column Chromatography (CC)

Column chromatography was performed on silica gel 60 (Merck) using a 10 x 0.7 cm, 20 x 0.7 cm, 25

x 2.4 cm, 40 x 2.4 cm or 50 x 7 cm column (length x inner diameter). Flash column chromatography

was performed by using a column pressure of 0.05 to 0.2 bar. Fractions of 4 or 7 ml were collected

and investigated by TLC, GC or GC-EIMS.

7.1.10. Preparative Thin Layer Chromatography (TLC)

Preparative Thin Layer Chromatography was performed on glass plates coated with silica gel 60 F 254

(Merck). Spots were visualized by UV light (254 nm) or spraying with anisaldehyde – sulfuric acid

reagent and heating to 150°C. Compounds were extracted from the stationary phase using Et2O or

EtOAc and the extract was filtered over a small layer of Na2SO4 and concentrated in vacuum or a

stream of N2.


164

7.1.11. Polarimetry

Measurements were performed with a polarimeter 341 (Perkin-Elmer) at 589 nm and 20 °C. Due to

the small amounts of many isolated compounds only the sense of optical rotation could be determined.

7.1.12. Infrared Spectroscopy (IR)

Infrared Spectroscopy was carried out on a HP 5890 gas chromatograph (Hewlett Packard) equipped

with a 30 m Innowax fused-silica capillary coupled to an HP 5965A Infrared Detector (Hewlett

Packard).

7.1.13. Ultraviolet Spectroscopy (UV)

UV spectra were measured from 190 – 500 nm with a Lambda 20 UV/VIS-spectrometer (Perkin-

Elmer). Methanol was used as the solvent. Extinction coefficients were determined by using the

formula A = c * L (A = absorbance, = extinction coefficient, c = concentration, L = path length).

7.1.14. UV Induced (E/Z)-Isomerisations

Solutions of 50 – 100 µmol of the corresponding (E)-isomers in 5 ml benzene were degassed with

argon for 5 min and irradiated with UV light using a Rayonet™ RPR-100 photoreactor (The Southern

N.E. Ultraviolet Co.) equipped with (16) 350 nm lamps to give mixtures of (Z/E)-isomers.

7.1.15. Photography

Super-Macro photography was performed using an Olympus C-5060 WZ digital camera (5.1

Megapixel), or a binocular with 0.8 x, 1.2 x, 3.8 x, or 5.0 x magnification (Carl Zeiss AG, Germany).

Shown scalebars are approximate.

7.1.16. Microphotography

Microphotography was performed using an Axioskop 2 MOT (Carl Zeiss AG, Germany) with coupled

to an Olympus C-5060 WZ digital camera (5.1 Megapixel). Shown scalebars are approximate.

7.1.17. Fluorescence Micrsocopy

Fluorescence Microscopy was performed using Olympus BA2 (Olympus, Japan).

7.1.18. Scanning Electron Microscopy (SEM)

Scanning Electron Microscopy (SEM) of carbon coated Corsinia coriandrina spores was performed

using a Leo 1525 electron microscope with field emission cathode.

7.1.19. Quantum Mechanical Calculations

Semi-empirical quantum mechanical calculations were performed using the Chem3D Ultra 9.0

software (CambridgeSoft) as an interface to MOPAC Pro 2002 Version 9.0 (J. J. P. Stewart, Fujitsu

Limited, Tokyo, Japan). Both AM1 and PM3 potential functions were used along with Restricted

Hartree-Fock (RHF) or Unrestricted Hartree-Fock (UHF) matrices depending on the electron spin


165

configuration. Different geometries were investigated using a molecular mechanics Force field (MM2)

and resulting models were refined to low gradient norms (< 0.001) with the semi-empirical methods

by using the PRECISE command.

7.2. Plant Materials & Extracts

7.2.1. Origin of Plant Materials

Fresh plant material of dioecious Corsinia coriandrina (Sprengler) Lindberg 1877 was collected by

the late W.A. König at an elevation of 500 m altitude near Tarifa, Andalusia, Spain in March 2003 and

at 350 m near St. Bartolomeo, Andalusia, Spain in April 2004. Additional material was collected at

100 m near Serpa, Ribeira da Lima, Alentejo, Portugal in Febuary 2004 (C. Sergio), at 170 m near

Safara, Ribeira de Safareja and at 100 m near Pulo de Lombo, Rio de Guadiana, Alentejo, Portugal

and at 140 m near Estremadura, Serra da Arrábida, Portugal in March 2005 (H. Muhle, S. von Reuß),

on the Island of La Palma in January 2006, and on the Island of Madeira in March 2007 (H. Muhle).

Most of the plant material was identified by H. Muhle (Institut für Systematische Botanik und

Ökologie, University Ulm, Germany). Herbarium material from Italy collected near “Pallanga” in

1887 (HBG 6457) and near “Florentice” in 1888 (HBG 6458) was obtained from the Herbarium

Hamburgense.

Herbarium material of Cronisia weddellii (Mont.) Grolle 1977, collected July 13. 1990 at an altitude

of 530 m ca. 60 km north of Caruarú, Brazil, on earth in open grasslands by A. Schäfer Verwimp (Nr.

12980) was kindly provided by R.S. Gradstein.

Fresh plant material of Fossombronia angulosa (Dicks.) Raddii was collected on the Island of

Tenerife, Spain, in April 2003, and on the Island of Madeira, Portugal, in March 2007 (H. Muhle).

Fresh plant material of Riccardia chamedryfolia (With.) Grolle was collected at Lydford Gorge near

Dartmoor, Devon, Great Britain in June, 2007 and at 900 m alt. at La Galga near Cube de la Galga, La

Palma, in January, 2006 (H. Muhle).

Fresh plant material of Tritomaria exsecta (Schrad.) Loeske, Jungermanniaceae, was collected in July

2003 at an elevation of 1500 m in Zillertaler Alps, Austria (H. Muhle).

Hydrodistillates of Pellia epiphylla (L.) Corda, Metzgeriales, were provided by W. A. König.

7.2.2. Deposition of Plant Material

A voucher specimen of Corsinia coriandrina has been deposited at the Herbarium Hamburgense

(HBG 6459). An in vitro culture of the monoclonal haploid C. coriandrina strain CC1 used in the

application experiments was provided to the Culture Collection of Autotrophic Organisms (CCALA,

Trebon, Czech Republic) and is kept at the Biozentrum Klein Flottbek, University of Hamburg,

Germany.

7.2.3. Hydrodistillation

Carefully cleaned fresh plant material was mixed with distilled H2O (1:1, v/v) and homogenated using

a blender. The resulting mass was diluted with distilled H2O, treated with boiling beads and ca. 0.5 g


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polyethylene glycol (PEG) to avoid foaming, and hydrodistilled for 2 – 4 h using a modified

Clevenger-type apparatus. Hydrodistillation products were collected in hexane (p.a.) or pentane (p.a.),

separated from the aqueous phase and dried by passing through a small layer of Na2SO4.

7.2.4. Extraction of Air Dried Plant Material

Carefully cleaned air dried plant material was crushed to a powder using pistil and mortar. The

resulting mass was extracted 2 – 5 times with organic solvents (5 – 10:1, v/v) like pentane, Et2O,

DCM, or EtOAc for 2 – 5 h. The combined extract was filtered over a small layer of Na2SO4 and

concentrated in vacuum or a stream of N2 to give green oil.

7.2.5. Extraction of Fresh Plant Material

Carefully cleaned fresh plant material was placed on filter paper to remove adhesive H2O. Plants were

cooled to – 195 °C with liquid N2, crushed to a powder using pistil and mortar, and mixed with excess

Na2SO4 (1 – 3:1, v/v) while still frozen. The resulting mass was extracted 2 – 5 times with Et2O or

pentane (5 – 10:1, v/v) for 5 – 10 min. The combined extract was filtered over a small layer of Na2SO4

and concentrated in vacuum or a stream of N2 to give green oil.

7.3. Axenic In Vitro Cultures of Corsinia coriandrina

Axenic in vitro cultures of monoclonal haploid Corsinia coriandrina were established in cooperation

with Dr. Klaus von Schwartzenberg at the Biozentrum Klein Flottbek, University of Hamburg,

Germany. In vitro cultured polyploid Corsinia coriandrina strain Lorbeer/33 was obtained from the

Culture collection of Autotrophic organisms (CCALA, Trebon, Czech Republic) in June 2006.

7.3.1. Plant media

Knop basal medium was prepared by dissolving 1000 mg/l Ca(NO3)2 x 4H2O, 250 mg/l KH2PO4, 250

mg/l KCl, 250 mg/l MgSO4 x 7H2O, 37 mg/l FeNaEDTA, 1 ml trace elements stock solution in sterile

H2O.

Gamborg B5 (GB) basal medium was prepared by dissolving 2500 mg/l KNO3, 250 mg/l MgSO4 x

7H2O, 150 mg/l CaCl2 x 2H2O, 150 mg/l NaH2PO4 x H2O, 134 mg/l (NH4)2SO4, 37 mg/l FeNaEDTA,

1 ml trace elements stock solution in sterile H2O.

Murashige Skoog (MSK) basal medium was prepared by dissolving 1650 mg/l NH4NO3, 1900 mg/l

KNO3, 440 mg/l CaCl2 x 2H2O, 370 mg/l MgSO4 x 7H2O, 170 mg/l KH2PO4, 37 mg/l FeNaEDTA, 1

ml trace elements stock solution in sterile H2O.

1/2, 1/5 and 1/10 diluted media was prepared using the corresponding equivalents.

Diluted MSKP/5 medium optimized for Corsinia coriandrina was prepared using 330 mg/l NH4NO3,

380 mg/l KNO3, 290 mg fumaric acid, 280 mg KOH, 90 mg/l CaCl2 x 2H2O, 75 mg/l MgSO4 x 7H2O,

140 mg/l KH2PO4, 37 mg/l FeNaEDTA, 1 ml/l trace elements stock solution and 4.5 g/l D-glucose in

sterile H2O.

Trace elements were identical for all media and consisted of 614 µg/l H3BO3 (9.93 µmol/l), 389 µg/l

MnCl2 x 4H2O (1.97 µmol/l), 55 µg/l KAl(SO4)2 x 12H2O (116 nmol/l), 55 µg/l CuSO4 x 5H2O (220

nmol/l), 55 µg/l CoCl2 x 6H2O (231 nmol/l), 55 µg/l ZnSO4 x 7 H2O (191 nmol/l), 28 µg/l LiCl (661

nmol/l), 28 µg/l KI (169 nmol/l), and 28 µg/l SnCl2 (148 nmol/l) available as a 1000x stock solution.


167

D-glucose or D-saccharose was supplemented at 25, 50 or 100 mM. Solid media were obtained by

adding 1 % agar (w/w). Media were adjusted to pH 5.7 with 1 M KOH or HCl solution and sterilized

by autoclaving at 120 °C at 1.2 bar for 20 minutes.

7.3.2. Establishment of Axenic In Vitro Cultures

Approximately 80 spores of Corsinia coriandrina (Spreng.) Lindb. were collected in June from a

single sporogon of a dioecious haploid specimen collected by the late W. A. König at 350 m near

St. Bartolomeo, Andalusia, Spain in April 2004. Spore surfaces were sterilized with 0.5 ml 1 % freshly

prepared calcium hypochlorite solution for 5 min, washed with 5 x 1 ml sterile H2O and centrifuged,

and stored in sterile H2O at 7 °C. Spores germinated within 3 – 4 weeks at 15 °C on a modified Knop

agar (10 ml) supplemented with 10 µl ampicilline (1000 U/ml), 10 µl amphotecerin B (250 µg/ml),

and 10 µl nystatine (1000 U/ml). Single plantlets (approximately 500 µm in diameter) were transferred

to petridishes with modified Knop agar (10 ml) supplemented with abovementioned antibiotics and

cultivated at 15 °C. After sub cultivating every month for 3 month, antibiotics were withdrawn and

axenic conditions were regularly checked by transfer to LB and PDA test media cultivated at 23 °C for

1 week. Propagation of three monoclonal strains (CC1 – 3) was performed by using excised plant

fragments of single plantlets as new inoculums. Agar cultures were kept in Petridishes sealed with

Nescofilm® to avoid contamination and rapid drying. Aerated liquid cultures were established in 250,

500 or 1000 ml glass vessels by bubbling filter sterilized air into the media via a glass tube (ca. 50

ml/min). Temporarily immersed cultures were established using RITA® vessels, purchased from

VITROPIC (ZAE Des Avants 34270 Saint-Mathieu-de-Tréviers, France; http://www.vitropic.fr/).

Immersion times of 3½ min every 6 hours were applied by using a timer controlled aquarium pump.

Plant cultures were kept in a growth chamber (KBK/LS 4330, Ehret; or Rumed, Rubarth Apparate

GmbH) at 15 °C with artificial light (10 µmol*m-2

*s-1

for agar cultures and 25 µmol*m-2

*s-1

for liquid

cultures) and a 16/8 day night cycle.

7.3.3. Flow Cytometry

Plant material was cut into small pieces (< 0.5 mm) and suspended in 500 µl DPA solution (Partec

GmbH, Münster). The suspension was passed through a 50 µm Filter (Partec Cell Trics™) which was

rinsed with 500 µl DPA. A Ploidy Analyser (Partec) was used at a flow rate of 0.8 µl/sec to measure

the relative DNA content per nucleus.

7.3.4. Application Experiments

Standard application experiments at c = 0.2 mM were performed by dissolving 30 µmol of the labelled

precursors (4.5 mg [2-D]-93, 5.0 mg [2-D]-152, 5.0 mg [2,3‟,5‟-D3]-152, 5.0 mg L-[3,3-D2]-153,

5.0 mg D-[3,3-D2]-153, 5.7 mg L-[U-D7]-154, 5.5 mg DL-[2-D]-154, 5.5 mg DL-[2,3-threo-D2]-154,

5.9 mg L-[3,3-D2]-155, 5.9 mg DL-[2-D]-155, 5.9 mg L-[CD3]-155, 4.6 mg [3,3-D2]-156, 5.1 mg

[2,2,3,3,3‟-D5]-157, 5.0 mg (±)-[2,3-threo-D2]-157, 4.2 mg [2,2,3‟-D3]-158, or 4.6 mg [CD3]-168) in

10 – 50 ml liquid MSKP/5 medium and re-adjusting to pH 5.7 using 1 M KOH or HCl solution.

Resulting solutions were filter sterilized and added to 2 cm3

of fresh tissue of monoclonal haploid

Corsinia coriandrina strain CC1 and adjusted to 150 ml liquid medium. Samples of 0.5 cm3 were

collected after (7), 14, 21 and 28 days and their Et2O / Na2SO4 extracts investigated by GC-EIMS with

splitless injection.

For NMR investigation 120 µmol of the labelled phenylpropanoid precursor (17.9 mg [2-D]-93,

20.0 mg [2,3‟,5‟-D3]-152, 20.0 mg L-[3,3-D2]-153, 20.5 mg [2,2,3,3,3‟-D5]-157, 20.2 mg (±)-[2,3-

threo-D2]-157, c = 0.4 mM) was applied at c = 0.4 mM to ca. 20 cm3 of fresh C. coriandrina tissue in


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300 ml liquid MSKP/5 medium for 1 month. 36.5 mg [2-13

C]-glycine (480 µmol, [2-13

C]-177),

149.4 mg [2-13

C]-sodium acetate (1.8 mmol, [2-13

C]-189), or 100.8 mg [1,2-13

C2]-sodium acetate (1.2

mmol, [1,2-13

C2]-189) were applied at concentrations of c = 1.6 mM, 6 mM and 4 mM for one month,

respectively. 1.53 g [D3]-potassium acetate (18 mmol, [D3]-189) was applied in 3 portions at c = 20

mM over 3 month.

7.4. Fossombronia angulosa

7.4.1. Isolation of C11 Hydrocarbons from Fossombronia angulosa

The crude hydrodistillates of Fossombronia angulosa were fractionated by column chromatography

on silica gel using a pentane - Et2O gradient. The hydrocarbon fractions were separated by semi

preparative gas chromatography using a 50 m FFAP thickfilm capillary (temperature program: 100 –

125 °C at 2 °C/min) to give (–)- -sabinene (20, RT = 3.4 min), (3R,5R)-(+)-dictyopterene A (29, RT =

7.9 min), (R)-(–)-dictyotene (32, RT = 10.2 min) and (S)-(+)-ectocarpene (30, RT = 10.7 min) .

(3R,5R)-(+)-dictyopterene A (29): C11H18; colourless oil; RI 1099; (+)-sense of optical rotation

(C6D6); 1H NMR (500.1 MHz, C6D6): = 0.67 (2H, t, J = 6.9 Hz, 4-CH2), 0.86 (3H, t, J = 7.3 Hz, 11-

CH3), 1.22 - 1.36 (6H, m, 3-H, 5-H, 9-CH2, 10-CH2), 1.96 (2H, dt, J = J = 6.6 Hz, 8-CH2), 4.88 (1H,

dd, J = 10.4 Hz, J = 1.6 Hz, 1-Hanti), 4.97 (1H, dd, JE = 15.5 Hz, J = 7.6 Hz, 6-H), 5.05 (1H, dd, J =

17.0 Hz, J = 1.6 Hz, 1-Hsyn), 5.31 (1H, ddd, J = 17.0 Hz, J = 10.4 Hz, J = 8.2 Hz, 2-H), 5.46 (1H, dt, JE

= 15.4 Hz, J = 6.6 Hz, 7-H); 13

C PENDANT (100.6 MHz, C6D6): = 14.2 (q, 11-CH3), 14.9 (t, 4-

CH2), 22.6 (t, 10-CH2), 23.9 (d, 3-CH), 24.6 (d, 5-CH), 32.2 (t, 9-CH2), 32.6 (t, 8-CH2), 112.0 (t, 1-

CH2), 129.1 (d, 7-CH), 132.1 (d, 6-CH), 141.0 (d, 2-CH); EIMS (70 eV) m/z (%) = 150 (3) [M], 121

(3), 107 (6), 105 (5), 93 (31), 91 (32), 80 (36), 79 (100), 77 (31), 67 (28), 66 (20), 65 (13), 55 (13), 53

(14), 51 (9), 43 (9), 41 (56), 39 (45).

(S)-(+)-ectocarpene (30): C11H16; colourless oil; RI 1147; (+)-sense of optical rotation (C6D6); 1H

NMR (500.1 MHz, C6D6): = 0.87 (3H, t, J = 7.6 Hz, 11-CH3), 1.97 (2H, dq, J = 7.3 Hz, J = 7.6 Hz,

10-CH2), 2.21 (1H, m, 6-H), 2.30 (1H, m, 6-H‟), 2.61 (1H, d.br, 2J = 19.9 Hz, 3-H), 2.81 (1H, d.br,

2J

= 19.9 Hz, 3-H‟), 3.52 (1H, s.br., 7-H), 5.33 (1H, dt, JZ = 9.8, J = 7.3 Hz, 9-H), 5.48 (1H, dd, J = J =

10.1 Hz, 8-H), 5.56 (1H, m, 2-H), 5.64 (2H, m, 1,4-H), 5.73 (1H, m, 5-H).; EIMS (70 eV) m/z (%) =

148 (11) [M], 133 (4), 131 (2), 119 (24), 117 (8), 115 (6), 107 (10), 105 (37), 103 (6), 92 (27), 91

(100), 82 (21), 79 (95), 78 (28), 77 (44), 66 (36), 55 (16), 53 (21), 51 (21), 41 (65), 39 (66).

(R)-(–)-dictyotene (32): C11H18; colourless oil; RI 1155; (–)-sense of optical rotation (C6D6); 1H

NMR (500.1 MHz, C6D6): = 0.87 (3H, t, J = 7.3 Hz, 11-CH3), 1.24 (6H, s.br, 8-CH2, 9-CH2, 10-

CH2), 2.13 (2H, m, 6-CH2), 2.43 (1H, s.br., 7-H), 2.58 (1H, d.br, 2J = 19.5 Hz, 3-H), 2.86 (1H, d.br,

2J

= 19.2 Hz, 3-H‟), 5.61 (3H, m, 1-H, 2-H ,4-H), 5.73 (1H, m, 5-H); EIMS (70 eV) m/z (%) = 150 (18)

[M], 135 (2), 121 (5), 107 (14), 105 (4), 94 (25), 93 (68), 91 (56), 80 (45), 79 (100), 77 (56), 67 (21),

66 (19), 65 (19), 57 (5), 55 (11), 53 (13), 51 (12), 41 (49), 39 (43).

7.4.2. Synthesis of (±)-1-Phenylpentan-1-ol (41)

A solution of 106 mg freshly distilled benzaldehyde (40) (1 mmol) in 2 ml dry hexane at 0 °C was

treated with 1 ml of a 1.6 M n-butyl lithium solution (1.6 mmol) in hexane. After stirring for 2 h 2 g

ice was added and the organic phase was separated, dried over Na2SO4 and concentrated in a stream of

N2. Column chromatography on silica gel with a hexane - Et2O mixture (10:1, v/v, UV detection)

afforded 130 mg racemic (±)-1-phenylpentan-1-ol (41) (793 µmol, 79 % yield).

(±)-1-phenylpentan-1-ol (41): C11H16O; colourless oil; RI 1330; 1H NMR (500.1 MHz, CDCl3): =

0.88 (3H, t, J = 7.3 Hz, 11-CH3), 1.20 - 1.43 (4H, m, 9,10-CH2), 1.69 (1H, m, 8-H), 1.78 (1H, m, 8-


169

H‟), 4.62 (1H, dt, J = 1.3 Hz, J = 6.0 Hz, 7-H), 7.25 (1H, m, 4-H), 7.32 (4H, m, 2,3,5,6-H); 13

C

PENDANT (100.6 MHz, CDCl3): = 14.0 (q, 11-CH3), 22.6 (t, 10-CH2), 28.0 (t, 9-CH2), 38.8 (t, 8-

CH2), 74.7 (d, 7-CH), 125.9 (d, 2,6-CH), 127.4 (d, 4-CH), 128.4 (d, 3,5-CH), 145.0 (s, 1-C); EIMS

(70 eV) m/z (%) = 164 (13) [M], 146 (9), 129 (2), 117 (24), 115 (10), 107 (100) [M – C4H9], 105 (19),

91 (13), 79 (67), 77 (42), 65 (3), 51 (12), 39 (7).

7.4.3. Synthesis of (E)-1-Phenylpent-1-ene (42)

A solution of 120 mg (±)-phenylpentan-1-ol (41) (730 µmol) in 10 ml DCM was treated with 100 mg

Amberlyst® 15. After stirring at room temperature for 6 h the mixture was filtered, dried over Na2SO4

and concentrated in vacuum. Column chromatography on silica gel using hexane afforded 70 mg (E)-

1-phenylpent-1-ene (42) (480 mmol, 66 % yield).

(E)-1-phenylpent-1-ene (42): C11H14; colourless oil; RI 1212; 1H NMR (500.1 MHz, CDCl3): =

0.95 (3H, t, J = 7.3 Hz, 11-CH3), 1.50 (2H, m, 10-CH2), 2.19 (2H, m, 9-CH2), 6.22 (1H, dt, JE = 15.8

Hz, J = 6.9 Hz, 8-H), 6.38 (1H, d, JE = 15.8 Hz, 7-H), 7.18 (1H, t, J = 7.3 Hz, 4-H), 7.28 (2H, t, J = 7.3

Hz, 3,5-H), 7.34 (2H, d, J = 7.3 Hz, 2,6-H); 13

C PENDANT (100.6 MHz, CDCl3): = 13.7 (q, 11-

CH3), 22.6 (t, 10-CH2), 35.1 (t, 9-CH2), 125.9 (d, 2,6-CH), 126.8 (d, 4-CH), 128.5 (d, 3,5-CH), 129.9

(d, 7-CH), 131.0 (d, 8-CH), 138.0 (s, 1-C); EIMS (70 eV) m/z (%) = 146 (37) [M], 128 (4), 117 (100)

[M – C2H5], 115 (43), 104 (30), 91 (27), 77 (6), 65 (7), 51 (6), 39 (7).

7.4.4. Synthesis of n-Pentylbenzene (31)

A solution of 50 mg (E)-1-phenylpent-1-ene (42) (342 µmol) in 5 ml hexane was treated with 10 mg

palladium on carbon (10 % Pd, w/w) and stirred under H2 atmosphere for 2 h. The mixture was filtered

and concentrated in a stream of N2. Column chromatography on silica gel using a pentane - Et2O

mixture (10:1, v/v) afforded 46 mg n-pentylbenzene (31) (308 µmol, 90 % yield) identical to the

natural product from Fossombronia angulosa.

pentylbenzene (31): C11H16; colourless oil; RI 1150; 1H NMR (500.1 MHz, CDCl3): = 0.89 (3H, t,

J = 6.9 Hz, 11-CH3), 1.33 (4H, m, 9,10-CH2), 1.62 (2H, m, 8-CH2), 2.60 (2H, t, J = 7.9 Hz, 7-CH2),

7.17 (3H, m, 2,4,6-H), 7.27 (2H, t, J = 6.9 Hz, 3,5-H); 13

C PENDANT (100.6 MHz, CDCl3): = 14.0

(q, 11-CH3), 22.6 (t, 10-CH2), 31.2 (t, 9-CH2), 31.5 (t, 8-CH2), 36.0 (t, 7-CH2), 125.6 (d, 4-CH), 128.2

(d, 3,5-CH), 128.4 (d, 2,6-CH), 143.0 (s, 1-C); EIMS (70 eV) m/z (%) = 148 (29) [M], 133 (2), 119

(2), 105 (11), 92 (71), 91 (100) [M – C4H9], 78 (6), 65 (13), 57 (3), 51 (4), 41 (8).

7.5. Riccardia chamedryfolia

7.5.1. Isolation of Prenylindoles (14 and 15)

The crude extracts of plant material of Riccardia chamedryfolia (With.) Grolle were fractionated by

column chromatography on silica gel 60 using a hexane - Et2O gradient and pure 7-prenylindole (14)

and 6-prenylindole (15) were isolated by column chromatography on silica gel 60 using a hexane -

DCM gradient.

7-Prenylindole (14): C13H15N, white solid, RI 1657; 1

H NMR (500.1 MHz, C6D6): = 1.57 (3H, s,

11-CH3), 1.61 (3H, s, 12-CH3), 3.32 (2H, d, J = 6.9 Hz, 8-CH2), 5.34 (1H, m, 9-CH), 6.56 (1H, s.br.,

3-CH), 6.65 (1H, s.br., 2-CH), 7.08 (1H, d, J = 7.3 Hz, 6-CH) 7.19 (1H, dd, J = J = 7.3 Hz, 5-CH),

7.25 (1H, s.br., NH), 7.63 (1H, d, J = 7.2 Hz, 4-CH); 13

C PENDANT (100.6 MHz, C6D6): = 17.8 (q,

11-CH3), 25.6 (q, 12-CH3), 30.7 (t, 8-CH2), 103.3 (d, 3-CH), 119.3 (d, 4-CH), 120.5 (d, 5-CH), 121.7

(d, 6-CH), 122.8 (d, 9-CH), 123.8 (d, 2-CH), 128.3 (s, 3a-C), 132.8 (s, 10-C), 135.5 (s, 7-C), 135.6 (s,


170

7a-C); EIMS (70 eV) m/z (%) = 185 (100) [M], 170 (84), 155 (24), 143 (10), 130 (67), 117 (29), 103

(6), 89 (7), 77 (16), 63 (7), 51 (5), 39 (9).

6-Prenylindole (15): C13H15N, white solid, RI 1758; 1H NMR (500.1 MHz, C6D6): = 1.67 (3H, s,

11-CH3), 1.72 (3H, s, 12-CH3), 3.53 (2H, d, J = 7.3 Hz, 8-CH2), 5.55 (1H, m, 9-CH), 6.49 (1H, s.br.,

3-CH), 6.59 (1H, s.br., 2-CH), 6.77 (1H, s.br., NH), 7.00 (1H, s, 7-CH), 7.09 (1H, d, J = 7.9 Hz, 5-

CH) 7.64 (1H, d, J = 8.2 Hz, 4-CH); 13

C PENDANT (100.6 MHz, C6D6): = 17.8 (q, 11-CH3), 25.9

(q, 12-CH3), 35.2 (t, 8-CH2), 102.6 (d, 3-CH), 110.6 (d, 7-CH), 120.9 (d, 4-CH), 121.4 (d, 5-CH),

123.6 (d, 2-CH), 125.1 (d, 9-CH), 126.8 (s, 3a-C), 131.5 (s, 10-C), 135.8 (s, 6-C), 136.8 (s, 7a-C);

EIMS (70 eV) m/z (%) = 185 (85) [M], 170 (100), 155 (41), 143 (16), 130 (30), 117 (26), 103 (6), 89

(7), 77 (18), 63 (6), 51 (5), 39 (7).

7.5.2. Synthesis of 3-Chloro-7-prenylindole (49)

A solution of 9.3 mg 7-prenylindole (14) (50 µmol) in 1 ml DCM was treated with 7.3 mg N-chloro

succinimide (55 µmol) in 1 ml DCM. After 30 min the solution was washed with 0.2 ml 1 M sodium

dithionite solution, dried over Na2SO4, concentrated in vacuum and the residue immediately

chromatographed on silica gel using a hexane - Et2O mixture (2:1, v/v) to give 9.3 mg 3-chloro-7-

prenylindole (49) (42.5 µmol, 85 % yield) identical to the natural product from Riccardia

chamedryfolia.

3-chloro-7-prenylindole (49); C13H14NCl, white solid, RI 1873; 1

H NMR (500.1 MHz, C6D6): =

1.53 (3H, s, 11-CH3), 1.61 (3H, s, 12-CH3), 3.18 (2H, d, J = 6.9 Hz, 8-CH2), 5.24 (1H, t, J = 6.9 Hz, 9-

CH), 6.53 (1H, s.br., 2-CH), 6.91 (1H, s.br., NH), 7.01 (1H, d, J = 7.3 Hz, 6-CH), 7.12 (1H, dd, J =

7.3 Hz, J = 7.9 Hz, 5-CH), 7.71 (1H, d, J = 8.2 Hz, 4-CH); 13

C PENDANT (100.6 MHz, C6D6): =

17.7 (q, 11-CH3), 25.6 (q, 12-CH3), 30.0 (t, 8-CH2), 107.0 (s, 3-CCl), 116.7 (d, 4-CH), 120.8 (d, 2-

CH), 121.2 (d, 6-CH), 122.2 (d, 5-CH), 122.8 (d, 9-CH), 124.3 (s, 3a-C), 133.1 (s, 10-C), 134.3 (s, 7a-

C) 135.7 (s, 7-C); EIMS (70 eV) m/z (%) = 219 [M] (75), 204 (100) [M – CH3], 169 (45), 164 (90),

154 (20), 151 (10), 141 (10), 128 (20), 115 (10), 101 (10), 83 (10), 77 (10), 63 (5).

7.5.3. Synthesis of 3-Chloro-6-prenylindole (50)

A solution of 6.1 mg 6-prenylindole (15, 33 µmol) was treated with 4.8 mg N-chlorosuccinimide (36

µmol) in 1 ml DCM. After 30 min the solution was washed with 0.2 ml 1 M sodium dithionite

solution, dried over Na2SO4, concentrated in vacuum and the residue immediately chromatographed on

silica gel using a hexane - Et2O mixture (2:1, v/v) to give 5.8 mg 3-chloro-6-prenylindole (50, 26.5

µmol, 80 % yield) identical to the natural product from Riccardia chamedryfolia.

3-chloro-6-prenylindole (50); C13H14NCl, white solid, RI 1992; 1H NMR (500.1 MHz, C6D6): =

1.63 (3H, s, 11-CH3), 1.70 (3H, s, 12-CH3), 3.43 (2H, d, J = 7.3 Hz, 8-CH2), 5.46 (1H, t, J = 7.2 Hz, 9-

CH), 6.38 (1H, s.br., NH), 6.44 (1H, d, J = 2.5 Hz, 2-CH), 6.83 (1H, s, 7-CH), 7.03 (1H, d, J = 7.6 Hz,

5-CH), 7.72 (1H, d, J = 7.9 Hz, 4-CH); 13

C PENDANT (100.6 MHz, C6D6): = 17.7 (q, 11-CH3),

25.8 (q, 12-CH3), 34.7 (t, 8-CH2), 106.2 (s, 3-CCl), 110.6 (d, 7-CH), 118.3 (d, 4-CH), 120.3 (d, 2-CH),

121.9 (d, 5-CH), 124.0 (s, 3a-C), 124.3 (d, 9-CH), 131.8 (s, 10-C), 135.6 (s, 7a-C), 137.0 (s, 6-C);

EIMS (70 eV) m/z (%) = 219 [M] (95), 204 (60), 184 (10), 169 (100) [M – CH3 – Cl], 168 (50), 164

(15), 154 (20), 151 (15), 141 (5), 128 (5), 115 (5).

