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Isolation and characterization of actively anabolized dilignol rhamnosides in the leaves of western red… Manners, Gary Duane 1970

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ISOLATION AMD CHARACTERIZATION OF ACTIVELY ANABOLIZED DILIGNOL RflAMNOSIDES IN THE LEAVES OF WESTERN RED CEDAR (THUJA PLICATA DONN) by GARY DUANE MANNERS B.S. (For.), Oregon State University, 1962 B.S. (Chern.), Oregon State University, 1963 M.S., Oregon State University, 1965 A THESIS SUBMITTED IN PARTIAL FULFILMEl'ff OF THE REQUIREC-ENT FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in the Department of Forestry We accept this thesis as conforming to the required standard. THE UNIVERSITY OF BRITISH COLUMBIA December, 1970 In presenting th i s thesis in pa r t i a l f u l f i lmen t of the requirements fo r an advanced degree at the Un ivers i ty of B r i t i s h Columbia, I agree that the L ibrary sha l l make i t f r ee l y ava i l ab le fo r reference and study. I fur ther agree that permission for extens ive copying of th i s thes i s for scho lar ly purposes may be granted by the Head of my Department or by his representat ives. It is understood that copying or pub l i ca t i on of th i s thes is fo r f i nanc ia l gain sha l l not be allowed without my wr i t ten permiss ion. Department The Univers i ty of B r i t i s h Columbia Vancouver 8, Canada i . ABSTRACT Three di l i g n o l rhamnosides were isolated i n 0.15 to 0.^0$ yield from the ethyl acetate solubles of a methyl alcohol extract of western red cedar (Thuja plicata Donn) leaves using s i l i c i c acid and Sephadex LH-20 pressure column chromatography. One of the three d i l i g n o l rhamnosides was characterized as l-(3'-methoxy-^ '-hydroxyphenyl)-2-0-l"-[2"-hyclroxy-4"-(pro-pane-3"'-a-L--rhamnoside)phenyl]-propane-lj3 d i o l , using NMR and chem-i c a l degradation i n conjunction with mass.spectral techniques on the compound and i t s derivatives. The other d i l i g n o l rhamnosides were not completely characterized, but were shown to be chemically related to the di l i g n o l identified. Based upon NMR, chemical and_ mass spectral data, the uncharacterized d i l i g n o l rhamnosides are speculated to contain phenylcoumaran and guaiacyl benzdioxane structures. The characterized d i l i g n o l rhamnoside represents the f i r s t reported occurrence of a free dil i g n o l glycoside i n plant tissues. The unusual a-L-rhamnose moiety of the di l i g n o l occurs i n a previously unreported linkage to the n-propyl hydroxyl group uncommon in lignin. The rhamnoside also displays the previously unreported guaiacylglycerol~catechol--8-aryl ether structure rather than the commonly observed guaiacylglycerol-guaiacyl~3-aryl ether structure. i i . A new combustion-absorption technique was developed and validated which allows high efficiency evaluation of low ac-t i v i t y radioactive products separated on thin layer cellulose chromatography plates. The technique was applied to an analysis of the anabolic products of an infusion feeding of U- C--L-phenyl-alanine to western red cedar leaves. Facile imbibition of U--14 C-L-phenylalahine occurs within ten hours. Maximum incorpor-ation of 0.30%. and 0.^0% of the available radioactivity occurs i n the characterized d i l i g n o l rhamnoside, and i t s suspected phenyl-coumaran homolog respectively, at the three to five hour period of the infusion feeding. The incorporation results indicate the participation of the d i l i g n o l rhamnosides i n aromatic metabolism in the leaves of western red cedar. This feeding experiment i s preliminary to future detailed biosynthetic studies i n the leaf tissue. The combustion-absorption technique i s limited to combustible sample weights of 7 nig. i i i . TABLE OF CONTENTS PAGE ABSTRACT '. i TABLE OF CONTENTS .: . i i i LIST OF TABLES. ..... v i LIST OF FIGURES............ .. v i i ACKNOlttEDGEMENT ix INTRODUCTION 1 LITERATURE REVIEW 4 Definitions.. 4 The Status of Lignin /....... 5 The .Location of Lignin 6 The Isolation of Lignin 8 The Characterization of Lignin 9 The Biosynthesis of Lignin.. 16 Aromatic Constituents Related to Lignin 26 Aromatics of Western Red Cedar 30 Metabolism and Lignin Formation 33 Literature Summary and Observations 35 MATERIALS AND METHODS 37 Collection of Leaves. 37-Leaf Extraction. 37 Chromatography 38 Thin layer cellulose chromatography 38 Thin layer s i l i c a gel chromatography....., 40 Column chromatography 40 Derivative Preparations 43 Acetylation 43 O-methylation. 43 Hydrogenolysis 44 Degradative Techniques 45 Alkaline nitrobenzene oxidation 45 Ethanolysis 46 , MATERIALS AND METHODS (Cont'd) i v Periodate oxidation 46 Hydrolysis •.. 47 Liquid S c i n t i l l a t i o n Counting of Low . .Activity Chromatograohic Samoles. .... 47 i l l , Leaf C Feeding .... .. 51 Spectral Techniques... • 54 Ultraviolet and infrared 54 Nuclear magnetic resonance . . 55 Mass spectroscopy 55 ' RESULTS ' . 56 Isolation of Compounds • 56 Compounds A 3B 3 and C - Isolation 57 Compounds A 3B, and C - Properties 60 Compounds A 3B, and C - Derivatives.... 62 Methylation , 62 Acetylation 63 Hydrogen.olysis ' 64 Compounds A ,B, and C - Degradative Studies 64 Hydrolysis 64 Ethanolysis .... 65 Alkaline nitrobenzene oxidation.. 66 Periodate oxidation 66 Liquid S c i n t i l l a t i o n Technique 67 l 2 |C Feeding ' 70 DISCUSSION '. 73 Structural Studies of the Dilignols. 73 Compound A 73 Chemical characterization. 75 NMR spectra of Compound A and i t s acetate.... 78 Mass spectrum of Compound A acetate. 84 Compound B 89 NMR spectra of Compound B and i t s acetate 91 Mass spectrum of Compound B acetate 95 Compound C • 98 NMR spectra of Compound C and i t s derivatives 99 DISCUSSION (Cont'd) v. Mass spectrum of Compound C acetate 105 Lignin Biosynthesis • ' 110 S c i n t i l l a t i o n - Chromatography Technique 113 Precursor Feeding Study • 119 CONCLUSION 126 Future Research 128 Literature Cited 130 v i . LIST OF TABLES TABLE • , PAGE 1. Sample preparation and results of li q u i d s c i n t i l l a t i o n counting comparison of scraping and combustion methods applied to samples from thin'layer cellulose chromato-graphic plates ............ 49 2. Properties of Compounds A,B, and C ........ 6 l 14 3. - Uptake of U- C-L-phenylalanine and i t s inccporation into Compounds A and B i n the leaves of v;estern red cedar .............. 71 v i i . LIST OF FIGURES FIGURE ' PAGE 1. S h i k i m i c a c i d pathway to the for m a t i o n of aromatic amino a c i d s p h e n y l a l a n i n e and t y r o s i n e 18 • 2 . Resonance forms of the f r e e r a d i c a l d e r i v e d from i o n i z e d c o n i f e r y l a l c o h o l 23 3 . Scheme f o r the e x t r a c t i o n and s e p a r a t i o n of components from western r e d cedar l e a v e s . . . . 39 4. A schematic r e p r e s e n t a t i o n of the pr e s s u r e column chromatography system 42 5. A schematic chromatogram of the c l a r i f i e d e t h y l a c e t a t e e x t r a c t from western r e d cedar leaves (spray, DSA) 51 6. T y p i c a l e l u t i o n curve of the c l a r i f i e d e t h y l a c e t a t e e x t r a c t from western r e d cedar leaves as run on LH-20 [CHCl^ :EtOH(4 :1)]. . 58 7. The NMR spectrum of Compound A 79 8. The NMR spectrum of the ac e t a t e d e r i v a t i v e of Compound A 80. 9 . The mass spectrum of the a c e t a t e d e r i v a t i v e of Compound A 85 10. The proposed mass s p e c t r a l f r a g m e n t a t i o n of rhamnose t r i a c e t a t e 87 11. The NMR spectrum of Compound B 9 2 12. The NMR spectrum of the ac e t a t e d e r i v a t i v e of Compound B 93... 1 3 . The mass spectrum of the a c e t a t e d e r i v a t i v e of Compound B 96 14. The NMR spectrum of Compound C 100 15. The NMR spectrum of the a c e t a t e d e r i v a t i v e of Compound C • . 101" LIST OF FIGURES (cont'd)_ v i i i . FIGURE PAGE 16 . The NMR spectrum of the aglycone of Compound .C... . .103 1 7 . The NMR spectrum of the a c e t a t e d e r i v a t i v e of the aglycone of Compound C 104 1 8 . The mass spectrum of the a c e t a t e d e r i v a t i v e of Compound C ' 106 1 9 . The e f f e c t of an e x t e r n a l quenching compound on the d e t e r m i n a t i o n of a c t i v i t y of scraped chromatographic samples...... 114 2 0 . The e f f e c t of an e x t e r n a l quenching compound on the d e t e r m i n a t i o n . o f a c t i v i t y of com-busted chromatographic samples 115 2 1 . The e f f e c t of chromatographic spot s i z e on l i q u i d s c i n t i l l a t i o n c o unting of com-b u s t i o n samples 116 2 2 . Autoradiogram of the gross e t h y l a c e t a t e s o l u b l e s from U- 4 C - L - p h e n y l a l a n i n e f e d western r e d cedar ( s o l v e n t - B E ) . . . . . . . 122 1 4 . 2 3 . Uptake of U-C-r-L-pheny l a l a n i n e by western red cedar leave's 124 24. I n c o r p o r a t i o n of U-^C-L-pheny l a l a n i n e i n t o Compounds A and B i n ffhe leaves of western r e d cedar 124 ACKNOWLEDGEMENT The author.wishes to g r a t e f u l l y acknowledge Dr. E.P. Swan,'part-time A s s o c i a t e P r o f e s s o r , F a c u l t y of F o r e s t r y , U n i v e r s i t y of B r i t i s h Columbia f o r his, p a t i e n t guidance i n the p l a n n i n g and experimental stages of t h i s study, and f o r h i s a s s i s t a n c e i n the p r e p a r a t i o n of t h i s t h e s i s . P a r t i c u l a r r e c o g n i t i o n must go t o the s t a f f of the Vancouver F o r e s t Products L a b o r a t o r y , Department of F i s h e r i e s and F o r e s t r y where a l l experimental phases of t h i s study were performed. S p e c i a l acknowledgement must be extended to the wood chemistry s e c t i o n of the Vancouver F o r e s t Products Lab. f o r t h e i r a s s i s t a n c e throughout the study, and i n p a r t i c u l a r to Dr. J.F. M a n v i l l e f o r h i s a i d i n the i n t e r p r e t a t i o n of the i n c l u d e d n u c l e a r magnetic resonance s p e c t r a . The author i s f u r t h e r indebted t o Dr. J.W. Wilson, P r o f e s s o r , F a c u l t y of F o r e s t r y , U n i v e r s i t y of B r i t i s h Columbia f o r h i s s u g g e s t i o n s , c r i t i c i s m s and a i d i n the pr e p a r a t o r y phases of t h i s t h e s i s . Acknowledgement as w e l l must go to the U n i v e r s i t y of B r i t i s h Columbia f o r the . f i n a n c i a l support extended f o r the d u r a t i o n of the academic program. X;. F i n a l - acknowledgement must go to my w i f e , C a r o l e , without whom the l a t t e r stages of the work would not have been p o s s i b l e . INTRODUCTION L i g n i n has g e n e r a l l y been d e f i n e d as a t r i d i -mensional phenylpropanoid polymer e x i s t i n g i n p l a n t t i s s u e s p r i m a r i l y f o r s t r u c t u r a l support. The p o l y m e r i c nature of t h i s substance suggests i t s m u l t i - s t a g e develop-ment over a wide range of phenylpropanoid m e t a b o l i z i n g t i s s u e s . The l i g n i n r e p r e s e n t s as much as 30% of the c o n s t i t u e n t s of c o n i f e r o u s plan.t stems, and this high content has i n i t i a t e d many i n v e s t i g a t i o n s i n t o the "where" and "how" of i t s f o r m a t i o n . The q u e s t i o n of where l i g n i n i s formed has r e s u l t e d i n many macro and micro q u a n t i t a t i v e analyses i n woody stems. These i n v e s t i g a t i o n s of amount and l o c a t i o n of l i g n i n i n the wood, bark, cambium, c e l l w a l l , and c e l l cytoplasm have produced evidence, but no c l e a r answers,"as to where the l i g n i n polymer i s formed. The v a r i a t i o n i n q u a n t i t a t i v e r e s u l t s suggests that the d e t e r m i n a t i o n of where l i g n i n i s formed may depend upon the l i g n i n d e f i n i -t i o n , J The d e f i n i t i o n of l i g n i n need not be c o n f i n e d to i t s p o l y m e r i c form. I t Is p o s s i b l e to expand t h i s d e f i n i -t i o n to i n c l u d e those monomeric, dimeric and o l i g o m e r i c phenylpropanoid p r e c u r s o r s of l i g n i n ( l i g n o l s ) as l i g n i n . The i n c l u s i o n of l i g n o l s i n t o the l i g n i n d e f i n i t i o n allows the d e t e r m i n a t i o n of where l i g n i n i s formed to be a s s o c i a t e d more s p e c i f i c a l l y to t hat p o i n t where the l i g n o l s f i r s t appear. I t can be f u r t h e r i n -f e r r e d t h a t the i n i t i a l p o i n t of l i g n i n f o r m a t i o n i s c o i n c i d e n t with the appearance of the m o n o l i g n o l s . How-e v e r , these same monolignols may serve as precursors to other n o n - l i g n i n phenylpropanoid compounds. T h e r e f o r e , the f i r s t t r u e l i g n i n c h a r a c t e r i s d i m e r i c ( d i l i g n o l ) i n the present, d e f i n e d nature of the chemical c h a r a c t e r of l i g -n i n , i . e . , a g u a i a c y l - g - a r y l e t h e r . i n most conifers. D i l i g n o l s have been l o c a t e d i n the cambial r e g i o n (36) and are c o n s i d e r e d to be formed from aromatic p r e -c u r s o r s o r i g i n a t i n g from t r a n s l o c a t e d sugars ( 7 8 ) . T h i s t r a n s f o r m a t i o n appears to be a l o c a l i z e d b i o s y n t h e t i c mechanism a s s o c i a t e d with the v i t a l i t y and s p e c i a l i z a t i o n of the t i s s u e . I t may be f u r t h e r suggested t h a t other h i g h l y a c t i v e t i s s u e s d i s p l a y s i m i l a r l o c a l i z e d b i o s y n t h e -t i c mechanisms.-T h i s i n v e s t i g a t i o n examines the hypothesis that the leaves of western red cedar (Thuja p l i c a t a Donn) may serve as a metabolic t i s s u e important i n the f o r m a t i o n of d i l i g n o l s . Such a h y p o t h e s i s n e c e s s a r i l y r e q u i r e s the development of techniques capable of I s o l a t i n g d i l i g n o l s f o r c h a r a c t e r i z a t i o n . V e r i f i c a t i o n .of the d i l i g n o l s ' p a r t i c i p a t i o n i n the. metabolism of the a c t i v e t i s s u e may be achieved through r a d i o a c t i v e t r a c e r s t u d i e s . The a n t i c i p a t e d low l e v e l s of i n c o r p o r a t e d r a d i o a c t i v i t y a s s o c i a t e d w i t h these t r a c e r s t u d i e s w i l l r e q u i r e new techniques f o r t h e i r measurement. 