PHENOLIC CONSTITUENTS of WESTERN HEMLOCK WOOD (Tsuga Heterophylla (Raf.) Sarg.) by VALERIA M. CSIZMADIA - BUDAI Dipl. Chem. Eng. Polytechnical University of Budapest, 1956 A Thesis Submitted in Partial Fulfilment of the Requirements for the Degree of MASTER OF SCIENCE in the Department of CHEMISTRY We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA December 1961 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of C\-«^^J^*r-^^ The University of British Columbia, Vancouver 8, Canada. Date 2-l> ^^-^^o-^r—y^ ^ nk'iL, (ii) . ABSTRACT The phenolic extractives from western hemlock wood (Tsuga heterophylla (Raf.) Sar.) have been examined The total extractive content of the wood amounted to 1.5$ of the dry weight. A leucoanthocyanidin and two lignans, conidendrin and hydroxymatairesinol, were isolated from the phenolic fraction by precipitation of a methanol solution into peroxide-free ether followed by separation on silicic acid-calcium sulphate chromatobars. The pigment produced by acid treatment of the isolated leucoantho• cyanidin was shown by spectral studies and alkaline degradation to be a mixture of cyanidin and an unidentified anthocyanidin. The two anthocyanidins had identical R^ values in different solvents and similar ultra-violet spectra in ethanol-hydrochloric acid solution but the shift of the absorption maxima caused by addition of aluminium chloride was negligible in the case of the unknown compound and amounted to 30 mji for cyanidin. Similar separations of the absorption maxima after complexing with aluminium ion were observed with the 3-methyl and 3-isopropyl ethers of the two anthocyanidins. The alkaline degradation products from the leucoanthocyanidin contained protocatechuic acid but no phloroglucinol. Degradation products of phloroglucinol, however, were present in the reaction mixture. These results suggested that the leuco• anthocyanidin occurred in the wood in dimeric form and that alkaline degradation of this structure produced a symmetrical hexahydroxyhenzophenone derivative which split up directly into fragments identical to those obtained from phloro• glucinol under the same conditions. New information on the structure of hydroxymatairesinol was obtained by comparison of the infrared spectra of the fully acetylated hydroxymatairesinol with that of the reduced compound and by neutral potassium permanganate oxidation of trimethylhydroxymatairesinol. The results obtained were in good (iii) agreement with only one of the two structures previously proposed for hydroxymatairesinol by other workers. The NMR spectra of hydroxymatairesinol and structurally related com• pounds were compared, but the interpretation of the spectrum of hydroxy• matairesinol proved to be difficult because broad, incompletely resolved lines were obtained due to the complexity and asymmetry of the molecule. (iv) ACKNOWLEDGEMENTS I would like to express my sincere thanks and appreciation to Dr. L. D. Hayward for his help and encouragement during the course of the research and the preparation of this thesis. I wish to thank the following for gifts of chemicals: Professor T. R. Seshadri, University of Delhi; Dr. J. A. F. Gardner and Mr. G. Barton, Forest Products Laboratory, Vancouver, B. C; Dr. H. L. Hergert, Rayonier Corporation Shelton, Washington; •"•Dr/'Rj. .iD. Bennett, Du Pont Company of Canada, Montreal; Dr. J. L. Keays, .Powell River Company, B. C; Dr. K. G. Booth, Crown-Zellerbach Corporation, Camas, Washington; Dr. A. S. Gregory, Weyerhauser Timber Company, Longview, Washington; Dr. E. L. Lovell, Alaska Pine and Cellulose Company, Vancouver, B.C. I wish also to express my thanks to Dr. D. McGreer, of this Department for his help in the analysis of NMR spectra, to Mr. B. I. Nist, Department of Chemistry, University of Washington, Seattle, for taking the NMR measurements, and to Drs.O. Goldschmid and H. L. Hergert for the opportunity to read the---- manuscript of their paper on hemlock lignin precursors in advance of publication. (v) TABLE OF CONTENTS TITLE PAGE (i) ABSTRACT (ii) ACKNOWLEDGEMENTS (iv) TABLE OF CONTENTS (v) LIST OF FIGURES (vii) LIST OF TABLE (ix) GENERAL INTRODUCTION 1 The Chemical Constitution of Wood '2 HISTORICAL INTRODUCTION 6 Leucoanthocyan(id)ins 7 Lignans 17 I. The Chemistry of Conidendrin 21 II. The Chemistry of Matairesinol 23 III. The Stereochemistry of Conidendrin and Matairesinol .. 27 IV. The Chemistry of Hydroxymatairesinol 29 V. The Lignan-Lignin Interrelationship in Biosynthesis .. 