7.5.4. Isolation of Chamedryfolian (51)

Chamedryfolian was isolated from an enriched fraction by repeated column chromatography using a

hexane – DCM gradient and preparative thin-layer chromatography on glass plates coated with silica


171

60 F254 using a hexane – EtOAc mixture (3:1, v/v; UV detection, Rf = 0.27) to give ca. 0.2 mg

chamedryfolian (51).

chamedryfolian (51); C13H13NO, white solid, RI 1676; 1H NMR (500.1 MHz, C6D6): = 1.58 (3H, s,

12-CH3), 2.73 (1H, dd, 2J = 14.8 Hz,

3J = 3.8 Hz, 8-H), 2.92 (1H, dd,

2J = 14.8 Hz,

3J = 7.9 Hz, 8-H‟),

4.38 (1H, dd, 3J = 7.9 Hz,

3J = 3.8 Hz, 9-H), 4.79 (1H, s, 11-H), 4.82 (1H, s, 11-H‟), 6.55 (1H, s.br, 3-

H), 6.72 (1H, s.br, 2-H), 6.92 (1H, d, J = 7.3 Hz, 6-H), 7.13 (1H, m, 5-H), 7.64 (1H, d, J = 7.9 Hz, 4-

H); 13

C NMR (125.6 MHz, C6D6) from HSQC and HMBC spectra (125 MHz, C6D6) = 34.5 (t, 8-

CH2), 90.1 (d, 9-CH), 102.8 (d, 3-CH), 113.2 (t, 11-CH2), 119.7 (d, 5-CH), 119.8 (d, 4-CH), 123.1 (d,

6-CH), 123.8 (d, 2-CH) [quaternary carbons 3a-C, 7-C, 7a-C, 10-C not detected]; EIMS (70 eV) m/z

(%) = 199 (27) [M], 184 (1), 171 (1), 156 (6), 130 (100) [M – C4H5O], 103 (10), 77 (12), 69 (4), 51

(1), 41 (6); HREIMS (70 eV): obs. m/z 199.0986 [M], calc. for C13H13NO: 199.0997, 1.1 mmu; obs.

m/z 130.0654, calc. for C9H8N: 130.0657, 0.2 mmu.

7.5.5. Chemical Correlation of Chamedryfolian (51)

A solution of 50 µg chamedryfolian (51) in 1 ml Et2O was stirred with 2 mg Pd/C (10 %, w/w) under

hydrogen atmosphere to afford a mixture of 7-(4-methylbutyl)indole (55) and 7-(3-methylbutyl)-

indoline (56), identical to those formed upon hydrogenation of 0.5 mg 7-prenylindole (14).

7-(3-methylbutyl)indole (55): C13H17N; EIMS (70 eV) m/z (%) = 187 (56) [M], 144 (3), 131 (100)

[M – C4H8], 130 (92) [M – C4H9], 117 (4), 109 (3), 103 (3), 89 (1), 77 (10).

7-(3-methylbutyl)indoline (56): C13H19N; EIMS (70 eV) m/z (%) = 189 (61) [M], 146 (3), 133 (51)

[M – C4H8], 132 (100) [M – C4H9], 117 (6), 109 (3), 105 (4), 91 (2), 77 (8).

7.5.6. Chemical Correlation of 7-(3-Methylbutadienyl)indole (52)

A solution of 50 µg 7-(3-methylbutadienyl)indole (52) in 1 ml Et2O was stirred with 2 mg Pd/C (10 %,

w/w) under hydrogen atmosphere to afford the same 7-(3-methylbutyl)indole (55) and 7-(3-methyl-

butyl)indoline (56) as obtained with 7-prenylindole (14).

7-(3-methylbutadienyl)indole (52): C13H13N; RI 1780; EIMS (70 eV) m/z (%) = 183 (42) [M], 182

(38) [M – H], 168 (100) [M – CH3], 167 (71) [M – H – CH3], 154 (4), 141 (11), 115 (9), 91 (9), 84

(10), 77 (6), 63 (5), 51 (2).

7.6. Corsinia coriandrina

7.6.1. Isolation of Secondary Metabolites

7.6.1.1. Isolation of ( )- -Pinene (18)

( )- -pinene (18) was isolated by a combination of column chromatography on silica gel using a

hexane – Et2O gradient and preparative gas chromatography using an SE-30 column at 80 °C

isothermally.

(4S,6S)-( )- -pinene (18): C10H16, colourless oil; RI 936; (–)-sense of optical rotation in C6D6, ee =

100 %; 1H NMR (500.1 MHz, C6D6): = 0.89 (3H, s, 8-CH3 syn), 1.24 (3H, s, 9-CH3 anti), 1.25 (1H, m,

5-Hsyn), 1.63 (3H, d, J = 1.9 Hz, 10-CH3), 1.90 (1H, m, 6-H), 2.03 (1H, s.br, 4-H), 2.16 (1H, d.br, 2J =

17.2 Hz, 3-H), 2.23 (1H, d.br, 2J = 17.3 Hz, 3-H‟), 2.32 (1H, dt, J = 8.5 Hz, J = 5.7 Hz, 5-Hanti), 5.20

(1H, s.br, 2-H); 13

C PENDANT (100.6 MHz, C6D6): = 21.0 (q, 8-CH3), 23.1 (q, 10-CH3), 26.5 (q, 9-

CH3), 31.6 (t, 3-CH2), 31.8 (t, 5-CH2), 38.1 (s, 7-C), 41.1 (d, 4-CH), 47.4 (d, 6-CH), 116.5 (d, 2-CH),


172

144.5 (s, 1-C); EIMS (70 eV): m/z (%): 136 (7) [M], 121 (12), 105 (11), 93 (100), 77 (28), 67 (8), 53

(6), 41 (12).

7.6.1.2. Isolation of ( )-(E)-Nerolidol (64)

(–)-(E)-nerolidol (64) was obtained as a pure fraction upon column chromatography on silica gel using

a hexane – Et2O gradient.

(R)-(–)-(E)-nerolidol (64): C15H26O, colourless oil, RI 1553; (–)-sense of optical rotation in C6D6, ee

= 80 %; 1H NMR (500.1 MHz, C6D6): = 1.12 (3H, s, 15-CH3), 1.44 – 1.54 (2H, m, 4-CH2), 1.56

(3H, s,14-CH3),1.59 (3H, s, 13-CH3), 1.68 (3H, s, 12-CH3), 2.05 – 2.18 (6H, m, 5,8,9,-CH2), 4.96 (1H,

dd, 3JZ = 10.7 Hz,

2J = 1.6 Hz, 1-Hanti), 5.20 (1H, dd,

3JE = 17.3 Hz,

2J = 1.6 Hz, 1-Hsyn), 5.24 (2H, m,

6,10-H), 5.75 (1H, dd, 3JE = 17.3 Hz,

3JZ = 10.7 Hz, 2-H);

13C PENDANT (100.6 MHz, C6D6): =

16.1 (q, 14-CH3), 17.7 (q, 13-CH3), 23.1 (t, 5-CH2), 25.9 (q, 12-CH3), 27.1 (t, 9-CH2), 28.0 (q, 15-

CH3), 40.2 (t, 8-CH2), 42.5 (t, 4-CH2), 73.5 (s, 3-C), 111.7 (t, 1-CH2), 124.9 (d, 10-CH), 125.0 (d, 6-

CH), 131.3 (s, 11-C), 135.3 (s, 7-C), 145.3 (d, 2-CH); EIMS (70 eV) m/z (%) = 204 (3) [M – H2O],

189 (5), 161 (14), 136 (22), 121 (13), 107 (36), 93 (69), 81 (30), 69 (100), 55 (34), 41 (80).

7.6.1.3. Isolation of (E,E)- -Springene (66)

-Springene (66) was isolated by a repeated column chromatography on silica gel using a hexane –

Et2O gradient and a hexane – DCM mixture (10:1, v/v).

(6E,10E)- -springene (66): C20H32, colourless oil; RI 1889; 1H NMR (500.1 MHz, C6D6): = 1.56

(6H, s, 18,19-CH3), 1.60 (3H, s, 17-CH3), 1.68 (3H, s, 16-CH3), 2.09 (4H, m, CH2), 2.18 (4H, m, CH2),

2.26 (4H, s, CH2), 4.98 (1H, d, JZ = 10.7 Hz, 1-Hanti), 5.00 (2H, s, 20-CH2), 5.22 (1H, d, JE = 17.7 Hz,

1-Hsyn), 5.27 (3H, m, 6,10,14-H), 6.37 (1H, dd, JE = 17.7 Hz, JZ = 10.7 Hz, 2-H); 13

C PENDANT

(100.6 MHz, C6D6): = 14.3, 16.1, 17.7 (3q, 17- CH3, 18- CH3, 19-CH3), 25.4 (q, 16-CH3), 27.1 , 27.3

(2t, 4-CH2, 5-CH2), 31.9 (2t, 9-CH2, 13-CH2), 40.2 (2t, 8-CH2, 12-CH2), 113.1 (t, 1-CH2), 116.0 (t, 20-

CH2), 124.6, 124.8, 125.0 (3d, 6-CH, 10-CH, 14-CH), 130.9, 134.7, 135.6, (3s, 7-C, 11-C, 15-C),

139.5 (d, 2-CH), 146.1 (s, 3-C); EIMS (70 eV) m/z (%) = 272 (7) [M], 257 (2), 229 (3), 204 (4), 187

(6), 175 (2), 161 (11), 147 (8), 133 (24), 120 (19), 107 (18), 93 (46), 81 (39), 69 (100), 55 (13), 41

(28).

7.6.1.4. Isolation of (Z)-Coriandrin ((Z)-65)

(Z)-Coriandrin ((Z)-65) was obtained as a pure fraction upon column chromatography on silica gel

using a hexane – Et2O gradient or isolated by thin layer chromatography using a hexane – EtOAc

mixture (4:1, Rf = 0.55). For NMR and EIMS data see: 7.6.2.7.

7.6.1.5. Isolation of (Z)-O-Methyltridentatol B ((Z)-72)

(Z)-O-Methyltridenatatol B ((Z)-72) was isolated by a combination of column chromatography on

silica gel using a hexane – Et2O gradient and thin layer chromatography using a hexane – EtOAc

mixture (5:1). For NMR and EIMS data see: 7.6.2.8.

7.6.1.6. Isolation of (R)-( -Corsifuran A (73)

(R)-(–)-Coriandrin (73) was obtained as a pure fraction upon column chromatography on silica gel

using a hexane – Et2O gradient or isolated by thin layer chromatography using a hexane – EtOAc

mixture (5:1, Rf = 0.33). For NMR and EIMS data see: 7.6.2.28.


173

7.6.1.7. Isolation of Corsifuran C (74)

Corsifuran C (74) was isolated by a combination of column chromatography on silica gel using a

hexane – Et2O gradient and thin layer chromatography using a hexane – EtOAc mixture (5:1, Rf =

0.35). For NMR and EIMS data see: 7.6.2.29.

7.6.1.8. Isolation of (E)-Corsistilbene ((E)-68)

Corsifuran C ((E)-68) was isolated by a combination of column chromatography on silica gel using a

hexane – Et2O gradient and thin layer chromatography using a hexane – EtOAc mixture (5:1, Rf =

0.40). For NMR and EIMS data see: 7.6.2.33.

7.6.1.9. Isolation of 4-Methoxyphenylethanal (59)

4-Methoxyphenylethanal (59) was isolated by a combination of column chromatography on silica gel

using a hexane – Et2O gradient and thin layer chromatography using a hexane – EtOAc mixture (5:1,

UV detection).

4-methoxyphenylethanal (59): C9H10O2, colourless oil; RI 1260; 1H NMR (500.1 MHz, CDCl3): =

3.54 (2H, d, 3J = 2.5 Hz, 7-H), 3.72 (3H, s, OCH3), 6.81 (2H, d,

3J = 8.7 Hz, 3-H, 5-H), 7.04 (2H, d,

3J

= 8.7 Hz, 2-H, 6-H), 9.63 (1H, t, 3J = 2.5 Hz, CHO); EIMS (70 eV) m/z (%) = 150 (14) [M], 121 (100)

[M – CHO], 91 (12), 77 (16), 65 (3), 51 (6); HREIMS (70 eV): obs. m/z 150.0652 [M], calc. for

C9H10O2: 150.0681, ∆ 2.9 mmu; obs. m/z 121.0644 [M – CHO], calc. for C8H9O: 121.0653, ∆ 0.9

mmu.

7.6.2. Synthesis of Secondary metabolites from Corsinia coriandrina

7.6.2.1. Synthesis of 2-Bromo-1-(4-methoxyphenyl)ethanone (76)

A solution of 15.0 g 1-(4-methoxyphenyl)ethanone (75, 100 mmol) in 60 ml CHCl3 was treated drop

wise with 5.1 ml bromine (99 mmol) in 40 ml CHCl3. After 30 min the solution was washed with 2 x

100 ml 1 M NaHCO3 soln., 2 x 50 ml 0.5 M sodium thiosulfate solution and 100 ml H2O, dried over

Na2SO4 and concentrated in vacuum. Recrystallization from Et2O afforded 19.4 g 2-bromo-1-(4-

methoxyphenyl)ethanone (76, 85 mmol, 85 % yield).

2-bromo-1-(4-methoxyphenyl)ethanone (76): C9H9O2Br, white solid; 1H NMR (400.1 MHz,

CDCl3): = 3.99 (3H, s, OCH3), 4.52 (2H, s, 2-H), 7.07 (2H, d, J = 8.9 Hz, 3‟,5‟-H), 8.08 (2H, d, J =

9.2 Hz, 2‟,6‟-H); 13

C PENDANT (100.6 MHz, CDCl3): = 31.2 (t, 2-CH2), 55.98 (q, OCH3), 114.5

(d, 3‟,5‟-CH), 127.3 (s, 1‟-C), 131.8 (d, 2‟,6‟-CH), 164.6 (s, 4‟-C), 190.3 (s, 1-C); EIMS (70 eV,

direct inlet) m/z (%) = 230 (45) [M + 2], 228 (46) [M], 135 (100) [M – CH2Br], 121 (27), 107 (19), 93

(38), 77 (41), 64 (22), 50 (11), 38 (6).

7.6.2.2. Synthesis of 2-Azido-1-(4-methoxyphenyl)ethanone (77)

A solution of 5.5 g NaN3 (84 mmol) in 75 ml H2O and 50 ml acetone was treated with a solution of

19.2 g 2-bromo-1-(4-methoxyphenyl)ethanone (76, 84 mmol) in 50 ml acetone. After stirring for 1 h

100 ml Et2O and 200 ml H2O were added. The aqueous phase was extracted with 100 ml Et2O and the

combined organic phases were dried over Na2SO4, concentrated in vacuum at 20 °C and dried at high

vacuum to afford 14.5 g 2-azido-1-(4-methoxyphenyl)ethanone (77, 76 mmol, 90 % yield).

2-azido-1-(4-methoxyphenyl)ethanone (77): C9H9N3O2, yellowish crystals; 1H NMR (400.1 MHz,

CDCl3): = 3.89 (3H, s, OCH3), 4.51 (2H, s, 2-H), 6.96 (2H, d, J = 8.9 Hz, 3‟,5‟-H), 7.89 (2H, d, J =


174

8.9 Hz, 2‟,6‟-H); 13

C PENDANT (100.6 MHz, CDCl3): = 55.0 (t, 2-CH2), 56.0 (q, OCH3), 114.6 (d,

3‟,5‟-CH), 127.8 (s, 1‟-C), 130.7 (d, 2‟,6‟-CH), 164.7 (s, 4‟-C), 192.1 (s, 1-C); EIMS (70 eV, direct

inlet) m/z (%) = 191 (7) [M], 163 (3), 135 (100) [M – CH2N3], 121 (5), 107 (20), 92 (34), 77 (36), 64

(17), 50 (6), 38 (4).

7.6.2.3. Synthesis of (±)-2-Amino-1-(4-methoxyphenyl)ethanol (78)

Under N2 atmosphere at 0 °C a suspension of 5.0 g LiAlH4 (131.8 mmol) in 100 ml dry Et2O was

treated drop wise with a solution of 10.9 g 2-azido-1-(4-methoxyphenyl)ethanone (77, 57.0 mmol) in

150 ml dry Et2O. After stirring at room temperature for 3 h the mixture was heated under reflux for 1

h. The ice cooled mixture was treated with 17.0 g powdered Na2SO4 x 10H2O (52.7 mmol) and stirred

at room temperature for 2 h until H2 evolution ceased. The organic phase was separated and the

residue suspended in 50 ml MeOH for 30 min and filtered. The combined organic phases were

concentrated in vacuum, the residue was taken up in Et2O, dried over Na2SO4 and concentrated in

vacuum to give 7.8 g 2-amino-1-(4-methoxyphenyl)ethanol (78, 46.6 mmol, 82 % yield).

(±)-2-amino-1-(4-methoxyphenyl)ethanol (78): C9H13NO2, white crystals; 1H NMR (400.1 MHz,

acetone-d6): = 2.80 (1H, dd, 2J = 11.7 Hz,

3J = 7.1 Hz, 2-H), 3.46 (1H, dd,

2J = 11.9 Hz,

3J = 6.4 Hz,

2-H‟), 3.27 (3H, s, OCH3), 4.77 (1H, dd, 3J =

3J = 6.9 Hz, 1-H), 6.88 (2H, d, J = 8.7 Hz, 3‟,5‟-H), 7.26

(2H, d, J = 8.7 Hz, 2‟,6‟-H); 13

C PENDANT (100.6 MHz, acetone-d6): = 55.9 (q, OCH3), 56.0 (t, 2-

CH2), 79.8 (d, 1-CH), 114.8 (d, 3‟,5‟-CH), 128.1 (d, 2‟,6‟-CH), 136.3 (s, 1‟-C), 160.2 (s, 4‟-C); EIMS

(70 eV, direct inlet) m/z (%) = 167 (16) [M], 149 (2), 137 (100) [M – CH2NH2], 121 (3), 109 (32), 94

(24), 77 (23), 66 (7), 51 (2), 39 (3).

7.6.2.4. Synthesis of (±)-2-Hydroxy-2-(4-methoxyphenyl)ethylammonium Chloride (79)

A solution of 7.6 g (±)-2-amino-1-(4-methoxyphenyl)ethanol (78, 45.5 mmol) in 100 ml propan-2-ol

at 0 °C was treated drop wise with 3.8 ml 37 % HCl until the solution was neutral to external pH-

paper. The solution was added to 150 ml Et2O. After 1 h at 7 °C the resulting crystals were obtained

by filtration, washed with Et2O, and dried in vacuum to afford 8.8 g racemic 2-hydroxy-2-(4-

methoxyphenyl)ethylammonium chloride (79, 43.2 mmol, 95 % yield).

(±)-2-hydroxy-2-(4-methoxyphenyl)ethylammonium chloride (79): C9H14NO2Cl, white crystals; 1H

NMR (400.1 MHz, D2O): = 3.24 (1H, dd, 2J = 13.0 Hz,

3J = 8.7 Hz, 1-H), 3.30 (1H, dd,

2J = 13.0

Hz, 3J = 4.3 Hz, 1-H‟), 3.86 (3H, s, OCH3), 4.98 (1H, dd,

3J = 8.7 Hz,

3J = 4.3 Hz, 2-H), 7.07 (2H, d,

3J = 8.9 Hz, 3‟,5‟-H), 7.42 (2H, d,

3J = 8.9 Hz, 2‟,6‟-H);

13C PENDANT (100.6 MHz, D2O): = 45.6

(t, 1-CH2), 55.8 (q, OCH3), 69.6 (d, 2-CH), 114.8 (d, 3‟,5‟-CH), 127.9 (d, 2‟,6‟-CH), 132.5 (s, 1‟-C),

159.5 (s, 4‟-C); EIMS (70 eV, direct inlet) m/z (%) = 167 (7) [M – HCl], 149 (1), 137 (100) [M – HCl

– CH2NH2], 121 (2), 109 (21), 94 (17), 77 (18), 66 (5), 51 (2), 39 (6), 36 (17).

7.6.2.5. Synthesis of (±)-2-Chloro-2-(4-methoxyphenyl)ethylammonium Chloride (80)

A suspension of 6.2 g (±)-2-hydroxy-2-(4-methoxyphenyl)ethylammonium chloride (79, 30.4 mmol)

in 50 ml CHCl3 at 0 °C was treated drop wise with a solution of 8.2 g SOCl2 (68.9 mmol) and 100 µl

dimethylformamide in 20 ml CHCl3. After stirring for 14 h solvent and excess reagent were removed

in vacuum at 20 °C. The residue was washed with 2 x 30 ml cold EtOH and 20 ml Et2O, and dried in

vacuum to afford 5.4 g racemic (±)-2-chloro-2-(4-methoxyphenyl)ethylammonium chloride (80, 24.3

mmol, 80 % yield).

(±)-2-chloro-2-(4-methoxyphenyl)ethylammonium chloride (80): C9H13NOCl2, white solid; 1

H

NMR (400.1 MHz, DMSO-d6): = 2.87 (1H, m, 1-H), 2.98 (1H, m, 1-H‟), 3.80 (3H, s, OCH3), 4.88


175

(1H, dd, J = 9.7 Hz, J = 3.0 Hz, 2-H), 6.99 (1H, d, J = 8.8 Hz, 3‟,5‟-H), 7.36 (1H, d, J = 8.5 Hz, 2‟,6‟-

H); 13

C PENDANT (100.6 MHz, DMSO-d6): = 46.3 (t, 1-CH2), 55.5 (q, OCH3), 69.0 (d, 2-CH),

114.1 (d, 3‟,5‟-CH), 127.5 (d, 2‟,6‟-CH), 134.3 (s, 1‟-C), 159.1 (s, 4‟-C); EIMS (70 eV, direct inlet)

m/z (%) = 187 (13) [M + 2 – HCl], 185 (40) [M – HCl], 156 (91), 155 (100) [M – HCl – CH2NH2],

150 (51), 134 (47), 121 (49), 91 (33), 77 (32), 65 (12), 44 (14), 36 (51).

7.6.2.6. Synthesis of (±)-2-Chloro-2-(4-methoxyphenyl)ethyl Isothiocyanate (81)

A suspension of 445 mg (±)-2-chloro-2-(4-methoxyphenyl)ethylammonium chloride (80, 2 mmol) in 8

ml diethyether at 0 °C was treated with 1 ml N-ethyldiisopropylamin (6 mmol), stirred for 1 h and

added drop wise to a solution of 400 µl thiophosgene (thiocarbonyl dichloride, 4 mmol) in 2 ml Et2O

at 0 °C. After 30 min 2 g ice were added and the organic phase separated, dried over Na2SO4, and

concentrated in vacuum. Column chromatography on silica gel using a mixture of hexane and EtOAc

(8:1, v/v; UV detection, Rf = 0.25) afforded 163 mg racemic (±)-2-chloro-2-(4-methoxyphenyl)ethyl

isothiocyanate (81, 0.72 mmol, 36 % yield).

(±)-2-chloro-2-(4-methoxyphenyl)ethyl isothiocyanate (81): C10H10NOSCl, colourless crystals; RI

1806; 1H NMR (400.1 MHz, C6D6): = 2.96 (1H, dd,

2J = 14.5 Hz, J = 6.4 Hz, 1-H), 3.07 (1H, dd,

2J

= 14.7 Hz, J = 7.1 Hz, 1-H‟), 3.20 (3H, s, OCH3) 4.26 (1H, dd, J = J = 6.7 Hz, 2-H), 6.61 (2H, d, J =

8.7 Hz, 3‟,5‟-H), 6.86 (2H, d, J = 8.7 Hz, 2‟,6‟-H); 13

C PENDANT (100.6 MHz, C6D6): = 52.1 (t, 1-

CH2), 55.1 (q, OCH3), 60.5 (d, 2-CH), 114.7 (d, 3‟,5‟-CH), 129.0 (d, 2‟,6‟-CH), 129.8 (s, 1‟-C), 135.2

(s, NCS), 160.9 (s, 4‟-C); EIMS (70 eV) m/z (%) = 227 (8) [M], 191 (63) [M – HCl], 176 (36), 157

(56), 155 (100) [M – CH2NCS], 148 (9), 134 (45), 121 (20), 119 (22), 104 (12), 91 (40), 77 (23), 65

(25), 63 (26), 51 (22), 45 (8), 39 (21); HREIMS (70 eV): obs. m/z 227.9170 [M], calc. for

C10H11NOS35

Cl: 277.0172, 0.2 mmu.

7.6.2.7. Synthesis of (Z)- and (E)-Coriandrins (65)

A solution of 100 mg (±)-2-chloro-2-(4-methoxyphenyl)ethyl isothiocyanate (81, 440 µmol) in 2.0 ml

Et2O was dehydrohalogenated in 100 µl portions at 210 °C in the injector port of a preparative GC.

The reaction products were separated by preparative GC using a SE 30 column (155 °C isothermally)

to give 32 mg (Z)-2-(4-methoxyphenyl)ethenyl isothiocyanate ((Z)-65, 168 µmol, 38 % yield) and 50

mg (E)-2-(4-methoxyphenyl)ethenyl isothiocyanate ((E)-65, 262 µmol, 60 % yield) identical to the

natural products from Corsinia coriandrina.

(Z)-coriandrin [(Z)-2-(4-methoxyphenyl)ethenyl isothiocyanate] ((Z)-65): C10H9NOS, yellowish

oil; RI 1708; 1H NMR (500.1 MHz, C6D6): = 3.20 (3H, s, OCH3), 5.18 (1H, d, JZ = 8.2 Hz, 1-H),

5.45 (1H, d, JZ = 8.2 Hz, 2-H), 6.60 (2H, d, J = 8.8 Hz, 3‟,5‟-H), 7.37 (2H, d, J = 8.8 Hz, 2‟,6‟-H); 13

C

PENDANT (100.6 MHz, C6D6): = 55.0 (q, OCH3), 111.3 (d, 1-CH), 114.6 (d, 3‟,5‟-CH), 126.9 (s,

1‟-C), 127.8 (d, 2-CH), 130.6 (d, 2‟,6‟-CH), 135.6 (s, NCS), 160.7 (s, 4‟-C); GC/FTIR (cm-1

) :

2200, 2096 s (N=C=S), 2058 s (N=C=S), 1609 (C=C), 1512 (C=CAr), 1398, 1260 (C-O-C), 1177,

1042, 832; EIMS (70 eV) m/z (%) = 191 (100) [M], 176 (54), 161 (6), 148 (14), 132 (4), 121 (25), 116

(6), 104 (14), 96 (4), 89 (16), 77 (10), 63 (13), 51 (8), 45 (5); HREIMS (70 eV) obs. m/z 191.0415

[M], calc. for C10H9NOS: 191.0405, ∆ 1.0 mmu, obs. m/z 176.0149, calc. for C9H6NOS: 176.0170, ∆

2.1 mmu, obs. m/z 161.0268, calc. for C9H7NS: 161.0299, ∆ 3.1 mmu, obs. m/z 148.0206, calc. for

C8H6NS: 148.0221, ∆ 1.5 mmu.

(E)-coriandrin [(E)-2-(4-methoxyphenyl)ethenyl isothiocyanate] ((E)-65): C10H9NOS, yellowish

oil; RI 1780; 1H NMR (400.1 MHz, C6D6): = 3.22 (3H, s, OCH3), 5.52 (1H, d, JE = 13.7 Hz, 1-H),

5.98 (1H, d, JE = 13.7 Hz, 2-H), 6.60 (2H, d, J = 8.9 Hz, 3‟,5‟-H), 6.74 (2H, d, J = 8.7 Hz, 2‟,6‟-H); 13

C PENDANT (100.6 MHz, C6D6): = 55.0 (q, OCH3), 113.5 (d, 1-CH), 114.7 (d, 3‟,5‟-CH), 128.1


176

(d, 2‟,6‟-CH), 130.6 (s, 1‟-C), 131.2 (d, 2-CH), 134.2 (s, NCS), 160.9 (s, 4‟-C); GC/FTIR (cm-1

) :

2096 s (N=C=S), 2055 s (N=C=S), 1610 (C=C), 1513 (C=CAr), 1251 (C-O-C), 1176, 1042; EIMS (70

eV) m/z (%) = 191 (100) [M], 176 (58), 161 (6), 148 (16), 132 (5), 121 (30), 116 (7), 104 (19), 96 (7),

89 (24), 77 (17), 63 (44), 51 (17), 45 (13).

7.6.2.8. Synthesis of (Z)- and (E)-O-Methyltridentatols (72)

A solution of 20 mg (Z/E)-2-(4-methoxyphenyl)ethenyl isothiocyanate ((Z/E)-65, 105 µmol) in 500 µl

hexane was added drop wise to a solution of 75 mg sodium methanethiolate (1.1 mmol) and 290 mg

[18]-crown-6 (1.1 mmol) in 500 µl dry THF. After stirring for 30 minutes 160 mg methyl iodide (1.1

mmol) was added. After 3 h 3 ml Et2O was added and the solution was washed with 2 x 3 ml H2O.

The organic phase was dried over Na2SO4 and concentrated in a stream of N2. Column

chromatography on silica gel using a hexane – Et2O gradient afforded a mixture of (Z)- and (E)-O-

methyltridentatol, which were separated by preparative gas chromatography at 180 °C isothermally

using 6-TBDMS-2,3-Me- -CD as the stationary phase to afford 12 mg (Z)-O-methyltridentatol B ((Z)-

72, 47 µmol 45 % yield) and 14 mg (E)-O-methyltridentatol A (55 µmol) ((E)-72, 55 µmol, 52 %

yield) identical to the natural products from Corsinia coriandrina.

(Z)-O-methyltridentatol B [(Z)-S,S-dimethyl-2-(4-methoxyphenyl)ethenyl iminodithiocarbonate]

((Z)-72): C12H15NOS2, yellow oil; RI 2192; 1H NMR (500.1 MHz, C6D6): = 2.15 (6H, s.br., 2 x

SCH3), 3.31 (3H, s, OCH3), 5.98 (1H, d, JZ = 8.5 Hz, 2-H), 6.84 (2H, d, J = 8.8 Hz, 3‟,5‟-H), 7.16 (1H,

d, JZ = 8.5 Hz, 1-H), 7.86 (2H, d, J = 8.8 Hz, 2‟,6‟-H); 13

C PENDANT (100.6 MHz, C6D6): = 15.1

(q, SCH3), 55.0 (q, OCH3), 114.1 (d, 3‟,5‟-CH), 121.9 (d, 2-CH), 130.5 (s, 1‟-C), 131.4 (d, 1-CH),

132.0 (d, 2‟,6‟-CH), 159.3 (s, 4‟-C), 162.0 (s, N=C(SR)2); EIMS (70 eV) m/z (%) = 253 (87) [M], 238

(1), 206 (100) [M – SCH3], 191 (49), 176 (21), 158 (45), 148 (3), 133 (36), 118 (8), 103 (10), 96 (14),

89 (19), 77 (13), 63 (12), 51 (7), 45 (10), 39 (7); HREIMS (70 eV): obs. m/z 253.0636 [M], calc. for

C12H15NOS2: 253.0595, ∆ 4.1 mmu, obs. m/z 206.0649, calc. for C11H12NOS: 206.0640, ∆ 0.9 mmu,

obs. m/z 191.0426, calc. for C10H9NOS: 191.0405, ∆ 2.1 mmu, obs. m/z 176.0149, calc. for C9H6NOS:

176.0170, ∆ 2.1 mmu, obs. m/z 158.0608, calc. for C10H8NO: 158.0606, ∆ 0.2 mmu.

(E)-O-methyltridentatol A [(E)-S,S-dimethyl-2-(4-methoxyphenyl)ethenyl iminodithiocarbonate]

((E)-72): C12H15NOS2, yellow oil; RI 2241; 1H NMR (500.1 MHz, C6D6): = 2.11 (3H, s.br, SCH3),

2.27 (3H, s.br, SCH3), 3.27 (3H, s, OCH3), 6.70 (2H, d, J = 8.8 Hz, 3‟,5‟-H), 6.96 (1H, d, JE = 13.2

Hz, 2-H), 7.28, (2H, d, J = 8.5 Hz, 2‟,6‟-H) 7.93 (1H, d, JE = 13.2 Hz, 1-H); 13

C PENDANT (100.6

MHz, C6D6): = 15.1 (q, SCH3), 55.0 (q, OCH3), 114.8 (d, 3‟,5‟-C), 127.8 (d, 2-CH), 128.1 (d, 2‟,6‟-

CH), 130.2 (s, 1‟-C), 132.9 (d, 1-CH), 159.6 (s, N=C(SR)2), 159.8 (s, 4‟-C); EIMS (70 eV) m/z (%) =

253 (85) [M], 238 (1), 206 (100) [M – SCH3], 191 (49), 176 (22), 158 (36), 148 (3), 133 (43), 118 (9),

103 (11), 96 (13), 89 (20), 77 (13), 63 (12), 51 (7), 45 (10), 39 (7); HREIMS (70 eV): obs. m/z

253.0621 [M], calc. for C12H15NOS2: 253.0595, ∆ 2.6 mmu, obs. m/z 206.0642, calc. for C11H12NOS:

206.0640, ∆ 0.2 mmu.

7.6.2.9. Synthesis of (±)-5-(4-Methoxyphenyl)-2-S-methylthio-4,5-dihydrothiazole (82)

A suspension of 111 mg (±)-2-chloro-2-(4-methoxyphenyl)ethylammonium chloride (80, 0.5 mmol) in

5 ml dry Et2O was treated with 60 µl carbon disulfide (1 mmol) and 260 µl N-ethyldiisopropylamine

(1.5 mmol). After 1h, 60 µl methyl iodide (1 mmol) was added and after an additional hour the

resulting solution was washed with 3 x 5 ml H2O, dried over Na2SO4, concentrated in vacuum; and

chromatographed on silica gel using hexane - EtOAc (4:1, v/v; UV detection) to afford 109 mg (±)-5-

(4-methoxyphenyl)-2-S-methylthio-4,5-dihydrothiazole (82, 456 µmol, 91 % yield).