4. LITERATURE REVIEW D e f i n i t i o n s " . Three g e n e r a l c l a s s e s of compounds are c o n s i d e r e d i n t h i s d i s s e r t a t i o n ; l i g n i n , l i g n o l and l i g n a n . The c l o s e r e l a t i o n s h i p i n nomenclature between these compounds r e q u i r e s t h e i r d e f i n i t i o n p r i o r t o the ensuing d i s c u s s i o n . L i g n i n : L i g n i n has never been s p e c i f i c a l l y d e f i n e d . I t may be g e n e r a l l y d e f i n e d as a widely o c c u r r i n g t r i -d i m e n s i o n a l biopolymer p r i m a r i l y composed of phenylpropa-noid u n i t s . T h i s biopolymer Is e s s e n t i a l l y i n s o l u b l e i n n e u t r a l s o l v e n t s and e x i s t s w i t h i n and bonded to p l a n t c e l l w a l l s f o r the primary purpose of s t r u c t u r a l support. A l l r e f e r e n c e to l i g n i n i n t h i s d i s s e r t a t i o n w i l l be to naturally occurring lignin. S y n t h e t i c a l l y produced l i g n i n w i l l be I n d i c a t e d as such. Lignols: L i g n o l i s a s p e c i f i c term i n t r o d u c e d by Freudenberg (35) to d e s c r i b e the "trapped" i n t e r m e d i a t e s i n the enzymatic dehydrogenation of c o n i f e r y l a l c o h o l to form s y n t h e t i c l i g n i n . Monomeric products from c o n i f e r y l a l c o h o l are d e s i g n a t e d " m o n o l i g n o l s " , d i m e r i c products " d i l i g n o l s " , t r i m e r i c products " t r i l i g n o l s " , e t c . The terms d i l i g n o l , t r i l i g n o l . . . o l i g o l i g n o l as used i n t h i s d i s s e r t a t i o n w i l l i n c l u d e those d i m e r i c , t r i m e r i c . . . 5. oligomeric phenylpropanol derivatives, both synthetic and natural, which exhibit alkyl-aryl ether and/or carbon-carbon bonds between the propanol chain of one unit and the aromatic nucleus of the next unit. Such derivatives are not restricted to dehydrogenation products of coniferyl alcohol. These lignols, as Freudenberg's, are optically inactive. .Lignan: This term defines a natural dimeric compound which i s o b t a i n e d through j o i n i n g t o g e t h e r two phenylpropanoid u n i t s i n a carbon-carbon bond between the middle ( 3 - 3 ' ) carbons of t h e i r p r o p y l s i d e c h a i n s . Such a combination i s formed s t e r e o - s p e c i f i c a l l y as an o p t i c a l l y a c t i v e l i g n a n which commonly has the 2-L,' 3-"TJ~cbnfiguration. The Status of L i g n i n Since I t s f i r s t r e c o g n i t i o n by Payen i n 1 8 3 3 , l i g -n i n has been the s u b j e c t of e x t e n s i v e chemical and biochemi-c a l i n v e s t i g a t i o n s . The net r e s u l t of these i n v e s t i g a t i o n s has been a c l e a r e r ( i f incomplete) understanding of l i g n i n f o r m a t i o n and s t r u c t u r e , coupled w i t h f r u s t r a t i o n i n a t -tempts to achieve s i g n i f i c a n t economic e x p l o i t a t i o n of l i g -n i n based p r o d u c t s . To those i n v o l v e d i n the chemical con-v e r s i o n of f o r e s t p r o d u c t s , l i g n i n r e p r e s e n t s a n o n u t i l i -zable waste product adding to ever e n l a r g i n g p o l l u t i o n . T h i s l i g n i n comprises 22-34% of most woods and w i l l occur as waste, i n excess of 50 m i l l i o n t o n s , from the world's, chemical p u l p i n g i n s t a l l a t i o n s i n 1970 (48). H a r k i n (48) 6. f e e l s t h a t the l i m i t s of accomplishment i n l i g n i n r e s e a r c h , as r e g u l a t e d by present methods, has been approached. He suggests that the wood based i n d u s t r i e s must improve, t h e i r l i g n i n performance through t h e . a p p l i c a t i o n of the knowledge gained i n s t r u c t u r a l e l u c i d a t i o n of l i g n i n to date. The L o c a t i o n of L i g n i n L i g n i n i s found i n the f i b e r c e l l w a l l s , f r u i t s , s t o n es, r o o t s , b a s t , p i t h , and cork c e l l s of the m a j o r i t y of members of the p l a n t kingdom,, except some lower forms of p l a n t l i f e ( i . e . , f u n g i ) . The d i f f e r i n g b a s i c chemical nature of l i g n i n has enabled a f u r t h e r d i v i s i o n as: gymnospermous,dicotyledonous and monocotyledonous l i g n i n a . T h i s d i v i s i o n i s based upon the predominant c h a r a c t e r of aromatic s u b s t i t u t i o n i n the phenylpropane monomers polymerized to the r e s p e c t i v e l i g n i n . The t y p i c a l gymnospermous ( c o n i f e r o u s ) l i g n i n contains guaiacylpropane ( 3-methoxy-4-hydroxyphenylpropane) monomers, while the di c o t y l e d o n o u s (hardwoods) l i g n i n c o n t a i n s y r i n g y l p r o p a n e ( 3 , 5-dimethoxy-4-hydroxyphenylpropane) monomers and gua i a c y l p r o p a n e . The monocotyledons (annual p l a n t s and gras s e s ) e x h i b i t the 4-hydroxyphenylpropane monomers as w e l l . C o n i f e r o u s l i g n i n has r e c e i v e d the g r e a t e s t amount of a t t e n t i o n owing to i t s g r e a t e r homogeneity and longer .history as a r e s i d u e of the chemical p u l p i n g i n d u s t r y . D i s t r i b u t i o n a l s t u d i e s of l i g n i f i c a t i o n i n woody 7. stems of Japanese r e d pine (Pinus d e n s i f l o r a S i e b . et Zucc. (12) have r e v e a l e d that there i s an i n c r e a s e In l i g n i n content v e r t i c a l l y i n the t r e e while the l i g n i n content i n the wood and bark i n c r e a s e s c e n t r i p e t a l l y with distance--from the cambial l a y e r . S i m i l a r i n v e s t i g a t i o n s i n eucalyptus (Eucalyptus regnans T. Muell.) by Stewart et a l . (91), i n c l u d i n g mono- and p o l y s a c c h a r i d e d e t e r m i n a t i o n s , are i n agree-ment. The p o l y s a c c h a r i d e r e s u l t s i n d i c a t e t h a t more than h a l f of the c e l l u l o s e and most of the h e m i c e l l u l o s e Is l a i d down i n the secondary w a l l before the l i g n i n i s d e p o s i t e d . The sequence of deposition was later substantiated i n an u l t r a v i o l e t m i c r o s c o p i c study of l i g n i f i c a t i o n d u r i n g x y l a r l y d i f f e r e n t i a t i o n i n Monterey pine (Pinus r a d i a t a D. Don) (101). T h i s study determined t h a t l i g n i n appears i n i t i a l l y d e p o s i t e d i n the primary w a l l near the c e l l c o r -ners d u r i n g the f o r m a t i o n of the S^ l a y e r . Subsequently, d u r i n g the f o r m a t i o n of the l a y e r , l i g n i n i s l a i d down along the i n t e r c e l l u l a r l a y e r and then s u c c e s s i v e l y In the t a n g e n t i a l and r a d i a l w a l l s . These r e s u l t s i n d i c a t e that the m a j o r i t y of l i g n i n appears i n the l a y e r d u r i n g or a f t e r f o r m a t i o n of the S^ l a y e r while l i g n i n Is c o n t i n u -o u s l y d e p o s i t e d i n the i n t e r c e l l u l a r and o u t e r l a y e r s of the c e l l w a l l . Recent work by Fergus e_t §JL. (25) has ex-amined the d i s t r i b u t i o n of l i g n i n across the c e l l w a l l s of early--and latew.ocd c e l l s i n b l a c k - spruce ,(Pice.a mariana . M i l l . ) u s i n g u l t r a v i o l e t microscropy and d e n s i t o m e t r i c analysis... T h e i r r e s u l t s show that i n the eariywcod c e l l w a l l , 72% of the t o t a l volume of l i g n i n i s i n the secondary w a l l compared to 28% o c c u r r i n g i n the middle l a m e l l a , In the latewood, 82% was found to occur i n the secondary w a l l while 18$ o c c u r r e d i n the middle l a m e l l a . The I s o l a t i o n of L i g n i n The chemical i s o l a t i o n of l i g n i n i n v o l v e s e i t h e r the removal of the carbohydrate with severe a c i d treatment l e a v i n g the i n s o l u b l e l i g n i n , or s o l u t i o n of the l i g n i n i n a p r o t i c s o l v e n t and i t s subsequent p r e c i p i t a t i o n ( 7 3 ) • Both of these methods produce l i g n i n which may or may not resemble the o r i g i n a l l i g n i n . However, the p o l y m e r i c na-t u r e of the. l i g n i n and i t s environment s e r i o u s l y r e s t r i c t o t h e r a l t e r n a t i v e s . o f i s o l a t i o n . Physical, and b i o l o g i c a l methods of i s o l a t i o n are a l s o a v a i l a b l e to separate l i g n i n from the carbohydrate m a t r i x . The p h y s i c a l methods employ the g r i n d i n g of wood i n a b a l l m i l l with or without n o n s w e l l i n g s o l v e n t s and oxygen ( 1 0 , 1 6 , 1 7 ) . B i o l o g i c a l methods u t i l i z e brown-r o t t i n g fungi.: to d i g e s t p l a n t p o l y s a c c h a r i d e s l e a v i n g the l i g n i n more a c c e s s i b l e to s o l v e n t e x t r a c t i o n ( 8 3 ) . I s o l a -ted c e l l u l a s e s have a l s o been.used to give high y i e l d s , of l i g n i n which has been s l i g h t l y a l t e r e d and w i l l d i s s o l v e i n p o l a r o r g a n i c s o l v e n t s ( 7 7 ) . A l l i s o l a t i o n techniques are l i m i t e d by t h e i r p r o p e n s i t y to produce l i g n i n which may be only p a r t i a l l y r e p r e s e n t a t i v e of n a t u r a l l i g n i n . The C h a r a c t e r i z a t i o n of L i g n i n The c h a r a c t e r i z a t i o n of l i g n i n as i t i s p r e s e n t l y understood has evolved from d e g r a d a t i v e and s y n t h e t i c i n -v e s t i g a t i o n s of l i g n o l s . The d i v e r s e nature of l i g n i n has s e v e r e l y l i m i t e d d e g r a d a t i o n techniques which have been s u c c e s s f u l l y a p p l i e d to other n a t u r a l polymers (e .g_. , h y d r o l y s i s of p r o t e i n s ) . P a r t i a l breakdown of l i g n i n has been accomplished, and the nature of the d e g r a d a t i o n pro-ducts formed i s dependent upon the s e v e r i t y of t h e . c o n d i -t i o n s used. M i l d o x i d a t i o n techniques a p p l i e d to l i g n i n ( a l k a -l i n e nitrobenzene or the oxides of copper, mercury, or s i l v e r i n a i r ) have produced s u b s t a n t i a l y i e l d s of aromatic aldehydes ( 9 , 7 5 , 9 8 ) . Three major aromatic aldehydes are obtained i n these d e g r a d a t i o n s : v a n i l l i n (3-methoxy-4-hy-droxybenzaldehyde), s yrInga ldehyde ( 3 , 5 dimethoxy - 4-hydroxy-benzaldehyde), and p-hydroxybenzaldehyde. M i l d o x i d a t i o n of c o n i f e r o u s bark l i g n i n has produced the forementioned aldehydes p l u s protocatechualdehyde ( 3 , 4 - d i h y d r o x y b e n z a l -dehyde) (5Q) . V a n i l l i n c o n t a i n s the g u a i a c y l nucleus which 10. i s predominant i n the c o n i f e r o u s s p e c i e s , while both v a n i l l i n and s y r i n g a l d e h y d e are found i n d i c o t y l e d o n o u s l i g n i n . The monocotyledonous l i g n i n c o n t a i n mainly the p-hydroxybenzaldehyde n u c l e u s . The dimer dehydrodivanillin''(.I) CHO CHO I has been o b t a i n e d as a minor product of t h i s o x i d a t i o n s u g g e s t i n g the e x i s t e n c e of some a r y l - a r y l bonds i n l i g n i n . S tronger o x i d a t i v e t e c h n i q u e s , u s i n g permanganate on methylated wood ( 3 0 , 59) y i e l d a l a r g e r number of a l i p h a t i c and m e t h o x y l - s u b s t i t u t e d aromatic a c i d s from-the o x i d i z e d l i g n i n . . Freudenberg et a l . (37, 39, 40, 4l) used the perman^-ganate o x i d a t i o n technique to o b t a i n v a l u a b l e l i g n i n s t r u c -t u r a l i n f o r m a t i o n from t h e . r a d i o a c t i v e l y l a b e l l e d o x i d a t i o n products of wood c o n t a i n i n g r a d i o a c t i v e l y l a b e l l e d l i g n i n . Reductive and s o l v o l y s i s methods a p p l i e d to l i g n i n (79) (such as r e d u c t i o n by c a t a l y t i c hydrogenation or s o l v o l y s i s w i t h a l k a l i metals i n l i q u i d ammonia) y i e l d d e r i v a t i v e s of p r o p y l c y q l o h e x a n o l or p r o p y l p h e n o l . A c i d c a t a l y z e d - e t h a n o l y s i s of l i g n i n produces H i b b e r t ' s ketones (58, 74), which i n d i c a t e the occurrence of a a Lnd/or 3-ether 11 . l i n k a g e s i n l i g n i n . The r e c e n t h y d r o g e n o l y s i s of the l i g n i n o f Hondo spruce [ P i c e a j e z o e n s i s ( S i e b . and Zucc.)] (63) r e s u l t e d i n the i s o l a t i o n of a carbon-carbon bonded dimer from the h y d r o g e n o l y s i s products which was i d e n t i f i e d as 1 - ( 3'-methoxy - 4'-hydroxypheny 1 ) - 2 ( 2"-hydroxy-3 "-methoxy-5"-n-propyl)-n-propane ( I I ) . Hydrogenolysis of d e h y d r o d i i -soeugenol ( I I I ) under the same c o n d i t i o n s gave q u a n t i t a t i v e cle.avage of the phenylcoumaran r i n g to yield II. Nimz ( 6 8 ) , has s u c c e s s f u l l y degraded l i g n i n u s i n g a c e t y l bromide or a c e t i c anhydride with boron t r i f l u o r i d e and t h i o a c e t i c a c i d with boron t r i f l u o r i d e (72) to l i b e r a t e l i g n o l fragments. These fragments were shown to undergo cleavage of the phenylcoumaran r i n g d u r i n g i s o l a t i o n ( 6 8 ) . I A t r i m e r i c - l i g n o l (IV) i n c o r p o r a t i n g a p h e n y l -coumaran s t r u c t u r e was c o n s i d e r e d to occur i n "spruce" l i g n i n based upon the i d e n t i f i c a t i o n of w-hydroxyguaiacyl-acetone ( V ) , the s u b s t i t u t e d phenylcoumarone ( V I ) , and II I I I • 12. the s u b s t i t u t e d stilb'ene (VII) i n the r e a c t i o n products of the a c i d o l y s i s of Bjorkman "spruce" l i g n i n and r e l a t e d a c i d o l y s i s products from model compounds ( 1 ) . CHJOH oca. IV OH V CH2C CH2OH OCH^ OH VI o CH2C CH2OH OCH, The products (VII and VI) were con s i d e r e d to a r i s e from compound IV v i a a coumaran r i n g opening and l o s s of formal-dehyde to form V I I j and the r e a c t i o n of the i n t e r m e d i a t e i n a r e v e r s e P r i n z r e a c t i o n to form the i s o l a t e d product VI. A comparison of the hig h y i e l d of the oi-hydroxyguai-a c y l a c e t o n e (V) with the y i e l d of the compounds o b t a i n e d 1 3 . i n the a c i d o l y s i s of 3-ary.l ether model compounds was c o n s i d e r e d c o n c l u s i v e evidence i n support of the e x i s t e n c e of a r y l g l y c e r o l 3 - a r y l e t h e r bonds i n l i g n i n . The d e s i r e to o b t a i n l i g n o l fragments from l i g -n i n has l e d to the a p p l i c a t i o n of m i l d h y d r o l y s i s t e c h -n i q u e s . Nimz (69) concluded that i t was necessary to e l i m i n a t e a c i d i c and a l k a l i n e h y d r o l y t i c c o n d i t i o n s , i n order to prevent condensation r e a c t i o n s ' of the very r e -a c t i v e benzyl, h y d r o x y l and b e n z y l e t h e r groups of l i g n i n . He p e r c o l a t e d "spruce" wood i n water at 1 0 0°C, and observed t h a t 10% of the l i g n i n went i n t o s o l u t i o n . These r e s u l t s were i n agreement w i t h those of Freudenberg e_t a l . (33) ,who observed that m i l d h y d r o l y s i s c o n d i t i o n s would cle a v e b e n z y l a r y l e t h e r groups i n "spruce" l i g n i n . Nimz (69) c o n s i d e r e d the d i s s o l v e d l i g n i n to r e p r e s e n t the ex-posed p o r t i o n of l i g n i n polymer which was probably bound to the " c o r e " l i g n i n by b e n z y l - a r y l e t h e r bonds. Monomeric, d i m e r i c , and o l i g o m e r i c p h e n o l i c d e g r a d a t i o n products were ob t a i n e d and t h e i r ' s-tructures e l u c i d a t e d (VIII to. XV). Two d i m e r i c dehydrogenation products of c o n i f e r y l a l c o h o l (XVI) had been p r e v i o u s l y o b t a i n e d i n a m i l d methyl a l c o h o l -h y d r o c h l o r i c a c i d h y d r o l y s i s of "spruce" l i g n i n ( 3 1 ) . These were D , L - p i n o r e s i n o l (XIII) and d e h y d r o d i c o n i f e r y l . a l c o h o l ( X I ) . The t r i m e r i c (XIV) and t e t r a m e r i c (XV) • lignols, isolated and characterized by Nimz (69) had . 