32 RESULTS AND DISCUSSION 36 I. Isolation of Phenolic Constituents form Western Hemlock Wood 37 II. Hemlock Leucoanthocyanidin 38 III. Hemlock Lignans 5^ A. Separation of Lignans 5k B. Structural Analysis of Hydroxymatairesinol (HMR).- 63 SUGGESTIONS FOR, FURTHER WORK 72 I. Leucoanthocyanidins 73 II. Lignans 73 EXPERIMENTAL 79 I. Materials 80 A. Solvents 80 B. Reagents 80 C. Reference Compounds 8l (vi) II. Analyses 8k A. Melting Points 8k B. Methoxyl Determination 8k C. Specific Rotation 8k D. Colour Tests 8k III. Spectra 86 A. Ultraviolet Spectra 86 B. Infrared Spectra 86 C. Nuclear Magnetic Resonance Spectra 86 IV. Chromatography 87 V. Extraction of Western Hemlock Wood 92 A. Collection and Preparation of Wood Samples 92 B. Extraction of Fats, Waxes, and Related Compounds. 93 C. Extraction of Phenolic Constituents 95 VI. Separation of Lignans on Cylindrical Chromatobars .. 99 VII. Reaction and Derivatives .' 105 A. Western Hemlock Leucoanthocyanidin 105 (i) Attempted Isolation of Anthocyanidin... 105 (ii) Alkaline Degradation of Hemlock Leucoanthocyanidin 105 B. Lignans 106 (i) Tri-0-acetyl-hydroxymatairesinol 106 (ii) Tri-O-benzoyl-hydroxymatairesinol 106 (iii) Reduction of Hydroxymatairesinol 106 (iv) Acetylation of Reduced HydroxymataireT sinol 106 (v) Conversion of Hydroxymatairesinol to ' Conidendrin 107 (vi) Dimethylhydroxymatairesinol 107 (vii) Trimethylhydroxymatairesinol 107 (viii) Potassium Permanganate Oxidation of Trimethylhydroxymatairesinol and Related Compounds 107 APPENDICES Ill I. Calculation of the Proton Nuclear Resonance Shielding Values (T) Ill II. Infrared Spectra Ilk REFERENCES 120 (vii) LIST OF FIGURES 1. The Range of Oxidation Level in the C-^ Group of Flavonoids .. 5 2. Synthesis of Dimethyl Aromatized LIX 2h 3- Synthesis of Dimethyl Aromatized LX 26 h. Rf values of Cyanidin and Its Derivatives Obtained by Acid Treatment of the Leucoanthocyanidin of Hemlock Wood in Various Solvents 39 5- Absorption Spectra of Dahlia (D) and Synthetic (S) Cyanidin and Their Aluminium Complexes 1+3 6. Absorption Spectra of Wood Anthocyanidin (W) and Aluminium Complexed Wood Anthocyanidin (c) hk 7- Absorption Spectra of the 3-Isopropyl Ether of the Wood Antho• cyanidin (PW) and the Corresponding Aluminium Complex (CPW) . 1+5 8. Difference Spectrum of Hemlock Anthocyanidin 1+6 9. Paperchromatogram of the Alkaline Degradation Products 1+8 10. Proposed Alkaline Degradation of a Dimeric Leucoanthocyanidin. 50 i 11. Suggested Structure and Degradation Products of Hemlock Leucoanthocyanidin 53 12. Separation of Lignans with Time of Development 55 13• Separation of Lignans by Two-dimensional Chromatography 57 Ik. Separation of Lignans by Counter-current Distribution 59 15- Thin Layer Chromatography of Lignans in Solvent S-2k 60 l6. Separation of Conidendrin and Hydroxymatairesinol on Chromatobars , 62 17- Infrared Spectra of Hydroxymatairesinol and Derivatives 66 18. Infrared Spectra of HMR, Reduced HMR, and Their Acetate Derivatives 68 19• Nuclear Magnetic Resonance Spectra of HMR and Structurally Related Known Compounds 69 20. Suggested Synthetic Route A to Dimethyl Hydroxymatairesinol. 75 21. Suggested Synthetic Route B to Dimethyl Hydroxymatairesinol . 77 22. Distributor for Chromatobars 91 (viii) 23. Two Kilogram Capacity Extractor 9^ 2k. Fractionation of the Methanol Extract of Hemlock Wood 96 25 • First Separation of Fraction C on Chromatobars 101 26. Combined Fractions I to IV of Lignans from First Chromatobar Separation 102 21. Thin Layer Chromatography of Fractions from Chromatobars I to II 103 28. Thin Layer Chromatography of Fractions from Chromatobars III and IV 10k 29. Infrared Spectra of Lignans from Hemlock Wood 115 30. Infrared Spectra of Hydroxymatairesinol (HMR) and HMR-benzoate. Il6 31. Infrared Spectra of HMR and Derivatives 117 32. Infrared Spectra of Hemlock Leucoanthocyanidin and Related Compounds 118 (ix) LIST OF TABLES I. Percentage Composition of Typical Plants 3 II. Leucoanthocyanidins 8 III. Lignans 20 LIV. Rf Values of Anthocyanidins from Hemlock Wood Prepared in Various Solvents 40 V. Absorption Maxima of Anthocyanidin hi VI. Alkaline Degradation Products 49 VII. R„ Values of Lignans in the Methanol Extract of Hemlock Wood 54 rV VIII. Colors and Rf Values of Lignans in TLC with Three Different Spray Reagen ts 6l IX. Chromatographic Tests of Hydroxymatairesinol (HMR) 63 X. Supporting Materials and Adsorbents for Chromatography 88 XI. Solvents for Chromatography 89 XII. Spray Reagents for Chromatography 90 XIII. Composition and Dimensions of Chromatoplates and Chromatobars 90 XIV.