(±)-5-(4-methoxyphenyl)-2-S-methylthio-4,5-dihydrothiazole (82): C11H13NOS2, white solid; 1H

NMR (500.1 MHz, C6D6): = 2.27 (3H, s, SCH3), 3.24 (3H, s, OCH3), 4.20 (1H, dd, 2J = 14.8 Hz, J =


177

6.3 Hz, 4-H), 4.21 (1H, dd, 2J = 14.8 Hz, J = 7.9 Hz, 4-H‟), 4.61 (1H, dd, J = 6.3 Hz,

J = 7.9 Hz, 5-H),

6.62 (2H, d, J = 8.8 Hz, 3‟,5‟-H), 7.00 (2H, d, J = 8.5 Hz, 2‟,6‟-H); 13

C PENDANT (100.6 MHz,

C6D6): = 15.2 (q, SCH3), 55.0 (q, OCH3), 57.4 (d, 5-CH), 72.9 (t, 4-CH2), 114.4 (d, 3‟,5‟-CH), 128.8

(d, 2‟,6‟-CH), 133.8 (s, 1‟-C), 160.0 (s, 4‟-C), 165.1 (s, 2-C); EIMS (70 eV) m/z (%) = 239 (100) [M],

206 (3), 192 (11), 166 (76), 158 (9), 151 (20), 148 (17), 135 (45), 121 (33), 91 (25), 87 (94), 77 (13),

72 (72), 65 (18), 59 (10), 51 (23), 45 (15), 39 (14); HREIMS (70 eV): obs. m/z 239.0457 [M], calc.

for C11H13NOS2: 239.0439, ∆ 1.8 mmu.

7.6.2.10. Synthesis of O-Methyltridentatol C (71)

A solution of 48 mg (±)-5-(4-methoxyphenyl)-2-S-methylthio-4,5-dihydrothiazole (82, 200 µmol) in 2

ml 1,4-dioxane was treated with 50 mg 2,3-dichloro-5,6-dicyano-p-benzoquinone (220 µmol) and

heated to 100 °C for 14 h. The solvent was evaporated in vacuum and the residue submitted to column

chromatography on silica gel using hexane - EtOAc (4:1, v/v; UV detection, Rf = 0.25) to afford 44

mg 5-(4-methoxyphenyl)-2-methylthio-1,3-thiazole (71, 186 µmol, 93 % yield) identical to O-methyl-

tridentatol C from Corsinia coriandrina.

O-methyltridentatol C [5-(4-methoxyphenyl)-2-methylthio-1,3-thiazole] (71): C11H11NOS2, white

solid; RI 2037; 1H NMR (500.1 MHz, C6D6): = 2.29 (3H, s, SCH3), 3.25 (3H, s, OCH3), 6.64 (2H, d,

J = 8.8 Hz, 3‟,5‟-H), 7.14 (2H, d, J = 8.8 Hz, 2‟,6‟-H), 7.68 (1H, s, 4-H); 13


C6D6): = 16.0 (q, SCH3), 54.8 (q, OCH3), 114.8 (d, 3‟,5‟-CH), 124.3 (s, 5-C), 128.0 (d, 2‟,6‟-CH),

137.6 (d, 4-CH), 138.9 (s, 1‟-C), 160.0 (s, 4‟-C), 164.1 (s, 2-C); EIMS (70 eV) m/z (%) = 237 (100)

[M], 222 (12), 204 (72), 189 (3), 178 (15), 164 (8), 149 (15), 132 (5), 121 (10), 108 (3), 89 (6), 77 (7),

63 (8), 51 (4), 45 (13), 39 (5); HREIMS (70 eV): obs. m/z 237.0294 [M], calc. for C11H11NOS2:

237.0282, ∆ 1.2 mmu; obs. m/z 204.0498, calc. for C11H10NOS: 204.0483, ∆ 1.5 mmu; obs. m/z

178.0331, calc. for C9H8NOS: 178.0321, ∆ 1.0 mmu; obs. m/z 164.0297, calc. for C9H8OS: 164.0296,

∆ 0.1 mmu; obs. m/z 132.0579, calc. for C9H8O: 132.0575, ∆ 0.4 mmu; obs. m/z 121.0117, calc. for

C7H5S: 121.0106, ∆ 1.1 mmu; UV (MeOH): max = 312 nm, = 28.850.

7.6.2.11. Synthesis of (±)-2-Chloro-2-(4-methoxyphenyl)ethyl Isocyanate (87)

A suspension of 111 mg (±)-2-chloro-2-(4-methoxyphenyl)ethylammonium chloride (80, 0.5 mmol) in

5 ml toluene was treated with 50 mg triphosgene (167 µmol), and 160 µl pyridine (2 mmol) in 1 ml

toluene was added drop wise. After 1 h, the resulting mixture was heated under reflux for 30 min,

diluted with 10 ml ice H2O, extracted with DCM, dried over Na2SO4, and concentrated in vacuum.

Flash column chromatography on silica gel using hexane - EtOAc (1:1, v/v; UV detection) afforded 39

mg (±)-2-chloro-2-(4-methoxyphenyl)ethyl isocyanate (87, 185 µmol, 37 % yield).

(±)-2-chloro-2-(4-methoxyphenyl)ethyl isocyanate (87): C10H10NO2Cl, colourless oil; 1H NMR

(500.1 MHz, C6D6): = 2.86 (1H, dd, 2J = 13.7 Hz, J = 6.0 Hz, 1-H), 3.05 (1H, dd,

2J = 13.7 Hz, J =

7.4 Hz, 1-H‟), 3.23 (3H, s, OCH3), 4.33 (1H, dd, J = J = 6.7 Hz, 2-H), 6.62 (2H, d, J = 8.5 Hz, 3‟,5‟-

H), 6.89 (2H, d, J = 8.5 Hz, 2‟,6‟-H); 13

C PENDANT (100.6 MHz, C6D6): = 49.9 (t, 1-CH2), 54.8 (q,

OCH3), 62.1 (d, 2-CH), 114.4 (d, 3‟,5‟-CH), 124.8 (s, NCO), 128.8 (d, 2‟,6‟-CH), 130.0 (s, 1‟-C),

160.6 (s, 4‟-C); EIMS (70 eV) m/z (%) = 213 (3) [M + 2], 211 (9) [M], 176 (14) [M – Cl], 175 (29)

[M – HCl], 157 (33), 155 (100) [M – CH2NCO], 134 (11), 132 (20), 121 (10), 119 (7), 112 (5), 104

(11), 91 (25), 89 (9), 77 (23), 65 (9), 63 (10), 51 (20), 44 (8); HREIMS (70 eV): obs. m/z 211.0412

[M], calc. for C10H10NO235

Cl: 211.0400, ∆ 1.2 mmu.


178

7.6.2.12. Synthesis of (Z)- and (E)-Corsinians (63)

A solution of 2 mg (±)-2-chloro-2-(4-methoxyphenyl)ethyl isocyanate (87) (9.5 µmol) in 200 µl Et2O

was dehydrohalogenated in two portions at 250 °C in the injector port of a preparative gas

chromatograph equipped with a SE-30 column to afford ca. 0.5 mg of a 1:1 mixture of (Z)- and (E)-2-

(4-methoxyphenyl)ethenyl isocyanates ((Z/E)-63) (2.9 µmol, 30 % yield) identical to (Z)- and (E)-

corsinian from Corsinia coriandrina. For EIMS, HREIMS and NMR data see: 7.6.2.16.

7.6.2.13. Synthesis of (E)-4-Methoxycinnamic acid (89) by Knoevenagel

A mixture of 2.7 g 4-methoxybenzaldehyde (58) (20 mmol) and 3.25 g malonic acid (88) (30 mmol) in

20 ml pyridine was treated with 50 µl piperidine and kept at 90 °C for 6 h. The resulting mixture was

poured into 50 ml ice cold 5 M HCl and the precipitating solids were removed by filtration, washed

with 50 ml 5 M HCl and 20 ml H2O, recrystallized from water / ethanol and dried in vacuum to afford

3.0 g (E)-4-methoxycinnamic acid (89) (16.8 mmol, 84 % yield).

(E)-4-methoxycinnamic acid [(E)-3-(4-methoxyphenyl)propenoic acid] (89): C10H10O3, white

solid; 1H NMR (400 MHz, CDCl3): = 3.85 (3H, s, OCH3), 6.33 (1H, d,

3JE = 16.0 Hz, 2-H), 6.92

(2H, d, 3J = 8.9 Hz, 3‟-H, 5‟-H), 7.51 (2H, d,

3J = 8.9 Hz, 2‟-H, 6‟-H), 7.75 (1H, d,

3JE = 15.8 Hz, 3-

H); 13

C NMR (100 MHz, CDCl3): = 55.4 (q, OCH3), 114.4 (d, 3‟,5‟-CH), 114.6 (d, 2-CH), 126.8 (s,

1-C), 130.1 (d, 2‟,6‟-CH), 146.7 (d, 3-CH), 161.8 (s, 4‟-C), 172.2 (s, COOH); EIMS (70 eV) m/z (%)

= 178 (100) [M], 161 (28), 147 (2), 133 (14), 121 (3), 118 (5), 107 (3), 89 (11), 77 (11), 63 (8), 51 (4),

39 (4).

7.6.2.14. Synthesis of (E)-3-(4-Methoxyphenyl)propenoyl Chloride (90)

A suspension of 1.25 g (E)-3-(4-methoxyphenyl)propenoic acid (89, 7.0 mmol) in 20 ml CHCl3 was

treated with 1.2 g SOCl2 (10 mmol) and heated under reflux for 30 min. Solvent and excess reagent

were evaporated in vacuum and the residue was recrystallized from toluene to afford 1.32 g (E)-3-(4-

methoxyphenyl)propenoyl chloride (90, 6.7 mmol, 96 % yield).

(E)-3-(4-methoxyphenyl)propenoyl chloride (90): C10H9O2Cl, white solid; 1H NMR (400.1 MHz,

C6D6): = 3.17 (3H, s, OCH3), 6.19 (1H, d, JE = 15.5 Hz, 2-H), 6.50 (2H, d, J = 8.9 Hz, 3‟,5‟-H), 6.82

(2H, d, J = 8.6 Hz, 2‟,6‟-H), 7.55 (1H, d, JE = 15.5 Hz, 3-H); 13


55.2 (q, OCH3), 114.9 (d, 3‟,5‟-CH), 120.1 (d, 2-CH), 126.2 (s, 1‟-C), 131.5 (d, 2‟,6‟-CH), 150.5 (d, 3-

CH), 163.2 (s, 4‟-C), 165.8 (s, 1-COCl); EIMS (70 eV, direct inlet) m/z (%) = 196 (10) [M], 161 (100)

[M – Cl], 133 (18), 118 (4), 102 (2), 90 (8), 87 (4), 63 (6); HREIMS (70 eV): obs. m/z 196.0305 [M],

calc. for C10H9O235

Cl: 196.0291, ∆ 1.4 mmu.

7.6.2.15. Synthesis of (E)-3-(4-Methoxyphenyl)propenoyl Azide (91)

A solution of 1.20 g (E)-3-(4-methoxyphenyl)propenoyl chloride (90, 6.1 mmol) in 10 ml acetone at

0 °C was treated with a solution of 0.40 g NaN3 (6.1 mmol) in a mixture of 6 ml H2O and 6 ml

acetone. The precipitating product was filtered and dried in vacuum at 0 °C to afford 1.10 g (E)-3-(4-

methoxyphenyl)propenoyl azide (91, 5.4 mmol, 80 % yield).

(E)-3-(4-methoxyphenyl)propenoyl azide (91): C10H9N3O2, off-white solid; 1H NMR (400.1 MHz,

C6D6): = 3.17 (3H, s, OCH3), 6.19 (1H, d, JE = 15.9 Hz, 2-H), 6.53 (2H, d, J = 8.9 Hz, 3‟,5‟-H), 6.98

(2H, d, J = 8.7 Hz, 2‟,6‟-H), 7.70 (1H, d, JE = 15.9 Hz, 3-H); 13


54.8 (q, OCH3), 114.6 (d, 3‟,5‟-CH), 116.9.1 (d, 2-CH), 126.9 (s, 1‟-C), 130.6 (d, 2‟,6‟-CH), 146.3 (d,

3-CH), 162.3 (s, 4‟-C), 171.6 (s, 1-CON3); EIMS (70 eV, direct inlet) m/z (%) = 203 (27) [M], 175

(100) [M – N2], 161 (32) [M – N3], 160 (33), 146 (8), 132 (85), 120 (16), 104 (27), 91 (14), 77 (26), 63


179

(13), 51 (14), 39 (14); HREIMS (70 eV): obs. m/z 203.0708 [M], calc. for C10H9N3O2: 203.0695, ∆

1.3 mmu.

7.6.2.16. Synthesis of (Z)- and (E)-Corsinians (63) by Curtius Rearrangement

Under N2 atmosphere a solution of 406 mg (E)-3-(4-methoxyphenyl)propenoyl azide (91, 2 mmol) in

10 ml dry toluene was heated under reflux for 3 h. The solvent was removed in vacuum to afford 350

mg (E)-2-(4-methoxyphenyl)ethenyl isocyanate ((E)-63, 2 mmol, 99 % yield). UV irradiation at 350

nm for 6 h afforded a 2:3 mixture of (Z)- and (E)-2-(4-methoxyphenyl)ethenyl isocyanates ((Z)-63 and

(E)-63) identical to (Z/E)-corsinians from Corsinia coriandrina and Cronisia weddellii.

(Z)-corsinian [(Z)-2-(4-methoxyphenyl)ethenyl isocyanate] ((Z)-63): C10H9NO2, yellowish oil; RI

1460; 1H NMR (500.1 MHz, C6D6): = 3.29 (3H, s, OCH3), 5.27 (1H, d, JZ = 8.2 Hz, 1-H), 5.46 (1H,

d, JZ = 8.2 Hz, 2-H), 6.71 (2H, d, J = 8.8 Hz, 3‟,5‟-H), 7.43 (2H, d, J = 8.8 Hz, 2‟,6‟-H); 13

C

PENDANT (100.6 MHz, C6D6): = 54.8 (q, OCH3), 113.7 (d, 1-CH), 114.1 (d, 3‟,5‟-CH), 123.0 (d,

2-CH), 125.4 (s, NCO), 127.4 (s, 1‟-C), 130.3 (d, 2‟,6‟-CH), 159.8 (s, 4‟-C); EIMS (70 eV) m/z (%) =

175 (100) [M], 160 (23), 146 (4), 132 (49), 120 (13), 104 (19), 91 (13), 77 (26), 63 (9), 51 (25), 39 (7);

HREIMS (70 eV): obs. m/z 175.0645 [M], calc. for C10H9NO2: 175.0633, ∆ 1.2 mmu, obs. m/z

160.0398, calc. for C9H6NO2: 160.0382, ∆ 1.6 mmu, obs. m/z 132.0470, calc. for C8H6NO: 132.0449,

∆ 2.1 mmu, obs. m/z 104.0517, calc. for C7H6N: 104.0500, ∆ 1.7 mmu.

(E)-corsinian [(E)-2-(4-methoxyphenyl)ethenyl isocyanate] ((E)-63): C10H9NO2, yellowish solid,

RI 1492; 1H NMR (500.1 MHz, C6D6,): = 3.27 (3H, s, OCH3), 5.62 (1H, d, JE = 13.7 Hz, 1-H), 6.04

(1H, d, JE = 13.7 Hz, 2-H), 6.68 (2H, d, J = 8.7 Hz, 3‟,5‟-H), 6.86 (2H, d, J = 8.7 Hz, 2‟,6‟-H); 13

C

PENDANT (100.6 MHz, C6D6): = 55.0 (q, OCH3), 114.7 (d, 3‟,5‟-CH), 116.1 (d, 1-CH), 125.2 (s,

NCO), 126.7 (d, 2-CH), 127.8 (d, 2‟,6‟-CH), 130.9 (s, 1‟-C), 160.2 (s, 4‟-C); EIMS (70 eV) m/z (%) =

175 (100) [M], 160 (26), 146 (5), 132 (51), 120 (14), 104 (18), 91 (13), 77 (27), 63 (12), 51 (28), 39

(8); HREIMS (70 eV): obs. m/z 175.0642 [M], calc. for C10H9NO2: 175.0633, ∆ 0.9 mmu.

7.6.2.17. Synthesis of (Z)- and (E)-Corsiandrens (67)

Under N2 atmosphere a solution of 140 mg sodium methanethiolate (2 mmol) in 10 ml dry DMSO was

treated with 175 mg (E)-2-(4-methoxyphenyl)ethenyl isocyanate ((E)-63, 1 mmol) in 5 ml toluene.

After 1 h, 10 ml Et2O and 15 ml H2O were added, the organic phase separated, washed with 5 x 30 ml

H2O, and dried over Na2SO4. The solvent was evaporated in a stream of N2 and the residue

chromatographed on silica gel using an hexane - EtOAc mixture (1:1, v/v; UV detection, Rf = 0.52) to

give 93 mg S-methyl (E)-2-(4-methoxyphenyl)ethenyl thiocarbamate ((E)-67, 417 µmol, 42 % yield).

UV irradiation at 350 nm for 6 h afforded a 3:1 mixture of (Z)- and (E)-2-(4-methoxyphenyl)ethenyl

S-methyl thiocarbamates ((Z)-67 and (E)-67) identical to (Z/E)-corsiandrens from Corsinia

coriandrina.

(Z)-corsiandren [(Z)-2-(4-methoxyphenyl)ethenyl-S-methylthiocarbamate] ((Z)-67): C11H13NO2S,

yellowish crystalls; RI 1907; 1H NMR (500.1 MHz, acetone-d6): = 2.08 (3H, s, SCH3), 3.26 (3H, s,

OCH3), 5.40 (1H, d, JZ = 9.5 Hz, 2-H), 6.61 (2H, d, J = 8.5 Hz, 3‟,5‟-H), 6.92 (2H, d, J = 8.5 Hz,

2‟,6‟-H), 6.96 (1H, s.br, 1-H); 13

C PENDANT (100.6 MHz, acetone-d6): = 11.9 (q, SCH3), 55.5 (q,

OCH3), 110.6 (d, 2-CH), 114.9 (d, 3‟,5‟-CH), 121.7 (d, 1-CH), 128.7 (s, 1‟-C), 130.4 (d, 2‟,6‟-CH),

158.9 (s, 4‟-C), 167.2 (s, COS); EIMS (70 eV) m/z (%) = 223 (34) [M], 175 [M – HSCH3] (100), 160

(19), 148 (20), 132 (58), 120 (11), 104 (30), 91 (15), 77 (36), 63 (13), 51 (26), 47 (19); HREIMS (70

eV): obs. m/z 223.0676 [M], calc. for C11H13NO2S: 223.0667, ∆ 0.9 mmu, obs. m/z 175.0647, calc. for

C10H9NO2: 175.0633, ∆ 1.4 mmu, obs. m/z 160.0400, calc. for C9H6NO2: 160.0382, ∆ 1.8 mmu, obs.


180

m/z 148.0776, calc. for C9H10NO: 148.0762, ∆ 1.6 mmu, obs. m/z 132.0467, calc. for C8H6NO:

132.0449, ∆ 1.8 mmu.

(E)-corsiandren [(E)-2-(4-methoxyphenyl)ethenyl-S-methylthiocarbamate] ((E)-67): C11H13NO2S,

white crystalls; RI 2078; 1H NMR (500.1 MHz, acetone-d6): = 2.34 (3H, s, SCH3), 3.78 (3H, s,

OCH3), 6.18 (1H, d, JE = 14.5 Hz, 2-H), 6.86 (2H, d, J = 8.8 Hz, 3‟,5‟-H), 7.27 (2H, d, J = 8.8 Hz,

2‟,6‟-H), 7.32 (1H, s.br, 1-H); 1H NMR (500 MHz, C6D6): 2.13 (3H, s, SCH3), 3.29 (3H, s, OCH3),

5.32 (1H, d, JE = 14.5 Hz, 2-H), 6.16 (1H, s.br, 1-H), 6.70 (2H, d, J = 8.8 Hz, 3‟,5‟-H), 6.97 (2H, d, J

= 8.5 Hz, 2‟,6‟-H), 7.39 (1H, s.br, NH); 13

C NMR (100 MHz, C6D6): 12.2 (q, SCH3), 54.8 (q,

OCH3), 112.1 (d, 2-CH), 114.5 (d, 3‟,5‟-CH), 121.3 (d, 1-CH), 126.9 (d, 2‟,6‟-CH), 128.3 (s, 1‟-C),

159.1 (s, 4‟-C), 164.7 (s, CO); 13

C PENDANT (100.6 MHz, acetone-d6): = 12.0 (q, SCH3), 55.5 (q,

OCH3), 112.1 (d, 2-CH), 115.0 (d, 3‟,5‟-CH), 122.6 (d, 1-CH), 127.3 (d, 2‟,6‟-CH), 130.4 (s, 1‟-C),

159.0 (s, 4‟-C), 165.8 (s, COS); EIMS (70 eV) m/z (%) = 223 (32) [M], 175 (100) [M – HSCH3], 160

(19), 148 (21), 132 (57), 120 (11), 104 (32), 91 (14), 77 (37), 63 (12), 51 (27), 47 (18); HREIMS (70

eV): obs. m/z 223.0679 [M], calc. for C11H13NO2S: 223.0667, ∆ 1.2 mmu; UV (MeOH): max = 288

nm, = 25.700.

7.6.2.18. Synthesis of (Z)- and (E)-Corsiandrenins (92)

Under N2 atmosphere a solution of 140 mg sodium methanethiolate (2 mmol) in 10 ml dry DMSO was

treated with 175 mg (E)-2-(4-methoxyphenyl)ethenyl isocyanate ((E)-63, 1 mmol) in 5 ml toluene.

After stirring for 1 h 102 mg Ac2O (1 mmol) in 1 ml DMSO was added drop wise. After 1 h, 30 ml

Et2O and 50 ml H2O was added, the organic phase separated, washed with 5 x 30 ml H2O, and dried

over Na2SO4. The solvent was evaporated in vacuum and the residue chromatographed on silica gel

using an hexane - EtOAc gradient to give 103 mg (E)-2-(4-methoxyphenyl)ethenyl N-acetyl-S-methyl

thiocarbamate ((E)-92, 390 µmol, 39 % yield). UV irradiation afforded a 3:1 mixture of (Z/E)-isomers

((Z)-92 and (E)-92) of which the (Z)-isomer was identical to (Z)-corsiandrenin ((Z)-92) from polyploid

Corsinia coriandrina strain Lorbeer/33.

(Z)-corsiandrenin [(Z)-2-(4-methoxyphenyl)ethenyl N-acetyl-S-methyl thiocarbamate] ((Z)-92):

C13H15NO3S, beige solid; 1H NMR (500.1 MHz, C6D6): = 1.93 (3H, s, SCH3), 2.14 (3H, s, COCH3),

3.17 (3H, s, OCH3), 5.97 (1H, d, JZ = 8.4 Hz, 2-H), 6.18 (1H, d, JZ = 8.7 Hz, 1-H), 6.60 (2H, d, J = 8.9

Hz, 3‟,5‟-H), 7.18 (2H, d, J = 8.9 Hz, 2‟,6‟-H); 13

C PENDANT (100.6 MHz, C6D6): = 13.6 (q,

SCH3), 25.7 (q, COCH3), 54.7 (q, OCH3), 114.5 (d, 3‟,5‟-CH), 122.3 (d, 1-CH), 125.6 (s, 1‟-C), 130.3

(d, 2‟,6‟-CH), 132.9 (d, 2-CH), 160.3 (s, 4‟-C), 170.6 (s, COCH3), 171.6 (s, COSCH3); EIMS (70 eV)

m/z (%) = 265 (23) [M], 223 (100) [M – CH2CO], 195 (2), 180 (17), 175 (69), 160 (8), 148 (32), 132

(22), 121 (11), 104 (8), 91 (6), 77 (13), 63 (4), 51 (10), 43 (73); HREIMS (70 eV): obs. m/z 265.0784

[M] , calc. for C13H15NO3S: 265.0773, ∆ 1.1 mmu; obs. m/z 223.0675, calc. for C11H13NO2S:

223.0667, ∆ 0.8 mmu; obs. m/z 175.0643, calc. for C10H9NO2: 175.0633, ∆ 1.0 mmu; obs. m/z

148.0775, calc. for C9H10NO: 148.0762, ∆ 1.3 mmu.

(E)-corsiandrenin [(E)-2-(4-methoxyphenyl)ethenyl N-acetyl-S-methyl thiocarbamate] ((E)-92):

C13H15NO3S, white crystals; 1H NMR (500.1 MHz, C6D6): = 1.98 (3H, s, SCH3), 2.29 (3H, s,

COCH3), 3.26 (3H, s, OCH3), 6.31 (1H, d, JE = 14.2 Hz, 2-H), 6.49 (1H, d, JE = 14.2 Hz, 1-H), 6.68

(2H, d, J = 8.8 Hz, 3‟,5‟-H), 7.11 (2H, d, J = 8.8 Hz, 2‟,6‟-H); 13

C PENDANT (100.6 MHz, C6D6):

= 14.0 (q, SCH3), 26.3 (q, COCH3), 54.8 (q, OCH3), 114.5 (d, 3‟,5‟-CH), 121.7 (d, 1-CH), 127.1 (s, 1‟-

C), 128.6 (d, 2‟,6‟-CH), 135.4 (d, 2-CH), 160.7 (s, 4‟-C), 170.6 (s, COCH3), 172.1 (s, COSCH3);

EIMS (70 eV) m/z (%) = 265 (27) [M], 223 (100) [M – CH2CO], 195 (4), 180 (16), 175 (63), 160 (9),

148 (34), 132 (23), 121 (12), 104 (17), 91 (8), 77 (16), 63 (6), 51 (12), 43 (68); HREIMS (70 eV):

obs. m/z 265.0788 [M] , calc. for C13H15NO3S: 265.0773, ∆ 1.5 mmu.


181

7.6.2.19. Synthesis of (E)-Cinnamic Acid (93)

A mixture of 2.1 g benzaldehyde (40) (20 mmol) and 3.25 g malonic acid (88) (30 mmol) in 20 ml

pyridine was treated with 50 µl piperidine and kept at 90 °C for 6 h. The resulting mixture was poured

into 50 ml ice cold 5 M hydrochloric acid and the precipitating solids were removed by filtration,

washed with 50 ml 5 M hydrochloric acid and 20 ml H2O, recrystallized from water / ethanol and

dried in vacuum to afford 2.5 g (E)-cinnamic acid (93) (17.2 mmol, 86 % yield).

(E)-cinnamic acid [(E)-3-phenylpropenoic acid] (93): C9H8O2; white solid; 1H NMR (500.1 MHz,

CDCl3): = 6.47 (1H, d, 3JE = 16.1 Hz, 2-H), 7.41 (3H, m, 3‟,4‟,5‟-H), 7.56 (2H, m, 2‟,6‟-H), 7.81

(1H, d, 3JE = 16.1 Hz, 3-H);

13C PENDANT (100.6 MHz, CDCl3): = 117.3 (d, 2-CH), 128.4 (d,

2‟,6‟-CH), 129.0 (d, 3‟,5‟-CH), 130.8 (d, 4‟-CH), 134.0 (s, 1‟-C), 147.1 (d, 3-CH), 172.3 (s, 1-

COOH); EIMS (70 eV, direct inlet) m/z (%) = 148 (92) [M], 147 (100) [M – H], 131 (23), 120 (6),

103 (45), 102 (26), 91 (19), 77 (37), 63 (6), 51 (24).

7.6.2.20. Synthesis of (E)-3-Phenylpropenoyl Chloride (94)

A suspension of 1.5 g (E)-3-phenylpropenoic acid (93, 10 mmol) in 10 ml CHCl3 was treated with 1.7

g SOCl2 (14 mmol) and heated under reflux for 30 min. Solvent and excess reagent was evaporated in

vacuum and the residue was recrystallized from toluene to afford 1.6 g (E)-3-phenylpropenoyl

chloride (94, 9.6 mmol, 96 % yield).

(E)-3-phenylpropenoyl chloride (94): C9H7OCl; white solid; 1H NMR (400.1 MHz, C6D6): = 6.22

(1H, d, JE = 15.8 Hz, 2-H), 6.91 – 7.01 (5H, m, 2„,3„,4„,5„,6„-H), 7.63 (1H, d, JE = 16.1 Hz, 3-H); 13

C

PENDANT (100.6 MHz, C6D6): = 122.5 (d, 2-CH), 129.0 (d, 2‟,6‟-CH), 129.2 (d, 3‟,5‟-CH), 131.4

(d, 4‟-CH), 132.9 (s, 1‟-C), 150.2 (d, 3-CH), 172.3 (s, 1-COCl); EIMS (70 eV, direct inlet) m/z (%) =

168 (3) [M + 2], 166 (8) [M], 131 (100) [M – Cl], 103 (51), 77 (33), 51 (30).

7.6.2.21. Synthesis of (E)-3-Phenylpropenoyl Azide (95)

A solution of 1.2 g (E)-3-phenylpropenoyl chloride (94, 6.1 mmol) in 10 ml acetone at 0 °C was

treated with a solution of 0.4 g NaN3 (6.1 mmol) in a mixture of 6 ml H2O and 6 ml acetone. The

precipitating product was filtered and dried in vacuum at 0 °C to afford 1.1 g (E)-3-(4-

methoxyphenyl)propenoyl azide (95, 5.4 mmol, 80 % yield).

(E)-3-phenylpropenoyl azide (95): C9H7N3O; white solid; 1H NMR (400.1 MHz, C6D6): = 6.22

(1H, d, JE = 15.8 Hz, 2-H), 6.91 – 7.01 (5H, m, 2„,3„,4„,5„,6„-H), 7.63 (1H, d, JE = 16.1 Hz, 3-H); 13

C

PENDANT (100.6 MHz, C6D6): = 119.5 (d, 2-CH), 128.7 (d, 2‟,6‟-CH), 129.0 (d, 3‟,5‟-CH), 130.9

(d, 4‟-CH), 134.2 (s, 1‟-C), 146.5 (d, 3-CH), 171.4 (s, 1-CON3); EIMS (70 eV, direct inlet) m/z (%) =

173 (43) [M], 145 (16), 131 (100) [M – N3], 117 (22), 103 (64), 90 (70), 77 (38), 63 (20), 51 (45).

7.6.2.22. Synthesis of (E)-2-Phenylethenyl Isocyanate (96)

Under N2 atmosphere a solution of 690 mg (E)-3-phenylpropenoyl azide (95, 4 mmol) in 10 ml dry

toluene was heated under reflux for 5 h. The solvent was removed in vacuum to afford 580 mg (E)-2-

phenylethenyl isocyanate (96, 4 mmol, 99 % yield).

(E)-2-phenylethenyl isocyanate (96): C9H7NO; white solid; 1H NMR (500.1 MHz, C6D6): = 5.67

(1H, d, JE = 13.9 Hz, 2-H), 6.00 (1H, d, JE = 13.9 Hz, 1-H), 6.90 (2H, d, J = 7.3 Hz, 2„,6„-H), 6.99 –

7.07 (3H, m, 3„,4„,5„-H); 13

C PENDANT (100.6 MHz, C6D6): = 117.9 (d, 1-CH), 125.1 (s, NCO),

126.3 (d, 2‟,6‟-CH), 126.7 (d, 2-CH), 128.0 (d, 4‟-CH), 128.8 (d, 3‟, 5‟-CH), 135.0 (s, 1‟-C); EIMS

(70 eV) m/z (%) = 145 (100) [M], 117 (63), 90 (96), 89 (55), 63 (19), 51 (7), 39 (6).


182

7.6.2.23. Synthesis of Dehydroniranin A (97)

Under N2 atmosphere a solution of 580 mg (E)-2-phenylethenyl isocyanate (96, 4 mmol) in 5 ml

toluene was added drop wise to a solution of 560 mg sodium methanethiolate (8 mmol) in 10 ml dry

DMSO. After 30 min, 1010 mg dimethyl sulfate (8 mmol) was added drop wise. After 1 h 20 ml Et2O

and 20 ml H2O were added, the organic phase washed with 3 x 20 ml H2O, dried over Na2SO4 and

concentrated in vacuum. Column chromatography on silica gel using hexane - Et2O (20:1, v/v)

afforded 335 mg (E)-2-phenylethenyl-N,S-dimethyl thiocarbamate (1.6 mmol, 40 % yield) identical to

dehydroniranin A (97) from Glycosmis cyanocarpa (Greger et al., 1996).

dehydroniranin A [(E)-2-phenylethenyl-N,S-dimethyl thiocarbamate] (97): C11H13NOS,

colourless oil, 1H NMR (500.1 MHz, C6D6): = 2.15 (3H, s, SCH3), 2.73 (3H, s.br, NCH3), 5.62 (1H,

d, JE = 14.5 Hz, 2-H), 7.02 (1H, m, J = 7.2 Hz, 4‟-H), 7.12 (2H, m, J = 7.6 Hz, 2‟,6‟-H), 7.18 (2H, m,

3‟,5‟-H) 8.08 (1H, s.br, 1-H); 13

C PENDANT (100.6 MHz, C6D6): = 13.1 (q, SCH3), 31.5 (q,

NCH3), 111.1 (d.br, 1-CH), 125.9 (d, 2‟,6‟-CH), 126.6 (d, 4‟-CH), 127.9 (d, 2-CH), 129.0 (d, 3‟,5‟-

CH), 137.1 (s, 1‟-C), 168.0 (s, CO); EIMS (70 eV) m/z (%) = 207 (100) [M], 192 (1), 160 (7), 150

(55), 132 (23), 117 (24), 103 (6), 91 (22), 75 (21), 65 (7), 42 (14); HREIMS (70 eV): obs. m/z

207.0727 [M], calc. for C11H13NOS: 207.0718, ∆ 0.9 mmu.