1.4. HO XIV XV XVI g l y c e r o l 3 - a r y l ether and g l y c e r o l 3 - a l k y l - a r y l l i n k a g e s . G u a i a c y l g l y c e r o l (VIII) and the g u a i a c y l g l y c e r o l - 3 - 0 - 4 ' -c o n i f e r y l a l c o h o l (IX) o c c u r r e d i n racemic m i x t u r e s . Com-pounds X and XIV have a l s o been i s o l a t e d and i d e n t i f i e d i n the dioxane:water h y d r o l y s i s of a Hondo spruce wood meal ( 8 l ) . Nimz (70) I s o l a t e d and i d e n t i f i e d the t r i m e r i c (XIV) and t e t r a m e r i c (XV) l i g n o l s i n the water p e r c o l a t i o n products of " p i n e " wood. The m i l d , a c i d i c h y d r o l y s i s products have f u r n i s h e d c o n v i n c i n g evidence on the s t r u c t u r e of l i g n i n . I t i s r e a l i z e d t h a t these h y d r o l y s i s products r e p r e s e n t the i d e n t i f i c a t i o n of only the low molecular weight p e r i p h e r a l groups of l i g n i n . The core of the l i g n i n should c o n t a i n s i m i l a r groupings whose h y d r o l y z a b l e ether bonds are l e s s a c c e s s i b l e and consequently not c l e a v e d . S u b s t a n t i a t i o n of t h i s l i g n i n c h a r a c t e r can be found i n the work of Forss et a l . ( 2 8 ) , who proposed a b a s i c polymeric guaiacylpropane r e p e a t i n g u n i t f o r l i g n i n . T h i s b a s i c u n i t was thought to be internally bound by alkyl-aryl ether and a r y l - a r y l 3 alkyl bonds and externally by alkyl-aryl ether bonds only. It was the external alkyl ether bonds which were selectively hydrolyzed under mild acidic conditions. Assurance of the r e l i a b i l i t y of these mild acidic hydrolysis results was strengthened by the characterization of similar products i n studies of alkaline hydro-l y s i s of l i g n i n (100). 16. The B i o s y n t h e s i s of L i g n i n The s t r u c t u r e of l i g n i n has been c l a r i f i e d by s i m u l a t e d b i o s y n t h e s i s , and v e r i f i e d through chemical d e g r a d a t i o n ( 3 5 ) . This, approach i s c o n t r a r y to normal chemical c h a r a c t e r i z a t i o n which would r e q u i r e the r e v e r s e . The unorthodox procedure r e s u l t s from the nature and en-vironment of the l i g n i n , and the d i f f i c u l t i e s i t imposes upon o r d e r l y d e g r a d a t i v e a n a l y s i s . These d i f f i c u l t i e s f o s t e r e d the concept of " t r a p p i n g " a r t i f i c i a l l y , b i o s y n -t h e s i z e d l i g n i n i n t e r m e d i a t e s d u r i n g the growth of the biopolymer under s i m u l a t e d n a t u r a l c o n d i t i o n s . However, such an.approach r e q u i r e d the p r i o r c l a r i f i c a t i o n of a r o -matic b i o s y n t h e s i s and the i s o l a t i o n and i d e n t i f i c a t i o n of chemical and enzymatic i n t e r m e d i a t e s necessary to the b i o s y n t h e t i c p r o d u c t i o n of l i g n i n . The e x t e n s i v e use of r a d i o a c t i v e t r a c e r s , enzymatic and chromatographic techniques have been the primary f a c -t o r s i n the e l u c i d a t i o n of the b i o s y n t h e s i s of aromatic compounds from atmospheric carbon d i o x i d e . Two r e c e n t r e -views ( 1 3 , 35) examine the r o l e of aromatic b i o s y n t h e s i s i n l i g n i n f o r m a t i o n . These reviews and more rec e n t work of s p e c i f i c i n t e r e s t are summarized below. The c o n v e r s i o n of carbon d i o x i d e to carbohydrate v i a p h o t o s y n t h e s i s i s w e l l understood, and i s presented 17. i n a t e x t examining p l a n t b i o c h e m i s t r y ( 8 ) . The carbohy-d r a t e p r e c u r s o r s known to i n i t i a t e b i o s y n t h e t i c f o r m a t i o n of aromatic compounds o r i g i n a t e from carbohydrate metabolism (EMP pathway or pentose phosphate shunt) or photosynthe-s i z e d COg. The c o n v e r s i o n of the carbohydrate p r e c u r s o r s (phosphoenol-pyruvate and D-erythrose-4-phosphate; to the aromatic amino a c i d s p h e n y l a l a n i n e and t y r o s i n e i s a l s o well understood. This pathway (Figure 1) was elucidated i n nutri-tional studies with the bacteria E. c o l i and has been reviewed(88). Chorismic a c i d was the l a s t i n t e r m e d i a t e to be i d e n t i f i e d (42, 43) and serves as the branch p o i n t to the f o r m a t i o n of prephenic a c i d or a n t h r a n i l i c a c i d i n the a r o m a t i z a t i o n sequence. Although t h i s pathway was determined i n b a c t e r i a , subsequent s t u d i e s (3, 4, 5) have shown the pathway to be a p p l i c a b l e to h i g h e r p l a n t s as w e l l . Brown and Neish (14, 15) noted t h a t p h e n y l a l a n i n e and, to a l i m i t e d extent t y r o s i n e , were good p r e c u r s o r s to l i g n i n i n numerous p l a n t s i n d i c a t i n g the c o n v e r s i o n of s h i k i m i c a c i d - d e r i v e d aromatic amino a c i d s i n t o l i g n i n . In 1961, Koukol and Conn (55) were able to i s o l a t e and c h a r a c t e r i z e the enzyme, p h e n y l a l a n i n e a m m o n i a - l y a s e w h i c h c a t a l y z e d the deamination of p h e n y l a l a n i n e to cinnamic a c i d . The concurrent i s o l a t i o n of t y r o s i n e ammonia-lyase i n grass (66) e s t a b l i s h e d the s i m i l a r deamination of 1 8 . COOH I -COPO3H, C H , Phosphoenol pyruvic acid C H O I H - C - O H ' I H - C - O H I C H 2 0 P 0 3 H A D-Erythrose-4-phosphate C O O H I c=o I C H , I H O - C - H • I H - C - O H I H - C - C H C H 2 0 P 0 3 H 2 3-Deoxy-D-arabino heptulosinic acid -7-phosphate V^COOH KOH J HoV-^ OH 5-Dehydro quinic acid ^ HC>j—ADH Quinic acid C O O H H^PO C O O H 6 H C B , ' C O O H C O O H 3-(Enolpyruvate ether) of phosphoshikimic acid HjOjPCT Y OH O H 5-Phosphoshikimic acid HC^ 5-Dehydro-shikimic acid Shikimic acid H O O C C H , - C O - C O O H : H J - C O - C O O H C H J - C H - C O O * " N H , OH Prephenic acid A n t h r a n i i i c a c x d . Phenylpyruvic acid L-Phenylalanine CH3-CO-COOH CH2-CH-COO" ' < "^' OH p-Hydroxyphenyl-pyruvic acid L-Tyrosine F i g u r e 1. S h i k i m i c a c i d pathway to the fo r m a t i o n of aromatic amino a c i d s p h e n y l a l a n i n e and t y r o s i n e ( 3 5 ) . t y r o s i n e to.p-hydroxycinnamic a c i d . With the establishment of the t r a n s f o r m a t i o n of the aromatic amino a c i d to c i n n -amic a c i d d e r i v a t i v e , i t became necessary t o prove that these cinnamic a c i d d e r i v a t i v e s could be f u r t h e r metabolized to e v e n t u a l l y f o r m ' l i g n i n . Smith and Neish (87) claimed to 19. .demonstrate the•irreversibility of cinnamic acid formation ' i when • they showed that a l l of the carbon atoms of l a b e l l e d cinnamic a c i d were i n c o r p o r a t e d i n t o "spruce" and "aspen" twig l i g n i n r a t h e r than being i n c o r p o r a t e d i n t o cinnamic a c i d p r e c u r s o r s . In a t a b u l a t i o n of t r a c e r ex-periments u t i l i z i n g cinnamic, p-coumaric,. c a f f e i c , f e r u l i c , and s i n a p i c a c i d s by Neish (35) , i t was demonstrated t h a t a l l of these cinnamic a c i d d e r i v a t i v e s were good p r e c u r s o r s to l i g n i n , with s i n a p i c a c i d being an e f f e c t i v e p r e c u r s o r i n those s p e c i e s (angiosperms) with s y r i n g y l l i g n i n . V e r i -f i c a t i o n of the cinnamic a c i d metabolism.route to l i g n i n u t i l i z e d these r i n g s u b s t i t u t e d cinnamic a c i d d e r i v a t i v e s , yet the mechanism of r i n g h y d r o x y l a t i o n and/or methoxylation had only been i n f e r r e d because of the l i m i t e d data a v a i l a b l e r e g a r d i n g h y d r o x y l a t i o n and m e t h y l a t i o n . Although i t had been demonstrated i n 1954 (18) that methionine serves as a methyl group donor i n l i g n i n forma-t i o n , the o r i g i n of the r e q u i r e d h y d r o x y l a t e d s u b s t r a t e and m e t h y l a t i n g enzyme were not determined u n t i l l a t e r . In 1 9 6 5 , cinnamic a c i d hydroxylase was d i s c o v e r e d i n spinach acetone powders (64) which was capable of forming p-hydroxy-cinnamic a c i d from cinnamic a c i d . Recently ( 9 9 ) , a p u r i f i e d phenolase from s p i n a c h l e a f has been shown to c a t a l y z e the p r o d u c t i o n of c a f f e i c a c i d from p-coumaric a c i d . M e t h y l a t -i n g enzymes have now been found which are capable of 2 0 . m e t h y l a t i n g c a f f e i c a c i d i n the presence of S-adenosyl-methionine i n grasses and woody shrubs ( 2 6 , 2 7 ) . H i g u c h i , et a l . (51) have a l s o d e s c r i b e d an enzyme p r e p a r a t i o n ( S - a d e n o s y l m e t h i o n i n e : c a t e c h o l - O - m e t h y l t r a n s f e r a s e ) which s e l e c t i v e l y methylated the meta-hydroxyl of c a f f e i c and 5 - h y d r o x y f e r u l i c a c i d s i n bamboo shoots. L i t t l e evidence i s a v a i l a b l e r e g a r d i n g f u r t h e r h y d r o x y l a t i o n and methyla-t i o n of t h e ' g u a i a c y l nucleus l e a d i n g to the form a t i o n of the. s y r i n g y l n u c l e u s . I t i s e v i d e n t , however, t h a t meta-b o l i c sequences l e a d to the s u b s t i t u t e d cinnamic a c i d s p r e v i o u s l y c o n s i d e r e d as good p r e c u r s o r s to l i g n i n . Such a sequence may be con s i d e r e d as: p h e n y l a l a n i n e -* cinnamic a c i d -»• p-coumaric a c i d -* c a f f e i c a c i d -* f e r u l i c a c i d ->-s i n a p i c a c i d . C o n i f e r i n ( 4 - 0 - c o n i f e r y l a l c o h o l 3-D-glucoside) has been shown to be an e x c e l l e n t p r e c u r s o r to l i g n i n i n c o n i f e r s (38) s u g g e s t i n g r e d u c t i o n of a cinnamic a c i d c a r b o x y l f u n c t i o n p r i o r t o . l i g n i f i c a t i o n . Experiments i n tobacco leaves (89) have e s t a b l i s h e d the b i o s y n t h e t i c path-way to the fo r m a t i o n of the q u i n l c a c i d e s t e r of c a f f e i c a c i d ( c h l o r o g e n i c a c i d ) from cinnamic a c i d , c o n c l u d i n g as w e l l that the c h l o r o g e n i c a c i d does not take part i n p o l y -phenol or l i g n i n b i o s y n t h e s i s . E l - B a s y o u n i and .co-workers ( 2 3 , 24) demonstrated the occurrence of m e t a b o l i c a l l y ac-t i v e i n t e r m e d i a t e s (acetone and a l c o h o l i n s o l u b l e ) , d e r i v e d 21. from p h e n y l a l a n i n e , which were r e a d i l y i n c o r p o r a t e d i n t o the l i g n i n of wheat and. b a r l e y p l a n t s . These i n t e r m e d i a t e s were b e t t e r l i g n i n p r e c u r s o r s than s o l u b l e analogs and produced hydroxycinnamic a c i d s upon h y d r o l y s i s . Bland and Logan (11) found, i n l i g n i f i c a t l o n . s t u d i e s on E u c a l y p t u s spp. shoots, that h y d r o x y l a t e d cinnamic a c i d s were con-v e r t e d to glucose * e s t e r s p r i o r to t h e i r i n c o r p o r a t i o n i n t o l i g n i n . H l g u c h i and Brown ( 4 9 ) , s t u d y i n g wheat p l a n t l i g -n i f i c a t i o n , concluded that c o n i f e r i n was not an o b l i g a t o r y p r e c u r s o r to l i g n i n f o r m a t i o n , based upon the o b s e r v a t i o n s t h a t : ( a ) ' c o n i f e r y l a l c o h o l c o u l d d i l u t e the. l i g n i f i c a t i o n i n t e r m e d i a t e formed from f e r u l i c acid., and (b) that c o n i -f e r a l d e h y d e and c o n i f e r y l a l c o h o l were obtained i n f e r u l i c 14 a c i d - C feedings r a t h e r than c o n i f e r i n . These data suggest the importance of e s t e r i f i e d cinnamic a c i d i n t e r m e d i a t e s i n l i g n i n f o r m a t i o n . In summary, 'the b i o s y n t h e t i c pathway to l i g n i n from C 0 2 may be c o n s i d e r e d to be: C 0 2 P h o t o s y n t h e s ^ s , , , , . s h i k i m i c a c i d pathway , . . . , . carbonydrates £ — — — £ - — ^ a r o m a t i c amino a c i d s deamination, h y d r o x y l a t i o n , m e t h y l a t i o n , , ., . , , . —- 2 — 2 ,—£ s u b s t i t u t e d c m -e s t e r i f I c a t i o n namic a c i d s > i n s o l u b l e e s t e r s of cinnamic . , r e d u c t i o n ,, , „ . . .' -, , , a c i d c- a l k y l ethers of cinnamic a l c o h o l s p o l y m e r i z a t i o n ,. - — 1* l i g n i n . The c o n c l u d i n g step i n the above scheme has been the b a s i s f o r f o r m a t i o n s t u d i e s on a r t i f i c i a l l i g n i n . T h i s 22. concept of determining l i g n i n s t r u c t u r e i s now almost synonymous with Freudenberg and the H e i d e l b e r g s c h o o l of l i g n i n r e s e a r c h . Freudenberg, a f t e r Klason, used c o n i f e r y l a l c o h o l as a s u b s t r a t e i n c o n j u n c t i o n with an enzyme pr e -p a r a t i o n from a common f i e l d mushroom P s a l l i o t a campestris to produce ,at n e u t r a l pH ,a polymerized substance which c l o s e l y resembled l i g n i n (34). In the l a s t 20 years of work on t h i s e n z y m a t i c a l l y produced polymer, i n t e r m e d i a t e s of the .condensation r e a c t i o n s have been i s o l a t e d 'and i d e n -t i f i e d thereby r e v e a l i n g the mechanism of l i g n i f i c a t i o n and the s t r u c t u r e of t h i s l i g n i n . Freudenberg's most r e -cent comprehensive review of h i s work and r e l a t e d work . t r a c e s t h i s e v o l u t i o n (35) which culminated with schematic formulae f o r l i g n i n based on the compiled r e s u l t s . The process of p o l y m e r i z a t i o n of p-hydroxycinnamyl a l c o h o l s to l i g n i n i s now c o n s i d e r e d to procede v i a the phenoxide forms which give metastable f r e e r a d i c a l s with f o u r p r i n c i p a l mesomers ( F i g u r e 2 ) . H a r k i n (48) has c l a s s i -f i e d the r e a c t i o n w i t h which these f r e e r a d i c a l s combine to f o r m ' s t a b i l i z e d forms by the f o l l o w i n g f i v e mechanisms at l e a s t : 1. M o l e c u l a r growth by f r e e - r a d i c a l p a i r i n g to form u n s t a b l e quinone methides. 