AN ABSTRACT OF THE THESIS OF NONG-KEUN SONG for the degree of DOCTOR OFPHILOSOPHY (Name of student) (Degree) in FOREST PRODUCTS presented on December 16. 1987 (Major department) (Date) Title: CHEMISTRY OF PHENOLIC STAINS IN DOUGLAS-FIR SAPWOOD. Abstract approved: Signature redacted for privacy. Mdrray L. Laver Dihydroquercetin [(2,3-trans)-3,3',4',5,7-pentahydroxy- flavanonol] was shown to be the precursor to the phenolic stains that often develop in Douglas-fir [Pseudotsuga menziesii (Mirb.) Franco] sapwood. Douglas-fir sapwood was extracted with methanol and the methanol solubles were fractionated by solvent partition, thin-layer chromato- graphy, silica-gel column chromatography, and Sephadex gel- permeation chromatography. Tyrosinase, a commercially available catechol oxidase enzyme, was used to locate and trace the precursors to color formation in the solubilized materials. Careful control of the separations and careful testing of the fractions for color formation resulted in the identification and isolation of dihydroquercetin as the precursor of the stains. It was established that neither oregonin, the color precursor of stains in red alder wood and bark, nor catechin, a flavonoid that has been associated with stains in western hemlock wood, were involved in the staining of Douglas-fir sapwood. The inhibitors, ethylenedinitrillotetraacetic acid disodium salt (EDTA), phenylthiourea, mercaptobenzothiazole, and Chloramine-T proved ineffective in preventing color formation when the methanol-extracted materials from Douglas-fir sapwood were treated with tyrosinase. An improved method of isolating dihydroquercetin from Douglas-fir bark was developed. This pure crystalline dihydroquercetin was reacted with tyrosinase to simulate the reactions that might lead to color formation in Douglas-fir sapwood. The reaction mixture became brown in color and a dark brown polymeric precipitate formed. The brown polymer was recovered and was shown to have a number of characteris- tics similar to the stains in Dotglas-fir sapwood by ultraviolet and infrared spectroscopy. The compounds 3,4- dihydroxybenzoic acid (protocatechuic acid), 3,4-dihydroxy- phenylacetic acid, and quercetin were found in the reaction products of dihydroquercetin and tyrosinase. They were considered to be degradation products of dihydroquercetin and possible intermediates in polymeric stain formation. Thus, a relationship between the presence of dihydroquer- cetin, enzyme action, and the phenolic stains that often develop in Douglas-fir sapwood was established. Chemistry of Phenolic Stains in Douglas-Fir Sapwood by Hong-Keun Song A THESIS submitted to Oregon State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy December 16, 1987 Commencement June 1988 APPROVED: Signature redacted for privacy. Associate Professor of Forest Products Chemistry in charge of major Signature redacted for privacy. arC Head of the Department of Forest Products Signature redacted for privacy. Dean of Gr.fl ate Scho Date thesis is presented December 16, 1987 Typed by Hong-Keun Song for Hong-Keun Song ACKNOWLEDGEMENTS I wish to express my deepest, and most sincere thanks and gratitude to my major professor and counselor Dr. Murray L. Laver whose interest, advice, and guidance during the course of this work has been a constant inspiration. I also wish to thank Dr. Robert L. Krahmer