7.6.2.24. Synthesis of (Z)- and (E)-Tuberines (98)

Under N2 atmosphere at 0 °C a solution of 175 mg (E)-corsinian ((E)-63, 1 mmol) in 1 ml dry THF

was added drop wise to 280 mg lithium tris-(tert-butyloxy)aluminium hydride (1.1 mmol) in 5 ml dry

THF. After 6 h the mixture was treated with 20 ml Et2O and 10 ml H2O, the organic phase washed

with 2 x 20 ml H2O, dried over Na2SO4 and concentrated. Column chromatography on silica gel using

hexane – EtOAc (3:1, v/v) afforded 152 mg (E)-tuberine ((E)-98, 860 µmol, 86 % yield). UV

irradiation at 350 nm for 6 h to afford a 2:3 mixture of (Z)- and (E)-tuberines ((Z)-98 and (E)-98)

identical to the natural products from Corsinia coriandrina. For syn-anti isomerism of N-formamide

group in (E)-98 see: Aguirre et al., 2003.

(E)-tuberine [(E)-2-(4-methoxyphenyl)ethenyl N-formamide] ((E)-98): C10H11NO2, white solid; 1H NMR (500.1 MHz, C6D6): syn-amide (ca. 75 %) = 3.28 (3H, s, OCH3), 5.65 (1H, d, JE = 14.5 Hz,

2-H), 5.69 (1H, s.br., NH), 6.69 (2H, d, J = 8.8 Hz, 3‟,5‟-H), 7.02 (2H, d, J = 8.8 Hz, 2‟,6‟-H), 7.50

(1H, dd, JE = 14.5 Hz, J = 11.0 Hz, 1-H), 7.54 (1H, s.br, CHO); anti-amide (ca. 25 %): = 3.33 (3H, s,

OCH3), 5.48 (1H, d, JE = 14.2 Hz, 2-H), 6.11 (1H, dd, JE = 14.2 Hz, J = 11.0 Hz, 1-H), 6.77 (2H, d, J

= 8.5 Hz, 3‟,5‟-H), 6.93 (2H, d, J = 8.5 Hz, 2‟,6‟-H), 7.36 (1H, s.br, NH), 7.78 (1H, d, J = 11.0 Hz,

CHO); 13

C PENDANT (100.6 MHz, C6D6): = 55.0 (q, OCH3), 113.8 (d, 2-CH), 114.8 (d, 3‟,5‟-CH),

119.7 (d, 1-CH), 128.6 (d, 2‟,6‟-CH), 129.1 (s, 1‟-C), 158.6 (d, CHO), 159.5 (s, 4‟-C); EIMS (70 eV)

m/z (%) = 177 (100) [M], 162 (3), 148 (10), 134 (76), 132 (28), 121 (21), 117 (18), 104 (12), 89 (11),

77 (29), 63 (14), 51 (24), 39 (14); HREIMS (70 eV): obs. m/z 177.0804 [M], calc. for C10H11NO2:

177.0790, ∆ 1.4 mmu.

(Z)-tuberine [(Z)-2-(4-methoxyphenyl)ethenyl N-formamide] ((Z)-98): C10H11NO2, glassy solid; 1H

NMR (500.1 MHz, C6D6): = 3.31 (3H, s, OCH3), 5.43 (1H, d, JZ = 9.5 Hz, 2-H), 6.74 (2H, d, J = 8.8

Hz, 3‟,5‟-H), 6.89 (2H, d, J = 8.5 Hz, 2‟,6‟-H), 7.05 (1H, d, JZ = 9.5 Hz, 1-H), 7.44 (1H, s, CHO); 13

C

PENDANT (100.6 MHz, C6D6): = 54.6 (q, OCH3), 110.2 (d, 2-C), 114.4 (d, 3‟,5‟-CH), 119.3 (d, 1-

CH), 128.2 (d, 2‟,6‟-CH) 129.4 (s, 1‟-C), 157.6 (d, CHO), 159.9 (s, 4‟-C); EIMS (70 eV) m/z (%) =

177 (100) [M], 162 (3), 148 (9), 134 (78), 132 (27), 121 (21), 117 (11), 104 (14), 89 (13), 77 (26), 63

(16), 51 (24), 39 (19); HREIMS (70 eV): obs. m/z 177.0806 [M], calc. for C10H11NO2: 177.0790, ∆

1.6 mmu; obs. m/z 148.0776, calc. for C9H10NO: 148.0762, ∆ 1.4 mmu; obs. m/z 121.0659, calc. for

C8H9O: 121.0648, ∆ 1.1 mmu;


183

7.6.2.25. Synthesis of (Z)- and (E)-Corsicillins (99)

Under N2 atmosphere at –70 °C a solution of 177 mg O,O-diethyl isocyanomethylene phosphonate

(102, 1 mmol) in 5 ml THF was treated with 550 µl 2 M sodium hexamethyldisilazane (1.1 mmol) in

THF. After 1 h, a solution of 136 mg 4-methoxybenzaldehyde (58, 1 mmol) in 1 ml THF was added

drop wise. After 12 h at RT, 10 ml Et2O and 5 ml H2O was added, the organic phase washed with 2 x

10 ml H2O, dried over Na2SO4 and concentrated in vacuum. Column chromatography on silica gel

using hexane - Et2O (20:1, v/v) afforded 137 mg of a 1:1.7 mixture of (Z)- and (E)-2-(4-

methoxyphenyl)ethenyl isocyanides ((Z/E)-99, 860 µmol, 86 % yield) identical to the (Z/E)-

corsicillins from Corsinia coriandrina

(Z)-corsicillin [(Z)-2-(4-methoxyphenyl)ethenyl isocyanide] ((Z)-99): C10H9NO, glassy solid; 1

H

NMR (500.1 MHz, C6D6): = 3.21 (3H, s, OCH3), 5.02 (1H, d, J = 9.1 Hz, 2-H), 5.61 (1H, s.br., 1-

H), 6.61 (2H, d, J = 8.8 Hz, 3‟,5‟-H), 7.50 (2H, d, J = 8.8 Hz, 2‟,6‟-H); 13


C6D6): = 54.8 (q, OCH3), 106.6 (d as triplet, JCN = 11.2 Hz, 1-CH), 114.4 (d, 3‟,5‟-CH), 125.8 (s, 1‟-

C), 131.1 (d, 2-CH), 131.2 (d, 2‟,6‟-CH), 160.9 (s, 4‟-C), 172.1 (s.br, NC); EIMS (70 eV) m/z (%) =

159 (100) [M], 144 (32), 129 (7), 116 (50), 102 (4), 89 (35), 75 (4), 63 (15), 51 (8), 39 (14); HREIMS

(70 eV): obs. m/z 159.0692 [M], calc. for C10H9NO: 159.0684, ∆ 0.8 mmu; obs. m/z 144.0460, calc.

for C9H6NO: 144.0449, ∆ 1.1 mmu; obs. m/z 116.0509, calc. for C8H6N: 116.0495, ∆ 1.4 mmu; obs.

m/z 89.0398, calc. for C7H5: 89.0386, ∆ 1.2 mmu.

(E)-corsicillin [(E)-2-(4-methoxyphenyl)ethenyl isocyanide] ((E)-99): C10H9NO, glassy solid; 1

H

NMR (500.1 MHz, C6D6): = 3.23 (3H, s, OCH3), 5.45 (1H, d, J = 14.2 Hz, 2-H), 6.32 (1H, d, J =

14.2 Hz, 1-H), 6.57 (2H, d, J = 8.5 Hz, 3‟,5‟-H), 6.70 (2H, d, J = 8.8 Hz, 2‟,6‟-H); 13

C PENDANT

(100.6 MHz, C6D6): = 54.8 (q, OCH3), 109.0 (d as triplet, JCN = 12.8 Hz, 1-CH), 114.5 (d, 3‟,5‟-CH),

125.6 (s, 1‟-C), 128.3 (d, 2‟,6‟-CH), 135.7 (d, 2-CH), 161.2 (s, 4‟-C), 167.8 (s.br, NC); EIMS: see

(Z)-isomer ((Z)-99); HREIMS (70 eV): obs. m/z 159.0697 [M], calc. for C10H9NO: 159.0684, ∆ 1.3

mmu.

7.6.2.26. Synthesis of (E)-4-Methoxycinnamonitrile (100)

A solution of 6.3 ml Aliquat® 336 (N-methyl-N,N,N-triocytlammonium chloride) in 250 ml acetonitrile

(101, 4.8 mol) was treated with 20.8 g freshly powdered KOH (37.1 mmol) and heated under reflux

for 30 min. A solution of 42.8 g 4-methoxybenzaldehyde (58, 315 mmol) in 50 ml acetonitrile (101, 1

mol) was added drop wise and the mixture was heated under reflux for 15 min, cooled to RT and

poured onto 500 g ice. The mixture was extracted with 2 x 100 ml DCM, and the organic phase was

washed with 2 x 100 ml H2O, dried over Na2SO4 and concentrated in vacuum. The residue was

dissolved in boiling EtOH, filtered, and stored at –20°C. The precipitating solids were repeatedly

recrystallized from EtOH, washed with cold EtOH and dried in vacuum to afford 10.9 g (E)-4-

methoxycinnamonitrile (100, 68.6 mmol, 22 % yield).

(E)-4-methoxycinnamonitrile [(E)-3-(4-methoxyphenyl)propenonitrile] (100): C10H9NO; off-white

solid; 1H NMR (400.1 MHz, CDCl3): = 3.84 (3H, s, OCH3), 5.71 (1H, d,

3JE = 16.8 Hz, 2-H), 6.91

(2H, d, 3J = 8.9 Hz, 3‟,5‟-H), 7.33 (1H, d,

3JE = 16.5 Hz, 3-H), 7.39 (2H, d,

3J = 8.6 Hz, 2‟,6‟-H);

13C

PENDANT (100.6 MHz, CDCl3): = 55.5 (q, OCH3), 93.3 (d, 2-CH), 114.5 (d, 3‟,5‟-CH), 118.7 (s,

1-CN), 126.3 (s, 1‟-C), 129.1 (d, 2‟,6‟-CH), 150.0 (d, 3-CH), 162.1 (s, 4‟-C); EIMS (70 eV) m/z (%) =

159 (100) [M], 144 (46), 129 (10), 116 (59), 102 (6), 89 (49), 75 (6), 63 (23), 51 (11), 39 (19).

7.6.2.27. Synthesis of (±)-Corsifuran B (114)

A solution of 270 mg 1,4-benzoquinone (113, 2.5 mmol) in 10 ml acetonitrile was treated with 13.5

mg FeCl3 hexahydrate (50 µmol) and a solution of 335 mg 4-methoxyphenylethene (112, 2.5 mmol) in


184

1 ml acetonitrile was added drop wise. After stirring at room temperature for 6 h the reaction mixture

was poured into 15 ml H2O and extracted with 2 x 15 ml DCM. The organic phase was dried over

Na2SO4 and concentrated in vacuum. Column chromatography on silica gel with DCM (UV detection,

Rf = 0.22) afforded 230 mg racemic (±)-5-hydroxy-2-(4-methoxyphenyl)-2,3-dihydrobenzofuran (0.95

mmol, 38 % yield) identical to corsifuran B (114) detected in Corsinia coriandrina.

(±)-corsifuran B [(±)-5-hydroxy-2-(4-methoxyphenyl)-2,3-dihydrobenzofuran] (114): C15H14O3;

white solid; 1H NMR (500.1 MHz, C6D6): = 2.84 (1H, dd,

2J = 15.8 Hz,

3J = 8.5 Hz, 3-H), 3.02 (1H,

dd, 2J = 15.8 Hz,

3J = 9.1 Hz, 3-H‟), 3.27 (3H, s, OCH3), 5.44 (1H, t,

3J = 8.8 Hz, 2-H), 6.33 (1H, dd,

3J = 8.6 Hz,

4J = 2.5 Hz, 6-H), 6.45 (1H, d,

4J = 2.5 Hz, 4-H), 6.73 (1H, d,

3J = 8.8 Hz, 7-H), 6.74 (2H,

d, 3J = 8.8 Hz, 3‟,5‟-H), 7.17 (2H, d,

3J = 8.8 Hz, 2‟,6‟-H);

13C PENDANT (100.6 MHz, C6D6): =

38.9 (t, 3-CH2), 54.8 (q, OCH3), 84.3 (d, 2-CH), 109.5 (d, 7-CH), 112.5 (d, 4-CH), 114.2 (d, 3‟,5‟-

CH), 114.6 (d, 6-CH), 127.5 (d, 2‟,6‟-CH), 128.3 (s, 3a-C), 134.7 (s, 1‟-C), 150.6 (s, 5-C), 154.3 (s,

7a-C), 159.9 (s, 4‟-C); EIMS (70 eV) m/z (%) = 242 (100) [M], 227 (8), 211 (4), 199 (7), 181 (16),

171 (4), 165 (4), 152 (10), 141 (5), 134 (13), 128 (8), 121 (7), 115 (17), 91 (9), 77 (14), 65 (11), 55

(22), 51 (17), 39 (15). HREIMS (70 eV): obs. m/z: 242.0961 [M], calc. for C15H14O3: 242.0943, ∆ 1.8

mmu; UV: max (MeOH) = 302 nm, = 5.200 M-1

cm-1

.

7.6.2.28. Synthesis of (±)-Corsifuran A (73)

A solution of 140 mg (±)-5-hydroxy-2-(4-methoxyphenyl)-2,3-dihydrobenzofuran (114; 580 µmol) in

3 ml acetone was treated with 500 mg anhydrous K2CO3 (3.6 mmol) and 170 mg methyl iodide (1.2

mmol). After heating at reflux for 12 h the mixture was filtered, dried, concentrated and

chromatographed on silica gel with of hexane / EtOAc mixture (5:1, v/v; UV detection, Rf = 0.33) to

give 140 mg racemic (±)-5-methoxy-2-(4-methoxyphenyl)-2,3-dihydrobenzofuran (550 µmol, 95 %

yield) identical to corsifuran A (73) isolated from Corsinia coriandrina.

(±)-corsifuran A [(±)-5-methoxy-2-(4-methoxyphenyl)-2,3-dihydrobenzofuran] (73): C16H16O3;

white solid; RI 2193; 1H NMR (500.1 MHz, C6D6): = 2.90 (dd, 1H,

2J = 15.4 Hz,

3J = 8.5 Hz, 3-H),

3.09 (dd, 1H, 2J = 15.8 Hz,

3J = 9.1 Hz, 3-H‟), 3.28 (s, 3H, 4‟-OCH3), 3.38 (s, 3H, 5-OCH3), 5.48 (t,

1H, 3J = 8.6 Hz, 2-H), 6.62 (dd, 1H,

3J = 8.5 Hz,

4J = 2.8 Hz, 6-H), 6.70 (d, 1H,

4J = 2.8 Hz, 4-H), 6.75

(d, 2H, 3J = 8.8 Hz, 3‟,5‟-H), 6.83 (d, 1H,

3J = 8.5 Hz, 7-H), 7.20 (d, 2H,

3J = 8.8 Hz, 2‟,6‟-H);

13C

PENDANT (100.6 MHz, C6D6): = 39.1 (t, 3-CH2), 54.8 (q, 4‟-OCH3), 55.6 (q, 5-OCH3), 84.3 (d, 2-

CH), 109.5 (d, 7-CH), 111.7 (d, 4-CH), 113.2 (d, 6-CH), 114.2 (d, 3‟,5‟-CH), 127.5 (d, 2‟,6‟-CH),

128.3 (s, 3a-C), 134.8 (s, 1‟-C), 154.6 (s, 7a-C), 154.9 (s, 5-C), 159.9 (s, 4‟-C); EIMS (70 eV) m/z (%)

= 256 (100) [M], 241 (6), 225 (2), 213 (3), 198 (4), 181 (4), 171 (2), 165 (2), 148 (6), 141 (3), 128 (4),

115 (6), 91 (3), 77 (5), 55 (8), 51 (6); HREIMS (70 eV): obs. m/z: 256.1091 [M], calc. for C16H16O3:

256.1099, ∆ 0.8 mmu; UV: max (MeOH) = 300 nm, = 4.900 M-1

cm-1

7.6.2.29. Synthesis of (±)-[5-OCD3]-Corsifuran A ([5-OCD3]-73)

A solution of 121 mg (±)-5-hydroxy-2-(4-methoxyphenyl)-2,3-dihydrobenzofuran (114; 0.5 mmol) in

3 ml acetone was treated with 415 mg anhydrous K2CO3 (3.0 mmol) and 145 mg [D3]-methyl iodide

(1.0 mmol). After heating at reflux for 12 h the mixture was filtered, dried, concentrated and

chromatographed on silica gel with of hexane / EtOAc mixture (5:1, v/v; UV detection, Rf = 0.33) to

give 118 mg (±)-[5-OCD3]-5-methoxy-2-(4-methoxyphenyl)-2,3-dihydrobenzofuran ([5-OCD3]-73,

455 µmol, 91 % yield).

(±)-[5-OCD3]-corsifuran A [(±)-5-trideuteromethoxy-2-(4-methoxyphenyl)-2,3-dihydrobenzo-

furan] ([5-OCD3]-73): C16H13D3O3; white solid; 1H NMR (500.1 MHz, C6D6): = 2.90 (dd, 1H,

2J =

15.4 Hz, 3J = 8.5 Hz, 3-H), 3.09 (dd, 1H,

2J = 15.8 Hz,

3J = 9.1 Hz, 3-H), 3.28 (s, 3H, 4‟-OCH3), 3.38


185

(m, < 0.01H, residual 5-OCH3), 5.48 (t, 1H, 3J = 8.6 Hz, 2-H), 6.62 (dd, 1H,

3J = 8.5 Hz,

4J = 2.8 Hz,

6-H), 6.70 (d, 1H, 4J = 2.8 Hz, 4-H), 6.75 (d, 2H,

3J = 8.8 Hz, 3‟,5‟-H), 6.83 (d, 1H,

3J = 8.5 Hz, 7-H),

7.20 (d, 2H, 3J = 8.8 Hz, 2‟,6‟-H);

13C PENDANT (100.6 MHz, C6D6): = 39.1 (t, 3-CH2), 54.8 (q,

4‟-OCH3), 84.3 (d, 2-CH), 109.5 (d, 7-CH), 111.7 (d, 4-CH), 113.2 (d, 6-CH), 114.2 (d, 3‟,5‟-CH),

127.5 (d, 2‟,6‟-CH), 128.3 (s, 3a-C), 134.8 (s, 1‟-C), 154.6 (s, 7a-C), 154.9 (s, 5-C), 159.9 (s, 4‟-C),

[signal for 5-OCD3 missing]; EIMS (70 eV) m/z (%) = 259 (100) [M], 241 (6) [M–CH3], 225 (2) [M–

OCH3], 213 (3) [M–CH3–CO], 198 (4), 181 (4), 171 (2), 165 (2), 148 (6), 141 (3), 128 (4), 115 (6), 91

(3), 77 (5), 55 (8), 51 (6).

7.6.2.30. Synthesis of Corsifuran C (74)

A solution of 51 mg (±)-5-methoxy-2-(4-methoxyphenyl)-2,3-dihydrobenzofuran (73) (200 µmol) in 1

ml 1,4-dioxane was treated with 48 mg 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) (210

µmol) and heated to 100 °C for 14 h. Crystals formed after cooling were washed with hexane and the

combined solution concentrated in vacuum. The residue was chromatographed on silica gel using

DCM (UV detection, Rf = 0.64) to give 48 mg 5-methoxy-2-(4-methoxyphenyl)benzofuran (190

µmol, 95 % yield) identical to corsifuran C (74) from Corsinia coriandrina.

corsifuran C [5-methoxy-2-(4-methoxyphenyl)benzofuran] (74): C16H14O3; off-white solid; RI

2239; 1H NMR (500.1 MHz, C6D6): = 3.27 (3H, s, 4‟-OCH3), 3.44 (3H, s, 5-OCH3), 6.61 (1H, d, J =

0.8 Hz, 3-H), 6.79 (2H, d, J = 8.9 Hz, 3‟,5‟-H), 6.84 (1H, dd, J = 8.9 Hz, J = 2.5 Hz, 6-H), 6.96 (1H, d,

J = 2.5 Hz, 4-H), 7.30 (1H, d, J = 8.9 Hz, 7-H), 7.75 (2H, d, J = 8.9 Hz, 2‟,6‟-H). 13

C PENDANT

(100.6 MHz, C6D6) = 55.1 (q, 5-OCH3), 55.7 (q, 4‟-OCH3), 100.7 (d, 3-CH), 104.0 (d, 4-CH), 112.0

(d, 7-CH), 113.1 (d, 6-CH), 114.9 (d, 3‟,5‟-CH), 124.2 (s, 1‟-C), 127.0 (d, 2‟,6‟-CH), 130.9 (s, 3a-C),

150.6 (s, 7a-C), 157.1 (s, 5-C), 157.6 (s, 2-C), 160.8 (s, 4‟-C); EIMS (70 eV) m/z (%) = 254 (100)

[M], 239 (44), 223 (2), 211 (18), 196 (5), 183 (7), 168 (20), 152 (10), 139 (16), 127 (7), 114 (6), 63

(7); HR-EIMS (70 eV): obs. m/z 254.0965 [M+], calc. for C15H14O3: 254.0943, ∆ = 2.2 mmu; UV:

max (MeOH) = 316 nm, = 52.900 M-1

cm-1

7.6.2.31. Synthesis of 4-Methoxybenzyl chloride (126)

A solution of 1.2 g SOCl2 (10 mmol) in 5 ml CHCl3 was added drop wise to a solution of 1.0 g

4-methoxybenzylalcohol (125) (7.2 mmol) in 10 ml CHCl3 at 0 °C. After 12 h gas evolution ceased

and solvent and excess reagent were removed in vacuum to afford 1.05 g 4-methoxybenzyl chloride

(126) (6.7 mmol, 93 % yield).

4-methoxybenzyl chloride (126): C8H9OCl; white solid; 1

H NMR (400.1 MHz, CDCl3): = 3.81

(3H, s, OCH3), 4.57 (2H, s, 7-CH2), 6.88 (2H, d, 3J = 8.6 Hz, 3,5-H), 7.32 (2H, d,

3J = 8.6 Hz, 2,6-H);

13C PENDANT (100.6 MHz, CDCl3): = 46.72 (t, 7-CH2), 55.74 (q, OCH3), 114.56 (d, 3,5-CH),

130.13 (s, 1-C), 130.48 (d, 2,6-CH), 160.11 (s, 4-C); EIMS (70 eV) m/z (%) = 158 (4) [M + 2], 156

(12) [M], 141 (1), 121 (100) [M – Cl], 106 (3), 91 (8), 78 (24), 63 (8), 51 (19), 39 (8).

7.6.2.32. Synthesis of Diethyl-4-methoxybenzyl phosphonate (127)

A mixture of 1.0 g 4-methoxybenzyl chloride (126; 6.4 mmol) and 1.7 g triethylphosphite (10 mmol)

was heated under reflux at 160 °C for 5 h. Excess reagent was removed in vacuum and the residue

chromatographed on silica gel using CHCl3 to afford 1.03 g diethyl-4-methoxybenzyl phosphonate

(127, 4.0 mmol, 62 % yield).

Diethyl-4-methoxybenzyl phosphonate (127): C12H19O4P; colourless liquid; 1H NMR (400.1 MHz,

CDCl3): = 1.24 (6H, t, RCH3), (2H, d, 2JH,P = 21.1 Hz, 7-CH2), 3.78 (3H, s, OCH3), 4.00 (4H, m,


186

POCH2R), 6.84 (2H, d, J = 8.7 Hz, 3,5-H), 7.20 (2H, d, J = 8.7 Hz, 2,6-H); 13

C PENDANT (100.6

MHz, CDCl3): = 16.7 (2q, 3JC,P = 6.1 Hz, RCH3), 32.6 (2t,

1JC,P = 38.3 Hz, 7-CH2), 55.5 (q, OCH3),

62.4 (2t, 2JC,P = 7.1 Hz, -POCH2R), 114.3 (2d,

4JC,P = 3.1 Hz, 3,5-CH), 123.7 (2s,

2JC,P = 9.2 Hz, 1-C),

131.1 (2d, 3JC,P = 6.1 Hz, 2,6-CH), 158.9 (2s,

5JC,P = 3.6 Hz, 4-C); EIMS (70 eV, direct inlet) m/z (%)

= 258 (29) [M], 230 (3), 215 (1), 149 (1), 135 (2), 121 (100) [M – PO3Et2], 109 (2), 91 (5), 78 (11), 65

(2), 51 (3); HREIMS (70 eV): obs. m/z 258.1024, calc. for C12H19O4P: 258.1021, ∆ 0.3 mmu.

7.6.2.33. Synthesis of (E)-Corsistilbene ((E)-68)

Under N2 atmosphere a suspension of 980 mg diethyl-4-methoxybenzyl phosphonate (127, 3.8 mmol)

and 96 mg sodium hydride (4.0 mmol) in 20 ml dry THF was treated with 520 mg

3-methoxybenzaldehyd (128, 3.8 mmol) and heated under reflux for 12 h. The mixture was treated

with 50 ml 1 M HCl and extracted with 2 x 50 ml Et2O. The organic phase was washed with 20 ml

1 M NaHCO3 solution, 20 ml H2O, dried over Na2SO4 and concentrated in vacuum. Flash column

chromatography on silica gel using a hexane - EtOAc mixture (5:1, v/v; Rf = 0.40) afforded 575 mg

(E)-3,4‟-dimethoxystilbene (2.4 mmol, 63 % yield) identical to (E)-corsistilbene ((E)-68) isolated from

Corsinia coriandrina.

(E)-corsistilbene [(E)-3,4’-dimethoxystilbene] ((E)-68): C16H16O2; white solid; RI 2215; 1H NMR

(500.1 MHz, C6D6): = 3.32 (3H, s, 4‟-OCH3), 3.38 (3H, s, 3-OCH3), 6.74 (1H, dd, 3J = 8.2 Hz,

4J =

1.9 Hz, 4-H), 6.79 (2H, d, J = 8.8 Hz, 3‟,5‟-H), 6.96 (1H, d, JE = 16.4 Hz, 7-H), 7.07 (1H, d, J = 9.1

Hz, 6-H), 7.08 (1H, d, JE = 16.1 Hz, 8-H), 7.11 (1H, d, 4J = 2.2 Hz, 2-H), 7.15 (1H, dd, J = J = 7.9 Hz,

5-H), 7.29 (2H, d, J = 8.5 Hz, 2‟,6‟-H); 1H NMR (500.1 MHz, CDCl3): = 3.83, 3.84 (2 x 3H, s,

OCH3), 6.79 (1H, dd, J = 8.2 Hz, J = 2.5 Hz, 4-H), 6.90 (2H, d, J = 8.5 Hz, 3‟,5‟-H), 6.94 (1H, d, JE =

16.4 Hz, -H), 7.03 (1H, s.br, 2-H), 7.05 (1H, d, JE = 16.4 Hz, -H), 7.09 (1H, d, J = 7.9 Hz, 6-H),

7.26 (1H, t, J = J = 7.9 Hz, 5-H), 7.45 (2H, d, J = 8.8 Hz, 2‟,6‟-H); 13


C6D6): = 55.3, 55.3 (2q, 3,4‟-OCH3), 113.1 (d, 2-CH), 114.3 (d, 4-CH), 115.4 (d, 3‟,5‟-CH), 120.4

(d, 6-CH), 128.1 (d, -CH), 129.3 (d, 2‟,6‟-CH), 130.1 (d, -CH), 131.0 (d, 5-CH), 131.6 (s, 1‟-C),

140.8 (s, 1-C), 161.2 (s, 4‟-C), 161.9 (s, 3-C); EIMS (70 eV) m/z (%) = 240 (100) [M], 225 (11), 209

(18), 197 (15), 182 (14), 165 (33), 153 (19), 139 (6), 128 (5), 120 (4), 112 (6), 102 (4), 89 (5), 76 (6),

63 (6), 51 (8), 39 (9); HREIMS (70 eV): obs. m/z 240.1127, calc. for C16H16O2: 240.1150, ∆ 2.3 mmu;

UV: max = 320 nm, = 27.400 M-1

cm-1

7.6.2.34. Isomerisation to (Z)-Corsistilbene ((Z)-68)

A solution of 12 mg (E)-corsistilbene ((E)-68, 500 µmol) in 5 ml benzene was irradiated at 350 nm for

1 h to afford a 9:1 mixture of (Z)- and (E)-corsistilbene, respectively. The solution was concentrated in

vacuum and isomers were separated by column chromatography on silica gel using a hexane – EtOAc

mixture (5:1, v/v; UV detection, Rf = 0.48 and 0.40) to afford 10 mg (Z)-3,4‟-dimethoxystilbene ((Z)-

68, 417 µmol, 83 % yield) identical to the natural product from Corsinia coriandrina.

(Z)-corsistilbene [(Z)-3,4’-dimethoxystilbene] ((Z)-68): C16H16O2; white solid; RI 1950; 1H NMR

(500.1 MHz, C6D6): = 3.22 (3H, s, 4‟-OCH3), 3.24 (3H, s, 3-OCH3), 6.46 (1H, d, JZ = 12.3 Hz, -H),

6.49 (1H, d, JZ = 12.3 Hz, -H), 6.61 (2H, d, J = 8.8 Hz, 3‟,5‟-H), 6.67 (1H, ddd, J = 8.2 Hz, J = 2.5

Hz, J = 0.9 Hz, 4-H), 6.97 (1H, d, J = 7.6 Hz, 6-H), 7.00 (1H, s.br, 2-H), 7.03 (1H, t, J = 7.9 Hz, 5-H),

7.23 (2H, d, J = 8.8 Hz, 2‟,6‟-H); 13

C PENDANT (100.6 MHz, C6D6): = 54.6. 54.7 (2q, 3,4‟-OCH3),

113.5 (d, 4-CH), 114.0 (d, 3‟,5‟-CH), 114.3 (d, 2-CH), 121.8 (d, 6-CH), 129.1 (d, -CH), 129.7 (d, 5-

CH), 130.5 (d, -CH), 130.7 (d, 2‟,6‟-CH); EIMS (70 eV) m/z (%) = 240 (100) [M], 225 (10), 209


187

(16), 197 (13), 182 (15), 165 (30), 153 (17), 139 (4), 128 (3), 120 (5), 112 (6), 102 (3), 89 (5), 76 (5),

63 (6), 51 (4), 39 (4); HREIMS (70 eV): obs. m/z 240.1145, calc. for C16H16O2: 240.1150, ∆ 0.5 mmu.

7.6.2.35 Synthesis of O,O-Dimethyllunularin (69)

A solution of 12 mg (E)-3,4‟-dimethoxystilbene ((E)-68) (500 µmol) in 5 ml Et2O was treated with

5 mg palladium on carbon (10 %, w/w) and stirred under H2 atmosphere for 6 h. The solution was

filtered and concentrated in vacuum. Flash column chromatography on silica gel with a hexane /

EtOAc mixture (5:1, v/v; UV detection, Rf = 0.48) afforded 12 mg 3,4‟-dimethoxybibenzyl (69, 496

µmol, 99 % yield) identical to the natural product from Corsinia coriandrina.

O,O-dimethyllunularin [3,4’-dimethoxybibenzyl] (69): C16H18O2; white solid; RI 1988; 1H NMR

(500.1 MHz, CDCl3): = 2.86 (4H, s, -H2, -H2), 3.76 (3H, s, 3-OCH3), 3.77 (3H, s, 4‟-OCH3), 6.71

(1H, s.br, 2-H), 6.73 (1H, dd, J = 7.9 Hz, J = 2.8 Hz, 4-H), 6.77 (1H, d, J = 7.6 Hz, 6-H), 6.82 (2H, d,

J = 8.5 Hz, 3‟,5‟-H), 7.09 (2H, d, J = 8.8 Hz, 2‟,6‟-H), 7.18 (1H, t, J = J = 7.9 Hz, 5-H); 13

C

PENDANT (100.6 MHz, CDCl3): = 36.9 (t, -CH2), 38.3 (t, -CH2), 55.1, 55.2 (2q, 3,4‟-OCH3),

111.2 (d, 4-CH), 113.7 (d, 3‟,5‟-CH), 114.2 (d, 2-CH), 120.9 (d, 6-CH), 129.3 (d, 2‟,6‟-CH), 129.3 (d,

5-CH), 133.8 (s, 1‟-C), 143.5 (s, 1-C), 157.8 (s, 4‟-C), 159.6 (s, 3-C); EIMS (70 eV) m/z (%) = 242

(17) [M], 178 (1), 165 (2), 152 (1), 134 (1), 121 (100), 115 (1), 106 (2), 91 (15), 78 (19), 65 (8), 51

(5), 39 (5); HREIMS (70 eV): obs. m/z 242.1328, calc. for C16H16O2: 242.1307, ∆ 2.1 mmu.