2. I n t r a m o l e c u l a r rearrangements of some quinone methides to form phenoxides (phenols) that may undergo renewed o x i d a t i o n to f r e e r a d i c a l s . F i g u r e 2. Resonance forms of the f r e e r a d i c a l d e r i v e d from i o n i z e d c o n i f e r y l a l c o h o l . 3. S t a b i l i z a t i o n of some quinone methides by a d d i t i o n of e l e c t r o p h i l e s (plus protons) to reform phenoxides (phenols) a l s o capable of f u r t h e r o x i d a t i o n . 4. I n c i d e n t a l s i d e c h a i n o x i d a t i o n s due to f r e e - r a d i c a l t r a n s f e r s and subsequent d i s -p r o p o r t i o n a t i o n s . 5. S t a b i l i z a t i o n o f some quinone methides by s i d e - c h a i n e l i m i n a t i o n , a type of d i s p r o -p o r t i o n a t i o n . h'arkin (48) il l u s t r a t e s the formation of "lignin" pro-ducts via the five processes, through combinations of radicals (Figure 2). Process 1 (e.g_. 'R&+ P^, Rb+ R + F^, RQ+ R ) followed by Process 2 w i l l produce the di l i g n o l structures such as guaiacylglycerol-3-0-4'-coniferyl alcohol (IX) and pinoresinol (XIII).. 24. HO Process 3 i n t r o d u c e s water to form b e n z y l a l c o -h o l s and probably i s i n v o l v e d i n the c o v a l e n t l i g n i n -carbohydrate bond by condensation with carbohydrates. T h i s process a l s o may r e s u l t i n the branching of l i g n i n by the f o r m a t i o n of n o n c y c l i c b e n z y l - a r y l ethers a f t e r condensation with u n o x i d i z e d phenoxides. Process 4 g i v e s r i s e to c a r b o n y l f u n c t i o n s i n a l k y l s i d e c h a i n s , while Process 5 leads to s t r u c t u r e s such as d i a r y l p r o p a n e d i o l s , d i p h e n y l e t h e r s , e s t e r s , d l o x e p l n s , and a r y l g l y c e r i c a c i d or a r y l g l y c e r a l d e h y d e e t h e r s . T h e r e f o r e , most of the i s o l a t e d components of the s y n t h e t i c l i g n i n , and the de-g r a d a t i o n products o f . l i g n i n , can be e x p l a i n e d i n terms of f r e e r a d i c a l combinations and subsequent rearrangements. R e c e n t l y , Connors et §JL. (19) formed three d i l i g -n o l s from the d i m e r i z a t i o n of c o n i f e r a l d e h y d e i n the pre- . sence of peroxidase enzyme and hydrogen peroxide in. 2 5 . aqueous solution. The products were 2 ,3-diformyl-l ,4-di -5-guaia-cylbuta-133--diene (XVTI),'a-(4-3-fonnylvinyl~2~methoxyphenoxy) coniferylaldehyde. (XVIII).v a-.(5-3-foiiuylviriyI-2-hydroxy-3-methoxy- • phenyl) coniferylaldehyde (XIX). • ' These dimers can., be e x p l a i n e d v i a the f r e e r a d i c a l s of F i g u r e 2 . The a l d e h y d i c s i d e chains of two c o n i f e r y l -aldehydes, comprising the a, 3-enone system rearranges at the quinone methide stage to form a s t a b l e phenol, which through the l o s s of the a c i d i c p roton from the c a r -bon a to the c a r b o n y l , subsequently rearomatizes with the f i n a l p r o t o n a t i o n of the phenoxyl anion to y i e l d the d i m e r i c p r o d u c t s . At t h i s p o i n t i t i s important to con-s i d e r what f r e e p r e c u r s o r s (monomeric, d i m e r i c , e t c . ) have OH XVII XVIII XIX 2 6 . been i s o l a t e d unchanged from l i g n i f y i n g systems to f u r t h e r s u b s t a n t i a t e the c o n c l u s i o n s drawn from d e g r a d a t i v e and b i o s y n t h e t i c r e s u l t s . Aromatic C o n s t i t u e n t s .Related to L i g n i n p l a n t t i s s u e s have .considered aromatic c o n s t i t u e n t s . The cambial sap o f " s p r u c e " d e a c t i v a t e d with formaldehyde, has been shown to c o n t a i n the 3-D-glucosides of 4 - 0 - s i n a p y l and 4 - 0-p-coumaryl a l c o h o l ( 3 2 ) . C o n i f e r i n , v a r i o u s sugars, s m a l l amounts of c o n i f e r y l a l c o h o l , q u i n i c a c i d , and un-known substances of h i g h m o l e c u l a r weight which gave y e l -low or orange-yellow c o l o r r e a c t i o n s with d i a z o t i z e d s u l f a n i l i c a c i d , have a l s o been shown to be present i n d e a c t i v a t e d " spruce", sap ( 3 6 ) . The "spruce" sap, without formaldehyde t r e a t m e n t w a s a l s o shown to c o n t a i n : s h i k i m i c a c i d , p r o t o c a t e c h u i c a c i d , D , L - p i n o r e s i n o l ( X I I I ) , g u a i a c y l g l y c e r o l 3 - c o n i f e r y l ether ( I X ) , and d e h y d r o d i -c o n i f e r y l a l c o h o l ( X I ) . S e v e r a l i n v e s t i g a t i o n s of a c t i v e l y m e t a b o l i z i n g IX XI XIII 2 7 . In an examination of the cambium and sapwood of western hemlock (Tsuga heterophy 11a (Raf.) Sarg.) for l i g n i n p r e c u r s o r s , Goldschmid and Hergert (46) found many com-pounds i n c l u d i n g s e v e r a l suspected l i g n a n g l y c o s i d e s whose s t r u c t u r e s were not e l u c i d a t e d . Among the compounds de-t e c t e d i n the sapwood were: c o n i d e n d r i n (XX), hydroxy-m a t a i r e s i n o l (XXI), o x o m a t a i r e s i n o l ( X X I I ) , p i n o r e s i n o l ( X I I I ) , d e h y d r o d i c o n i f e r y l a l c o h o l ( I X ) , and s e v e r a l sus-pected l i g n a n g l y c o s i d e s . XX . XXI XXII Barton (6) has very r e c e n t l y s t u d i e d western hemlock sap-wood and r e p o r t e d the occurrence of l i o v i l (XXIII) and a no v e l d i l i g n o l 2 - ( a - h y d r o x y - v a n i l l y l ) - 5 - o j - h y d r o x y p r o p y l ) -7-methoxy coumaran (XXIV). T h i s compound (XXIV) e x h i b i t s a unique 3-y ;-linkage, and was a l s o thought to occur as an 28. a l i p h a t i c g l y c o s i d e CH2CH2CH2OH XXIII XXIV St u d i e s on- l e a f t i s s u e have mainly been concerned with carbohydrate content. However, i n a study of low molecular weight aromatic compounds i n the leaves of Scots pine "(Pinus sylvestris L.) (97), two glycosides of guaiacylglycerol . were i d e n t i f i e d . • The two g l y c o s i d e s were a-threo-3-D-g l u c o g u a i a c y l g l y c e r o l (XXV) and 13-threo-3-D-glucoguaiacyl-g l y c e r o l (XXVI). G H p H HG< HCOGIucose CHpH HCOGIucose I HCOH OCH-OH OH XXV XXVI 2 9 . In a study of the e x t r a c t i v e s of the leaves of tamarack (Larix l a r i c i n a (Du Roi) K. Koch), Niemann (67) found the 3 - g l u c o s i d e s .of v a n i l l i c and p-coumaric a c i d s and the a ~ g l u c o s i d e of p-hydroxybenzoic a c i d . Takahashi et a l . (96) have examined the p o l y p h e n o l i c s i n the leaves of one hundred c o n i f e r s n o t i n g p r i m a r i l y the occurrence of f l a v o n p i d s . A r e c e n t i n c o r p o r a t i o n study (80) u s i n g the leaves of Douglas f i r (Pseudotsuga m e n z e s i i (Mirb.) Franco) showed i n c o r p o r a t i o n of the T - g l u c o v a n i l l i n i n t o l i g n i n whereas T - f e r u l i c a c i d was not i n c o r p o r a t e d . . A recent examination (52) of p h e n y l a l a n i n e ammonia-lyase (PAL) activity i n eucalyptus (Eucalyptus" sieberi L. Johnson\ and " _(E. slderoxylori A. Cunn et Wools) leaves indicated-that glu-cose may serve as a b e t t e r p r e c u r s o r than p h e n y l a l a n i n e i n the f o r m a t i o n of polyphenols such as the s t i l b e n e s and f l a v a n o i d s . I t was s p e c u l a t e d that PAL a c t i v i t y may be more d i r e c t l y r e l a t e d t o l i g n i f i c a t i o n than p o l y p h e n o l b i o s y n t h e s i s . When p h e n y l a l a n i n e was f e d to the l e a v e s , p-coumarylquinic a c i d and c h l o r o g e n i c a c i d were the f i r s t m e t a bolic products to be formed while glucose f e e d i n g pro-duced c a t e c h i n and the g l y c o s i d e s of s t i l b e n e s and f l a -vonoids f i r s t . The f o r m a t i o n of the l i g n a n s i s another important d i m e r i z a t i o n r e a c t i o n of monomeric phenylpropane 3 0 . p r e c u r s o r s . No s p e c i f i c evidence i s a v a i l a b l e r e g a r d i n g the b i o s y n t h e s i s of l i g n a n s . Neish (65) has p o s t u l a t e d that the' l i g n a n s are probably formed through a r e d u c t i v e c o u p l i n g of cinnamyl a l c o h o l s through the 3 carbon atoms of the propylene s i d e c h a i n . Such a c o u p l i n g Is suggested to be s p e c i f i c a l l y enzyme c o n t r o l l e d , thereby p r o d u c i n g the common 2-L, 3-D c o n f i g u r a t i o n of the l i g n a n s . The" specific configuration and the lack of triphenylpropenoid lignans,•pre-cludes the quinone methide pathway s i n c e the p o l y m e r i z a t i o n of c o n i f e r y l a l c o h o l v i a f r e e r a d i c a l r e a c t i o n s , g i v e s o p t i c a l l y i n a c t i v e p r o d u c t s . The route of f o r m a t i o n o f l i g n a n s and l i g n i n , however, may be c l o s e l y r e l a t e d b i o -s y n t h e t i c a l l y s i n c e they both a r i s e from cinnamyl a l c o h o l c o u p l i n g r e a c t i o n s . AROMATICS OF WESTERN RED CEDAR .. -The study of lignans and tropolones has been extensive i n western red cedar (Thuja plicata Donn). Characterization of these components has played a primary role i n the u t i l i z a t i o n of the species (62). The tropolones (2-hydroxy-2,4,6-cycloheptatrl-en-l-ones) are non-benzenoid aromatics which are steam v o l i t i l e . Those which.have been isolated and characterized from western red cedar wood,according to Barton and MacDonald (7) , are: a-thuja-p l i c i n (XXVTI), 3-thujaplicin (XXVTII), y- thujaplicin (XXIX), . 3- thujaplicinol (XXX), 0- dolabrin (XXXI), together with 31. XXVII' XXVIII XXIX XXX XXXI XXXVa, R=H XXXVIa, R 1 , R 2 = H XXXVII XXXVb, R=OH XXXVIb, R 1=0H;R 2-H XXXVIc, R 1 = R 2 = O H 32. s i m i l a r compounds such- as nezukone (XXXII) / methyl t h u j a t e ('.XXXIV.), and t h u j i c a c i d (XXXIII). The l i g n a n s which have been i s o l a t e d and c h a r a c t e r i z e d i n western r e d cedar are e i t h e r d e r i v a t i v e s of 3,Y-dibenzylbutane: t h u j a p l i c a t i n (XXXVa), dihydroxy thuj a p l i c a t i n (XXXVb), t h u j . a p l i c a t i n methyl ether (XXXVIa), h y d r o x y t h u j a p l i c a t i n methyl ether (XXVIb), d i h y d r o x y t h u j a p l i c a t i n methyl e t h e r (XXVIc), or of t e t r a h y d r o n a p t h a l e n e : p l i c a t i c a c i d (XXXVII), p l i c a t i n (XXXVIII), p l i c a t i n a p t h o l (XXIX), and p l i c a t i n a p t h a l e n e ( X L ) . The l a r g e number of lignans. In western red cedar has generated i n t e r e s t i n t o t h e i r b i o s y n t h e s i s and s i t e of fo r m a t i o n . In a chromatographic study o f the l i g n a n s of western r e d cedar r e l a t e d to the sapwood-heartwood t r a n s -f o r m a t i o n , Swan e_t a l . (95) concluded that the major p o r t i o n of the l i g n a n s ( i n western r e d cedar) are formed i r i s i t u at the sapwood-heartwood boundary. The t r a n s f o r m a t i o n through h y d r o x y l a t i o n was co n s i d e r e d to continue i n t o the heartwood- f o r many years by the r o u t e : thuj a p l i c a t i n -> dih y d r o x y t h u j a p l i c a t i n ->- p l i c a t i n -> p l i c a t i c a c i d . In a l a t e r a d d i t i o n to t h i s p r o p o s a l , Swan and J i a n g (94) con-s i d e r e d that p l i c a t i n a p t h o l was d e r i v e d from p l i c a t i n through d e h y d r a t i o n and h y d r o x y l a t i o n with the t r a n s f o r m a t i o n extending- w e l l i n t o the newly formed heartwood. Hydroxy-l a t i o n of the tropone nezukone to form the t h u j . a p l i c i n s was observed to occur wholly at the sapwood-heartwood boundary. Mono- and d i h y d r o x y l a t e d t h u j a p l i c a t i n methyl ether d e r i v a t i v e s were c o n s i d e r e d to be formed i n a manner s i m i l a r to the f o r m a t i o n of p l i c a t i n . No r e c o g n i z e d • i n t e r m e d i a t e s which c o u l d d e f i n e the t r a n s f o r m a t i o n of phenylpropane to a l i g n a n have been i s o l a t e d . Swan (95) suggests that the search f o r such unknown i n t e r m e d i a t e s must extend to the sapwood and cambial areas'by u t i l i z i n g i n v i t r o or i n v i v o r a d i o a c t i v e t r a c e r experiments. Metabolism and L i g n i n Formation T h i s d i s c u s s i o n would be incomplete without an examination of the t o t a l " c o n s e c u t i v e u t i l i z a t i o n of nu-t r i e n t s l e a d i n g to l i g n i n f o r m a t i o n i n the l i v i n g p l a n t . Ik Coniferous l e a f p h o t o s y n t h e s i s s t u d i e s u s i n g CO ^  have shown r a d i o a c t i v i t y i n c o r p o r a t i o n i n t o s u c r o s e , r a f f i n o s e , D-glucose, and D - f r u c t o s e w i t h the m a j o r i t y of a c t i v i t y o c c u r r i n g i n sucrose ( 8 5 , 8 6 ) . A recent study of photo-a s s i m i l a t i o n of "^CO^ i n the branches of red pine (Pinus  r e s i n o s a Ait*);,/ (78) r e v e a l e d the d i s t r i b u t i o n of photo-synthate predominantly up the main stem to the a p i c a l growth r e g i o n . The r a d i o a c t i v i t y a s s i m i l a t i n g from the lower worls moved down the stem and could be d e t e c t e d i n the r o o t s . Autoradiography of i n t e r n o d a l s e c t i o n s r e -v e a l e d the c o n c e n t r a t i o n of l i g n i n p r e c u r s o r s ( s h i k i m i c a c i d , q u i n i c a c i d ) i n newly d i f f e r e n t i a t i n g t i s s u e . 