and Dr. Joseph J. Karchesy for their assistance, counsel and particularly for the use of some of their laboratory equipment. Acknowledgement is also made to Rodger L. Kohnert, Department of Chemistry, Oregon State University, for his assistance in obtaining the 400 MHz Nuclear Magnetic Resonance data. Lastly, I would like to express my appreciation to my parents and my family for their moral support, understanding, and patience throughout the past years. The research was supported by the Department of Forest Products, Oregon State University. TABLE OF CONTENTS INTRODUCTION 1 HISTORICAL REVIEW 3 EXPERIMENTAL 20 Bark of Red Alder 20 1.1. Collection of Bark 20 1.2. Extraction 20 1.3. Separation of Oregonin 21 1.3.1. Chromatography 21 1.3.1.1. Thin-Layer Chromatography 21 1.3.1.2. Enzymatic Detection of Color Precursors 21 1.3.1.3. Column Chromatography 22 1.3.2. Analysis of Oregonin by HPLC 23 1.3.2.1 Column and Equipment 23 1.3.2.2 Optimum Operating Conditions 25 1.4. Identification of Oregonin. 25 Sapwood of Douglas-fir 27 2.1. Collection 27 2.2. Extraction 28 2.3. Color Test of Solubles with Tyrosinase 28 2.3.1. Test of Inhibitors to Color Formation 31 2.4. Separation of the Stain Precursors 31 2.4.1. Thin-Layer Chromatography 31 2.4.2. Silica-Gel Column Chromatograpy 32 2.4.2.1. Packing Material, Columns and Separations 32 2.5. Isolation of Dihydroquercetin (DHQ) 33 2.5.1. Isolation 33 2.5.2. Identification of Dihydroquercetin 35 2.6. Isolation of a Glycoside of Dihydroquercetin 36 2.6.1. Liquid Chromatography on Silica-Gel Columns 36 2.6.2. Liquid Chromatography on Sephadex LH-20 Columns 38 2.6.3. Identification 38 Preparation of Dihydroquercetin from Douglas-fir Bark 41 3.1. Bark Extraction 41 3.2. Purification of Dihydroquercetin 43 3.3. Crystallization of Dihydroquercetin 43 Reaction of Dihydroquercetin with Tyrosinase 44 4.1. Reaction of Dihydroquercetin with Tyrosinase 44 4.2. Separation of Reaction Products 45 4.2.1 Gel-Permeation Chromatography 46 4.3. Identification of Reaction Products 53 4.4. The Reaction of Dihydroquercetin with Tyrosinase on Douglas-fir Sapwood 56 RESULTS AND DISCUSSION Bark of Red Alder 58 1.1. Collection of Bark 58 1.2. Extraction 58 1.3. Separation of Oregonin 59 1.3.1. Chromatography 59 1.3.1.1. TLC Chromatography 59 1.3.1.2. Enzymatic Detection of Color Precursors 60 1.3.1.3. Column Chromatography 62 1.3.2. Analysis of Oregonin by HPLC 63 1.3.2.1. Column and Equipment 63 1.3.2.2. Optimun Operating Conditions 63 1.4. Identification of Oregonin. 63 Sapwood of Douglas-fir 70 2.1. Collection 71 2.2. Extraction 71 2.3. Test of Inhibitors to Color Formation 74 2.4. Separation of the Stain Precursors 75 2.4.1. Thin-Layer Chromatography 75 2.4.2. Silica-Gel Column Chromatography 79 2.4.2.1. Silica-Gel Column Chromatography 79 2.5. Isolationof Dihydroquercetin 81 2.5.1. Isolation 81 2.5.2. Identification of Dihydroquercetin 84 2.6. Isolation of a Glycoside of Dihydroquercetin 89 2.6.1. Liquid Chromatography on Silica-Gel Columns 89 2.6.2. Liquid Chromatography on Sephadex LH-20 Columns 90 2.6.3. Identification 90 Preparation of Dihydroquercetin from Douglas-fir Bark 101 3.1. Bark Extraction 101 3.2. Purification of Dihydroquercetin 102 Reaction of Dihydroquercetin with Tyrosinase 103 4.1. Reaction of Dihydroquercetin with Tyrosinase 103 4.2. Separation of Reaction Products 105 4.2.1. Gel-Permeation Chromatography 105 4.3. Identification of Reaction Products 108 4.4. Reaction of Dihydroquercetin with Tyrosinase on Douglas-fir Sapwood 134 4.5. Discussion 134 SUMMARY AND CONCLUSIONS 138 BIBLIOGRAPHY 141 LIST OFFIGURES Figure Page Structural transformation reactions of anthocyanidins. 