7.6.2.36. Synthesis of 2-Hydroxy-5,4’-dimethoxybibenzyl (70)

A solution of 13 mg (±)-corsifuran A (73) (500 µmol) in 5 ml Et2O was treated with 5 mg palladium

on carbon (10 %, w/w) and stirred under H2 atmosphere for 6 h. The solution was filtered and

concentrated. Flash column chromatography on silica gel with a hexane - EtOAc mixture (5:1, v/v;

UV detection, Rf = 0.21) afforded 12 mg 2-hydroxy-5,4‟-dimethoxybibenzyl (70, 465 µmol, 93 %

yield) identical to the natural product from Corsinia coriandrina.

2-hydroxy-5,4’-dimethoxybibenzyl (70): C16H18O3; white solid; 1H NMR (500.1 MHz, C6D6): =

2.86 (4H, s.br, 7,8-CH2), 3.31 (3H, s, 4‟-OCH3), 3.35 (3H, s, 5-OCH3), 6.32 (1H, dd, 3J = 8.5 Hz,

4J =

2.2 Hz, 3-CH), 6.58 (1H, dd, 3J = 8.5 Hz,

4J = 3.2 Hz, 4-H), 6.70 (1H, d,

4J = 3.2 Hz, 6-H), 6.75 (2H,

d, J = 8.5 Hz, 3‟,5‟-H), 6.99 (2H, d, J = 8.5 Hz, 2‟,6‟-H); 13


33.2 (t, 7-CH2), 35.7 (t, 8-CH2), 54.8 (q, 4‟-OCH3), 55.3 (q, 5-OCH3), 112.4 (d, 4-CH), 114.2 (d, 3‟,5‟-

CH), 116.1 (d, 6-CH), 116.4 (d, 3-CH),129.4 (s, 1-C), 129.8 (d, 2‟,6‟-CH), 134.3 (s, 1‟-C), 148.1 (s, 2-

C), 154.3 (s, 5-C), 158.6 (s, 4‟-C); EIMS (70 eV) m/z (%) = 258 (19) [M], 137 (5), 121 (100), 91 (3),

77 (5), 55 (2); HREIMS (70 eV): obs. m/z 258.1270, calc. for C16H18O3: 258.1256, ∆ 1.4 mmu.

7.6.3. Synthesis of Deuterium Labelled Precursors

7.6.3.1. Synthesis of (E)-Cinnamic Acid (93) by Perkin

A mixture of 7.95 g benzaldehyde (40, 75 mmol, freshly distilled) and 9.3 ml Ac2O (148, 125 mmol)

was treated with 5.5 g potassium acetate (56 mmol) (freshly melted and powdered) and heated under

reflux to 170 °C for 3 h. The reaction mixture was dissolved in 100 ml 2 M KOH solution, extracted

with Et2O, and filtered. The filtrate was acidified with conc. HCl and the precipitating solids were

collected by filtration, recrystallized from EtOH - H2O and dried in vacuum to afford 6.9 g cinnamic

acid (93, 46.5 mmol, 62 % yield).

(E)-cinnamic acid [(E)-3-phenylpropenoic acid] (93): C9H8O2, white solid; for NMR and EIMS data

see 7.6.2.19.


188

7.6.3.2. Synthesis of (E)-[2-D]-Cinnamic Acid ([2-D]-93) by Perkin

A mixture of 800 mg benzaldehyde (40, 7.5 mmol, freshly distilled) and 1 ml [D6]-Ac2O ([D6]-148,

13.8 mmol; > 0.98 D) was treated with 680 mg potassium acetate (6.9 mmol, freshly melted and

powdered) and heated under reflux to 170 °C for 3 h. The reaction mixture was dissolved in 10 ml 2 M

KOH solution, extracted with Et2O, and filtered. The filtrate was acidified with conc. HCl and the

precipitating solids were collected by filtration, recrystallized from EtOH - H2O and dried in vacuum

to afford 580 mg (E)-[2-D]-cinnamic acid ([2-D]-93, 3.9 mmol, 52 % yield) with > 0.74 D as

determined by 1H NMR (calc. 0.67 D for complete exchange).

(E)-[2-D]-cinnamic acid [(E)-2-deutero-3-phenylpropenoic acid] ([2-D]-93): C9H7DO2, 0.74 D,

white solid; 1H NMR (500.1 MHz, CDCl3): = 6.47 (0.26 H, d,

3JE = 16.1 Hz, residual 2-H), 7.41

(3H, m, 3‟,4‟,5‟-H), 7.56 (2H, m, 2‟,6‟-H), 7.81 (1H, s.br, 3-H); 13

C PENDANT (100.6 MHz, CDCl3):

= 128.4 (d, 2‟,6‟-CH), 129.0 (d, 3‟,5‟-CH), 130.8 (d, 4‟-CH), 134.0 (s, 1‟-C), 147.0 (d, 3-CH), 172.3

(s, 1-COOH) [signal for 2-CD missing]; EIMS (70 eV, direct inlet) m/z (%) = 149 (96) [M], 148 (100)

[M – H], 132 (24), 121 (7), 104 (42), 103 (24), 92 (13), 78 (24), 77 (19), 63 (3), 51 (16).

7.6.3.3. Synthesis of (E/Z)-Cinnamonitriles (149)

A solution of 2.1 ml phase-transfer catalyst Aliquat® 336 (methyltriocytlammonium chloride) in 80 ml

acetonitrile (101, 1.53 mol) was treated with 6.9 g freshly powdered KOH (125 mmol) and heated

under reflux for 30 min. A solution of 11.1 g benzaldehyde (40, 105 mmol) in 20 ml acetonitrile (101,

380 mmol) was added drop wise and the mixture was heated under reflux for 15 min, cooled to RT

and poured onto 200 g ice. The mixture was extracted with 2 x 50 ml DCM, and the organic phase was

washed with 2 x 50 ml cold H2O, dried over Na2SO4 and concentrated in vacuum. Flash column

chromatography on silica gel using a hexane - EtOAc mixture (10:1, v/v) afforded 1.9 g

cinnamonitrile (149, 14.7 mmol, 14 % yield) as a 5:1 mixture of the (E) and (Z) isomer.

(E)-cinnamonitrile [(E)-3-phenylpropenonitrile] ((E)-149): C9H7N, colourless oil; 1H NMR (500.1

MHz, C6D6): = 5.15 (1H, d, JE = 16.7 Hz, 2-CH), 6.71 (1H, d, JE = 17.7 Hz, 3-CH), 6.82 (2H, d, 3JAr

= 7.6 Hz, 2‟,6‟-CH), 6.97 (3H, m, 3‟,4‟,5‟-CH); 13

C PENDANT (100.6 MHz, CDCl3): = 96.4 (d, 2-

CH), 118.2 (s, 1-CN), 127.4 (d, 2‟,6‟-CH), 129.1 (d, 3‟,5‟-CH), 131.2 (d, 4‟-CH), 133.5 (s, 1‟-C),

150.6 (d, 3-CH); EIMS (70 eV) m/z (%) = 129 (100) [M], 102 (44) [M – HCN], 89 (2), 76 (12), 63

(8), 51 (19), 39 (7).

(Z)-cinnamonitrile [(Z)-3-phenylpropenonitrile] ((Z)-149): C9H7N, colourless oil; 1H NMR (500.1

MHz, C6D6): = 4.70 (1H, d, 3JZ = 12.0 Hz, 2-CH), 6.31 (1H, d,

3JZ = 12.0 Hz, 3-CH), 7.01 (3H, m,

3‟,4‟,5‟-CH), 7.58 (2H, d, 3JAr = 7.9 Hz, 2‟,6‟-CH);

13C PENDANT (100.6 MHz, CDCl3): = 95.1 (d,

2-CH), 118.2 (s, 1-CN), 128.9 (d, 2‟,6‟-CH), 129.0 (d, 3‟,5‟-CH), 131.0 (d, 4‟-CH), 133.2 (s, 1‟-C),

148.7 (d, 3-CH); EIMS (70 eV) m/z (%) = 129 (100) [M], 102 (45) [M – HCN], 89 (1), 76 (13), 63

(7), 51 (20), 39 (7).

7.6.3.4. Synthesis of (E/Z)-[2-D]-Cinnamonitriles ([2-D]-149)

Under N2 atmosphere a solution of 1.34 g [D3]-acetonitrile ([D3]-101, 0.98 D, 19.0 mmol) in 10 ml dry

THF at 0 °C was treated with 3.5 g sodium hexamethyldisilazane (NaHMDS) (19.1 mmol). After

stirring at 0°C for 30 min a solution of 2 g benzaldehyde (40) (18.9 mmol, freshly distilled) in 5 ml dry

THF was added drop wise and the solution slowly returned to RT, and heated under reflux for 1 h.

After addition of 50 ml H2O the product was extracted with 3 x 20 ml Et2O, dried over Na2SO4, and

concentrated in vacuum. Flash column chromatography on silica gel with a hexane - EtOAc mixture

(10:1, v/v) afforded 290 mg [2-D]-cinnamonitrile ([2-D]-149, 2.2 mmol, 12 % yield) as a 6:1 mixture


189

of the (E)- and (Z)-isomer. A deuterium enrichment of > 0.98 D (calc. 0.98 D) was determined by 1H

NMR.

(E)-[2-D]-cinnamonitrile [(E)-2-deutero-3-phenylpropenonitrile] ((E)-[2-D]-149): C9H6DN, > 0.98

D, colourless oil; 1H NMR (500.1 MHz, C6D6): = 5.15 (0.02H, d,

3JE = 16.7 Hz, residual 2-CH),

6.71 (1H, t, 3JH,D = 2.4 Hz, 3-CH), 6.82 (2H, d,

3JAr = 7.6 Hz, 2‟,6‟-CH), 6.97 (3H, m, 3‟,4‟,5‟-CH);

13C PENDANT (100.6 MHz, C6D6): = 118.2 (s, 1-CN), 127.4 (d, 2‟,6‟-CH), 129.1 (d, 3‟,5‟-CH),

131.2 (d, 4‟-CH), 133.5 (s, 1‟-C), 150.6 (d, 3-CH) [signal for 2-CD missing]; EIMS (70 eV) m/z (%) =

130 (100) [M], 103 (41) [M – HCN], 89 (2), 77 (10), 63 (6), 51 (20), 39 (7).

(Z)-[2-D]-cinnamonitrile [(Z)-2-deutero-3-phenylpropenonitrile] ((Z)-[2-D]-149): C9H6DN; > 0.98

D; colourless oil; 1H NMR (500.1 MHz, C6D6): = 4.70 (0.02H, d,

3JZ = 12.0 Hz, residual 2-CH),

6.31 (1H, s.br., 3-CH), 7.01 (3H, m, 3‟,4‟,5‟-CH), 7.58 (2H, d, 3JAr = 7.9 Hz, 2‟,6‟-CH);

13C

PENDANT (100.6 MHz, C6D6: = 118.2 (s, 1-CN), 128.9 (d, 2‟,6‟-CH), 129.0 (d, 3‟,5‟-CH), 131.0

(d, 4‟-CH), 133.2 (s, 1‟-C), 148.7 (d, 3-CH) [signal for 2-CD missing]; EIMS (70 eV) m/z (%) = 130

(100) [M], 103 (37) [M – HCN], 89 (2), 77 (9), 63 (6), 51 (19), 39 (6).

7.6.3.5. Attempted Synthesis of (E)-[2-D]-Cinnamic Acid ([2-D]-93)

A mixture of 144 mg (E/Z)-[2-D]-cinnamonitrile ([2-D]-149, > 0.98 D) (1.1 mmol) and 2 ml 5 M

aqueous KOH solution (10 mmol) was heated under reflux until NH3 evolution ceased (5 h). The

reaction mixture was extracted with Et2O and filtered. The filtrate was acidified with concentrated HCl

and the precipitating solids were collected by filtration, recrystallized from EtOH - H2O and dried in

vacuum to afford 104 mg unlabelled (E)-cinnamic acid (93) (700 µmol, 63 % yield) as shown by 1H

NMR.

(E)-cinnamic acid [(E)-3-phenylpropenoic acid] (93): C9H8O2; < 0.05 D, white solid; for NMR and

EIMS data see 7.6.2.19.

7.6.3.6. Attempted 1H/

2D Exchange in Potassium (E)-Cinnamate

A mixture of 15 mg cinnamic acid (93) (100 µmol) and 112 mg KOH (2.0 mmol) in 1 ml D2O (55.4

mmol, 0.99 D) was heated under reflux for 5 h to give unchanged potassium cinnamate as shown by 1H NMR spectroscopy of the crude reaction mixture.

7.6.3.7. Synthesis of (E)-[2-D]-Cinnamic Acid ([2-D]-93) by Hydrolysis with KOD

A mixture of 130 mg (E/Z)-cinnamonitrile (149) (1 mmol) and 112 mg KOH (2 mmol) in 2 ml D2O

(111 mmol, 0.99 D) was heated under reflux for 5 h. The reaction mixture was extracted with Et2O and

filtered over cotton wool. The filtrate was acidified with conc. HCl and the precipitating solids were

collected by filtration, recrystallized from EtOH - H2O and dried in vacuum to afford 130 mg [2-D]-

cinnamic acid ([2-D]-93, 870 µmol, 87 % yield). A deuterium enrichment of > 0.97 D (calc. 0.987 D)

was determined by 1H NMR.

(E)-[2-D]-cinnamic acid [(E)-2-deutero-3-phenylpropenoic acid] ([2-D]-93): C9H7DO2, > 0.97 D,

white solid; for NMR and EIMS data see 7.6.3.2.

7.6.3.8. Synthesis of (E)-[2-D]-Cinnamic Acid ([2-D]-93) by Hydrolysis with NaOD

Under N2 atmosphere and external cooling 46 mg sodium (2 mmol) was added to 2 ml frozen D2O

(111 mmol, 0.99 D). After the reaction was completed 130 mg (E/Z)-cinnamonitrile (149) (1.0 mmol)

was added and the mixture heated under reflux for 5 h. The reaction mixture was extracted with Et2O


190

and filtered over cotton wool. The filtrate was acidified with conc. HCl and the precipitating solids

were collected by filtration, recrystallized from EtOH - H2O and dried in vacuum to afford 83 mg [2-

D]-cinnamic acid ([2-D]-93, 560 µmol, 56 % yield) with > 0.82 D by 1H NMR (calc. 0.99 D).

(E)-[2-D]-cinnamic acid [(E)-2-deutero-3-phenylpropenoic acid] ([2-D]-93): C9H7DO2. > 0.82 D,

white solid; for 1H NMR,

13C NMR and EIMS data see: 7.6.3.2.

7.6.3.9. Synthesis of (E)-4-Methoxycinnamic Acid (89) by Hydrolysis

A mixture of 5.0 g (E)-4-methoxycinnamonitrile (100) (31.4 mmol) and 16.0 g (285 mmol) KOH in

50 ml H2O was heated under reflux for 5 h. The reaction mixture was extracted with Et2O and filtered

over cotton wool. The filtrate was acidified with conc. HCl and the precipitating solids were collected

by filtration, recrystallized from H2O and dried in vacuum to afford 5.2 g (E)-4-methoxycinnamic acid

(89, 29.2 mmol, 93 % yield).

(E)-4-methoxycinnamic acid [(E)-3-(4-methoxyphenyl)propenoic acid] (89): C10H10O3; white

solid; for NMR and EIMS data see: 7.6.2.13.

7.6.3.10. Synthesis of (E)-[2-D]-4-Methoxycinnamic acid ([2-D]-89) by Hydrolysis

A mixture of 159 mg (E)-4-methoxycinnamonitrile (100) (1.0 mmol) and 112 mg KOH (2.0 mmol) in

1 ml D2O (55.4 mmol, 0.99 D) was heated under reflux for 5 h. The reaction mixture was extracted

with Et2O and filtered over cotton wool. The filtrate was acidified with conc. HCl and the precipitating

solids were collected by filtration, washed with H2O and dried in vacuum to afford 133 mg (E)-[2-D]-

4-methoxycinnamic acid ([2-D]-89, 743 µmol, 74 % yield), with deuterium enrichment of > 0.84 D

(calc. 0.974 D) as determined by 1H NMR.

(E)-[2-D]-4-methoxycinnamic acid [(E)-2-deutero-3-(4-methoxyphenyl)propenoic acid] ([2-D]-

89): C10H9DO3, > 0.84 D, white solid; 1H NMR (400.1 MHz, CDCl3): = 3.85 (3H, s, OCH3), 6.33

(0.16 H, d, 3JE = 16.0 Hz, residual 2-H), 6.92 (2H, d,

3J = 8.9 Hz, 3‟,5‟-H), 7.51 (2H, d,

3J = 8.9 Hz,

2‟,6‟-H), 7.74 (1H, s.br, 3-H); 13

C PENDANT (100.6 MHz, CDCl3): = 55.4 (q, OCH3), 114.4 (d,

3‟,5‟-CH), 126.8 (s, 1‟-C), 130.1 (d, 2‟,6‟-CH), 146.6 (d, 3-CH), 161.8 (s, 4‟-C), 172.1 (s, COOH)

[signal for 2-CD missing]; EIMS (70 eV, direct inlet) m/z (%) = 179 (100) [M], 162 (31), 148 (1), 134

(12), 121 (3), 119 (5), 107 (4), 89 (10), 77 (13), 63 (6), 51 (4), 39 (5).

7.6.3.11. Synthesis of (E)-4-Hydroxycinnamonitrile (151)

A solution of 1.59 g 4-methoxycinnamonitrile (100) (10 mmol) in 20 ml DCM was treated with 20 ml

1 M BBr3 (20 mmol) in DCM. After stirring at room temperature for 72 h the mixture was diluted with

40 ml H2O and the aqueous phase extracted with 40 ml DCM. The combined organic phases were

washed with 3 x 50 ml H2O, dried over Na2SO4 and concentrated in vacuum. Repeated flash column

chromatography on silica gel 60 using a hexane - EtOAc mixture (1:1, v/v, UV detection, Rf = 0.39)

afforded 280 mg (E)-4-hydroxycinnamonitrile (151) (1.9 mmol, 19 % yield) and 1.13 g unchanged

(E)-4-methoxycinnamonitrile (100) (7.1 mmol).

(E)-4-hydroxycinnamonitrile [(E)-3-(4-hydroxyphenyl)propeno nitrile] (151): C9H7NO; white

solid; 1H NMR (500.1 MHz, CDCl3): = 5.47 (1H, s.br, OH), 5.71 (1H, d,

3JE = 16.4 Hz, 2-H), 6.86

(2H, d, 3J = 8.8 Hz, 3‟,5‟-H), 7.33 (1H, d,

3JE = 16.7 Hz, 3-H), 7.36 (2H, d,

3J = 8.8 Hz, 2‟,6‟-H);

13C

PENDANT (100.6 MHz, CDCl3): = 93.5 (d, 2-CH), 116.2 (d, 3‟,5‟-CH), 118.6 (s, CN), 126.6 (s, 1‟-

C), 129.3 (d, 2‟,6‟-CH), 150.1 (d, 3-CH), 158.5 (s, 4‟-C); EIMS (70 eV) m/z (%) = 145 (100) [M], 117

(43), 90 (28), 63 (14), 51 (6), 39 (10).


191

7.6.3.12. Synthesis of (E)-4-Coumaric Acid (152) by Hydrolysis

A mixture of 73 mg (E)-4-hydroxycinnamonitrile (151) (500 µmol) and 550 mg KOH (10 mmol) in

4.5 ml H2O (225 mmol) was heated under reflux for 5 h. The reaction mixture was extracted with Et2O

and filtered over cotton wool. The filtrate was acidified with conc. HCl and the precipitating solids

were collected by filtration, recrystallized from H2O and dried in vacuum to afford 42 mg (E)-

4-coumaric acid (152, 254 µmol, 51 % yield).

(E)-4-coumaric acid [(E)-3-(4-hydroxyphenyl)propenoic acid] (152): C9H8O3; white solid; 1H

NMR (500.1 MHz, acetone-d6): = 6.34 (1H, d, 3JE = 15.8 Hz, 2-H), 6.90 (2H, d,

3J = 8.5 Hz, 3‟,5‟-

H), 7.56 (2H, d, 3J = 8.5 Hz, 2‟,6‟-H), 7.62 (1H, d,

3JE = 15.8 Hz, 3-H);

13C PENDANT (100.6 MHz,

acetone-d6): = 115.8 (d, 2-CH), 116.6 (d, 3‟,5‟-CH), 127.1 (s, 1‟-C), 130.9 (d, 2‟,6‟-CH), 145.6 (d, 3-

CH), 160.4 (s, 4‟-C), 168.1 (s, 1-COOH); EIMS (70 eV, direct inlet) m/z (%) = 164 (100) [M], 147

(44), 118 (20), 117 (20), 107 (11), 91 (19), 65 (17).

7.6.3.13. Synthesis of (E)-[2-D]-4-Coumaric Acid ([2-D]-152) by Hydrolysis

A mixture of 145 mg (E)-4-hydroxycinnamonitrile (151) (1.0 mmol) and 112 mg KOH (2.0 mmol) in

1 ml D2O (55.4 mmol, 0.99 D) was heated under reflux for 10 h. The reaction mixture was extracted

with Et2O and filtered over cotton wool. The filtrate was acidified with conc. HCl and the precipitating

solids were collected by filtration, recrystallized from EtOH - H2O and dried in vacuum to afford 112

mg (E)-[2-D]-4-coumaric acid ([2-D]-152, 678 µmol, 68 % yield) with deuterium enrichment of >

0.87 D (calc. 0.965 D) as determined by 1H NMR.

(E)-[2-D]-4-coumaric acid [(E)-2-deutero-3-(4-hydroxyphenyl)propenoic acid] (152): C9H7DO3,

˂ 0.87 D, white solid; 1H NMR (500.1 MHz, acetone-d6): = 6.34 (0.13H, d,

3JE = 15.8 Hz, residual

2-H), 6.90 (2H, d, 3J = 8.5 Hz, 3‟,5‟-H), 7.56 (2H, d,

3J = 8.5 Hz, 2‟,6‟-H), 7.62 (1H, s.br, 3-H);

13C

PENDANT (100.6 MHz, acetone-d6): = 116.6 (d, 3‟,5‟-CH), 127.1 (s, 1‟-C), 130.9 (d, 2‟,6‟-CH),

145.6 (d, 3-CH), 160.4 (s, 4‟-C), 168.1 (s, 1-COOH) [signal for 2-CD missing]; EIMS (70 eV, direct

inlet) m/z (%) = 165 (100) [M], 148 (43), 119 (22), 117 (19), 107 (10), 91 (18), 65 (15).

7.6.3.14. Synthesis of [3,5-D2]-4-Hydroxybenzaldehyde ([D2]-150)

In a Reactivial® a mixture of 390 mg 4-hydroxybenzaldehyde (150) (3.2 mmol) and 0.45 ml

triethylamine (3.3 mmol) in 2.5 ml D2O (139 mmol, 0.99 D) was heated at 110 °C for 6 days. The

solution was diluted with 50 ml H2O, acidified with 5 M HCl and the product extracted with 3 x 30 ml

EtOAc. The organic phase was washed with H2O, dried over Na2SO4 and concentrated in vacuum. To

increase [D]-enrichment the procedure was repeated. Column chromatography on silica gel using a

hexane - EtOAc mixture (5:1, v/v; UV detection, Rf = 0.08) afforded 350 mg [3,5-D2]-4-hydroxy-

benzyldehyde ([D2]-156, 2.8 mmol, 88 % yield) with > 0.65 D as determined by 1H NMR

spectroscopy (calc. > 0.98 D).

[3,5-D2]-4-hydroxybenzaldehyde ([D2]-150): C7H4D2O2, > 0.65 D, beige solid; 1H NMR (400.1

MHz, CDCl3): = 6.98 (0.70H, d, 3JAr = 9.0 Hz, residual 3,5-H), 7.82 (2H, s.br, 2,6-H), 9.87 (1H, s,

CHO); 13

C PENDANT (100.6 MHz, CDCl3): = 116.0 (d, residual 3,5-CH); 132.4 (d, 2,6-CH); 161.6

(s, 4-C), 191.1 (d, 7-CHO); EIMS (70 eV, direct inlet) m/z (%) = 124 (87) [M], 123 (100) [M – H], 95

(32), 67 (30).

4-hydroxybenzaldehyde (150): C7H6O2, beige solid; 1H NMR (400.1 MHz, CDCl3): = 6.98 (2H, d,

3JAr = 9.0 Hz, 3,5-H), 7.82 (2H, d,

3JAr = 8.9 Hz, 2,6-H), 9.87 (1H, s, CHO);

13C PENDANT (100.6

MHz, CDCl3): = 116.0 (d, 3,5-CH); 132.4 (d, 2,6-CH); 161.6 (s, 4-C), 191.1 (d, 7-CHO); EIMS (70

eV, direct inlet) m/z (%) = 122 (93) [M], 121 (100) [M – H], 93 (35), 65 (47), 63 (16), 53 (8), 50 (9),

39 (48).


192

7.6.3.15. Synthesis of [D4]-Malonic Acid ([D4]-88)

A solution of 1040 mg malonic acid (88, 10 mmol) in 5 ml D2O (278 mmol, 0.99 D) was kept at 80°C

for 24 h. After lyophilisation the procedure was repeated to afford 1030 mg [D4]-malonic acid ([D4]-

88, 9.5 mmol, 95 % yield) with an average enrichment of 0.76 D (calc. 0.93 D) as determined by MS.

[D4]-malonic acid ([D4]-88): C3D4O4, > 0.76 D, white solid; EIMS (70 eV, direct inlet) m/z (%) = 107

(1) [M], 89 (8) [M – OD], 64 (28) [M – CO2 for D4], 63 (46) [M – CO2 for D3], 62 (25) [M – CO2 for

D2], 61 (1) [M – CO2 for D1], 46 (55) [M – CD2COOD], 44 (100) [C2D2O].

malonic acid (88): C3H4O4, white solid; EIMS (70 eV, direct inlet) m/z (%) = 104 (2) [M], 87 (9) [M

– OH], 60 (79) [M – CO2], 45 (62) [M – CH2COOH], 42 (100) [C2H2O].

7.6.3.16. Synthesis of (E)-[2,3’,5’-D3]-4-Coumaric Acid ([D3]-152) by Knoevenagel

Under N2 atmosphere a mixture of 186 mg [3,5-D2]-4-hydroxybenzaldehyde ([D2]-150) (1.5 mmol,

> 0.65 D) and 325 mg [D4]-malonic acid ([D4]-88) (3.0 mmol, > 0.76 D) in 2 ml pyridine was treated

with 5 µl piperidine and kept at 90 °C for 6 h. The resulting mixture was poured into 5 ml ice cold 5 M

HCl and the precipitating solids were removed by filtration, washed with 5 ml 5 M HCl and 2 ml H2O,

recrystallized from H2O / EtOH and dried in vacuum to afford 140 mg (E)-[2,3‟,5‟-D3]-4-coumaric

acid ([D3]-152, 0.84 mmol, 56 % yield). 1H NMR spectroscopy indicated > 0.78 D for the 2-position

and > 0.66 D for the aromatic 3‟,5‟-positions.

(E)-[2,3’,5’-D3]-4-coumaric acid [(E)-2-deutero-3-(3,5-dideutero-4-hydroxyphenyl)propenoic

acid] ([D3]-152): C9H5D3O3, white solid; 1H NMR (500.1 MHz, acetone-d6): = 6.34 (0.22H, d,

3JE =

15.8 Hz, residual 2-H), 6.90 (0.68H, d, 3JAr = 8.5 Hz, residual 3‟,5‟-H), 7.56 (2H, s.br., 2‟,6‟-H), 7.62

(1H, s.br, 3-H); 2D NMR (61.4 MHz, acetone): = 6.32 (2-D), 6.89 (3‟,5‟-D);

13C PENDANT (100.6

MHz, acetone-d6): = 127.1 (s, 1‟-C), 130.9 (d, 2‟,6‟-CH), 145.6 (d, 3-CH), 160.4 (s, 4‟-C), 168.1 (s,

1-COOH) [signals for 2-CD and 3‟,5‟-CD missing]; EIMS (70 eV, direct inlet) m/z (%) = 167 (86)

[M] for D3, 166 (100) [M] for D2, 165 (59), 150 (28), 149 (34), 148 (15), 121 (26), 109 (7), 95 (12), 79

(19), 66 (10), 52 (12).

7.6.3.17. Synthesis of L- and D-[3,3-D2]-Phenylalanine (L-[3,3-D2]-153, D-[3,3-D2]-153)

In a sealed tube under H2 atmosphere (10 ml, 444 µmol) a mixture of 83 mg D-(+)- or L-(–)-

phenylalanine (153, 500 µmol) and 10 mg Pd/C (10 %, 9.4 µmol Pd) in 2 ml D2O (111 mmol, 0.99 D)

was heated at 110°C for 5 h. The solution was filtered hot and allowed to cool. Lyophilisation afforded

74 mg L-[3,3-D2]-phenylalanine (L-[D2]-153, 443 µmol, 87 % yield) with deuterium enrichment of

0.98 D for 3-Pro-R-H and 0.92 D for 3-Pro-S-H or 76 mg D-[3,3-D2]-phenylalanine (D-[D2]-153, 455

µmol, 91 % yield) with deuterium enrichment of 0.98 D for 3-Pro-S-H and 0.94 D for 3-Pro-R-H,

respectively.

L-phenylalanine [(S)-(–)-2-amino-3-phenylpropanoic acid] (L-153): C9H11NO2, white solid; 1H

NMR (500.1 MHz, D2O): = 3.05 (1H, dd, 2J = 14.5 Hz,

3J = 7.9 Hz, 3-Pro-R-H), 3.22 (1H, dd,

2J =

14.5 Hz, 3J = 5.0 Hz, 3-Pro-S-H), 3.92 (1H, dd,

3J = 7.9 Hz,

3J = 5.0 Hz, 2-H), 7.21 (2H, d,

3J = 7.6

Hz, 2‟,6‟-H), 7.28 – 7.38 (3H, m, 3‟,4‟,5‟-H); MS (FAB) m/z 166 [M + H]; D (c = 0.5 in 1 M HCl) =

– 9.4°

L-[3,3-D2]-phenylalanine [(S)-(–)-2-amino-3,3-dideutero-3-phenylpropanoic acid] (L-[D2]-153):

C9H9D2NO2, white solid; 1H NMR (500.1 MHz, D2O): = 3.05 (0.02H, s.br, residual 3-Pro-R-H),

3.22 (0.08H, s.br, residual 3-Pro-S-H), 3.91 (1H, s.br, 2-H), 7.25 (2H, d, 3J = 7.6 Hz, 2‟,6‟-H), 7.28 –

7.38 (3H, m, 3‟,4‟,5‟-H); MS (FAB) m/z 168 [M + H]; D (c = 0.5 in 1 M HCl) = – 9.1°

D-[3,3-D2]-phenylalanine [(R)-(+)-2-amino-3,3-dideutero-3-phenylpropanoic acid] (D-[D2]-153):

C9H9D2NO2, white solid; 1H NMR (500.1 MHz, D2O): = 3.05 (0.02H, s.br, residual 3-Pro-S-H),


193

3.22 (0.06H, s.br, residual 3-Pro-R-H), 3.91 (1H, s.br, 2-H), 7.25 (2H, d, 3J = 7.6 Hz, 2‟,6‟-H), 7.28 –

7.38 (3H, m, 3‟,4‟,5‟-H); MS (FAB) m/z 168 [M + H]; D (c = 0.5 in 1 M HCl) = + 9.3°

7.6.3.18. Synthesis of L-[3,3-D2]-Tyrosine (L-[3,3-D2]-154)

In a sealed tube under H2 atmosphere 90 mg L-tyrosine (154, 0.5 mmol) and 10 mg Pd/C (10 %, 9.4

µmol Pd) in 2 ml D2O (111 mmol, 0.99 D) was heated at 110°C for 5 h. The solution was filtered hot

and allowed to cool. Lyophilisation afforded 52 mg L-[3,3-D2]-tyrosine (L-[3,3-D2]-154, 285 µmol,

57 % yield) with deuterium enrichment of 0.42 D for 3-Pro-R-H and 0.25 D for 3-Pro-S-H.

L-[3,3-D2]-tyrosine [(S)-(–)-2-amino-3,3-dideutero-3-(4-hydroxyphenyl)propanoic acid] (L-[3,3-

D2]-154): C9H9D2NO3; white solid; 1H NMR (500.1 MHz, D2O): = 2.99 (0.58H, dd,

2J = 14.8 Hz,

3J

= 7.6 Hz residual 3-Pro-R-H), 3.14 (0.75H, dd, 2J = 14.8 Hz,

3J = 5.0 Hz, residual 3-Pro-S-H), 3.86

(1H, m, 2-H), 6.83 (1.7H, d, 3JAr = 8.5 Hz, 3‟,5‟-H), 7.13 (2H, d,

3JAr = 8.5 Hz, 2‟,6‟-H); MS (FAB)

m/z = no [M + H] detected.

L-tyrosine [(S)-(–)-2-amino-3-(4-hydroxyphenyl)propanoic acid] (L-154): C9H11NO3; white solid; 1H NMR (500.1 MHz, D2O): = 2.99 (1H, dd,

2J = 14.8 Hz,

3J = 7.6 Hz, 3-Pro-R-H), 3.14 (1H, dd,

2J

= 14.8 Hz, 3J = 5.0 Hz, 3-Pro-S-H), 3.86 (1H, m, 2-H), 6.83 (1.71H, d,

3JAr = 8.5 Hz, 3‟,5‟-H), 7.13

(2H, d, 3JAr = 8.5 Hz, 2‟,6‟-H); MS (FAB) m/z = no [M + H] detected.