34. P r e f e r e n t i a l l a b e l i n g of l i g n i n over c e l l u l o s e , suggested that l i g n i n b i o s y n t h e s i s competed s u c c e s s f u l l y f o r new photos-ynthate d u r i n g growth. The data i n d i c a t e s the t r a n s l o c a t i o n of carbohydrates ( p a r t i c u l a r l y sucrose) from the leaves to the cambial t i s s u e s f o r f u r t h e r meta-b o l i s m . Stewart ( 9 0 ) , i n a summary of evidence r e l a t i n g to the s e q u e n t i a l f o r m a t i o n of secondary t i s s u e s i n t r e e s , notes t h a t the cambium converts d i s a c c h a r i d e s and o l i g o -s a c c h a r i d e s to.monosaccharides f o r f u r t h e r metabolism by the d i f f e r e n t i a t i n g c e l l . The bul k of the a v a i l a b l e con-v e r t e d carbohydrate i s i n c o r p o r a t e d i n t o the primary w a l l i n the b i o s y n t h e s i s of p o l y s a c c h a r i d e s c o n t r o l l e d by the p r o t o p l a s t . The s h i k i m i c a c i d pathway i s probably i n i t i a t e d at t h i s stage. During the c y t o p l a s m i c stages of p o l y s a c c h a r i d e s y n t h e s i s , the aromatic amino a c i d s produced by the s h i k i -mic a c i d pathway, p a r t i c i p a t e i n aminotransferase and aminohydrolase r e a c t i o n s to produce the'hydroxyaromatic a c i d s . These i n t u r n are converted to e s t e r i f i e d p r e c u r -sors of the polyphenols and l i g n i n , as p r e v i o u s l y d e s c r i b e d . These e s t e r i f i e d p r e c u r s o r s may then be e x c r e t e d i n t o the c e l l v a c u o l e , e x c r e t e d i n t o the t r a n s l o c a t o r y systems of the r a y s , or i n c o r p o r a t e d i n t o the c e l l w a l l . In the l a s t i n s t a n c e , the g l y c o s i d e s are h y d r o l y z e d , and converted to l i g n i n through the a c t i o n of an o x i d o r e d u c t a s e . As the f i b e r enters senescence, the c o n c e n t r a t i o n of carbohy-d r a t e s decreases c a u s i n g a breakdown of the cytoplasmic membrane, and a consequent b l e n d i n g of vacuole components with those of the g e n e r a l cytoplasm. During t h i s stage much of the p h e n o l i c e x c r e t o r y c o n s t i t u e n t s d i f f u s e through, the c e l l w a l l . I t i s t h i s change i n b i o l o g i c a l a c t i v i t y a s s o c i a t e d with senescence which i s probably r e s p o n s i b l e f o r more than 50% of the l i g n i n b e i n g depo-s i t e d a f t e r most of the c e l l u l o s e has been l a i d down. L i t e r a t u r e Summary and Observations I t i s apparent from the l i t e r a t u r e t h a t the pro-cess of l i g n i n f o r m a t i o n has been e x t e n s i v e l y s t u d i e d from s y n t h e t i c , b i o s y n t h e t i c , and degradative v i e w p o i n t s . The composite view, of l i g n i n f o r m a t i o n i n v o l v e s a carbohydrate to aromatic monomer to polymer r e l a t i o n s h i p which c o r r e -sponds to a l e a f to cambium to c e l l l o c a t i o n a l sequence i n woody stems. The nature of i n t e r m e d i a t e s at a l l e a r l y stages allows t h e i r e f f i c i e n t t r a n s p o r t and e v e n t u a l t r a n s f o r m a t i o n to l i g n i n . Most s t u d i e s of l i g n i n f ormation i n woody stems have been concerned with cambial or sapwood r e g i o n s with s e v e r a l important monomeric i n t e r m e d i a t e s l o c a t e d and c h a r a c t e r i z e d . C o n s i d e r a b l y fewer f r e e d i l i g n o l s to o l i g o l i g n o l s have been l o c a t e d or c h a r a c t e r i z e d i n a l l 36. . p l a n t systems, although d i l i g n o l s have been shown as e f f e c t i v e p r e c u r s o r s to l i g n i n f ormation i n s y n t h e t i c s t u d i e s . The l e a f - b r a n c h r e l a t i o n s h i p p r o v i d e s a p r o m i s i n g area f o r the l o c a t i o n of s t a b l e i n t e r m e d i a t e s r e l a t e d to the b i o s y n t h e s i s of p o l y a r o m a t i c compounds. The study of a c t i v e l y m e t a b o l i z i n g l e a f t i s s u e s o f f e r s the added advan-tage of ease of experimental m a n i p u l a t i o n i n r a d i o a c t i v e and enzymatic s t u d i e s i n comparison to woody t i s s u e s . De-velopment of a n a l y t i c a l techniques capable of a c c e l e r a t e d a n a l y s i s of t e s t r e s u l t s are necessary, i n order to o b t a i n experimental data c o n s i s t e n t with the metabolic c a p a b i l i t y of the t i s s u e . I t i s remarkable t h a t so l i t t l e r e s e a r c h has been done on the aromatic c o n s t i t u e n t s of c o n i f e r o u s , p l a n t l e a v e s . The leaves of western red cedar o f f e r par-t i c u l a r p o t e n t i a l f o r l i g n i n s t u d i e s , s i n c e the species con-t a i n s a h i g h e r than average amount of both l i g n i n and l i g n a n s . 37-. MATERIALS AND METHODS ; C o l l e c t i o n of Leaves Leaf samples were obtained from low l e v e l branches of f i v e or more randomly s e l e c t e d western r e d cedar (Thuja p l i c a t a Donn) t r e e s on the U n i v e r s i t y of B r i t i s h Columbia campus. C o l l e c t i o n s were made i n January 1 9 6 9 , A p r i l 1 9 6 9 , June 1 9 6 9 , October 1 9 6 9 , and June 1 9 7 0 . The f i r s t t h ree c o l l e c t i o n dates p r o v i d e d samples f o r the de- . velopment of i s o l a t i o n t e c h n i q u e s . The t h i r d c o l l e c t i o n was used f o r f e e d i n g experiments and the i s o l a t i o n of g l y -c o s i d e s . The f i n a l c o l l e c t i o n s served to repeat the i s o -l a t i o n procedures and to develop improvements. The leaves were cut from the t r e e s t a k i n g care to a v o i d branch m a t e r i a l , cones or f l o w e r s . The samples were immediately p l a c e d i n a p l a s t i c bag, s e a l e d , and t r a n s p o r t e d to the l a b o r a t o r y . Leaf E x t r a c t i o n 14 F o l l o w i n g the removal of l e a f samples f o r C f e e d -i n g experiments and moisture -determination, the remaining leaves were chopped i n a l a r g e Waring b l e n d e r without s o l v -ent . The chopped leaves were weighed and p l a c e d i n a l a r g e Soxhlet e x t r a c t o r f o r . e x t r a c t i o n w i t h methyl a l c o h o l over a 72 hour p e r i o d . A s i n g l e e x t r a c t i o n was decid e d 38. upon because the h i g h percentage of moisture i n the leaves prevented t r u e s e q u e n t i a l e x t r a c t i o n i n a p i l o t attempt. The methyl a l c o h o l e x t r a c t which was o b t a i n e d was worked up a c c o r d i n g to F i g u r e 3. The e t h y l a c e t a t e e x t r a c t obtained contained leaf phenolic glycosides without interfering chlorophylls. c Chromatography T h i n l a y e r c e l l u l o s e chromatography T h i n l a y e r c e l l u l o s e p l a t e s were prepared u s i n g 25 grams of A v i c e l m i c r o c r y s t a l l i n e c e l l u l o s e (EMC Corp. Div. of American V i s c o s e Co.), i n 85 ml of water f o l l o w e d by homogenization of f i f t e e n seconds i n a Waring b l e n d e r . The r e s u l t a n t s l u r r y ( t h i c k n e s s 0.25 nim) was a p p l i e d t o f i v e 8" x 8" g l a s s p l a t e s i n a Shandon t h i n l a y e r a p p l i c a t o r . The p l a t e s were allowed t o a i r - d r y o v e r n i g h t . Four s o l v e n t systems \\rere used i n t h i n l a y e r c e l -l u l o s e chromatography: (1) n-butanol: c h l o r o f o r m : a c e t i c a c i d : water (4:1:1:1) (BCAW); (2) lower l a y e r c h l o r o f o r m : a c e t i c a c i d : water (2:3:1.5) (CAW); (3) 2% aqueous a c e t i c a c i d (2%); and (4) isopropanol:ammonia:water (20:1:2) (IAW). The f i r s t two s o l v e n t s were c o n s i d e r e d as the r e f e r e n c e system f o r the study. T h i n l a y e r c e l l u l o s e chromatography of sugar r e s i d u e s used e t h y l a c e t a t e : p r y i d i n e : water (12:5:4) as the d e v e l o p i n g s o l v e n t . 39. " Western Red Cedar Leaves -Waring blender Chopped leaves Leaf samples f o r 14 C f e e d i n g study D i s c a r d leaves - E x t r a c t i o n with methyl a l c o h o l 72 hours Methyl a l c o h o l s o l u b l e s - F i l t e r through c e l i t e . Methyl a l c o h o l f i l t r a t e 1) Evaporate to dryness 2) Wash with 1 l i t e r c h l o r o f o r m Chloroform i n s o l u b l e s 1) Add 1.5 l i t e r s and c e l i t e to form t h i c k s l u r r y 2) F i l t e r through c e l i t e Chloroform washings-d i s c a r d e d Water i n s o l u b l e s - d i s c a r d e d Water s o l u b l e s 1) Concentrate on evaporator 2) E x t r a c t with e t h y l ether E t h y l e t h e r s o l u b l e s 0.8% - not ana l y z e d Water s o l u b l e s E x t r a c t with e t h y l a c e t a t e Water s o l u b l e s -d i s c a r d e d E t h y l a c e t a t e s o l u b l e s 3.9% F i g u r e 3 . . Scheme f o r the e x t r a c t i o n and s e p a r a t i o n of components from western red cedar l e a v e s . 40. D e t e c t i o n of the compounds on the c e l l u l o s e p l a t e s p r i m a r i l y u t i l i z e d d i a z o t i z e d s u l f a n i l i c a c i d . . Other s p r a y s , i n c l u d i n g d i a z o t i z e d p - n i t r o a n i l i n e and Barton's reagent ( F e C l ^ 'Fel^IN^ ) ) were used, to d e t e c t p h e n o l i c s , while sugar r e s i d u e s were, l o c a t e d with p - a n i s i d i n e hydro-c h l o r i d e . • T h i n l a y e r s i l i c a g e l chromatography S i l i c a , gel. plates'were prepared u s i n g a. s l u r r y of 35 g of Merck S i l i c a Gel G In 70 ml of water. This ' s l u r r y was a p p l i e d at a t h i c k n e s s of 0.25 mm or 0 . 5 0 mm ( f o r p r e p a r a t i v e chromatography) on f i v e 8" x 8" glass' p l a t e s u s i n g a Shandon a p p l i c a t o r . The p l a t e s were then p l a c e d -i n a 130°.C oven f o r !•£ hours f o r a c t i v a t i o n . P l a t e s not used-immediately -were s t o r e d i n ' a 70°C oven to prevent d e a c t i v a t i o n . 1 Two s o l v e n t s were used f o r t h i n l a y e r s i l i c a p l a t e s : (1) chloroform:methyl a l c o h o l (4 : 1 ) (CM), and (2) benzene: e t h y l a l c o h o l ( 9 : 1 ) (BE). A spray of concentrated s u l f u r -i c a c i d , c o n c e n t r a t e d n i t r i c a c i d ( 1 : 1 ) f o l l o w e d by heat-i n g was used to detect compounds on the p l a t e s . A l t e r n a -t i v e l y , d i a z o t i z e d s u l f a n i l i c a c i d was a l s o used -as a d e t e c t i o n reagent.- . • Column chromatography Column chromatography was the most important means of i s o l a t i n g the p h e n o l i c g l y c o s i d e s In t h i s i n v e s t i g a t i o n . 41. S i l i c i c a c i d ( F i s h e r ) and Sephadex LH-20 (Pharmacia) were the two major chromatographic media used. The s e l e c t i o n of LH-20 as a g e l - f i l t r a t i o n medium r e s u l t e d from p r i o r chromatographic experiments (79) and with p i l o t s t u d i e s on the e t h y l a c e t a t e e x t r a c t . E a r l i e r t r i a l s , using. • Sephadex G-10 and G - 2 5 , p r o v i d e d e x c e l l e n t r e s u l t s i n the s e p a r a t i o n of the then unknown d i l i g n o l g l y c o s i d e s from other mono- and p o l y p h e n o l i c g l y c o s i d e s . These g e l s were e l u t e d with water or water-methanol and the d e s i r e d g l y -cosides were r e c l a i m e d by ethyl, a c e t a t e e x t r a c t i o n . Sepha-dex LH-20"was used with comparable r e s u l t s u s i n g o r g a n i c s o l v e n t e l u t i o n s . The s o l v e n t system which proved the most e f f e c t i v e on LH-20 was c h l o r o f o r m : e t h y l a l c o h o l ( 4 : 1 ) (CE). C e l l u l o s e column chromatography was a l s o ex-amined and although i t p r o v i d e d s e p a r a t i o n of the g l y c o s i d e s , the s e p a r a t i o n was much l e s s d e f i n i t i v e and;, again r e q u i r e d e t h y l a c e t a t e e x t r a c t i o n of the water e l u a n t . D e a c t i v a t e d s i l i c i c a c i d was used as the other chromatographic medium with e i t h e r CM or 3E as e l u a n t s . These s o l v e n t s are c o i n c i d e n t with those used on s i l i c a g e l p l a t e s and the s e p a r a t i o n seen on the p l a t e s was g e n e r a l l y r e p r o d u c i b l e on the s i l i c i c a c i d columns. T h i s medium was used p r i m a r i l y i n the f i n a l stages of separa-t i o n of the p h e n o l i c g l y c o s i d e s . 4 2 . In an e f f o r t to m i t i g a t e . t h e o f t e n long and l a -bo r i o u s task of column chromatography, newer methods of pre s s u r e chromatography (47) and u l t r a v i o l e t m o n i t o r i n g were used. The schematic diagram ( F i g u r e 4) d e p i c t s the chromatographic apparatus used i n the i n v e s t i g a t i o n . . Recorder Ultraviol-Monifor Fraction Coll. Column 1 Column 2 Sample Fraction Inj. Coll. F i g u r e 4. A schematic r e p r e s e n t a t i o n of the pressure column chromatography system. The pump was a Milton-Roy Model 196-47 capable of de-l i v e r i n g 240 ml/hr at a di s c h a r g e pressure of 1000 p s i . T h i s pump was used to apply pressure flow to the chroma-t o g r a p h i c columns at 50 to 10.0. p s i . The u l t r a v i o l e t r e -c o r d i n g meter was a G i l s o n Model UV 280 IF coupled to a Leeds Northrup s t r i p r e c o r d e r . Column No. 1 had an LH-20 43. bed dimension of 76 cm x 2 . 5 cm with CE as the e l u a n t . Column No. 2 had a s i l i c i c a c i d packing (60 cm x 1 cm) and e i t h e r CM or BE were used as eluants . Flow r a t e s were, a d j u s t a b l e i n the L H - 2 0 column f o r 0-4 ml/min, while the s i l i c i c a c i d had a maximum flow r a t e of 1 ml/min due to p r e s s u r e l i m i t a t i o n s of i t s c o n n e c t i o n . These flow r a t e s r e p r e s e n t as much as 10 times t h a t a v a i l a b l e with g r a v i t y feed on the same columns. D e r i v a t i v e P r e p a r a t i o n s  A c e t y l a t i o n A c e t i c anhydride: p y r i d i n e ( 1 : 1 ) was used to a c e t y l a t e f r e e h y d r o x y l groups. The a c e t y l a t i o n mixture and compound were allowed to stand o v e r n i g h t at room tem-pera t u r e and the f o l l o w i n g morning they were warmed on a steam bath ( 100°C) f o r 1 nour. The excess reagent was removed from the mixture under vacuum. 