8 The enzymatic oxidation of catechol. 12 The isomeric tetrahydroxydiphenyls. 12 The yellow quinone. 12 The water-reaction mechanism of catechol. 13 The structures of the 3-isopropylcatechol -orcinol condensation products. 15 The polymerization of catechin autoxidation. 16 The mechanism of catechin oxidation. 17 Isolation of oregonin. 26 Isolation of dihydroquercetin. 29 Isolation of a dihydroquercetin glycoside. 39 Crystallization of dihydroquercetin. 42 Separation of the dihydroquercetin reaction products with tyrosinase. 47 TLC chromatogram of oregonin from red alder bark. 61 HPLC spectrum of oregonin on a C-18 reverse phase column. 64 ProtonNMRspectrum (acetone-DO of oregonin. 66 Carbon-13NMRspectrum (acetone-DO of oregonin. 67 DEPT of oregonin. 69 TLC chromatograms of the extracts of Douglas-fir sapwood. 78 TLC chromatogram of the ethyl acetate extract of Douglas-fir sapwood. 83 UV spectraofdihydroquercetin and quercetin in methanol. 85 ProtonNMRspectum (acetone-D6)of dihydroquercetin. 86 Carbon-13NMRspectrum (acetone-D6)of dihydroquercetin. 88 Paper and TLC chromatogramsofthe hydrolyzate of the dihydroquercetin glycoside. 92 UV spectraofthe dihydroquercetin glucoside with an alkaline shift reagent. 94 UV spectraofthe dihydroquercetin glucoside with aluminium chloride with and without hydrochloric acid. 95 UV spectra of the dihydroquercetin glucoside with sodium acetate with and without boric acid. 96 ProtonNMRspectrum (acetone D6) of dihydroquercetin-3'-0-glucopyranoside. 97 Carbon-13NMRspectrum (acetone D6) of dihydroquercetin-3'-0-glucopyranoside. 99 UV spectra of the polymer R resulting from the reactionofdihydroquercetin with tyrosinase, and the stained wood (SW). 110 IRspectrumofthe polymerR of dihydroquercetin reaction products with tyrosinase. 111 IRspectrumofstained wood. 112 IRspectrumofstained wood andofunstained Douglas-fir sapwood. 113 ProtonNMRspectrum (acetone D6)ofsample A-4 (3,4-dihydroxybenzoic acid). 114 Carbon-13 NMR spectrum (acetone D6)of sample A-4 (3,4-dihydroxybenzoic acid). 115 ProtonNMRspectrum (acetone D6)ofsample A-1 (3,4-dihydroxyphenylacetic acid and 3,4- dihydroxybenzoic acid). 116 Carbon-13NMRspectrum (acetone D6) of sample A-1 (3,4-dihydroxyphenylacetic acid and 3,4- dihydroxybenzoic acid). 117 Heteronuclear correlated (HETCOR) spectrum of sample A-1 (3,4-dihydroxybenzoic acid and 3,4-dihydroxyphenylacetic acid). 118 DEPT of sample A-1 (3,4-dihydroxybenzoic acid and 3,4-dihydroxyphenylacetic acid). 120 IR spectrum of sample A-4 (3,4-dihydroxybenzoic acid). 121 IR spectrum of sample A-1 (3,4- dihydroxyphenylacetic acid and 3,4-dihydroxybenzoic acid). 122 UV spectra of sample A-1 (3,4-dihydroxybenzoic acid) with sodium acetate with and without boric acid. 123 UV spectra of sample A-1 (3,4-dihydroxybenzoic acid and 3,4-dihydroxyphenylacetic acid) with and without boric acid. 124 Degradation pattern of dihydroquercetin by tyrosinase. 126 Proton NMR spectrum (methanol D4) of Q-2 (Figure 13). 127 Proton NMR spectrum (methanol D4) of Q-2 after standing for one weex. 129 GPC spectrum of Q-2 (Figure 13).