7.6.3.19. Synthesis of L-[3,3-D2]-O-Methyltyrosine (L-[3,3-D2]- 155)

In a sealed tube under H2 atmosphere 98 mg L-O-methyltyrosine (155, 500 µmol) and 10 mg Pd/C

(10 %, 9.4 µmol Pd) in 2 ml D2O (111 mmol, 0.99 D) was heated at 110°C for 5 h. The solution was

filtered hot and allowed to cool. Lyophilisation afforded 60 mg L-[3,3-D2]-O-methyltyrosine (L-[D2]-

155, 305 µmol, 61 % yield) with deuterium enrichment of 0.95 D for 3-Pro-R-H and 0.94 D for 3-Pro-

S-H.

L-[3,3-D2]-O-methyltyrosine [(S)-(–)-2-amino-3,3-dideutero-3-(4-methoxyphenyl) propanoic

acid] (L-[D2]-155): C10H11D2NO3, white solid; 1H NMR (500.1 MHz, D2O): = 3.02 (0.05H, s.br,

residual 3-Pro-R-H), 3.17 (0.06H, s.br, residual 3-Pro-S-H), 3.79 (3H, s, OCH3), 3.89 (1H, s.br, 2-H),

6.96 (2H, d, 3J = 8.2 Hz, 3‟,5‟-H), 7.21 (2H, d,

3J = 8.0 Hz, 2‟,6‟-H); MS (FAB) m/z 198 [M + H].

L-O-methyltyrosine [(S)-(–)-2-amino-3-(4-methoxyphenyl)propanoic acid] (L-155): C10H13NO3,

white solid; 1H NMR (500.1 MHz, D2O): = 3.02 (1H, dd,

2J = 14.5 Hz,

3J = 7.9 Hz, 3-Pro-R-H),

3.17 (1H, dd, 2J = 14.5 Hz,

3J = 4.9 Hz, 3-Pro-S-H), 3.79 (3H, s, OCH3), 3.90 (1H, dd,

3J = 7.9 Hz,

3J

= 4.9 Hz, 2-H), 6.96 (2H, d, 3J = 8.2 Hz, 3‟,5‟-H), 7.21 (2H, d,

3J = 8.0 Hz, 2‟,6‟-H); MS (FAB) m/z

196 [M + H].

7.6.3.20. Synthesis of [3,3-D2]-Dihydrocinnamic Acid ([3,3-D2]-156)

In a sealed tube under H2 atmosphere 75 mg dihydrocinnamic acid (156, 500 µmol) and 10 mg Pd/C

(10 %, 9.4 µmol Pd) in 2 ml D2O (111 mmol, 0.99 D) was heated at 110°C for 5 h. The solution was

filtered hot and allowed to cool. Lyophilisation afforded 62 mg [3,3-D2]-dihydrocinnamic acid ([3,3-

D2]-156, 408 µmol, 82 % yield) with deuterium enrichment of 0.89 D for benzylic 3-H‟s and 0.24 D

for 2-H‟s (D3/D2 = 0.5 by MS).

[3,3-D2]-dihydrocinnamic acid [3,3-dideutero-3-phenylpropanoic acid] ([3,3-D2]-156): C9H8D2O2,

white solid; 1H NMR (500.1 MHz, acetone-d6): = 2.59 (1.52H, s, residual 2-H), 2.89 (0.22H, s,

residual 3-H), 7.18 (1H, m, 4‟-H), 7.25 (4H, m, 2‟,3‟,5‟,6‟-H); 13

C PENDANT (100.6 MHz, acetone-

d6): = 35.9 (t, residual 2-CH2), 127.3 (d, 4‟-CH), 129.6 (2d, 2‟,3‟,5‟,6‟-CH), 142.3 (s, 1‟-C), 174.6 (s,


194

1-COOH), [signal for 3-CD2 missing]; EIMS (70 eV, direct inlet) m/z (%) = 153 (11) [M] for D3, 152

(22) [M] for D2, 106 (38), 93 (100) [C7H5D2].

dihydrocinnamic acid [3-phenylpropanoic acid] (156): C9H10O2, white solid; 1H NMR (500.1

MHz, acetone-d6): = 2.59 (2H, t, J = 7.5 Hz, 2-H), 2.89 (2H, t, J = 7.5 Hz, 3-H), 7.18 (1H, m, 4‟-H),

7.25 (4H, m, 2‟,3‟,5‟,6‟-H); 13

C PENDANT (100.6 MHz, acetone-d6): = 31.9 (t, 3-CH2), 35.9 (t, 2-

CH2), 127.3 (d, 4‟-CH), 129.6 (2d, 2‟,3‟,5‟,6‟-CH), 142.3 (s, 1‟-C), 174.6 (s, 1-COOH); EIMS (70 eV,

direct inlet) m/z (%) = 150 (14) [M], 104 (39), 91 (100) [C7H7].

7.6.3.21. Synthesis of [2,2,3,3,3’-D5]-Phloretic Acid ([D5]-157)

In a sealed tube under H2 atmosphere 83 mg phloretic acid (157, 500 µmol) and 10 mg Pd/C (10 %,

9.4 µmol Pd) in 2 ml D2O (111 mmol, 0.99 D) was heated at 110°C for 5 h. The solution was filtered

hot and allowed to cool. Lyophilisation afforded 78 mg [2,2,3,3,3‟-D5]-phloretic acid ([D5]-157,

458 µmol, 92 % yield) with deuterium enrichment of 0.98 D for benzylic 3-H‟s, 0.92 D for -CH

acidic 2-H‟s, and 0.29 D for aromatic 3‟,5‟-H‟s by 1H NMR (D5/D4 = 0.72 by MS).

[2,2,3,3,3’-D5]-phloretic acid [2,2,3,3-tetradeutero-3-(3-deutero-4-hydroxyphenyl)-propanoic

acid] ([D5]-157): C9H8D2O3, white solid; 1H NMR (500.1 MHz, acetone-d6): = 2.53 (0.16H, s,

residual 2-H), 2.78 (0.04H, s, residual 3-H), 6.76 (1.41H, d, J = 8.8 Hz, residual 3‟,5‟-H), 7.08 (2H, d,

J = 8.2 Hz, 2‟,6‟-H); 2D NMR (61.4 MHz, acetone): = 2.53 (2-D), 2.78 (3-D), 6.76 (3‟,5‟-D);

13C

PENDANT (100.6 MHz, acetone-d6): = 116.4 (d, 3‟,5‟-CH), 130.5 (d, 2‟,6‟-CH), 133.0 (s, 1‟-C),

157.0 (s, 4‟-C), 174.7 (s, 1-COOH) [signals for 2-CD2 and 3-CD2 missing]; EIMS (70 eV, direct inlet)

m/z (%) = 171 (21) [M] for D5, 170 (29) [M] for D4, 110 (88) [C7H4D3O], 109 (100) [C7H5D2O].

phloretic acid [(3-(4-hydroxyphenyl)-propanoic acid)] (157): C9H10O3, white solid; 1H NMR

(500.1 MHz, acetone-d6): = 2.56 (2H, t, J = 7.6 Hz, 2-H), 2.82 (2H, t, J = 7.6 Hz, 3-H), 6.76 (2H, d,

J = 8.5 Hz, 3‟,5‟-H), 7.08 (2H, d, J = 8.2 Hz, 2‟,6‟-H); 13

C PENDANT (100.6 MHz, acetone-d6): =

31.2 (t, 3-CH2), 36.8 (t, 2-CH2), 116.4 (d, 3‟,5‟-CH), 130.5 (d, 2‟,6‟-CH), 133.0 (s, 1‟-C), 157.0 (s, 4‟-

C), 174.7 (s, 1-COOH); EIMS (70 eV, direct inlet) m/z (%) = 166 (14) [M], 120 (8), 107 (100)

[C7H7O].

7.6.3.22. Synthesis of [2,2,3’-D3]-Tyramine ([2,2,3’-D3]-158)

In a sealed tube under H2 atmosphere 69 mg tyramine (158, 500 µmol) and 10 mg Pd/C (10 %, 9.4

µmol Pd) in 2 ml D2O (111 mmol, 0.99 D) was heated at 110°C for 5 h. The solution was filtered hot

and allowed to cool. After lyophilisation the residue was recrystallized from H2O - EtOH to afford

51 mg [2,2,3‟-D3]-tyramine ([2,2,3‟-D3]-158, 337 µmol, 67 % yield) with 0.90 D for benzylic 2-H‟s

and 0.50 D for aromatic 3‟,5‟-H‟s by 1H NMR (D3/D2 = 0.83 by MS).

[2,2,3’-D3]-tyramine [2,2-dideutero-2-(3-deutero-4-hydroxyphenyl)ethylamine] ([2,2,3’-D3]-172):

C8H9D2NO, white solid; 1H NMR (500.1 MHz, acetone-d6): = 2.75 (0.19H, m, residual 2-H), 3.37

(2H, s, 1-H), 6.74 (1.00H, d, J = 8.2 Hz, residual 3‟,5‟-H), 7.05 (2H, d, J = 8.5 Hz, 2‟,6‟-H); 13

C

PENDANT (100.6 MHz, acetone-d6): = 54.2 (t, 1-CH2), 115.8 (d, residual 3‟,5‟-CH), 130.6 (d,

2‟,6‟-CH), 132.4 (s, 1‟-C) 156.4 (s, 4‟-C), [signal for 2-CD2 missing]; EIMS (70 eV, direct inlet) m/z

(%) = 140 (10) [M] for D3, 139 (12) [M] for D2, 111 (100) [M – COH], 110 (85), 94 (4), 80 (24), 53

(12).

tyramine [2-(4-hydroxyphenyl)ethylamine] (158): C8H11NO, white solid; 1H NMR (500.1 MHz,

acetone-d6): = 2.75 (2H, t, J = 7.5 Hz, 2-H), 3.39 (2H, t, J = 7.5 Hz, 1-H), 6.74 (2H, d, J = 8.2 Hz,

3‟,5‟-H), 7.05 (2H, d, J = 8.4 Hz, 2‟,6‟-H); 13

C PENDANT (100.6 MHz, acetone-d6): = 37.2 (t, 2-

CH2), 54.2 (t, 1-CH2), 115.8 (d, 3‟,5‟-CH), 130.6 (d, 2‟,6‟-CH), 132.4 (s, 1‟-C) 156.4 (s, 4‟-C); EIMS


195

(70 eV, direct inlet) m/z (%) = 137 (29) [M], 108 (100) [M – COH], 107 (68), 91 (5), 77 (28), 63 (6),

51 (10).

7.6.3.23. Synthesis of L-N-Acetyltyrosine (159)

A suspension of 905 mg L-tyrosine (154, 5 mmol) in 5 ml H2O at 95 °C was treated with 3.75 ml

Ac2O (40 mmol) in 8 portions. The resulting solution was filtered and the solvent evaporated in

vacuum to give an oil which was dissolved in 5 ml acetone, filtered and concentrated in vacuum. The

residue was dissolved in 10 ml 1,4-dioxane and the solution slowly diluted with 10 ml hexane. The

precipitating solids were removed by filtration, washed with hexane and dried in vacuum to give 1030

mg L-N-acetyltyrosine (159, 4.6 mmol, 92 % yield).

L-N-acetyltyrosine [(S)-2-acetamido-3-(4-hydroxyphenyl)propanoic acid] (159): C11H13NO4,

white solid; 1H NMR (500.1 MHz, acetone-d6): = 1.90 (3H, s, NHCOCH3), 2.90 (1H, dd,

2J = 13.9

Hz, 3J = 7.9 Hz, 3-Pro-R-H), 3.08 (1H, dd,

2J = 14.2 Hz,

3J = 5.4 Hz, 3-Pro-S-H), 4.65 (1H, dd, J = J

= 6.7 Hz, 2-H), 6.75 (2H, d, 3J = 8.5 Hz, 3‟,5‟-H), 7.07 (2H, d,

3J = 8.5 Hz, 2‟,6‟-H);

13C PENDANT

(100.6 MHz, acetone-d6): = 23.0 (q, COCH3). 37.7 (t, 3-CH2), 54.9 (d, 2-CH), 116.3 (d, 3„,5„-CH),

129.2 (s, 1‟-C), 131.5 (d, 2„,6„-CH), 157.4 (s, 4‟-C), 170.7 (s, CONH), 173.6 (s, 1-COOH); EIMS (70

eV, direct inlet) m/z (%) = 223 (2) [M], 164 (68), 147 (4), 136 (6), 120 (3), 107 (100) [C7H7O], 91 (5),

77 (9), 60 (6), 43 (23).

7.6.3.24. Synthesis of [CD3]-Methyl L-N-Acetyl-[CD3]-O-methyltyrosinate ([CD3]-160)

A solution of 223 mg L-N-acetyl tyrosine (159, 1 mmol) in 5 ml acetone was treated with 280 mg

K2CO3 (2 mmol) and 430 mg [CD3]-methyl iodide (3 mmol, > 0.99 % D). The mixture was heated

under reflux for 60 h, filtered, concentrated in vacuum and the residue submitted to column

chromatography on silica gel 60 using a hexane – EtOAc mixture (3/1, v/v; UV detection) to give

202 mg [CD3]-methyl L-N-acetyl-[CD3]-O-methyltyrosinate ([CD3]-160, 786 µmol, 79 % yield).

[CD3]-methyl L-N-acetyl-[CD3]-O-methyltyrosinate [trideuteromethyl (S)-2-acetamido-3-(4-

trideuteromethoxyphenyl)propanoate] ([CD3]-160): C13H11D6NO4; white solid; 1H NMR (500.1

MHz, acetone-d6): = 1. 19 (3H, s, NHCOCH3), 2.94 (1H, dd, 2J = 14.2 Hz,

3J = 7.6 Hz, 3-Pro-R-H),

3.04 (1H, dd, 2J = 14.1 Hz,

3J = 5.4 Hz, 3-Pro-S-H), 3.76 (0.01H, m, residual OCH3), 4.61 (1H, dd,

3J

= 7.6 Hz, 3J = 5.4 Hz, 2-H), 6.84 (2H, d,

3J = 8.5 Hz, 3‟,5‟-H), 7.17 (2H, d,

3J = 8.5 Hz, 2‟,6‟-H);

13C

PENDANT (100.6 MHz, acetone-d6): = 22.6 (q, COCH3), 39.7 (t, 3-CH2), 54.7 (d, 2-CH), 114.4 (d,

3‟,5‟-CH), 129.9 (s, 1‟-C), 131.2 (d, 2‟,6‟-CH), 159.6 (s, 4‟-C), 169.9 (s, CONH), 173.0 (s, 1-COOR),

[signals for CD3 missing]; EIMS (70 eV, direct inlet) m/z (%) = 257 (2) [M], 198 (62), 181 (4), 164

(13), 153 (6), 124 (100) [C7H6OCD3], 107 (4), 91 (5), 78 (6), 43 (17).

7.6.3.25. Synthesis of L-[CD3]-O-Methyltyrosine Hydrochloride ([CD3]-155)

Under N2 a suspension of 154 mg [CD3]-methyl L-N-acetyl-[CD3]-O-methyltyrosinate ([CD3]-160,

0.6 mmol) in 6 ml 3 M HCl was heated under reflux for 72 h. The solution was filtered, decolourized

with 200 mg charcoal, filtered and concentrated in vacuum to 1 ml, which was treated with 100 ml

toluene and the remaining H2O removed by azeotropic distillation using a Dean Stark trap. The residue

was recrystallized from H2O - EtOH to give 124 mg L-[CD3]-O-methyltyrosine hydrochloride ([CD3]-

155, 528 µmol, 88 % yield).

L-[CD3]-O-methyltyrosine hydrochloride ([CD3]-155): C10H11D3NO3Cl; beige solid; 1H NMR

(500.1 MHz, D2O): = 3.12 (1H, dd, 2J = 14.8 Hz,

3J = 7.6 Hz, 3-Pro-R-H), 3.23 (1H, dd,

2J = 14.8

Hz, 3J = 5.7 Hz, 3-Pro-S-H), 3.79 (0.01H, s, residual OCH3), 4.21 (1H, dd, J = 7.3 Hz, J = 5.7 Hz, 2-


196

H), 6.95 (2H, d, J = 8.8 Hz, 3‟,5‟-H), 7.21 (2H, d, J = 8.8 Hz, 2‟,6‟-H); 13


D2O): = 35.2 (t, 3-CH2), 54.8 (d, 2-CH), 115.0 (d, 3‟,5‟-CH), 126.9 (s, 1‟-C), 131.1 (d, 2‟,6‟-CH),

158.8 (s, 4‟-C), 172.2 (s, 1-COOH), [signal for CD3 missing]; FAB-MS (70 eV) m/z 199 [M – Cl].

7.6.3.26. Conversion to L-[CD3]-O-Methyltyrosine ([CD3]-155)

A solution of 118 mg L-[CD3]-O-methyltyrosine hydrochloride ([CD3]-155, 500 µmol) in 3 ml H2O at

95 °C was treated with 0.5 ml 1M KOH solution. After 24 h at 7 °C the precipitating solids were

removed by filtration, washed with H2O and dried in vacuum to give 92 mg L-[CD3]-O-methyltyrosine

([CD3]-155, 460 µmol, 92 % yield).

L-[CD3]-O-methyltyrosine [(S)-(–)-2-amino-3-(4-trideuteromethoxyphenyl)propanoic acid]

([CD3]-155): C10H10D3NO3; white solid; 1H NMR (500.1 MHz, D2O): = 3.02 (1H, dd,

2J = 14.5 Hz,

3J = 7.9 Hz, 3-Pro-R-H), 3.17 (1H, dd,

2J = 14.5 Hz,

3J = 4.9 Hz, 3-Pro-S-H), 3.79 (0.01H, s, residual

OCH3), 3.90 (1H, t, 3J =

3J = 6.8 Hz, 2-H), 6.96 (2H, d,

3J = 8.2 Hz, 3‟,5‟-H), 7.21 (2H, d,

3J = 8.0 Hz,

2‟,6‟-H); MS (FAB) m/z 199 [M + H].

7.6.3.27. Synthesis of DL-[2-D]-N,O-Diacetyltyrosine ([2-D]-161)

A mixture of 450 mg L-N-acetyl tyrosine (159, 2 mmol) in 2 ml D2O (111 mmol, 0.99 D) was treated

with 100 mg NaOH (2.5 mmol) and 1 ml Ac2O and kept at 50 °C for 6 h. The resulting mixture was

placed in an ice bath and acidified to pH 2 with concentrated HCl. The precipiating product was

filtered, recrystallized from H2O and dried in vacuum to afford 450 mg DL-[2-D]-N,O-diacetyl

tyrosine ([2-D]-161, 1.7 mmol, 85 % yield) with ˂ 0.98 D by 1H NMR.

DL-[2-D]-N,O-diacetyltyrosine [(R/S)-(±)-2-acetamido-2-deutero-3-(4-acetoxyphenyl)propanoic

acid] ([2-D]-161): C13H14DNO5; > 0.98 D; white solid; 1H NMR (500.1 MHz, acetone-d6): = 1.86

(3H, s, NHCOCH3), 2.23 (3H, s, OCOCH3), 2.97 (1H, d, 2J = 13.9 Hz, 3-Pro-R-H), 3.16 (1H, d,

2J =

13.6 Hz, 3-Pro-S-H), 4.65 (0.01H, m, residual 2-H), 6.97 (2H, d, 3J = 8.2 Hz, 3‟,5‟-H), 7.26 (2H, d,

3J

= 8.2 Hz, 2‟,6‟-H), [assignment of anisochoric hydrogens for L-form]; 13


acetone-d6): = 21.4 (q, OCOCH3), 23.0 (q, NHCOCH3), 38.2 (t, 3-CH2), 122.5 (d, 3„,5„-CH), 131.6

(d, 2„,6„-CH), 137.7 (s, 1‟-C), 150.7 (s, 4‟-C), 170.4 (s, NHCOR), 171.0 (s, OCOR), 173.6 (s, 1-

COOH), [signal for 2-CD missing]; EIMS (70 eV, direct inlet) m/z (%) = 266 (2) [M], 207 (11), 165

(44), 107 (100) [C7H7O], 43 (49).

7.6.3.28. Synthesis of DL-[2-D]-Tyrosine Hydrochloride ([2-D]-154)

Under N2 atmosphere a suspension of 425 mg DL-[2-D]-N,O-diacetyltyrosine ([2-D]-161, 1.6 mmol)

in 16 ml 3 M HCl was heated under reflux for 72 h. The solution was filtered, decolourized with

200 mg charcoal, filtered and concentrated in vacuum to 1 ml, which was treated with 100 ml toluene

and the remaining H2O removed by azeotropic distillation. The residue was recrystallized from H2O -

EtOH to give 310 mg DL-[2-D]-tyrosine hydrochloride ([2-D]-154, 1.3 mmol, 82 % yield) with ˂

0.98 D by 1H NMR.

DL-[2-D]-tyrosine hydrochloride [(RS)-(±)-2-amino-2-deutero-3-(4-hydroxyphenyl)propanoic

acid HCl] ([2-D]-154): C9H11DNO3Cl, > 0.98 D, white solid; 1H NMR (500.1 MHz, D2O): = 3.09

(1H, d, 2J = 14.5 Hz, 3-Pro-R-H), 3.21 (1H, d,

2J = 14.5 Hz, 3-Pro-S-H), 4.20 (0.02H, s.br, residual 2-

H), 6.85 (2H, d, 3J = 7.8 Hz, 3‟,5‟-H), 7.14 (2H, d,

3J = 7.3 Hz, 2‟,6‟-H) [assignment of anisochoric

hydrogens for L-form]; MS (FAB) m/z 183 [M – Cl].


197

7.6.3.29. Synthesis of L-N-Acetyl-O-methyltyrosine (162)

A suspension of 400 mg L-O-methyltyrosine (155, 2 mmol) in 4 ml H2O at 95 °C was treated with

1.5 ml Ac2O (16 mmol) in 3 portions. The resulting solution was filtered and the solvent evaporated in

vacuum to give an oil which was dissolved in 5 ml acetone, filtered and concentrated in vacuum. The

residue was dissolved in 10 ml 1,4-dioxane and the solution slowly diluted with 10 ml hexane. The

precipitating solids were removed by filtration, washed with hexane and dried in vacuum to give

420 mg L-N-acetyl-O-methyltyrosine (162, 1.8 mmol, 90 % yield).

L-N-acetyl-O-methyltyrosine [(S)-2-acetamido-3-(4-methoxyphenyl)propanoic acid] (162):

C12H15NO4; white solid; 1H NMR (500.1 MHz, acetone-d6): = 1.89 (3H, s, NHCOCH3), 2.92 (1H,

dd, 2J = 13.9 Hz,

3J = 7.9 Hz, 3-Pro-R-H), 3.10 (1H, dd,

2J = 13.9 Hz,

3J = 5.4 Hz, 3-Pro-S-H), 3.76

(3H, s, OCH3), 4.65 (1H, dd, 3J = 7.9 Hz,

3J = 5.3 Hz, 2-H), 6.84 (2H, d,

3J = 8.5 Hz, 3‟,5‟-H), 7.17

(2H, d, 3J = 8.5 Hz, 2‟,6‟-H);

13C PENDANT (100.6 MHz, acetone-d6): = 23.0 (q, COCH3), 37.6 (t,

3-CH2), 54.9 (d, 2-CH), 55.8 (q, OCH3), 114.9 (d, 3„,5„-CH), 130.4 (s, 1‟-C), 131.5 (d, 2„,6„-CH),

159.9 (s, 4‟-C), 170.6 (s, CONH), 173.5 (s, 1-COOH); EIMS (70 eV, direct inlet) m/z (%) = 237 (1)

[M], 178 (55), 161 (2), 150 (3), 134 (3), 121 (100) [C8H9O], 108 (3), 91 (4), 77 (7), 65 (2), 43 (18).

7.6.3.30. Synthesis of DL-[2-D]-N-Acetyl-O-methyltyrosine ([2-D]-162)

A suspension of 240 mg L-N-acetyl-O-methyltyrosine (162, 1 mmol) in 1 ml D2O (55.4 mmol,

0.99 D) was treated with 50 mg NaOH (1.25 mmol) and 0.5 ml Ac2O and kept at 50 °C for 6 h. The

resulting mixture was placed in an ice bath and acidified to pH 2 with concentrated HCl. The

precipiating product was filtered, recrystallized from H2O and dried in vacuum to afford 195 mg

DL-[2-D]-N-acetyl-O-methyltyrosine ([2-D]-162, 0.81 mmol, 81 % yield) with ˂ 0.98 D by 1H NMR.

DL-[2-D]-N-acetyl-O-methyltyrosine [(RS)-(±)-2-acetamido-2-deutero-3-(4-methoxyphenyl)-

propanoic acid] ([2-D]-162): C12H14DNO4, ˂ 0.98 D, white solid; 1H NMR (500.1 MHz, acetone-d6):

= 1.89 (3H, s, NHCOCH3), 2.92 (1H, d.br, 2J = 13.9 Hz, 3-Pro-R-H), 3.10 (1H, d.br,

2J = 13.9 Hz,

3-Pro-S-H), 3.76 (3H, s, OCH3), 4.65 (0.01H, m, residual 2-H), 6.84 (2H, d, 3J = 8.5 Hz, 3‟,5‟-H), 7.17

(2H, d, 3J = 8.5 Hz, 2‟,6‟-H), [assignment of anisochoric hydrogens for L-form];

13C PENDANT

(100.6 MHz, acetone-d6): = 23.0 (q, COCH3). 37.6 (t, 3-CH2), 55.8 (q, OCH3), 114.9 (d, 3„,5„-CH),

130.4 (s, 1‟-C), 131.5 (d, 2„,6„-CH), 159.9 (s, 4‟-C), 170.6 (s, CONH), 173.5 (s, 1-COOH) [signal for

2-CD missing]; EIMS (70 eV, direct inlet) m/z (%) = 238 (1) [M], 179 (55), 162 (2), 151 (3), 135 (3),

121 (100) [C8H9O], 108 (3), 91 (4), 77 (7), 65 (2), 43 (18).

7.6.3.31. Synthesis of DL-[2-D]-O-Methyltyrosine Hydrochloride ([2-D]-155)

Under N2 atmosphere a suspension of 190 mg DL-[2-D]-N-acetyl-O-methyltyrosine ([2-D]-162,

0.8 mmol) in 8 ml 3 M HCl was heated under reflux for 72 h. The solution was filtered, decolourized

with 200 mg charcoal, filtered and concentrated in vacuum to 1 ml, which was treated with 100 ml

toluene and the remaining H2O removed by azeotropic distillation. The residue was recrystallized from

H2O - EtOH to give 120 mg DL-[2-D]-O-methyltyrosine hydrochloride ([2-D]-155, 0.61 mmol, 76 %

yield) with ˂ 0.98 D by 1H NMR.

DL-[2-D]-O-methyltyrosine hydrochloride [(RS)-(±)-2-amino-2-deutero-3-(4-methoxyphenyl)-

propanoic acid HCl ([2-D]-155): C10H13DNO3Cl, ˂ 0.98 D, white solid; 1H NMR (500.1 MHz,

D2O): = 3.12 (1H, d.br, 2J = 14.8 Hz, 3-Pro-R-H), 3.23 (1H, d.br,

2J = 14.8 Hz, 3-Pro-S-H), 3.79

(3H, s, OCH3), 4.21 (0.01H, dd, J = 7.3 Hz, J = 5.7 Hz, residual 2-H), 6.95 (2H, d, J = 8.8 Hz, 3‟,5‟-

H), 7.21 (2H, d, J = 8.8 Hz, 2‟,6‟-H) [assignment of anisochoric hydrogens for L-form]; MS (FAB)

m/z 197 [M – Cl].


198

7.6.3.32. Synthesis of (Z)-4-(4-Acetoxybenzylidene)-2-methyloxazol-5(4H)-one (164)

A mixture of 1.8 g 4-hydroxybenzaldehyde (150, 14 mmol), 2.0 g N-acetylglycine (163, 17 mmol),

and 1.5 g sodium acetate (19 mmol) in 6.7 ml Ac2O was heated at 120 °C for 2 h. The mixture was

quenched with 7 ml ice H2O and the solids collected by filtration. The crude product was washed in

20 ml 50 % aqueous EtOH, filtered and dried in vacuum to give 2.0 g (Z)-4-(4-acetoxybenzylidene)-

2-methyloxazol-5(4H)-one (164, 8.2 mmol, 59 % yield).

(Z)-4-(4-acetoxybenzylidene)-2-methyloxazol-5(4H)-one (164): C13H11NO4; yellow solid; 1H NMR

(500.1 MHz, CDCl3): = 2.32 (3H, s, CH3), 2.40 (3H, s, CH3), 7.11 (1H, s, 6-H), 7.19 (2H, d, 3J = 8.8

Hz, 3‟,5‟-H), 8.12 (2H, d, 3J = 8.8 Hz, 2‟,6‟-H);

13C PENDANT (100.6 MHz, CDCl3): = 15.7 (q, 2-

CH3), 21.2 (q, 4‟-OCOCH3), 122.1 (d, 3‟,5‟-CH), 130.2 (d, 6-CH), 130.9 (s, 5-C), 132.6 (s, 1‟-C),

133.5 (d, 2‟,6‟-CH), 152.6 (s, 4‟-C), 166.3 (s, 4-C), 167.7 (s, 1-CO), 168.9 (s, 4‟-OCOCH3); EIMS (70

eV, direct inlet) m/z (%) = 245 (15) [M], 203 (54), 175 (4), 133 (100), 118 (1), 105 (2), 89 (2), 77 (4),

63 (3), 51 (3), 43 (66).

7.6.3.33. Synthesis of (Z)-2-Acetamido-3-(4-acetoxyphenyl)propenoic Acid (165)

A suspension of 2.0 g (Z)-4-(4-acetoxybenzylidene)-2-methyloxazol-5(4H)-one (164, 8.2 mmol) in

100 ml of a H2O - acetone mixture (4:1, v/v) was treated with 45 mg sodium acetate (0.7 mmol) and

heated under reflux for 2 h. The solution was filtered hot and allowed to cool. After storing at 7 °C for

24 h the precipitating solids were collected by filtration and dried in vacuum to give 1.8 g (Z)-

2-acetamido-3-(4-acetoxyphenyl)propenoic acid (165, 6.8 mmol, 83 % yield).

(Z)-2-acetamido-3-(4-acetoxyphenyl)propenoic acid (165): C13H13NO5; yellow solid; 1H NMR

(500.1 MHz, DMSO-d6): = 2.00 (3H, s, NCOCH3), 2.29 (3H, s, 4‟-OCOCH3), 7.18 (2H, d, 3J = 8.5

Hz, 3‟,5‟-CH), 7.23 (1H, s, 7-CH), 7.66 (2H, d, 3J = 8.5 Hz, 2‟,6‟-CH), 9.48 (s, NH), 12.68 (1H, s.br,

1-COOH); 13

C PENDANT (100.6 MHz, DMSO-d6): = 21.3 (q, 4‟-OCOCH3), 22.9 (q, NHCOCH3),

122.2 (d, 3‟,5‟-CH), 122.5 (d, 3-CH), 127.7 (s, 2-C), 131.3 (d, 2‟,6‟-CH), 131.7 (s, 1‟-C), 151.1 (s, 4‟-

C), 166.7 (s, 1-COOH), 169.4 (s, NHCOCH3), 169.6 (s, OCOCH3); EIMS (70 eV, direct inlet) m/z

(%) = 263 (10) [M], 221 (32), 203 (6), 179 (100) [M – C2H2O – C2H2O], 133 (42), 107 (4), 77 (4), 43

(34).

7.6.3.34. Synthesis of DL-[2,3-threo-D2]-N,O-Diacetyltyrosine ([2,3-threo-D2]-161)

A solution of 130 mg (Z)-2-acetamido-3-(4-acetoxyphenyl)propenoic acid (165, 0.5 mmol) in 5 ml

ethanol-d1 was treated with 20 mg palladium on carbon (10 % Pd, w/w) and hydrogenated with

deuterium gas (> 0.98 D) at 5 bar for 48 h using an autoclave. The catalyst was removed by filtration

and washed with acetone. Concentration in vacuum afforded 130 mg DL-[2,3-threo-D2]-N,O-

diacetyltyrosine ([2,3-threo-D2]-161) (0.49 mmol, 98 % yield) with 0.91 D for the 3-Pro-S-position

and 0.94 D for the 2-position (assignment for L-form).