0 - m e t h y l a t i o n M e t h y l a t i o n of p h e n o l i c h y d r o x y l groups u t i l i z e d excess diazomethane i n methyl a l c o h o l . The compound to be methylated was d i s s o l v e d i n minimal methyl a l c o h o l , cooled to about - 10°C f o r 1 hour a f t e r which' c o l d diazomethane was added. The mixture was then r e t u r n e d to - 1 0°C o v e r n i g h t . The f o l l o w i n g morning the r e s i d u a l m e t h y l a t i n g agents were removed under vacuum. 44. Dimethyl s u l f a t e - p o t a s s i u m carbonate was used i n c o n j u n c t i o n with methyl i o d i d e - s i l v e r oxide In an attempt to f u l l y methylate one of the i s o l a t e d g l y c o s i d e s . Dimethyl s u l f a t e (50% excess) and potassium carbonate (50% excess) v;ere added i n p o r t i o n s to an anhydrous acetone s o l u t i o n of the g l y c o s i d e . T h i s s o l u t i o n was r e f l u x e d under n i t r o g e n f o r 5 hours with the e x c l u s i o n of moist a i r . The r e s u l t a n t mixture was cooled and f i l t e r e d . The i n o r g a n i c s a l t s were washed t h r i c e with acetone. The acetone s o l u t i o n was evaporated to dryness i n a r o t a r y evaporator and r e d i s s o l v e d i n dimethyIformamide. Methyl i o d i d e (3 e q u i v a l e n t s per OH group) and s i l v e r oxide (2 e q u i v a l e n t s per OH group) were added at room temperature and the mixture l e f t o v e r n i g h t . The f o l l o w i n g morning water was added t o the mixture and the methylated d e r i v a -t i v e was e x t r a c t e d from the aqueous s o l u t i o n with c h l o r o -form. Stronger m e t h y l a t i o n c o n d i t i o n s were r e q u i r e d to complete the t o t a l m e t h y l a t i o n procedure. These c o n d i -t i o n s u t i l i z e d d i m ethyl s u l f a t e - s o d i u m hydroxide i n a pro-cedure i d e n t i c a l to t h a t mentioned above without the' sub-sequent use of methyl i o d i d e / s i l v e r oxide. Hydrogenolysis I t was necessary to c a t a l y t i c a l l y hydrogenate the b e n z y l a l c o h o l group of one of the i s o l a t e d g l y c o s i d e s . 4 5 . '.This hydrogenolysis was done with a 20 mg samole in approximate-l y 5 ml of e t h y l a l c o h o l i n a test, tube to which 50-70 mg of c a t a l y s t [(PdCl^-BaSO^) A d l e r and Mar-ton (2)] was added The t e s t tube was then p l a c e d i n a.Parr low pressure hydro ge n a t i o n apparatus. The sample was f i r s t evacuated and then hydrogen was i n t r o d u c e d to a pressure of 40 p s i . The mixture was shaken f o r 15 min and the sample removed and f i l t e r e d . Degradative Techniques A l k a l i n e nitrobenzene o x i d a t i o n A l k a l i n e nitrobenzene o x i d a t i o n was used to d e t e r -mine the b a s i c aromatic n u c l e i and the manner of l i n k a g e of phenylpropane u n i t s i n the major o c c u r i n g g l y c o s i d e . The procedure of Stone and B l u n d e l l (92) w i t h the improve-ments of Pepper and Siddequeullah (76) was used. A 10 mg sample was o x i d i z e d 2 . 5 hours at l 6 0°C i n the presence of nitrobenzene and sodium hydroxide. The r e s u l t a n t mixture was e x t r a c t e d with e t h e r , a c i d i f i e d with h y d r o c h l o r i c a c i d and e x t r a c t e d with ether to o b t a i n the aromatic aldehydes. Q u a l i t a t i v e t h i n l a y e r c e l l u l o s e was run on the f i n a l products u s i n g CAW. The compounds were l o c a t e d on the t h i n l a y e r c e l l u l o s e chromatography p l a t e s u s i n g •. diazotized s u l f a n i l i c acid (DSA). 46. E t h a n o l y s i s E t h a n o l y s i s of Conpound A' • or f u l l y methylated Compound A was run on a 10 mg sample contained i n one ml of 2% h y d r o c h l o r i c a c i d - e t h y l a l c o h o l •. The sample tube was s e a l e d under n i t r o g e n and heated f o r one hour at 100°C. The e t h a n o l y s i s mixture was c o o l e d and n e u t r a l i z e d w i t h sodium b i c a r b o n a t e and run on t h i n l a y e r c e l l u l o s e i n n-b u t a n o l s a t u r a t e d with aqueous ammonia or on t h i n l a y e r s i l i c a g e l (BE) f o r the methylated d e r i v a t i v e . Compara-t i v e standard compounds were run.on the chromatogram to determine what fragments were formed i n the r e a c t i o n . P e r i o d a t e o x i d a t i o n P e r i o d a t e o x i d a t i o n was a p p l i e d to a methylated' derivative (aromatic hydroxyls) • of one of the compounds i s o l a t e d from the e t h y l a c e t a t e e x t r a c t of the western red cedar l e a v e s . T h i s o x i d a t i o n was run a c c o r d i n g to the procedure of Dyer ( 2 1 ) . The methylated d e r i v a t i v e ( 3 9 . 7 rng) was d i s s o l v e d i n 1 ml of e t h y l a l c o h o l , and water was ad-ded to a t o t a l volume of 5 ml. T h i s s o l u t i o n was added to 1 0 .ml of 0 . 0 6 9 3 M p e r i o d a t e s o l u t i o n and the s o l u t i o n was brought to 25 ml and s t o r e d at 0°C. A 5 nil a l i q u o t was withdrawn a f t e r 1 and 2 hours. Sodium b i c a r b o n a t e , excess sodium a r s e n i t e and s t a r c h i n d i c a t o r were added and the so-l u t i o n was t i t r a t e d with 0 . 0 5 3 4 M i o d i n e s o l u t i o n . to the s t a r c h end p o i n t . Determination of molar uptake was based on a p r i o r d e t e r m i n a t i o n of i o d i n e r e q u i r e d to t i t r a t e the a r s e n i t e a v a i l a b l e i n solution.. H y d r o l y s i s H y d r o l y s i s of the non-methylated g l y c o s i d e s was a t -tempted with 2% aqueous o x a l i c a c i d . The o x a l i c a c i d so-l u t i o n was added to an aqueous s o l u t i o n of the g l y c o s i d e and the mixture r e f l u x e d f o r 2 hours. F o l l o w i n g the r e f l u x time, the mixture was c o o l e d and t h r i c e e x t r a c t e d w i t h c h l o r o f o r m . The c h l o r o f o r m e x t r a c t was evaporated to dryness and chroma-tographed to determine the degree of h y d r o l y s i s . S e v e r a l of the compounds were d i f f i c u l t to h y d r o l y z e and r e q u i r e d r e -h y d r o l y s i s or h y d r o l y s i s under c o n d i t i o n s used f o r the methy-l a t e d d e r i v a t i v e s . H y d r o l y s i s of the methylated d e r i v a t i v e s was run i n 6% h y d r o c h l o r i c a c i d i n methanol. The h y d r o l y s i s s o l u t i o n was added to the g l y c o s i d e and r e f l u x e d f o r 2 hours. The c o o l e d s o l u t i o n was then e x t r a c t e d with c h l o r o f o r m : water and the c h l o r o f o r m s o l u b l e s were analyzed c h r o m a t o g r a p h i c a l -l y f o r the aglycone, while the water s o l u b l e s were d e i o n i z e d with a s t r o n g quaternary amine exchange r e s i n and chromato-g r a p h i c a l l y compared to standard sugars. L i q u i d S c i n t i l l a t i o n Counting of- Low A c t i v i t y  Chromatographic Samples A new method f o r determining l e v e l s of r a d i o a c t i v i t y •4-8.. of c h r o m a t o g r a p h i c a l l y separated compounds was developed i n t h i s study. Because the method i s a.new tec h n i q u e , i t was necessary to t e s t i t s v a l i d i t y and to compare i t to present methods which i t would p o t e n t i a l l y r e p l a c e . The proven technique c o u l d then be a p p l i e d i n a p i l o t f e e d i n g study i n the leaves of western red cedar. Table 1 denotes the sample p r e p a r a t i o n s used i n v a l i d a t i o n of the method. The samples f o r the experiment were taken from two c e l l u l o s e p l a t e s of a - f i v e p l a t e p r e -p a r a t i o n as p r e v i o u s l y d e s c r i b e d . On these p l a t e s , 1 cm diameter c i r c l e s were i n s c r i b e d through the c e l l u l o s e l a y e r to the g l a s s . The r a d i o a c t i v e samples were prepared by a p p l y i n g 10 u l of U - l i j C - L - p h e n y l a l a n i n e s o l u t i o n to the 1 cm c i r c l e s ( f i v e r e p l i c a t e s ) . Other n o n - r a d i o a c t i v e compounds and chromogenic sprays were then a p p l i e d to the samples. Those samples which were to be combusted were l i f t e d from the p l a t e s by a p p l y i n g a 3% w/v s o l u t i o n of c e l l u l o s e n i t r a t e ( P a r l o d i o n - M a l l i n k r o d t ) i n e t h y l a l c o h o l : ether (1:1) dropwise to j u s t cover each c i r c l e . The c e l -l u l o s e n i t r a t e s o l u t i o n a l s o c o ntained 3 ml/1 b l a c k mark-i n g i n k . As the c i r c l e s d r i e d , they c o u l d be l i f t e d c l e a n -l y from the g l a s s without l e a v i n g a r e s i d u e . The samples were then mounted on wire stands and p l a c e d i n s c i n t i l l a -t i o n v i a l s ready f o r combustion. 49. TABLE 1 Sample p r e p a r a t i o n and r e s u l t s of l i q u i d s c i n t i l l a t i o n c o u n t i n g comparison of s c r a p i n g and combustion methods a p p l i e d t o samples from t h i n l a y e r c e l l u l o s e p l a t e s . Compound added Amount added (Jug) Spray Combustion, dpm Scrape, dpm Activity ratio obs. result/ standard Standard — 2781 — — Standard — 1941 — phe.n alari y i -i n e 2 Ninh y d r i n 2332 1. 20 6 2255 1 . 16 12 2288 1.17 2 2463 .886 6 . 2492 .896 r 12 2423 .871 quer cet i n 2 no 2036 1 . 0 5 5 1923 .99 10 1675 .86 15 1533 • 79 2 ' 2781 1.00 5 2748 .99 10 2753 .99 15 2773 .99 2 B i s d i a z o . benz . 1530 .788 5 1389 .716 10 1181 .608 15 1327 .684 2 2570 . 9 2 3 5 2255 .813 10 2337 .837 15 i 2433 .853 2 Barton's 2053 1.06 5 1791 . 9 2 3 10 1652 .851 15 1388 .715 2 2657 .966 5 2730 .982 10 2711 .975 15 2695 .934 10 DSA 2480 .862 p i n o s y l v i n 2 no 2294 1.18 5 2418 1.24 10 2381 1.20 2 2746 .987 5 2685 .97 10 ' r 2 762 .99 blank - B i s D.B. 1577 .812 - 1 2424 .872 - N i n h y d r i n 2169 1.11 - 2736 .854 - Barton 1s 196 4 1.01 i - 1 2717 .977 50. J u s t p r i o r to combustion, 1 ml of c h i l l e d CO^ ab-sorbant [ethanolamine: ethylene g l y c o l monomethyl ether ( 1 : 2 ) ] was p l a c e d i n the bottom of each v i a l . Stopcock grease was spread t h i n l y about the l i p of the v i a l and the v i a l was then f l u s h e d with oxygen f o r 5 seconds. The cap was q u i c k l y t i g h t e n e d on the v i a l and the sample was com-busted u s i n g an e x t e r n a l focused i n f r a r e d l i g h t . Immediate-l y a f t e r combustion the sample was shaken and co o l e d at - 1 0°C f o r 10 minutes. T h i r t e e n ml of s c i n t i l l a t i o n cock-t a i l [ 5 . 5 g PPO i n t o l u e n e : C e l l o s o l v e (ethylene g l y c o l monomethyl-ether) ( 3 : 1 ) ] was then added and the sample was ready f o r s c i n t i l l a t i o n c o u n t i n g . Those samples r e q u i r i n g s c r a p i n g were removed from the p l a t e as c l e a n l y as p o s s i b l e u s i n g a s c a l p e l . The scraped sample was p l a c e d i n a s c i n t i l l a t i o n v i a l to which 14 ml of L i q u i f l u o r (New England Nuclear Corp.) was added. The v i a l s were shaken v i g o r o u s l y p r i o r to cou n t i n g . A l l sam-p l e s were counted ( 2 x 5 min) i n a Packard 1200 s c i n t i l l a -t i o n counter w i t h a c a l i b r a t e d e x t e r n a l s t a n d a r d . The r e s u l t s of t h i s experiment are i n c l u d e d i n Table 1. A separate experiment was run to determine the limits of spot size upon combustion efficiency. • 51. 14 Leaf C Feeding A p o r t i o n of the leaves c o l l e c t e d i n the f i e l d were 14 immediately prepared f o r a C f e e d i n g experiment. The purpose of t h i s experiment was t w o f o l d : ( l ) . t o f u r t h e r sub-s t a n t i a t e the a p p l i c a b i l i t y of the newly developed combus-t i o n technique and (2) to determine the r e l a t i v e a n a b o l i c a c t i v i t y of i s o l a t e d d i l i g n o l s as a p r e l i m i n a r y step to k i n e t i c s t u d i e s i n the l e a v e s . o f western red cedar. The c o l l e c t e d leaves were cut with s c i s s o r s i n t o p i e c e s averaging about 1/8 i n c h i n l e n g t h . These cut leaves were c a r e f u l l y i n s p e c t e d to e l i m i n a t e woody m a t e r i a l . A p o r t i o n of the leaves a v a i l a b l e f o r the f e e d i n g experiment were immediately weighed and p l a c e d i n a 105°C oven f o r 18 hours to determine oven-dry weight. The remaining leaves were weighed i n t o f o u r 2 g p o r t i o n s and p l a c e d i n . f o u r 14 p e t r i d i s h e s c o n t a i n i n g approximately 2 uc/ml of U- C-L-p h e n y l a l a n i n e i n 6 ml of s t e r i l i z e d water. Three 10 u l samples of the f e e d i n g s o l u t i o n were withdrawn from each p e t r i d i s h immediately a f t e r i n t r o d u c -t i o n of the samples. These samplesv.were p l a c e d i n cou n t i n g v i a l s c o n t a i n i n g L i q u i f l u o r (New England Nuclear Corp.) and were s u b j e c t e d to s c i n t i l l a t i o n c o unting to determine the amount of l a b e l e d p h e n y l a l a n i n e a v a i l a b l e to the leaves at the beginning of the f e e d i n g p e r i o d . The dishes were then 5 2 . covered and i l l u m i n a t e d by two 250 watt incandescent bulbs at a d i s t a n c e of approximately 3 f e e t . The four' d i s h e s , r e p r e s e n t e d f e e d i n g times of 1 , 3 , 5 and 10 hours. At the end of each time p e r i o d , 3 more 10 y l samples were w i t h -drawn from the i n d i v i d u a l f e e d i n g s o l u t i o n s and counted to determine the percent uptake of the l a b e l l e d s o l u t i o n with time. Upon completion of each feeding, the leaves were f i l t e r e d from the l a b e l l e d s o l u t i o n and washed wit h d i s t i l l e d water. The washed leaves were then p l a c e d i n i n d i v i d u a l -m i c r o - s o x h l e t s f o r e x t r a c t i o n with methyl a l c o h o l . E x t r a c -t i o n and workup a c c o r d i n g to the scheme d e p i c t e d i n F i g u r e 3 y i e l d e d a gross e t h y l a c e t a t e e x t r a c t . The e t h y l a c e t a t e e x t r a c t was taken to dryness under vacuum and t r a n s f e r e d i n e t h y l a l c o h o l to f o u r 2 ml volume-t r i c f l a s k s . Based upon e a r l i e r chromatographic evidence, i t was assumed at t h i s p o i n t that the compounds of i n t e r e s t would be present i n the e t h y l a c e t a t e e x t r a c t . Three 60 u'l r e p l i c a t e s of each f e e d i n g s o l u t i o n ( r e p r e s e n t i n g the f o u r time p e r i o d s ) were a p p l i e d to separate t h i n l a y e r c e l l u l o s e p l a t e s and developed two d i m e n s i o n a l l y with BCAW i n the f i r s t d i r e c t i o n f o l l o w e d by a i r - d r y i n g and development i n the second d i r e c t i o n with CAW.. The p l a t e s were then d r i e d , and sprayed w i t h d i a z o t i z e d s u l f a n i l i c a c i d . The compounds des i g n a t e d as A and B on the schematic chromatogram ( F i g u r e s ) 53. were then s t r i p p e d .from the p l a t e and counted a c c o r d i n g to the method p r e v i o u s l y d e s c r i b e d . Chromatographic spots w i t h i r r e g u l a r . s h a p e and s i z e r e q u i r e d c a r e f u l s u b d i v i s i o n , with a r a z o r blade f o l l o w i n g removal from the p l a t e s u r f a c e . T h i s s u b d i v i s i o n i n s u r e d t o t a l combustion of the s p o t s , with the count r a t e s of the i n d i v i d u a l s e c t i o n s being summed to o b t a i n the r e p r e s e n t a t i v e count r a t e f o r each spot. The absorbed r a d i o a c t i v i t y was counted u s i n g a Packard 1200 s c i n -t i l l a t i o n counter and the observed count r a t e s were r e l a t e d 14 to the o r i g i n a l C uptake to determine per cent i n c o r p o r a -t i o n i n each compound r e l a t e d to f e e d i n g time. " ' S p e c t r a l Techniques U l t r a v i o l e t and i n f a r e d U l t r a v i o l e t s p e c t r a were obtained f o r column chroma-t o g r a p h i c a l l y p u r i f i e d samples or methyl a l c o h o l e l u a n t s of spots scraped from c e l l u l o s e p l a t e s . The s p e c t r a were run i n methyl a l c o h o l i n a Beckman DK-2 r e c o r d i n g s p e c t r o -photometer u s i n g a p p r o p r i a t e b l a n k s . S p e c t r a l s h i f t s of the samples ..-.were determined i n the presence of 0.1 N sodium methoxide. U l t r a v i o l e t a n a l y s i s was a l s o used i n the de-t e r m i n a t i o n of p h e n o l i c h y d r o x y l a c c o r d i n g t o the method of . Goldschmid ( 4 5 ) . The method i n v o l v e s the d e t e r m i n a t i o n of d i f f e r e n c e s p e c t r a o b t a i n e d f o r the s h i f t of the phenoxide 54. F i g u r e 5: A schematic chromatogram of the c l a r i f i e d e t h y l acetate e x t r a c t from western r e d cedar l e a v e s . (spray -DSA), i o n formed with the a d d i t i o n of 0 . 1 N sodium methoxide. The extent of change of maxima was compared t o a eugenol s t a n -dard to determine f r e e p h e n o l i c h y d r o x y l content. I n f r a r e d ' s p e c t r a were obtained from samples i n potassium bromide p e l l e t s on a Perkin-Elmer 521 i n f r a r e d spectrophotometer. • Nuclear magnetic resonance A l l n u c l e a r magnetic resonance (NMR) s p e c t r a were, obtained on a V a r i a n HA - 1 0 0 , 100 mHz NMR spectrophotometer. The samples were prepared i n de u t e r o c h l o r o f o r m or de u t e r a t e d acetonei T e t r a m e t h y l s i l a n e was added as an i n t e r n a l s t a n -dard and lo c k s i g n a l , at t = 10. Normal sample d i l u t i o n was approximately 10 mg/ 300 y l of s o l v e n t . For samples of l e s s than 5 mg a m i c r o - c e l l was used with. the. d i l u t i o n of 1-5 mg/30 u-1 of s o l v e n t . Nuclear magnetic double r e s o -nance (NMDR) experiments were a p p l i e d to d e r i v a t i v e s to determine coupled s i g n a l s . Mass spectroscopy Mass spectra, were obtained f o r p u r i f i e d a c e t a t e d e r i v a t i v e s of d i l i g n o l rhamnosides. These s p e c t r a were run by Morgan-Schaffer C o r p o r a t i o n , M o n t r e a l , Quebec. High a m p l i f i c a t i o n s p e c t r a of hig h m o l e c u l a r weight f r a g -ments were i n c l u d e d . 56. RESULTS I s o l a t i o n of Compounds Y i e l d s of 1.47 g of e t h y l ether s o l u b l e s and 23.8 g of e t h y l a c e t a t e s o l u b l e s were obtained from 1.5 kg (58.6% moisture) of f r e s h western r e d cedar leaves t r e a t e d a c c o r d i n g to F i g u r e 3.. These e x t r a c t weights r e p r e s e n t y i e l d s of 0.76% ( e t h y l e t h e r ) and 3.86$ ( e t h y l a c e t a t e ) . b y weight of the oven-dry l e a v e s . Chromatographic examination of the gross e t h y l . a c e -t a t e e x t r a c t r e v e a l e d a wide range of p o l y p h e n o l i c s i n c l u d -i n g f l a v o n o i d s . S e v e r a l compounds which gave an orange, red-orange r e a c t i o n w i t h d i a z o t i z e d s u l f a n i l i c a c i d (DSA) ran near the s o l v e n t f r o n t when developed i n 2% aqueous ace-t i c a c i d on t h i n l a y e r c e l l u l o s e p l a t e s . T h i s r e a c t i o n with DSA i s i n d i c a t i v e of an alpha hydroxy g u a i a c y l nucleus ( 4 6 ) . Two dim e n s i o n a l chromatography u s i n g BCAW i n the f i r s t d i r e c t i o n and CAW i n the second d i r e c t i o n , with t h i n l a y e r c e l l u l o s e p l a t e s , r e s u l t e d i n good s e p a r a t i o n of these compounds (Figure 5, compounds A to I ) w i t h a wide range of Rf values i n CAW. The Rf values i n CAW are i n -v e r s e l y r e l a t e d to the number of f r e e h y d r o x y l groups of the chromatographed compounds ( 2 2 ) . 5(7. C o n s i d e r a t i o n of the two d i mensional chromatography-sprayed w i t h DSA i n d i c a t e d that Compound A occurred i n the h i g h e s t y i e l d and gave the very d i s t i n c t orange c o l o r r e -a c t i o n . S e v e r a l other compounds a l s o gave an orange c o l o r r e a c t i o n ( B ^ E j I ^ J ) but o c c u r r e d i n lower y i e l d . Some com-pounds a l s o gave a r e d or red-orange r e a c t i o n with DSA (C, D,K,F,G,H,L). . Other compounds (0 to T) a l s o r e a c t e d with DSA but were not i n v e s t i g a t e d beyond chromatographic det e c -t i o n . Compounds.A, B and C- I s o l a t i o n The gross e t h y l a c e t a t e e x t r a c t was found to f r a c -t i o n a t e on LH-20 with chloroform: e t h y l a l c o h o l (4:1) as the e l u t i n g s o l v e n t . P i l o t s t u d i e s a l s o determined that a p r e p a r a t i v e LH-20 s e p a r a t i o n of the e x t r a c t was a d v i s a b l e p r i o r to a q u a n t i t a t i v e , run. T h e r e f o r e , the crude e t h y l a c e t a t e e x t r a c t was d i s s o l v e d i n the e l u t i n g s o l v e n t , d i v i d e d i n t o two f r a c t i o n s (approximately 25 ml each and a p p l i e d s e p a r a t e l y to. a LH-20 column ( 3 . 5 cm x 40 cm). F i f t y ml f r a c t i o n s were taken and monitored one d i m e n s i o n a l -l y on t h i n l a y e r s i l i c a g e l p l a t e s (CM). The compounds under study were ob t a i n e d i n the f i r s t 500 ml of e l u a n t . The schematic chromatogram ( F i g u r e 5) r e v e a l s the s u b f r a c -t i o n now devoid of almost a l l of the u n d e s i r a b l e f l a v o n o i d and r e l a t e d compounds. The column was s t r i p p e d u s i n g c h l o r o -form: methyl a l c o h o l ( 3 : 7 ) , r e s w o l l e n to near the o r i g i n a l bed l e v e l , and the second h a l f of the e x t r a c t was a p p l i e d and run, again being monitored by t h i n l a y e r s i l i c a g e l p l a t e s . The e l u t i o n volume remained constant. The c l a r i f i e d e x t r a c t was now ready f o r a p p l i c a t i o n to the q u a n t i t a t i v e LH-20 column.. I t was c o n c e n t r a t e d i n the vacuum evaporator (estimated weight 10 to 15 g ) , d i v i d e d i n t o 4 equal p o r t i o n s and applied,under p r e s s u r e at a flow r a t e of 2 ml/minute, and 10 ml f r a c t i o n s were taken. The e l u a n t was monitored u s i n g the G i l s o n UV monitor and s i l i c a p l a t e s . The e l u t i o n curve of a t y p i c a l run i s shown i n F i g u r e 6. 0 100 200 300 400 500 600 • E l u t i o n volume, ml F i g u r e 6. T y p i c a l e l u t i o n curve of c l a r i f i e d e t h y l a c e t a t e e x t r a c t from western r e d cedar leaves as run-on LH-20 (CHCl^:Et0H(4:1)). T h i n l a y e r chromatography r e v e a l e d that the 220 to 280 ml f r a c t i o n c o n t a i n e d Compounds B,C,D as major c o n s t i t u e n t s while Compounds A and.J were the major c o n s t i t u e n t s of the 420 to.480 ml f r a c t i o n . A f r a c t i o n appearing near 100 ml appeared to be carbohydrate or a c y c l i t o l and was not i n -v e s t i g a t e d f u r t h e r . The f r a c t i o n s from the f o u r runs were separated a c c o r d i n g to the two major e l u t i o n peaks and concentrated to a s m a l l volume (5 to 10 m l ) . These f r a c t i o n s were now a p p l i e d to a p r e p a r a t i v e s i l i c a g e l column (3-5 cm x 40 cm) and e l u t e d w i t h CM. Ten ml f r a c t i o n s were taken up to a t o t a l e l u t i o n volume of 500 ml. The d e s i r e d com-pounds were e l u t e d from t h i s column with approximately 250 ml s o l v e n t . The column served two main purposes: f i r s t , any high m olecular non-phenolic compounds were e l i m i n a t e d e i t h e r by f a i l i n g to run on the column or by running near the f r o n t ; second,.some primary s e p a r a t i o n of the compounds B,C,D and A and J p r i o r t o . a p p l i c a t i o n to the semi-micro q u a n t i t a t i v e s i l i c a column was achieved. The f r a c t i o n s c o n t a i n i n g the compounds were concen-t r a t e d i n a r o t a r y evaporator and a p p l i e d i n 2 ml p o r t i o n s to column No. 2 under p r e s s u r e . The column was e l u t e d under constant pressure at a r a t e of 0.75 ml/min and 4 to 5 ml f r a c t i o n s were c o l l e c t e d . The compounds e l u t e d from the column i n the 35 to 50 ml range. Pure f r a c t i o n s were determined by t h i n l a y e r s i l i c a g e l chromatography and set 60. a s i d e . Those f r a c t i o n s not c o n t a i n i n g d e s i r a b l e compounds were d i s c a r d e d and the remaining f r a c t i o n s were recombined f o r other runs.By these procedures, chromatographically pure fractions of Compounds A, B and C were obtained. The y i e l d of Compound A (based on the oven-dry weight of l e a v e s ) was 0.15%. I t was estimated t h a t the y i e l d of a l l compounds g i v i n g d i s t i n c t i v e c o l o r r e a c t i o n s with DSA was approximately 0.5% of the weight of oven-dry l e a v e s . Compounds A, B and C - P r o p e r t i e s Table No. 1 p r e s e n t s the chromatographic, chromogenic, s p e c t r a l and p h y s i c a l p r o p e r t i e s of Compounds A, B, and C i n t a b u l a r form. In c o n s i d e r i n g chromatographic p r o p e r t i e s , both Rf values and chromogenic spray r e a c t i o n s are i n c l u d e d . I t should be noted t h a t the chromatographic s o l v e n t d e s i g n a t e d BCAW was used i n l i e u of the more u s u a l BAW [ b u t a n o l , a c e t i c a c i d , water (4 : 1 : 5)] because i t gave l e s s s t r e a k i n g while d u p l i c a t i n g the Rf values of the c l a s s i c s o l v e n t . In the d e t e r m i n a t i o n of an a l k a l i n e d i f f e r e n c e curve, a eugenol standard was run to check the procedure and reaffirm the calculated Ae value of 4.1 x 10° as being representative of one" max * free aromatic hydroxyl group. The major peaks of the compound's i n f r a -red spectra are presented i n Table 2. The NMR and mass spectra obtained for the compounds and their derivatives are represented i n Figures 7 to 9 and Figures 11 to 18. Figure 10 i s a specific rep-resentation of mass spectral fragmentation associated with the ace-tate derivative of Compound A. TABLE 2 PROPERTIES OF i COMPOUNDS A, B AND C CHROMATOGRAPHIC PROPERTIES CHROMOGENIC PROPERTIES SPECTRAL PROPERTIES (EXCLUDING NMR. MASS SPECTRAL) PHYSICAL PROPERTIES ACETATE DERIVATIVE ON CELLUL05E SILICA ON CELLULOSE Pi Tl TTA ULTRAVIOLET MAX ..... INFHARED BANDS cm _ x MalWt, CALC. C.HANAL m.p. " BCAW CAW 2* IAW BE CM DSA B pNA 5 ACID U.V. DSA JNMeOH MBOH+BASE it* BAND Don. S t r BAND DOO . S t r . MH/M Emp.Form %C %H |(un c o n . ) COMPOUND A 0.63 0.50 0.76 Q ,14 0.39 ( + ) R-0 (- FIT Y-0 279nm 289.5nn._ B04 10 J 3400 292B 1770 1605 1505 1449 1429 1370 1270 1230 0 B W a a m m m B m 1207 1123 1009 1042 978 909 . 877 805 781 m a a B W w w w w 804 -C-39H4B°1B 57.98 5.84 48-50* COMPOUND B 0.73 0.95 0.B7 0.23 0.51 P ( + ) , q PAT Y 280.5nn 2B8nro 248nn * 3400 292B 1700 1600 1502 1442 1436 1370 1268 1231 , s a w B B m m m a w 1120 100B 1040 977 902 871 805 ' 7B1 m a a w w w w w 70677447 « 58.08 6,32 • COMPOUND C 0.73 0.84 0.70 « 0.48 R, It) f H nbs, Q 281nn 2Blnm 251nn • 3420 2925 1470 1600 1503 1447 1434 1375 126B 1205 a B W a a w H w a w 1230 1121 1085 1040 974 909 B71 B05 790 , w m a a w w w w w 702 C35 H42°15-BCAW - b u t o n o l , c h l o r o f o r m , a c e t i c a c i d , water ( 4 : l i l d ) i DSA » d i a z o t i z e d s u l f o n i l i c a c i d CAU « c h l o r o f o r m , a c e t i c a c i d , water (2i3:1.5) I B • Barton'o reagent IAW - i s o p r o p o n o l , ammonia, water ( 2 0 i l t 2 ) B „ Schrooder'o reagent 2% = 2% aqueoua a c e t i c a c i d pNA m p - n i t r o o n o l i n e BE " benzene, e t h a n o l ( 9 i l ) CH - c h l o r o f o r m , n s t h a n o l (4«1) ' i. • n o t d o t o r a i n a d 0 ».Orange Y - y e l l o w R « red {•1 m p o a i t i v a r e a c t i o n br = b r i g h t fl - fluoreBcant ab8« aboarbo (-)- n e g a t i v o r e a c t i o n • 6 2 . F i g u r e s 9, 13, 18 are bar graph r e p r e s e n t a t i o n s of the mass s p e c t r a l f ragmentation of the a c e t a t e s of Compounds A, B and C. For convenience, the f i g u r e s have been d i v i d e d i n t o two p a r t s so that the hi g h mass end of the spectrum i s i n magni f i e d p r o p o r t i o n . Those mass fragments which appear as primary de-composition products have been l a b e l e d with t h e i r m o l e c u l a r i o n number. Metastable peaks have not been noted on these, s p e c t r a . In a l l three mass s p e c t r a the m/e 43 i o n was the 100% i o n . Compounds A, B and C - D e r i v a t i v e s M e t h y l a t i o n Aromatic-hydroxyl m e t h y l a t i o n of Compound A used diazomethane i n methyl a l c o h o l . T h i n l a y e r s i l i c a g e l chromatography (CM).of the m e t h y l a t i o n r e a c t i o n mixture showed the presence of two compounds (Rf 0.45, 0.55). The lower Rf compound d i s p l a y e d a p o s i t i v e r e a c t i o n . t o Barton's reagent ( i n d i c a t i v e of an aromatic h y d r o x y l ) , while the high Rf compound showed no r e a c t i o n . The methylated r e a c -t i o n mixture was separated on the s i l i c a g e l pressure c o l -umn u s i n g CM as the e l u a n t . The separated low Rf compound was c o l l e c t e d and s u b j e c t e d to r e - m e t h y l a t i o n with diazome-thane. T h i n l a y e r chromatography of the products from t h i s r e a c t i o n showed f u r t h e r formation of the Rf 0.55 compound. 