DL-[threo-2,3-D2]-N,O-diacetyltyrosine [(S,S/R,R)-(±)-2-acetamido-2,3-dideutero-3-(4-acetoxy-

phenyl)propanoic acid] ([2,3-threo-D2]-161): C13H13D2NO5, white solid; 1H NMR (500.1 MHz,

acetone-d6): = 1.89 (3H, s, NHCOCH3), 2.24 (3H, s, OCOCH3), 2.99 (1H, s.br, 3-Pro-R-H), 3.18

(0.09H, m, residual 3-Pro-S-H), 4.71 (0.06H, m, residual 2-H), 7.03 (2H, d, J = 8.5 Hz, 3‟,5‟-H), 7.28

(2H, d, J = 8.5 Hz, 2‟,6‟-H), [assignment for L-form]; 13

C PENDANT (100.6 MHz, acetone-d6): =

21.4 (q, OCOCH3), 23.0 (q, NHCOCH3), 38.2 (d as triplet, 1JC,D = 19.7 Hz, 3-CHD), 122.5 (d, 3„,5„-

CH), 131.6 (d, 2„,6„-CH), 137.7 (s, 1‟-C), 150.7 (s, 4‟-C), 170.4 (s, NHCOR), 171.0 (s, OCOR 173.6

(s, 1-COOH) [signal for 2-CD (54.9) missing]; EIMS (70 eV, direct inlet) m/z (%) = 267 (3) [M], 208

(11), 166 (45), 108 (100) [C7H6DO], 43 (51).


199

7.6.3.35. Synthesis of DL-[threo-2,3-D2]-Tyrosine Hydrochloride ([2,3-threo-D2]-154)

Under N2 atmosphere a suspension of 130 mg DL-[2,3-threo-D2]-N,O-diacetyl-tyrosine ([2,3-threo-

D2]-161, 0.5 mmol) in 5 ml 3 M HCl was heated under reflux for 72 h. The solution was filtered,

decolourized with 200 mg charcoal, filtered and concentrated in vacuum to 1 ml, which was treated

with 100 ml toluene and the remaining H2O removed by azeotropic distillation. The residue was

recrystallized from H2O - EtOH to give 90 mg DL-[2,3-threo-D2]-tyrosine hydrochloride ([2,3-threo-

D2]-154) (0.4 mmol, 80 % yield).

DL-[threo-2,3-D2]-tyrosine [(S,S/R,R)-(±)-2-amino-2,3-dideutero-3-(4-hydroxyphenyl) propanoic

acid] ([2,3-threo-D2]-154): C9H9D2NO3; white solid; 1H NMR (500.1 MHz, D2O): = 2.99 (1H, s.br,

3-Pro-R-H), 3.14 (0.09H, d, 2J = 14.8 Hz, residual 3-Pro-S-H), 3.86 (0.06H, s.br, residual 2-H), 6.83

(1.71H, d, 3JAr = 8.5 Hz, 3‟,5‟-H), 7.13 (2H, d,

3JAr = 8.5 Hz, 2‟,6‟-H) [assignment for L-form]; MS

(FAB) m/z 184 [M + H].

7.6.3.36. Synthesis of [CD3]-4-Methoxybenzaldehyde ([CD3]-58)

Under N2 atmosphere a mixture of 1.22 g 4-hydroxybenzaldehyde (150) (10 mmol) and 2.0 g K2CO3

(15 mmol) in 20 ml acetone was treated with 1.6 g [D3]-methyl iodide (11 mmol) and heated under

reflux for 2 days. The mixture was diluted with 50 ml H2O, acidified with 5 M HCl and the product

extracted with 3 x 30 ml Et2O. The organic phase was washed with H2O, dried over Na2SO4 and

concentrated in vacuum. Column chromatography on silica gel using a hexane - EtOAc mixture (5:1,

v/v; UV detection, Rf = 0.27) afforded 1.28 g [CD3]-4-methoxybenzaldehyde ([CD3]-58) (9.24 mmol,

92 % yield).

[CD3]-4-methoxybenzaldehyde [4-trideuteromethoxybenzaldehyde ([CD3]-58): C8H5D3O2, > 0.99

D, colourless oil; 1H NMR (400.1 MHz, CDCl3): = 3.80 (0.01H, s.br, residual OCH3), 6.92 (2H, d,

3J = 8.9 Hz, 3,5-H), 7.75 (2H, d,

3J = 8.9 Hz, 2,6-H), 9.80 (1H, s, CHO);

13C PENDANT (100.6 MHz,

CDCl3): = 114.3 (d, 3.5-CH), 130.0 (s, 1-C), 131.9 (d, 2,6-CH), 164.6 (s, 4-C), 190.7 (d, CHO)

[signal for OCD3 missing]; EIMS (70 eV) m/z (%) = 139 (68) [M], 138 (100) [M – H], 110 (10), 92

(23), 78 (27), 63 (18), 51 (7), 39 (19).

7.6.3.37. Synthesis of [CD3]-4-Methoxy- -nitrostyrene ([CD3]-167)

A solution of 280 mg [CD3]-4-methoxybenzaldehyde ([CD3]-58, 2 mmol) in 3 ml acetic acid

(35 mmol) was treated with 0.6 ml nitromethane (166, 11.2 mmol) and 0.3 ml benzylamine

(2.7 mmol) and heated at 100 °C for 2 h. The reaction mixture was diluted with H2O and precipitating

solids collected by filtration, recrystallized from MeOH – H2O (1:1, v/v) and dried in vacuum to afford

262 mg [CD3]-4-methoxy- -nitrostyrene ([CD3]-167) (1.44 mmol, 72 % yield).

(E)-[CD3]-4-methoxy- -nitrostyrene [(E)-1-[trideuteromethoxy-4-(2-nitrovinyl)benzene ([CD3]-

167)]: C9H6D3NO3, > 0.98 D, yellow crystals; 1H NMR (500.1 MHz, CDCl3): = 3.87 (0.01H, s,

residual OCH3), 6.96 (2H, d, 3JAr = 8.5 Hz, 3‟,5‟-H), 7.51 (2H, d,

3JAr = 8.5 Hz, 2‟,6‟-H), 7.52 (1H, d,

3JE = 13.5 Hz, 1-H), 7.98 (1H, d,

3JE = 13.5 Hz, 1-H);

13C PENDANT (100.6 MHz, CDCl3): = 114.9

(d, 3‟,5‟-CH), 122.6 (s, 1‟-C), 131.2 (d, 2‟,6‟-CH), 135.1 (d, 2-CH), 139.0 (d, 1-CH), 163.0 (s, 4‟-C)

[signal for OCD3 missing]; EIMS (70 eV, direct inlet) m/z (%) = 182 [M] (27), 135 (100) [M –

HNO2], 124 (39), 89 (45), 63 (39).

(E)-4-methoxy- -nitrostyrene [(E)-1-[methoxy-4-(2-nitrovinyl)benzene] (167): C9H9NO3; yellow

crystals; 1H NMR (400.1 MHz, CDCl3): = 3.87 (3H, s, OCH3), 6.96 (2H, d,

3JAr = 8.6 Hz, 3‟,5‟-H),

7.51 (2H, d, 3JAr = 8.6 Hz, 2‟,6‟-H), 7.53 (1H, d,

3JE = 14.1 Hz, 2-H), 7.98 (1H, d,

3JE = 13.7 Hz, 1-H);

13C PENDANT (100.6 MHz, CDCl3): = 55.5 (q, OCH3), 114.9 (d, 3‟,5‟-CH), 122.6 (s, 1‟-C), 131.2


200

(d, 2‟,6‟-CH), 135.1 (d, 2-CH), 139.0 (d, 1-CH), 163.0 (s, 4‟-C); EIMS (70 eV, direct inlet) m/z (%) =

179 (70) [M], 162 (9), 148 (4), 132 (100) [M – HNO2], 121 (29), 118 (25), 117 (21), 89 (35), 77 (30),

63 (23), 51 (11), 39 (5).

7.6.3.38. Synthesis of [CD3]-O-Methyltyramine ([CD3]-168)

Under N2 atmosphere a solution of 255 mg [CD3]-4-methoxy- -nitrostyrene ([CD3]-167, 1.4 mmol) in

5 ml dry THF was added drop wise to 160 mg LiAlH4 (4.2 mmol) in 10 ml dry THF at 0 °C. After

stirring for 15 min the mixture was heated under reflux for 5 h. Excess reagent was destroyed by the

addition of 645 mg Na2SO4 x 10H2O (2 mmol), the mixture filtered and the residue washed with Et2O.

The combined organic phases were dried over Na2SO4 and concentrated in vacuum. The oily residue

was dissolved in 5 ml propan-2-ol, neutralized with concentrated HCl, and added to 10 ml Et2O. After

24 h at – 20 °C the precipitating solids were collected by filtration, washed with Et2O and dried in

vacuum to give 210 mg [CD3]-O-methyltyramine hydrochloride ([CD3]-168, 1.1 mmol, 79 % yield).

[CD3]-O-methyltyramine HCl [2-(4-trideuteromethoxyphenyl)ethylammonium chloride] ([CD3]-

168): C9H10D3NO HCl, white solid; 1H NMR (500.1 MHz, D2O): = 2.89 (2H, t,

3J = 7.2 Hz, 2-CH2),

3.19 (2H, t, 3J = 7.2 Hz, 1-CH2), 3.78 (0.01H, s, residual OCH3), 6.96 (2H, d,

3JAr = 8.8 Hz, 3‟,5‟-H),

7.22 (2H, d, 3JAr = 7.5 Hz, 2‟,6‟-H);

13C PENDANT (100.6 MHz, D2O): = 32.2 (t, 2-CH2), 41.9 (t, 1-

CH2), 114.9 (d, 3‟,5‟-CH), 129.51 (s, 1‟-C), 130.5 (d, 2‟,6‟-CH), 158.4 (s, 4‟-C), [signal for OCD3

missing]; MS (FAB) m/z 155 [M – Cl].

O-methyltyramine HCl [2-(4-methoxyphenyl)ethylammonium chloride] (168): C9H13NO HCl,

white solid; 1H NMR (500.1 MHz, D2O): = 2.89 (2H, t,

3J = 7.2 Hz, 2-CH2), 3.19 (2H, t,

3J = 7.2

Hz, 1-CH2), 3.78 (3H, s, OCH3), 6.96 (2H, d, 3JAr = 8.8 Hz, 3‟,5‟-H), 7.22 (2H, d,

3JAr = 7.5 Hz, 2‟,6‟-

H); 13

C PENDANT (100.6 MHz, D2O): = 32.2 (t, 2-CH2), 41.9 (t, 1-CH2), 55.8 (q, OCH3), 114.9 (d,

3‟,5‟-CH), 129.51 (s, 1‟-C), 130.5 (d, 2‟,6‟-CH), 158.4 (s, 4‟-C); MS (FAB) m/z 152 [M – Cl].

7.6.3.39. Synthesis of (±)-[2,3-threo-D2]-Phloretic Acid ([2,3-threo-D2]-157)

A solution of 178 mg (E)-coumaric acid (152) (1 mmol) in 5 ml benzene was treated with ca. 10 mg

[RhCl(PPh3)3] catalyst and hydrogenated with deuterium gas at 3 atm for 2 days using an autoclave.

The solvent was removed in vacuum and the residue washed with petrol ether and recrystallized from

H2O - EtOH (1:1, v/v) to afford 163 mg (±)-[2,3-threo-D2]-phloretic acid ([2,3-threo-D2]-157)

(970 µmol, 97 % yield).

(±)-[2,3-threo-D2]-phloretic acid ((S,S/R,R)-(±)-2,3-dideutero-3-(4-hydroxyphenyl)propanoic

acid) ([2,3-threo-D2]-192): C9H8D2O3, white solid; 1H NMR (500.1 MHz, acetone-d6): = 2.52 (1H,

d.br., 3J = 5.9 Hz, 2-CHD), 2.79 (1H, d.br.,

3J = 5.9 Hz, 3-CHD), 6.75 (2H, d,

3JAr = 8.6 Hz, 3‟,5‟-H),

7.08 (2H, d, 3JAr = 8.4 Hz, 2‟,6‟-H);

1H NMR (500.1 MHz, CDCl3): = 2.62 (1H, d.br.,

3J = 6.3 Hz, 2-

CHD), 2.87 (1H, d.br., 3J = 6.3 Hz, 3-CHD), 6.76 (2H, d,

3JAr = 8.5 Hz, 3‟,5‟-H), 7.08 (2H, d,

3JAr =

8.2 Hz, 2‟,6‟-H); 13

C PENDANT (100.6 MHz, acetone-d6): = 30.5 (d as triplet, JCD = 19.7 Hz; 3-

CHD), 36.0 (d as triplet, JCD = 19.7 Hz, 2-CHD), 116.0 (d, 3‟,5‟-CH), 130.8 (d, 2‟,6‟-CH), 132.6 (s,

1‟-C), 156.6 (s, 4‟-C), 174.1 (s, 1-COOH); EIMS (70 eV, direct inlet) m/z (%) = 168 (20) [M], 108

(100) [C7H6DO], 78 (12).


201

7.7. (1S,2R)-(+)-spiroaxa-5,7-diene from Ulmus americana

7.7.1. Isolation of (–)-Axenol (211) from Juniperus oxycedrus

700 mg Juniperus oxycedrus wood oil (ca. 1.6 % axenol by GC) was chromatographed on silica gel

with hexane - EtOAc mixture (10:1, v/v). Preparative GC using a 2,6-Me-3-Pe- -CD column (135 °C

isothermally) afforded 9 mg (–)-axenol (211).

(–)-axenol [(1S,2R,5S,6R)-(–)-spiroaxa-7-en-6-ol] (211): C15H26O, colourless crystals; (–)-sense of

optical rotation (C6D6), RI 1575; 1H NMR (500.1 MHz, C6D6): = 0.83 (3H, d, J = 6.9 Hz, 14-CH3),

0.93 (3H, d, J = 6.9 Hz, 12-CH3), 0.94 (3H, d, J = 6.9 Hz, 13-CH3), 1.04 – 1.15 (2H, m, 3,5-H), 1.39

(1H, m, 4-H), 1.45 (1H, m, 3-H), 1.57 – 1.64 (2H, m, 4-H‟, 11-H), 1.62 (3H, d, J = 1.0 Hz, 15-CH3),

1.74 – 1.81 (2H, m, 2,10-H), 1.88 (1H, m, 10-H‟), 2.14 (2H, t.br, J = 7.9 Hz, 9-CH2), 3.55 (1H, s, 6-

H), 5.19 (1H, d, J = 1.6 Hz, 7-H); 13

C PENDANT (100.6 MHz, C6D6): = 16.9 (q, 14-CH3), 17.3 (q,

15-CH3), 21.2 (q, 13-CH3), 21.7 (q, 12-CH3), 25.0 (t, 4-CH2), 29.9 (d, 11-CH), 32.5 (t, 3-CH2), 34.5 (d,

2-CH), 34.5 (t, 10-CH2), 36.8 (t, 9-CH2), 46.1 (d, 5-CH), 59.7 (s, 1-C), 76.5 (d, 6-CH), 126.6 (d, 7-

CH), 142.9 (s, 8-C); EIMS (70 eV) m/z (%) = 222 (17) [M], 204 (4), 161 (12), 147 (4), 135 (11), 121

(97), 108 (47), 93 (45), 91 (47), 81 (100), 67 (22), 55 (41), 41 (83).

7.7.2. Amberlyst Catalyzed Rearrangement of (+)-Aromadendrene ((+)-216)

A solution of 5 mg (+)-aromadendrene ((+)-216) in 500 µl DCM was treated with 10 mg Amberlyst®

15. After stirring for 4 days the reaction mixture was filtered and concentrated in a stream of N2. Flash

column chromatography on silica gel with pentane and consecutive preparative GC using an 2,6-Me-

3-Pe- -CD column (140 °C to 160 °C) and 6-TBDMS-2,3-Me- -CD column (90 °C, isotherm)

afforded 0.5 mg (1R,2R)-(–)-1-epi-spiroaxa-5,7-diene (219).

(1R,2R)-(–)-1-epi-spiroaxa-5,7-diene (219): C15H24; colourless oil; (–)-sense of optical rotation

(C6D6); RI 1390; 1H NMR (500.1 MHz, C6D6): = 0.91 (3H, d, J = 6.9 Hz, 14-CH3), 1.01 (3H, d, J =

6.9 Hz, 12-CH3), 1.02 (3H, d, J = 6.9 Hz, 13-CH3), 1.39 (1H, m, 3-H‟), 1.60 (2H, m, 2,3-H), 1.66 (3H,

s.br., 15-CH3), 1.68 (1H, m, 10-H), 1.90 (2H, m, 4-CH2), 1.94 (1H, m, 10-H‟), 2.13 (1H, sept, J = 6.9

Hz, 11-H), 2.14 (1H, m, 9-H), 2.21 (1H, m, 9-H‟), 5.10 (1H, s.br., 7-H), 5.44 (1H, s.br., 6-H); 13

C

PENDANT (100.6 MHz, C6D6): = 16.4 (q, 14-CH3), 16.9 (q, 15-CH3), 21.9, 22.2 (2q, 12,13-CH3),

26.4 (t, 4-CH2), 29.3 (t, 3-CH2), 33.5 (t, 10-CH2), 35.5 (d, 11-CH), 37.6 (t, 9-CH2), 37.8 (d, 2-CH),

54.5 (s, 1-C), 129.0 (d, 6-CH), 133.9 (d, 7-CH), 139.6 (s, 8-C), 141.0 (s, 5-C); EIMS (70 eV) m/z (%)

= 204 (27) [M], 189 (2), 175 (1), 162 (79), 147 (100) , 133 (8), 119 (5), 105 (34), 93 (8), 91 (25), 81

(12), 79 (16), 77 (18), 69 (5), 67 (7), 65 (8), 55 (14), 53 (11), 51 (4), 43 (5), 41 (37), 39 (12).

7.7.3. Amberlyst Catalyzed Rearrangement of (+)-Ledene (218)

A solution of 5 mg (+)-ledene (218) in 500 µl DCM was treated with Amberlyst® 15 as described in

7.7.2. to afford 0.4 mg (1R,2R)-(–)-1-epi-spiroaxa-5,7-diene (219).

(1R,2R)-(–)-1-epi-spiroaxa-5,7-diene (219): for NMR and EIMS data see: 7.7.2.

7.7.4. Amberlyst Catalyzed Rearrangement of (–)-allo-Aromadendrene (217)

A solution of 5 mg (–)-allo-aromadendrene (217) in 500 µl DCM was treated with Amberlyst® 15 as

described in 7.7.2. to afford 0.4 mg (1R,2R)-(–)-1-epi-spiroaxa-5,7-diene (219).

(1R,2R)-(–)-1-epi-spiroaxa-5,7-diene (219): for NMR and EIMS data see 7.7.2.


202

7.7.5. Solid Superacid Catalyzed Rearrangement of (+)-Aromadendrene ((+)-216)

A solution of 5 mg (+)-aromadendrene ((+)-216) in 0.5 ml DCM was treated with 10 mg TiO2/SO42-

(precalcinated at 400 °C for 4 h). After 10 min. the reaction mixture was filtered over silica gel and

concentrated in a stream of N2. Flash-column chromatography on silica gel with pentane and semi-

preparative gaschromatography using a DB 1701 thickfilm capillary column (100 to 160 °C at

+ 3 °C/min, 5 min) afforded 2.4 mg (1R,2S)-(–)-ent-spiroaxa-5,7-diene ((–)-206), 1.2 mg (1S,2S)-(–)-

ent-spiroaxa-5,8-diene (221), 0.3 mg (1R,2S)-ent-spiroaxa-4,7-diene (222), and 0.2 mg (1R,2S)-ent-

spiroaxa-4,8-diene (223).

(1R,2S)-(–)-ent-spiroaxa-5,7-diene ((–)-206): C15H24; colourless oil; (–)-sense of optical rotation

(C6D6); RI 1388; 1H NMR (500.1 MHz, C6D6): = 0.90 (3H, d, J = 6.6 Hz, 14-CH3), 0.99 (6H, d, J =

6.9 Hz, 12,13-CH3), 1.40 (1H, m, 3-H), 1.57 (1H, m, 2-H), 1.58 (1H, m, 3-H‟), 1.61 (3H, d, J = 1.0 Hz,

15-CH3), 1.81 (1H, ddd, J = 13.3 Hz, J = 9.1 Hz, J = 5.4 Hz, 10-H), 1.89 (3H, m, 4-H, 4-H‟ 10-H‟),

2.13 (1H, sept, J = 6.9 Hz, 11-H), 2.16 (1H, m, 9-H), 2.21 (1H, m, 9-H‟), 5.11 (1H, s, 7-H), 5.35 (1H,

s, 6-H); 13

C PENDANT (100.6 MHz, C6D6): = 16.9 (2q, 14,15-CH3), 21.6 (q, 12-CH3), 21.9 (q, 13-

CH3), 25.7 (t, 4-CH2), 29.9 (t, 3-CH2), 35.3 (d, 11-CH), 36.5 (t, 9-CH2), 38.0 (d, 2-CH), 38.1 (t, 10-

CH2), 54.4 (s, 1-C), 128.3 (d, 6-CH), 130.2 (d, 7-CH), 139.4 (s, 8-C), 140.4 (s, 5-C); EIMS (70 eV)

m/z (%) = 204 (29) [M], 189 (2), 175 (1), 162 (81), 147 (100), 133 (8), 119 (56), 105 (30), 93 (10), 91

(27), 81 (11), 79 (15), 77 (16), 69 (4), 67 (6), 65 (7), 55 (13), 53 (9), 51 (3), 43 (7), 41 (29), 39 (10).

(1S,2S)-(–)-ent-spiroaxa-5,8-diene (221): C15H24; colourless oil; (–)-sense of optical rotation (C6D6);

RI 1395; 1H NMR (500.1 MHz, C6D6): = 0.90 (3H, d,

3J = 6.6 Hz, 14-CH3), 1.01 (6H, d,

3J = 6.6

Hz, 12,13-CH3), 1.38 (1H, m, 3-H), 1.57 (2H, m, 2,3-H‟), 1.63 (3H, s, 15-CH3), 1.90 (2H, m, 4-CH2),

1.97 (1H, d, 2J = 17.3 Hz, 10-H), 2.13 (1H, sept,

3J = 6.6 Hz, 11-H), 2.23 (1H, d,

2J = 17.3 Hz, 10-H‟),

2.37 (2H, s, 7-H), 5.25 (1H, s, 9-H), 5.55 (1H, s, 6-H); EIMS (70 eV) m/z (%) = 204 (100) [M], 189

(35), 175 (7), 161 (89), 147 (34), 136 (24), 133 (34), 121 (46), 119 (77), 105 (80), 93 (60), 91 (64), 81

(37), 79 (31), 77 (34), 69 (28), 67 (22), 65 (14), 55 (34), 53 (20), 43 (24), 41 (57), 39 (21).

(1R,2S)-ent-spiroaxa-4,7-diene (222): C15H24; colourless oil; RI 1400; 1H NMR (500.1 MHz, C6D6):

= 0.91 (3H, d, 3J = 6.6 Hz, 14- CH3), 1.02 (6H, d,

3J = 6.6 Hz, 12,13-CH3), 1.63 (1H, m, 2-H), 1.66

(1H, m, 10-H), 1.67 (3H, s, 15-CH3), 1.75 (1H, m, 3-H), 1.82 (1H, m, 10-H‟), 1.95 (2H, s, 6-CH2), 2.13

(2H, m, 3-H‟, 11-H), 2.20 (2H, t, J = 7.3 Hz, 9-CH2), 2.92 (1H, s, 7-H), 5.40 (1H, s, 4-H); EIMS (70

eV) m/z (%) = 204 (4) [M], 189 (1), 175 (1), 161 (3), 147 (2), 133 (2), 119 (3), 108 (100), 93 (24), 91

(10), 81 (7), 79 (8), 77 (6), 67 (3), 65 (2), 55 (4), 53 (4), 41 (10).

(1R,2S)-ent-spiroaxa-4,8-diene (223): C15H24; colourless oil; RI 1405; 1H NMR (500.1 MHz, C6D6):

= 0.87 (3H, d, 3J = 6.6Hz, 14-CH3), 0.99 (6H, d,

3J = 6.6 Hz, 12,13-CH3), 1.61 (1H, m, 2-H), 1.65

(3H, s, 15-CH3), 1.76 (1H, d.br, 2J = 17.3 Hz, 3-H), 1.84 (1H, d,

2J = 17.0 Hz, 10-H), 1.93 (1H, d,

2J =

17.0 Hz, 6-H), 2.00 (1H, d, 2J = 16.7 Hz, 6-H‟), 2.12 (1H, m, 1-H), 2.17 (2H, m, 3,10-H), 2.31 (1H,

d.br, 2J = 18.3 Hz, 7-H), 5.24 (1H, s, 9-H), 5.36 (1H, s, 4-H); EIMS (70 eV) m/z (%) = 204 (23) [M],

189 (5), 161 (18), 148 (9), 136 (9), 133 (8), 120 (100), 107 (42), 105 (42), 93 (93), 91 (37), 81 (17), 79

(25), 77 (26), 69 (11), 67 (11), 65 (9), 55 (18), 53 (13), 51 (4), 43 (21), 41 (38).

7.7.6. Solid Superacid Catalyzed Rearrangement of (+)-Ledene (218)

A solution of 1 mg (+)-ledene (218) in 0.5 ml DCM was treated with 2 mg TiO2/SO42–

as described in

7.7.5. to afford 0.3 mg (1R,2R)-(–)-1-epi-spiroaxa-5,7-diene (219). Minor amounts of 1-epi-spiroaxa-

5,8-diene (224), 1-epi-spiroaxa-4,7-diene (225), and 1-epi-spiroaxa-4,8-diene (226) were also detected

by GC-MS.



203

7.7.7. Solid Superacid Catalyzed Rearrangement of (–)-allo-Aromadendrene (217)

A solution of 1 mg (–)-allo-aromadendrene (217) in 0.5 ml DCM was treated with 2 mg TiO2/SO42–

as

described in 7.7.5. to afford 0.5 mg (1R,2R)-(–)-1-epi-spiroaxa-5,7-diene (219). Minor amounts of

1-epi-spiroaxa-5,8-diene (224), 1-epi-spiroaxa-4,7-diene (225), and 1-epi-spiroaxa-4,8-diene (226)

were also detected by GC-MS.


7.7.8. Isolation of (–)-ent-Aromadendrene ((–)-216) from Pellia epiphylla

The essential oil of Pellia epiphylla (L.) Corda, Metzgeriales, obtained by hydrodistillation of the

fresh plant material, was chromatographed on silica gel using a hexane - Et2O gradient. From the

hydrocarbon fraction about 1 mg (–)-ent-aromadendrene ((–)-216) was isolated by consecutive

preparative GC using 6-TBDMS-2,3-Me- -CD (110 °C, + 1 °C/min, to 130 °C) and 2,6-Me-3-Pe- -

CD (120 °C, + 1 °C/min, to 140 °C) as stationary phase. EIMS and NMR data were identical to those

previously reported (Joulain & König, 1998).

7.7.9. Solid Superacid Catalyzed Rearrangement of (–)-ent-Aromadendrene ((–)-216)

A solution of 2 mg (–)-ent-aromadendrene ((–)-216) in 100 µl DCM was treated with 4 mg TiO2/SO42–

as described in 7.7.5. to afford 0.8 mg (1S,2R)-(+)-spiroaxa-5,7-diene ((+)-206) identical to the

compound obtained from (–)- -cubebene. Traces of the corresponding (1R,2R)-(+)-spiroaxa-5,8-diene

(221), (1S,2R)-spiroaxa-4,7-diene (222), and (1S,2R)-spiroaxa-4,8-diene (223) were also detected by

GC-EIMS.

(1S,2R)-(+)-spiroaxa-5,7-diene ((+)-206): C15H24; colourless oil; (+)-sense of optical rotation (C6D6);

for EIMS and NMR data see: (1R,2S)-(–)-ent-206 in 7.7.5.

7.8. Serratia odorifera - Synthesis of Octamethylbicyclo[3.2.1]octadienes

7.8.1. Synthesis of 2,4-Dibromopentan-3-one (232)

A mixture of 25.5 g pentan-3-one (231, 0.26 mol) at 0 °C was treated with 0.5 g phosphorous

tribromide and 80 g bromine (0.5 mol) is added drop wise. Evolving hydrogen bromide is trapped in

sodium hydroxide solution. At the the end of the bromine addition the flask is evacuated and

immediately fractionally distilled under reduced pressure to give 44.4 g 2,4-dibromopentan-3-one

(232, 0.18 mol, 70 % yield) as a mixture of DL- and meso-isomers.

2,4-dibromopentan-3-one (232): C5H8OBr2, colourless liquid; 1H NMR (500 MHz, CDCl3): = 1.81

(6H, d, J = 6.6 Hz, CH3), 4.99 (2H, d, J = 6.6 Hz, CH) and 1.88 (6H, d, J = 6.9 Hz, CH3), 4.78 (2H, J

= 6.9 Hz, CH); 13

C PENDANT (100 MHz, CDCl3): = 20.1 (q, CH3), 44.3 (d, CH), 196.3 (s, CO) and

22.2 (q, CH3), 44.5 (d, CH), 198.7 (s, CO); EIMS (70 eV) m/z (%) = 244 (4) [M], 137 (38), 135 (40),

109 (64), 107 (67), 56 (100).

7.8.2. 1,2,4,5,6,7,8-Heptamethylbicyclo[3.2.1]oct-6-en-3-ones (236 - 238)

Under N2 atmosphere a solution of 272 mg 1,2,3,4,5-pentamethylcyclopentadiene (235, 2 mmol) in

1.4 ml acetonitrile (dried over mol sieve 3 A and distilled) was treated with 480 mg sodium iodide

(3.2 mmol; dried at 150 °C for 24 h and powdered) and 160 mg copper powder (2.4 mmol). A solution

of 200 mg 2,4-dibromopentan-3-one (232, 0.8 mmol) in 600 µl acetonitrile was added drop wise and

the resulting mixture stirred for 4 h. 10 ml diethyl ether were added and the resulting mixture added to


204

10 ml ice cold 15 % ammonia solution. The organic phase was separated and the aqueous phase

extracted with 3 x 5 ml diethyl ether. The combined organic phases were washed with > 3 x 10 ml ice

cold 5 % ammonia solution, until free of blue coloured copper salts and with > 5 x 10 ml ice-water

until pH was neutral. The organic phase was dried over sodium sulfate and the solvent removed in a

rotary vacuum evaporator under nitrogen at 30 °C at reduced pressure. The residue was cooled to 0°C,

the flask filled with nitrogen, and the residue was taken up in 1 ml pentane. Upon storage at – 20 °C

bisequatorial-238 precipitated. The solution was fractionated by column chromatography on silica gel

at 0 °C using pentane to afford 39 mg meso-8-anti-2,4-bisaxial-1,2,4,5,6,7,8-heptamethylbicyclo-

[3.2.1]oct-6-en-3-one (236, 0.18 mmol, 22 % yield) and 90 mg meso-8-anti-2,4-bisequatorial-

1,2,4,5,6,7,8-heptamethylbicyclo[3.2.1]oct-6-en-3-one (238, 0.4 mmol, 50 % yield).

meso-8-anti-2,4-bisaxial-1,2,4,5,6,7,8-heptamethylbicyclo[3.2.1]oct-6-en-3-one (236): C15H24O,

colourless liquid; 1H NMR (500 MHz, C6D6): = 0.63 (3H, d, J = 6.6 Hz, 16-CH3), 0.66 (6H, s, 9-

CH3, 13-CH3), 1.00 (6H, d, J = 7.6 Hz, 10-CH3, 12-CH3), 1.26 (6H, s, 14-CH3, 15-CH3), 2.03 (1H, q, J

= 6.7 Hz, 8-H), 2.16 (2H, q, J = 7.2 Hz, 2-CH, 4-CH). 13

C PENDANT (100 MHz, C6D6): = 9.5 (q,

14-CH3, 15-CH3), 10.7 (q, 16-CH3), 15.1 (q, 9-CH3, 13-CH3), 16.8 (q, 10-CH3, 12-CH3), 40.5 (d, 8-

CH), 49.8 (s, 1-C, 5-C), 51.9 (d, 2-CH, 4-CH), 136.1 (s, 6-C, 7-C), 216.0 (s, 3-C); EIMS (70 eV) m/z

(%) = 220 (19) [M], 205 (1), 163 (16), 149 (9), 136 (54), 135 (61), 134 (100), 121 (23), 119 (14), 105

(16), 91 (15), 85 (7), 77 (4), 67 (5), 55 (8).

meso-8-anti-2,4-bisequatorial-1,2,4,5,6,7,8-heptamethylbicyclo[3.2.1]oct-6-en-3-one (238):

C15H24O, white crystals; 1H NMR (500 MHz, C6D6): = 0.64 (3H, d, J = 6.9 Hz, 16-CH3), 0.81 (6H,

s, 9-CH3, 13-CH3), 1.14 (6H, d, J = 6.9 Hz, 10-CH3, 12-CH3), 1.37 (6H, s, 14-CH3, 15-CH3), 1.56 (1H,

q, J = 6.9 Hz, 8-H), 2.03 (2H, q, J = 6.9 Hz, 2-CH, 4-CH). 13

C PENDANT (100 MHz, C6D6): = 11.3

(q, 14-CH3, 15-CH3), 11.9 (q, 16-CH3), 12.8 (q, 9-CH3, 13-CH3), 16.9 (q, 10-CH3, 12-CH3), 52.2 (s, 1-

C, 5-C), 56.4 (d, 2-CH, 4-CH), 58.9 (d, 8-CH), 134.7 (s, 6-C, 7-C), 210.7 (s, 3-C=O); EIMS (70 eV)

m/z (%) = 220 (19) [M], 205 (1), 163 (16), 149 (9), 136 (54), 135 (61), 134 (100), 121 (23), 119 (14),

105 (16), 91 (15), 85 (7), 77 (4), 67 (5), 55 (8).

(±)-8-anti-2-equatorial-4-axial-1,2,4,5,6,7,8-heptamethylbicyclo[3.2.1]oct-6-en-3-one (237):

C15H24O, colourless oil; 1H NMR (500 MHz, C6D6): = 0.64 (3H, d, J = 6.9 Hz, 16-CH3), 0.65 (3H, s,

13-CH3), 0.83 (3H, s, 9-CH3), 0.97 (3H, d, J = 7.6 Hz, 12-CH3), 1.12 (3H, d, J = 7.2 Hz, 10-CH3), 1.27

(3H, s, 15-CH3), 1.39 (3H, s, 14-CH3), 1.83 (1H, q, J = 6.9 Hz, 8-H), 2.07 (1H, q, J = 7.2 Hz, 2-CH),

2.26 (1H, q, J = 7.0 Hz, 4-CH); 13

C PENDANT (100 MHz, C6D6): = 9.7, 10.9 (q, 14-CH3, 15-CH3),

11.4 (q, 16-CH3), 13.4 (q, 9-CH3, 13-CH3), 16.5, 16.9 (q, 10-CH3, 12-CH3), 50.1, 52.1 (s, 1-C, 5-C),

49.7 (d, 8-CH), 52.6, 53.3 (d, 2-CH, 4-CH), 133.9, 137.7 (s, 6-C, 7-C), 213.3 (s, 3-C=O); EIMS (70

eV) m/z (%) = 220 (19) [M], 205 (1), 163 (16), 149 (9), 136 (54), 135 (61), 134 (100), 121 (23), 119

(14), 105 (16), 91 (15), 85 (7), 77 (4), 67 (5), 55 (8).

7.8.3. bisaxial-1,2,4,5,6,7,8-Heptamethyl-3-methylenebicyclo[3.2.1]oct-6-ene (239)

Under N2 atmosphere a solution of 220 mg meso-8-anti-2,4-bisaxial-1,2,4,5,6,7,8-heptamethyl-

bicyclo[3.2.1]oct-6-en-3-one (236, 1 mmol) in 5 ml THF at 0 °C was treated with 3 ml 0.5 M Tebbe

reagent (1.5 mmol) in toluene. After stirring for 1 h the mixture was treated with 10 ml diethyl ether

and 5 ml 1 M sodium hydroxide solution, the organic phase washed with water, dried over Na2SO4,

and concentrated in vacuum. Column chromatography on silica gel using pentane afforded 170 mg

meso-8-anti-2,4-bisaxial-1,2,4,5,6,7,8-heptamethyl-3-methylenebicyclo[3.2.1]oct-6-ene (239, 780

µmol, 78 % yield).

meso-8-anti-2,4-bisaxial-1,2,4,5,6,7,8-heptamethyl-3-methylenebicyclo[3.2.1]oct-6-ene (239):

C16H26, white solid; 1H

NMR (500 MHz, C6D6): = 0.73 (3H, d, J = 6.9 Hz, 16-CH3), 0.83 (6H, s, 9-

CH3, 13-CH3), 1.12 (6H, d, J = 7.3 Hz, 10-CH3, 12-CH3), 1.41 (6H, s, 14-CH3, 15-CH3), 2.04 (1H, q, J


205

= 6.9 Hz, 8-H), 2.08 (2H, q, J = 7.2 Hz, 2-CH, 4-CH), 4.63 (2H, s, 11-H); 13

C PENDANT (100 MHz,

C6D6): = 9.6 (q, 14-CH3, 15-CH3), 11.0 (q, 16-CH3), 17.7 (q, 9-CH3, 13-CH3), 19.5 (q, 10-CH3, 12-

CH3), 39.9 (d, 8-CH), 44.1 (d, 2-CH, 4-CH), 51.5 (s, 1-C, 5-C), 111.1 (t, 11-CH2), 134.2 (s, 6-C, 7-C),

156.0 (s, 3-C); EIMS (70 eV) m/z (%) = 218 (30) [M], 203 (8), 189 (3), 162 (11), 148 (4), 136 (100),

135 (89), 134 (70), 121 (28), 105 (18), 91 (14), 83 (28), 67 (9), 55 (18), 41 (21), HREIMS (70 eV):

obs. m/z 218.2044 [M], calc. for C16H26: 218.2035, 0.9 mmu.

7.8.4. bisequatorial-1,2,4,5,6,7,8-Heptamethyl-3-methylenebicyclo[3.2.1]oct-6-ene (241)

Under N2 atmosphere a solution of 220 mg meso-8-anti-2,4-bisequatorial-1,2,4,5,6,7,8-

heptamethylbicyclo[3.2.1]oct-6-en-3-one (238, 1 mmol) in 5 ml THF at 0 °C was treated with 3 ml

0.5 M Tebbe reagent (1.5 mmol) in toluene. After stirring for 1 h the mixture was treated with 10 ml

diethyl ether and 5 ml 1 M sodium hydroxide solution, the organic phase washed with water, dried

over Na2SO4, and concentrated in vacuum. Column chromatography on silica gel using pentane

afforded 185 mg meso-8-anti-2,4-bisequatorial-1,2,4,5,6,7,8-heptamethyl-3-methylenebicyclo[3.2.1]-

oct-6-ene (241, 850 µmol, 85 % yield).

meso-8-anti-2,4-bisequatorial-1,2,4,5,6,7,8-heptamethyl-3-methylenebicyclo[3.2.1]oct-6-ene (241):

C16H26, colourless oil; 1H NMR (500 MHz, C6D6): = 0.73 (3H, d, J = 6.6 Hz, 16-CH3), 0.92 (6H, s,

9-CH3, 13-CH3), 1.07 (6H, d, J = 6.9 Hz, 10-CH3, 12-CH3), 1.47 (6H, s, 14-CH3, 15-CH3), 1.52 (1H, q,

J = 6.6 Hz, 8-H), 2.10 (2H, q, J = 6.9 Hz, 2-CH, 4-CH), 4.88 (2H, s, 11-H); 13

C PENDANT (100

MHz, C6D6): = 12.4 (q, 14-CH3, 15-CH3), 14.3 (q, 16-CH3), 15.3 (q, 9-CH3, 13-CH3), 17.2 (q, 10-

CH3, 12-CH3), 59.7 (d, 8-CH), 47.7 (d, 2-CH, 4-CH), 52.4 (s, 1-C, 5-C), 106.2 (t, 11-CH2), 133.1 (s, 6-

C, 7-C), 157.0 (s, 3-C); EIMS (70 eV) m/z (%) = 218 (30) [M], 203 (8), 189 (3), 162 (11), 148 (4),

136 (100), 135 (89), 134 (70), 121 (28), 105 (18), 91 (14), 83 (28), 67 (9), 55 (18), 41 (21).

7.8.5. 1,2,3,4,5,6,7,8-Octamethylbicyclo[3.2.1]oct-6-en-3-ol (242)

Under N2 atmosphere a solution of 440 mg meso-8-anti-2,4-bisequatorial-1,2,4,5,6,7,8-

heptamethylbicyclo[3.2.1]oct-6-en-3-one (238, 2 mmol) in 5 ml THF at 0 °C was treated with 2 ml

1.6 M methyl lithium (3.2 mmol) in diethyl ether. After stirring for 4 h the mixture was poured onto

20 cm3 ice and extracted with 3 x 20 ml diethyl ether. The organic phase was washed with water, dried

over Na2SO4 and concentrated in vacuum. Column chromatography afforded 260 mg meso-8-anti-2,4-

bisequatorial-1,2,3,4,5,6,7,8-octamethylbicyclo[3.2.1]oct-6-en-3-ol (242, 1.1 mmol, 55 % yield) as a

40:1 mixture of the diastereoisomers.

meso-8-anti-2,4-bisequatorial-1,2,3,4,5,6,7,8-octamethylbicyclo[3.2.1]oct-6-en-3-ol (242): C16H28O,

white crystals; 1H NMR (500 MHz, C6D6): = 0.65 (3H, d, J = 6.9 Hz, 16-CH3), 0.81 (6H, s, 9-CH3,

13-CH3), 1.10 (6H, d, J = 7.3 Hz, 10-CH3, 12-CH3), 1.21 (3H, s, 11-CH3), 1.29 (1H, q, J = 6.9 Hz, 8-

H), 1.42 (2H, q, J = 7.3 Hz, 2-CH, 4-CH). 1.43 (6H, s, 14-CH3, 15-CH3); 13

C PENDANT (100 MHz,

C6D6): = 11.7 (q, 10-CH3, 12-CH3), 12.1 (q, 16-CH3), 13.1 (q, 14-CH3, 15-CH3), 17.3 (q, 9-CH3, 13-

CH3), 28.3 (q, 11-CH3), 50.7 (s, 1-C, 5-C), 50.9 (d, 2-CH, 4-CH), 60.4 (d, 8-CH), 75.1 (s, 3-COH),

136.1 (s, 6-C, 7-C); EIMS (70 eV) m/z (%) = 236 (24), 218 (4), 203 (2), 189 (1), 163 (85), 152 (100),

149 (48), 136 (67), 134 (72), 121 (70), 105 (32), 91 (29), 79 (17), 73 (17), 67 (11), 55 (24), 43 (34).


206

7.8.6. 1,2,3,4,5,6,7,8-Octamethylbicyclo[3.2.1]octa-2,6-diene (243) and

1,3,4,5,6,7,8-Heptamethyl-2-methylenebicyclo[3.2.1]oct-6-ene (244)

A solution of 22 mg meso-8-anti-2,4-bisequatorial-1,2,3,4,5,6,7,8-octamethylbicyclo- [3.2.1]oct-6-en-

3-ol (100 µmol) in 5 ml hexane was treated with ca. 50 mg acidic ion exchange resin Amberlyst® 15.

After stirring for 4 h the mixture was filtered. Column chromatography on silica gel using pentane and

preparative GC using a SE 30 column (120 °C, isotherm) afforded 12 mg (55 % yield) of a 3:2 mixture

of (±)-8-anti-4-equatorial-1,2,3,4,5,6,7,8-octamethylbicyclo[3.2.1]octa-2,6-diene (243) and (±)-8-anti-

4-equatorial-1,3,4,5,6,7,8-heptamethyl-2-methylenebicyclo[3.2.1]oct-6-ene (244).

(±)-8-anti-4-equatorial-1,2,3,4,5,6,7,8-octamethylbicyclo[3.2.1]octa-2,6-diene (243): C16H26,

colourless oil; 1H NMR (500 MHz, C6D6): = 0.78 (3H, d, J = 7.3 Hz, 16-CH3), 0.86 (3H, d, J = 6.9

Hz, 12-CH3), 0.96, 0.97 (6H, s, 9,13-CH3), 1.51, 1.52 (6H, s, 14,15-CH3), 1.56, (6H, s, 11-CH3), 1.68

(6H, s, 10-CH3), 1.76 (1H, q, J = 7.2 Hz, 8-CH), 1.84 (1H, q, J = 6.9 Hz, 4-CH); 13

C PENDANT (100

MHz, C6D6): = 11.2 (q, 14/15*-CH3), 12.2 (q, 16-CH3), 13.3 (q, 14/15*-CH3), 14.6 (q, 12-CH3), 15.6

(q, 10-CH3), 16.0 (q, 9,13*-CH3), 17.7 (q, 11-CH3), 18.3 (q, 9/13*-CH3), 47.7 (q, 8-CH), 50.2, 51.0 (s,

1,5-C), 55.9 (q, 4-CH), 127.8 (s, 3-C), 129.2 (s, 6-C), 136.5 (s, 2-C), 140.0 (s, 7-C) [*

interchangeable]; EIMS (70 eV) m/z (%) = 218 (18) [M], 203 (3), 163 (28), 162 (41), 147 (100), 135

(21), 119 (5), 105 (5), 91 (8), 77 (3), 55 (5), 41 (8).

(±)-8-anti-4-equatorial-1,3,4,5,6,7,8-heptamethyl-2-methylenebicyclo[3.2.1]oct-6-ene (244):

C16H26, colourless oil; 1H NMR (500 MHz, C6D6): = 0.69 (3H, d, J = 7.3 Hz, 16-CH3), 0.78 (6H, d, J

= 7.3 Hz, 11,12-CH3), 0.96 (3H, s, 13-CH3), 1.11 (3H, s, 9-CH3), 1.21 (1H, m, 4-CH), 1.56 (3H, s, 14-

CH3), 1.73 (3H, s, 15-CH3), 1.75 (1H, m, 3-CH), 1.83 (1H, q, J = 7.3 Hz, 8-CH), 4.71, 5.07 (2H, s,

CH2); 13

C PENDANT (100 MHz, C6D6): = 8.9, 11.2 (q, 11,12-CH3), 12.2 (q, 16-CH3), 14.2 (q, 15-

CH3), 17.8 (q, 14-CH3), 21.9 (q, 13-CH3), 23.6 (q, 9-CH3), 44.0 (d, 8-CH), 46.8 (d, 4-CH), 50.0 (s, 5-

C), 51.4 (s, 1-C), 54.9 (d, 3-CH), 106.5 (t, 10-CH2), 127.8 (s, 7-C), 136.1 (s, 6-C), 150.9 (s, 2-C);

EIMS (70 eV) m/z (%) = 218 (100) [M], 203 (74), 189 (13), 173 (8), 163 (31), 147 (40), 136 (92),

121(33), 105 (20), 97 (18), 91 (20), 83 (59), 77 (10), 69 (12), 55 (33), 41 (27).

8. Hazardous chemicals

207


Risk – phrases Safety – phrases

acetic acid C 10-35 23.2-26-45

acetic anhydride C 10-34 26-45

acetic anhydride-d6 C 10-34 26-45

acetone F 11 9-16-23.2-33

acetone-d6 F 11 9-16-23.2-33

acetonitrile F, T 11-23/24/25 16-27-45

acetonitrile-d3 F, T 11-23/24/25 16-27-37-45

acetylchloride F, C 11-14-34 9-16-26-45

benzaldehyde Xn 22 24

benzene F, T 45-11 E48/23/24/25 53-45

benzene-d6 F, T 45-11 E48/23/24/25 53-45

1,4-benzoquinone T 23/25-36/37/38 26-28.1-45

benzylamine C 34 26-45

boron tribromide 1M in DCM C, T+ 14-26/28-35 9-26-28.6-36/37/39-45

bromine C, T+ 26-35 7/9-26-45

n-butyl lithium 1.6 M in hexane F, C 11-14/15-17-34-48/20 6.1-7/9-26-33-36/37/39-45

carbondisufide F, T 11-36/38-48/23-62-63 16-33-36/37-45

N-chlorosuccinimide Xi 38

cyclohexane F 11 9-16-33

cyclohexane-d12 F 11 9-16-33

cyclohexylamine C 10-21/22-34 36/37/39-45

deuterochloric acid C 34-37 26-36/37/39-45

deuterium (gas) F+ 12 2-9-16-33

2,3-dichloro-5,6-dicyano-1,4-

quinone (DDQ)

T 25-29 45

dichloromethane (DCM) Xn 40 23.2-24/25-36/37

diethyl ether F 12-19 9-16-29-33

dimethyl sulfate T+

45-E25-E26-34 53-45

dimethylsulfoxide - - 24-25

dimethylsulfoxide-d6 - - 24-25

1,4-dioxane F, Xn 11-19-36/37-40 16-36/37

ethanol F 11 7-16

ethanol-d1 F 11 7-16

ethyl acetate F 11 16-23.2-29-33

N-ethyldiisopropylamin F, Xi 11-36/38 -

ferrumtrichloride hexahydrate Xn 22-38-41 26-39

fumaric acid Xi 36 26

hexane F, Xn 11-48/20 9-16-24/25-29-51

hydrochloric acid C 34-37 26-36/37/39-45

hydrogen (gas) F+ 12 2-9-16-33

indole Xn 21/22 36/37

methyl iodide T 21-23/25-37-38-40 36/37-38-45

[D3]-methyl iodide T 21-23/25-37-38-40 36/37-38-45

lithiumaluminiumhydride F 15 7/8-24/25-43.6

malonic acid Xn 22-36 22-24

methanol F, T 11-23/25 7-16-24-45

methanol-d4 F, T 11-23/25 7-16-24-45

methanthiol (gas) F, Xn 12-20 16-25


208

Risk – phrases Safety – phrases

4-methoxybenzylalcohole Xn 22 24

4-methoxyphenylethanone Xn 22-38 -

methylithium in Et2O F, C 11-15-34 16-26-36/37/39-43-45

nitromethane Xn 5-10-22 41

pentane F 11 9-16-29-33

3-pentanone F 11 9-16-33

pentamethylcyclopentadiene - 10 -

phosgene T+ 26 7/9-24/25/45

phosphor tribromide C 14-34-37 26-45

piperidine F, T 11-23/24-34 16-26-27-45

potassium (metal) F, C 14/15-34 5.3-8-43.6-45

potassium carbonate Xn 22-36 22-26

potassium hydroxide C 35 26-37/39-45

propan-1-ol F 11 7-16

propan-2-ol F 11 7-16

pyridine F, Xn 11-20/21/22 26-28.1

sodium (metal) F, C 14/15-34 5.3-8-43.7-45

sodium azide T+

28-32 28.1-45

sodium carbonate Xi 36 22-26

sodium hexamethyldisilylamide C 11-34 16-26-33-36/37/39-45

sodium hydride F, C 15-34 7/8-26-36/37/39-43.6-45

sodium hydroxide C 35 26-37/39-45

sodium methanolate T 10-23/25-34 16-26-45

sodium methanethiolate C 29-34 26-36/37/39-45

sulfuric acid C 35 26-30-45

Tebbes reagent F, C 11-20-34 16-26-29-33-36/37/39-45

tetrahydrofurane (THF) F, Xi 11-19-36/37 16-29-33

tetramethylsilane (TMS) F 12 9-16-29-43.3

thiophosgene T 22-23-36/37/38 7-9-36/37-45

thionylchloride C 14-34-37 26-45

toluene F, Xn 11-20 16-25-29-33

trichloromethane Xn 22-38-40-48/20/22 36/37

trichloromethane-d1 Xn 22-38-40-48/20/22 36/37

triethylamine F, Xi 11-36/37 16-26-29

triethylphosphite Xi 10-36/37/38 26-36

triphosgene T+

26 7/9-24/25-45

tyramine Xi 36/37/38 26-36

9. Colour Plates

210

9. Colour Plates

Plate 1: Fossombronia angulosa thalli (a), sporogon (b), spores and elaters (c), spore (d),

and thallus cells showing chloroplasts and highly refractive oil bodies (e).

◄oil body ◄chloroplasts

25 µm

100 µm

a 1 cm b

d c

e

5 mm

25 µm

9. Colour Plates

211

Plate 2: Riccardia chamedryfolia (With.) Grolle from La Palma.

Plate 3: Oil bodies of Riccardia chamedryfolia (With.) Grolle from Palma

(composite image from two slides)

1 mm

25 µm

9. Colour Plates

212

Plate 4: Gametangia and sporangia of dioecious haploid Corsinia coriandrina from Palma.

Plate 5: Air pores and oil bodies in the thallus surface section of Corsinia coriandrina.

◄oil body

◄oil body

◄air pore

◄air pore

◄archegonium

▼sporangium

(diploid)

female

male

5 mm

antheridia ▼

9. Colour Plates

213

Plate 6: Cronisia weddellii (Mont.) Grolle from Brazil (rehydrated herbarium material).

Plate 7: Collection of Corsinia coriandrina

spores located at the decaying thallus end.

Plate 8: SEM images of Corsinia coriandrina

spores (distal, medial, and proximal face).

50 µm

9. Colour Plates

214

Plate 9: Germination of Corsinia coriandrina spore (left and enlargement) and small callus with

rhizoids (right) on Knop agar supplemented with antibiotics.

Plate 10: Establishment, regeneration and growth of monoclonal Corsinia coriandrina strain CC I

within 3 months on MSK/5 agar supplemented with 25 mM D-glucose.

◄ rhizoids

◄ callus spore ►

1 mm 1 mm

500 µm

9. Colour Plates

215

Plate 11: Corsinia coriandrina thalli grown on MSK/5 agar with 25 mM D-glucose (left)

and dedifferentiated calli grown on MSK/5 agar with 50 mM D-glucose (right).

Plate 12: Different plant materials of Corsinia coriandrina strain CC1 from aerated liquid in vitro

cultures using MSKP/5 medium supplemented with 25 mM D-glucose and 2.5 mM potassium

fumarate; undifferentiated globular propagules (left) and differentiated thalli (right).

1 c m

1 cm 1 cm

1 c m

9. Colour Plates

216

Plate 13: Differentiated thalli of Corsinia coriandrina strain CC1 from in vitro cultures using

MSKP/5 medium supplemented with 25 mM D-glucose and 2.5 mM potassium fumarate under TIS

conditions using the Recipient for Automated Temporary Immersion (RITA®).

Plate 14: Recipient for Automated Temporary Immersion (RITA®).

9. Colour Plates

217

Plate 15: Cross section of Corsinia coriandrina thallus from liquid in vitro cultures.

Plate 16: UV fluorescence microscopy of Corsinia coriandrina thallus slide showing fluorescent

and non-fluorescent oil bodies (arrows); and UV-B (350 nm) induced fluorescence of

chlorophylls (chl) and (E)-corsistilbene ((E)-68) isolated from Corsinia coriandrina.

▼ oil body

rhizoid ▲

air chamber ▼

scale ▲

chl (E)-68

chl (E)-68

500 µm

10. References

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240

Poster Presentations

Saritas, Y., von Reuß, S. H., König, W. A.; A reinvestigation of the essential oil of Petasites

hybridus. 29th

International Symposium on Essential Oils (ISEO 1998), Frankfurt, Germany.

von Reuß, S. H., Wu, C.-L., König, W. A.; Sesquiterpene constituents in Mylia taylorii and

Mylia nuda (Hepaticae). 32nd

International Symposium on Essential Oils (ISEO 2003),

Würzburg, Germany.

von Reuß, S. H., König, W. A.; Coridrin und Coriandrin - zwei schwefel- und

stickstoffhaltige Naturstoffe aus dem Lebermoos Corsinia coriandrina (Marchantiales). 16.te

Irseer Naturstofftage der Dechema e.V., Kloster Irsee, Germany. Febuary 2004

von Reuß, S. H., Wu, C.-L., Muhle, H., König, W. A.; New sesquiterpenoids from Mylia

taylorii and Mylia nuda (Hepaticae). Future Trends in Phytochemistry, Garngano, Italy. May

2004.

McLeod, G., Gries, R., von Reuß, S. H., König, W. A., McIntosh, R. L., Gries, G.; Dutch Elm

Disease: how a pathogen solves its transportation problem. Entomological Society of British

Columbia; Annual Meeting, Simon Fraser University, Burnaby, Canada. October 2004.

McLeod, G., Chatterton, M., Gries, R., von Reuß, S. H., Schaefer, P., Rahe, J., König, W. A.,

McIntosh, R., Higashiura, Y., Möller, K., Engelmann, A., Plettner, E., Gries, G.;

Communication Ecology of Dutch Elm Disease and Lymantriid Mots. 5th Asia Pacific

Congress of Entomology, APCE; Insects, Nature and Humans, Jeju, Korea, October 2005.

241

Oral Presentations

von Reuß, S. H.; New natural products from the liverworts Mylia taylorii, Mylia nuda and

Corsinia coriandrina. Department of Organic Chemistry, Tamkang University, Tamsui,

Taiwan, December 3, 2003.

von Reuß, S. H, König, W. A.; Coridrin und Coriandrin - zwei schwefel- und stickstoffhaltige

Naturstoffe aus dem Lebermoos Corsinia coriandrina. 16. Irseer Naturstofftage der Dechema

e.V., Kloster Irsee, Germany, Febuary 26, 2004.

von Reuß, S. H.; Wu, C.-L., Muhle, H., König, W. A.; New sesquiterpenoids from Mylia

taylorii and Mylia nuda (Hepaticae). Future Trends in Phytochemistry, Garngano, Italy, May

7, 2004.

von Reuß, S. H.; Identification of New Secondary Metabolites and Investigation of their

Biogenesis using Stable Isotope Labelled Precursors and Axenic In Vitro Cultures.

Department of Biology, Biozentrum Klein Flottbek, University of Hamburg, Germany,

August 31, 2006.

von Reuß, S. H; von Schwartzenberg, K.; König, W. A.; Arylethenyl isothiocyanates and

related compounds from the liverwort Corsinia coriandrina, Marchantiales. Glucosinolate

Biology, Chemistry and Biochemistry, and its Application to Human Health and Agriculture,

Jena, Germany, September 13, 2006.

von Reuß, S. H.; Octamethylbicyclo[3.2.1]octadiene aus Serratia odorifera – Identifizierung

und Synthese. Institut für Biowissenschaften, University of Rostock, Germany, April 1, 2009.

von Reuß, S. H.; von Schwartzenberg, K.; König, W. A.; New Secondary Metabolites from

Liverworts and Micro-organisms. Identification, Synthesis and Investigation of their

Biosynthesis. Boyce Thompson Institute for Plant Research, Cornell University, Ithaca, USA,

April 3, 2009.

242

Publications

Saritas, Y., von Reuß, S. H., König, W. A., 2002. Sesquiterpene constituents in Petasites

hybridus. Phytochemistry 59, 795-803

von Reuß, S. H., Wu, C.-L., Muhle, H., König, W. A., 2004. Sesquiterpene constituents from

the essential oils of the liverworts Mylia taylorii and Mylia nuda. Phytochemistry 65, 2277-

2291.

von Reuß, S. H., König, W. A., 2004. Corsifurans A–C, 2-arylbenzofurans of presumed

stilbenoid origin from Corsinia coriandrina (Hepaticae). Phytochemistry 65, 3113-3118.

von Reuß, S. H., König, W. A., 2005. Olefinic Isothiocyanates and Iminodithiocarbonates

from the Liverwort Corsinia coriandrina. European Journal of Organic Chemistry, 1184-

1188.

McLeod, G., Gries, R., von Reuß, S. H., Rahe, J. E., McIntosh, R., König, W. A., Gries, G.,

2005. The pathogen causing Dutch elm disease makes host trees attract insect vectors.

Proceedings of the Royal Society B 272, 2499-2503.

Schiestel, F.P., Steinebrunner, F., Schulz, C., von Reuß, S., Francke, W., Weymuth, C.,

Leuchtmann, A., 2006. Evolution Of 'Pollinator'-Attracting Signals In Fungi. Biology Letters

2, 401-404.

Baran, S., von Reuß, S. H., König, W. A., Kalemba, D., 2006. Composition of the Essential

Oil of Abies koreana Wils. Flavor and Fragrance Journal 22, 78-83.

Adio, A. M., von Reuß, S. H., Paul, C., Muhle, H., König, W. A., 2007. Sesquiterpenoid

constituents of the liverwort Marsupella aquatica. Tetrahedron: Asymmetry 18, 1245-1253.

Adams, H., Gilmore, N. J., Jones, S., Muldowney, M. P., von Reuß, S. H., Vemula, R., 2008.

Asymmetric synthesis of corsifuran A by an enantioselective oxazaborolidine reduction. Org.

Lett. 10, 1457 -1460.

243

Höckelmann, C., Becher, P., von Reuß, S.H., Jüttner, F., 2009. Sesquiterpenes of the

Geosmin-Producing Cyanobacterium Calothrix PCC 7507 and their Toxicity to Invertebrates.

Z. Naturforsch. 64c, 49-55.

244

CURRICULUM VITAE

Name Stephan Heinrich von Reuß

Date of Birth May 16. 1975

Place of Birth Dortmund, Germany

Nationality German

Institutions Attended

1980 – 1985 Primary school, Ernst-Abbe Schule, Kaufungen

1985 – 1991 Secondary school, Integrierte Gesamtschule, Kaufungen

1991 – 1994 Secondary school, Herderschule, Kassel, Abitur examinations in 1994.

1994 – 1998 Undergraduate studies in Chemistry at the University of Hamburg,

Vordiploma exams in Febuary 1998.

1998 – 2002 Undergraduate studies concluded by Diploma exams in December 2002.

2002 – 2003 Diploma Thesis in Organic Chemistry under the supervision of

Prof. W. A. König, University of Hamburg, Germany

Isolierung, Strukturaufklärung und Synthese sekundärer Naturstoffe aus den

Lebermoosen Mylia taylorii, Mylia nuda und Corsinia coriandrina

2004 – 2008 Doctoral Thesis in Organic Chemistry started under the supervision of

Prof. W. A. König (deceased November 19. 2004) and continued under the

supervision of Prof. W. Francke, University of Hamburg, Germany

Structure Elucidation and Synthesis of New Secondary Metabolites from

Liverworts and Microorganisms and Investigation of their Biogenesis

Experience

1999 – 2001 Research in Prof. W. A. Köngs lab, Synthesis of petasiten.

2000 One month ethnobotanical fieldwork in Papua New Guinea.

Dec. 2003 Research visit at the Department of Organic Chemistry, Tamkang University,

Taiwan, under the supervision of Prof. Chia-Li Wu.

2004 – 2007 „Wissenschaftlicher Mitarbeiter“ at the Department of Organic Chemistry,

University of Hamburg; Assitant to undergraduate lab work.

2005 Two months ethnobotanical fieldwork in Papua New Guinea

2007 – 2009 „Wissenschaftlicher Mitarbeiter “at the Department of Organic Chemistry,

University of Hamburg.

List of Abbreviations

245


separation factor

D specific optical rotation [°]

Ac2O acetic anhydride

AM1 Austin Model 1

br broad

C4H cinnamic acid-4-hydroxylase

CCALA Culture Colloection of Autotrophic Organisms

CHS chalcone synthase

COSY corellation spectroscopy 13

C {1H} NMR broadband decoupled carbon-13 NMR spectroscop y

C carbon-13 chemical shift [ppm]

D deuterium chemical shift [ppm]

H proton chemical shift [ppm]

difference

-isotope shift [ppm]

-isotope shift [ppm]

d doublet

D deuterium enrichment [atom %]

DCM dichloromethane

de diastereoisomeric excess [%]

DNA desoxyribonucleinic acid

DDQ 2,3-dichloro-5,6-dicyano-1,4-benzoquinone

DMAP 4-dimethylaminopyridine

DMPP dimethylallyl pyrophospahte

DMSO dimethyl sulfoxide

ee enantiomeric excess [%]

EAD electroannographic detection

eDNA environmental DNA

EDTA ethylenediamine tetraacetate

eGC enantioselective gas chromatography

EI electron impact

EIMS electron impact mass spectrometry

ep electron pair

Et2O diethyl ether

EtOAc ethyl acetate

EtOH ethanol

eV electron volt

FAB-MS fast atom bombardment mass spectrometry

FPP farnesyl pyrophosphate

FS flavanoid synthase

FTIR fourier-transform-infrared spectroscopy


246

GB Gamborg B5 medium

GC gas chromatography

GPP geranyl pyrophosphate

HREIMS high resolution electron impact mass spectrometry 1H NMR proton nuclear magnetic resonance spectroscopy

HMQC heteornuclear multiple quantum coherence

HMBC heteronuclear multiple bond correlation

HOMO highest occupied molecular orbital

HPLC high performance liquid chromatography

Hz hertz

IFS isoflavonoid synthase

IPP isopentenyl pyrophosphate

isnA isonitrile synthase A

isnB isonitrile synthase B 1J direct coupling constant [Hz]

2J geminal coupling constant [Hz]

3J vicinal coupling constant [Hz]

4J allylic coupling constant [Hz]

LB liquid browth

LUMO lowest unoccupied molecular orbital

max absorptium maximum [nm]

m multiplet

[M] molecular ion

MeOH methanol

MEP methylerythritol-4-phosphate

MM2 molecular model 2

MS mass spectrometry

MSK Murashige Skoog medium

MSKP phosphate enriched Murashige Skoog medium

MVA mevalonate

m/z mass charge ratio

n number

NADH -nicotineamid-adenine dinucleotide

nd not detected / not determined

NMR nuclear magnetic resonance

NOESY nuclear Overhauser effect spectroscopy

NOE nuclear Overhauser effect

P4H phenylalanine-4-hydroxylase

PAL phenylalanine ammonia Lyase

PDA potato-dextose agar

PENDANT polarisation transfer during attached nucleus testing

Pd/C palladium on carbon

PGC preparative gas chromatography


247

pH – log (cH3O+)

PHD phenylalanine dehydrogenase

PM3 parameterized model 3

ppm parts per million

RDA retro-Diels-Alder reaction

RHF restricted Hartree-Fock matrix

RI Kovats retention index (GC)

Rf retention factor (TLC)

RT retention time [min]

RT room temperature

RITA recipient for automated temporary immersion

STS stilbene synthase

t triplet

TAL tyrosine ammonia lyase

TAT tyrosine amino transferase

THF tetrahydrofuran

TIS temporary immersion system

TLC thin layer chromatography

TMS tetramethylsilane

TYDC tyrosine decarboxylase

UHF unrestricted Hartree-Fock matrix

UV ultraviolet spectroscopy

v/v volume per volume

w/w weight per weight

248

Hiermit erkläre ich an Eides statt, dass ich die vorliegende Arbeit selbstständig angefertigt und

keine anderen als die von mir angegebenen Hilfsmittel und Quellen verwendet habe. Ich

versichere weiterhin, dass die vorliegende Dissertation weder in gleicher noch in veränderter

Form bereits in einem anderen Prüfungsverfahren vorgelegen hat.

Stephan H. von Reuß

Hamburg, den 29. Mai 2009

Structure Elucidation and Synthesis of New Secondary ...

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