6 3 . Based upon the chromatographic behaviour and c o l o r r e a c -t i o n s , the Rf . 0 . 5 5 compound was co n s i d e r e d the f u l l y aroma-t i c h y d r o x y l methylated d e r i v a t i v e of Compound A, while the lower Rf ( 0 . 4 5 ) compound was c o n s i d e r e d to be a par-t i a l l y methylated d e r i v a t i v e of Compound A. • Complete ( a l l h y d r o x y l s ) m e t h y l a t i o n of Compound A r e q u i r e d more s t r i n g e n t c o n d i t i o n s . M e t h y l a t i o n was f i r s t attempted u s i n g dimethyl s u l f a t e with sodium carbo-nate f o l l o w e d by methyl i o d i d e / s i l v e r oxide i n dimethyl formamide. These c o n d i t i o n s were chosen to minimize r e -arrangements which might occur because' of the h i g h l y r e a c -t i v e b e n z y l h y d r o x y l group i n Compound A. T h i n l a y e r chromatography showed m e t h y l a t i o n t o be incomplete f o r these c o n d i t i o n s even a f t e r two attempts. I t was t h e r e f o r e necessary to apply the s t r o n g e r m e t h y l a t i o n c o n d i t i o n s on the p a r t i a l l y methylated product u s i n g dimethyl s u l f a t e with sodium hydroxide. V e r i f i c a t i o n of the complete methy-l a t i o n was ob t a i n e d by a negative r e a c t i o n of the methylated product to Barton's reagent and the l a c k of i n f r a r e d ab-sorbance i n the 2700 .nm to 2900 nm range. A c e t y l a t i o n Acetate d e r i v a t i v e s of these three compounds were formed a c c o r d i n g t o the procedure p r e v i o u s l y o u t l i n e d . T r i t u r a t e d a c e t a t e s were obtained_through s c r a t c h i n g of the 64; a c e t y l a t e d mixture under petroleum e t h e r ( 6 5 - 1 1 0°C) and c o o l i n g at 5°C. Hydrogenolysis C a t a l y t i c ' hydrogenolyis was attempted on Compound A u s i n g a c a t a l y s t (2) h i g h l y r e a c t i v e i n the r e d u c t i o n of b e n z y l h y d r o x y l groups. The r e a c t i o n was run with the com-pound d i s s o l v e d i n e t h y l a l c o h o l f o r 10 minutes and the product was c h r o m a t o g r a p h i c a l l y examined to determine the degree of hydrogenolysis . The 10 minute hydrogenolysis r e -s u l t e d i n the apparent c o n v e r s i o n o f Compound A to a second compound (Rf 0 . 5 1 i n CHCl^MeOH), Compound B, i n about 50% y i e l d . When i s o l a t e d , t h i s compound gave a ne g a t i v e G i e r e r t e s t f o r the b e n z y l h y d r o x y l group. • # Compounds A, B and C - Degradative S t u d i e s . H y d r o l y s i s H y d r o l y s i s of Compounds A, B and C was attempted u s i n g 2% aqueous o x a l i c a c i d or 6% h y d r o c h l o r i c a c i d i n methanol. H y d r o l y s i s of Compounds A and B was g e n e r a l l y c o n s i d e r e d u n s u c c e s s f u l because of the fo r m a t i o n of s e v e r a l by-products, however.hydrolysis of Compound C r e s u l t e d i n a low y i e l d of a s i n g l e product, the p u t a t i v e aglycone of the parent g l y c o s i d e . An examination of the h y d r o l y s i s products of Compound B r e v e a l e d the presence of Compound C, 65. i n d i c a t i n g a chemical r e l a t i o n s h i p existing between the two compounds under c o n d i t i o n s of h y d r o l y s i s . The primary presence of L-rhamnose and minor amounts of L~arabinose and D-xylose were determined by chromatography of the h y d r o l y s a t e of i n i t i a l l y separated compounds. Enzy-matic h y d r o l y s i s was a l s o a p p l i e d to Compound A. Chromato-graphy r e v e a l e d the probable f o r m a t i o n of some aglycone i n the enzyme h y d r o l y s i s but i n , y i e l d i n s u f f i c i e n t f o r e f f e c t i v e study. E t h a n o l y s i s Compound A was s u b j e c t e d to e t h a n o l y s i s as p r e v i o u s l y d e s c r i b e d . The r e s u l t i n g products were n e u t r a l i z e d with, sodium b i c a r b o n a t e and run on t h i n l a y e r c e l l u l o s e i n b u t y l a l c o h o l s a t u r a t e d with ammonia. Standard compounds p l a c e d on the p l a t e i n c l u d e d : l~guaiacylpropan-l,2--one, .. l-ethoxy-l-gualacylpropan-2-one, l-guaiacyl-2--ethoxypropan-l-one, and l-guaiacylpropan-2-ohe. • W h e n sprayed with DSA, the e t h a n o l y s i s products of Compound A showed 2 s p o t s , one at Rf 0 . 9 2 which d i s p l a y e d a red-orange c o l o r , and a second spot Rf 0 . 2 0 which d i s p l a y e d a lemon yellow c o l o r . The high Rf compound e x h i b i t e d e s s e n t i a l l y the same Rf value as ' l-ethoxy-l-guaiacylpropan-2-one or l-guaiacylpropan-2-one, with a color reaction similar to the former. • The DSA c o l o r r e a c t i o n of the spot o c c u r r i n g at Rf 0 . 2 0 i s c o n s i d e r e d t y p i c a l of p - h y d r o x y l b e n z y l groups (46). 66. The f u l l y methylated d e r i v a t i v e of Compound A was a l s o s u b j e c t e d to e t h a n o l y s i s . The r e s u l t i n g e t h a n o l y s i s product was n e u t r a l i z e d with sodium b i c a r b o n a t e and chroma-tographed i n comparison to d i h y d r o c o n i f e r y l a l c o h o l on t h i n l a y e r s i l i c a p l a t e s i n BE. One of the major c o n s t i t u e n t s of the e t h a n o l y s i s was shown to run at e x a c t l y the same Rf value as the d i h y d r o c o n i f e r y l a l c o h o l standard and d i s p l a y e d an i d e n t i c a l c o l o r r e a c t i o n with DSA. A l k a l i n e n i t r o b e n z e n e o x i d a t i o n Compound A was s u b j e c t e d to a l k a l i n e nitrobenzene o x i d a t i o n a c c o r d i n g to the method p r e v i o u s l y described..' The r e s u l t s of t h i s o x i d a t i o n ' showed two major spots when chroma-tographed i n CAW (Rf 0 . 9 5 and Rf 0 . 5 0 ) . The h i g h Rf compound gave an orange c o l o r when sprayed with DSA"while the lower Rf compound gave a l e s s d i s t i n c t c o l o r r e a c t i o n ( o f f .white to cream). Chromatography of the a l k a l i n e nitrobenzene pro-duct.,in comparison to the standards v a n i l l i n and p r o t o c a t e -chualdehyde, showed i d e n t i c a l c o l o r r e a c t i o n s and Rf v a l u e s . P e r i o d a t e o x i d a t i o n The p a r t i a l l y methylated d e r i v a t i v e of Compound A ( p h e n o l i c h y d r o x y l s only) was s u b j e c t e d to p e r i o d a t e o x i d a -t i o n a c c o r d i n g to the procedure of Dyer (21). The r e a c t i o n mixture was t i t r a t e d f o r excess a r s e n i t e a f t e r one and two hours. Consumption of p e r i o d a t e showed 2 moles consumed at 67. the end of one and two hours. T h e r e f o r e , the r e a c t i o n was c o n s i d e r e d complete. The r e a c t i o n mixture was a c i d i f i e d and e x t r a c t e d with c h l o r o f o r m . The c h l o r o f o r m e x t r a c t was con- • c e n t r a t e d to. dryness and taken up i n methyl a l c o h o l . An u l t r a v i o l e t spectrum of the product, showed a peak at 280 nm which gave no s h i f t upon the a d d i t i o n of 0.1 N sodium methoxide. L i q u i d S c i n t i l l a t i o n Technique T h i s study i n c l u d e s a p r e l i m i n a r y i n v e s t i g a t i o n of 14 the i n f u s i o n of U- C-L-phenylalanine i n t o the leaves of western r e d cedar.. The i n v e s t i g a t i o n was concerned p a r t i -c u l a r l y with the f o r m a t i o n of d i l i g n o l g l y c o s i d e s i n leaves f l o a t e d i n an aqueous s o l u t i o n of the r a d i o a c t i v e phenyala-n i n e . The f e e d i n g method and the chemical p r o p e r t i e s of the d i l i g n o l g l y c o s i d e s e s t a b l i s h e d that two primary f a c t o r s would govern the measurement of i n c o r p o r a t e d r a d i o a c t i v i t y . F i r s t , low l e v e l s of i n c o r p o r a t i o n (near 1 per cent) c o u l d be expected. Second, those compounds i n c o r p o r a t i n g r a d i o -a c t i v i t y c o u l d be c h r o m a t o g r a p h i c a l l y l o c a t e d s p e c i f i c a l l y through autoradiography or g e n e r a l l y with chromogenic spray r e a g e n t s . The experimental design r e q u i r e d e v a l u a t i o n of i n -c o r p o r a t i o n on c h r o m a t o g r a p h i c a l l y separated samples. Four methods were a v a i l a b l e f o r a n a l y s i s of the c h r o m a t o g r a p h i c a l l y separated r a d i o a c t i v e compounds: (a) autoradiography ( d e n s i -68. t o m e t r i c a n a l y s i s ) (b) chromatographic s t r i p c o unting ( t h i n window G e i g e r - M e u l l e r c o u n t e r ) , (c). combustion-absorption ( l i q u i d s c i n t i l l a t i o n a n a l y s i s ) , (d) suspension ( l i q u i d s c i n t i l l a t i o n a n a l y s i s ) . The a n t i c i p a t e d low l e v e l s of a c t i v i t y e f f e c t i v e l y e l i m i n a t e d autoradiography and s t r i p c o unting. A u t o r a d i o -graphy would r e q u i r e e x c e s s i v e time, while' s t r i p c o u n t i n g was of too low e f f i c i e n c y f o r measuring low a c t i v i t y . The p h e n o l i c nature of the compounds to be s t u d i e d , and t h e i r r e q u i r e d l o c a t i o n with chromogenic spray r e a g e n t s , could s e v e r e l y l i m i t , l i q u i d s c i n t i l l a t i o n a n a l y s i s of suspended low a c t i v i t y samples. Many p h e n o l i c compounds and t h e i r - c o l o r e d products (from v a r i o u s chromatographic spray reagents) a r e . r e c o g n i z e d to be e f f e c t i v e chemical quenchers d u r i n g l i q u i d s c i n t i l l a t i o n a n a l y s i s . I t has a l s o been shown by Houx (53) that scraped suspended s i l i c a g e l samples, a d v e r s e l y a f f e c t the recorded e x t e r n a l standard r a t i o r e g a r d l e s s of the chemi-c a l quenching agent.present. Of the a v a i l a b l e a l t e r n a t i v e s , combustion-absorption of chromatographic samples seemed the most p l a u s i b l e because i t would e f f e c t i v e l y e l i m i n a t e chemical and p h y s i c a l quench-m mg, while n e a r l y 100 per cent of the C l a b e l would be a v a i l a b l e f o r l i q u i d s c i n t i l l a t i o n analysis(£0). The l i m i t i n g f a c t o r t o a combustion technique was the e f f i c i e n t combustion of c h r o m a t o g r a p h i c a l l y separated samples. The combustion 69. would be most f e a s i b l e i f a s s o c i a t e d with paper, or t h i n l a y e r c e l l u l o s e chromatography. S i l i c a g e l was not c o n s i d e r e d because i t does not burn under normal combustion c o n d i t i o n s . • T h i n l a y e r c e l l u l o s e , chromatography o f f e r e d the most s u i t a b l e medium to produce s m a l l s i z e d chromatographic spots which co u l d be combusted. R a d i o a c t i v e sample p r e p a r a t i o n s from t h i n l a y e r c e l l u l o s e p l a t e s i n the past had p a r a l l e l e d those from t h i n l a y e r s i l i c a p l a t e s . These p r e p a r a t i o n s i n v o l v e s c r a p i n g of the chromatographic spot from the p l a t e and subsequently counting the sample as a suspension. Although the scraped sample can be burned, a c l e a n e r , more e f f i c i e n t method of p r e p a r a t i o n was d e s i r a b l e . The method used i n t h i s study allows the c l e a n removal of the chromatographic spots from the p l a t e a f t e r s p r a y i n g . These prepared samples are then combusted d i r e c t l y i n the s c i n t i l l a t i o n v i a l i n the presence of a absorbent, and then counted. In o r d e r to. s u b s t a n t i a t e the t e c h n i q u e , an e x p e r i -ment was designed to e s t a b l i s h : (1) the comparative e f f i -c i e n c y of the c e l l u l o s e n i t r a t e combustion versus the s c r a p -i n g - s u s p e n s i o n method, (2) changes i n e f f i c i e n c y due to chemical quenching f o r the scraped and combusted samples, (3) the e f f i c i e n c y changes as a r e s u l t of chromogenic spray r e a c t i o n s f o r the two methods^and (4) sample s i z e l i m i t a t i o n s i n combustion. 70. Table 1 denotes the samples prepared to t e s t the p r o p o s a l s and the c o u n t i n g r e s u l t s o b tained from f i v e r e p l i c a t i o n s of each sample. Q u e r c e t i n ( 3 , 5, 7, 3 ',4' pentahydroxyflavone) was used as a p h e n o l i c quenching agent because of i t s e x t e n s i v e c o n j u g a t i o n , c o l o r ^ a n d a v a i l a b i l i t y . P i n o s y l v i n (3,5 d i h y d r o x y s t i l b e n e ) was chosen as a chemical quenching agent because i t Is able to- absorb u l t r a v i o l e t r a d i a t i o n and f l u o r e s c e . The choice of Barton's reagent and b i s - d i a z o t i z e d b e n z i d i n e as sprays was p r i m a r i l y because of t h e i r common use i n d e t e c t i n g p h e n o l i c compounds. However, the sprays cause c o l o r r e a c t i o n s v i a d i f f e r e n t mechanisms. Barton's reagent depends upon c h e l a t i o n of phenols w i t h f e r r i c i r o n to produce a d i s t i n c t i v e blue complex; b i s - -d i a z o t i z e d b e n z i d i n e depends upon a z o - c o u p l i n g to p h e n o l i c compounds. The data o f Table l i s g r a p h i c a l l y r e c o r d e d i n F i g u r e s 19 and 2 0 . F i g u r e 21 g r a p h i c a l l y p r e s e n t s the r e -s u l t s of the separate d e t e r m i n a t i o n of chromatographic spot s i z e on counting e f f i c i e n c y of combusted samples. 14 C Feeding : The a n a l y s i s of s c i n t i l l a t i o n r e s u l t s o b tained i n the l e a f f e e d i n g experiment are summarized i n Table 3 . Figure 22 represents the preliminary autoradiographic verification of U-^C-L-phenylananine incorporation into Compounds A and B i n western red cedar leaves. The radioactivity uptake by the leaves during the later infusion study i s shown i n Figure 23. The relative incorporation of radioactive phenylalanine into Compounds A and B i s depicted graphically i n Figure 24. The results repre-iamnose Compound A Compound B (speculated) Feeding time hr. A c t i v i t y i n leaves dpm Inc.,% A c t i v i t y i n Compound A dpm Inc.,%. A c t i v i t y i n Compound B dpm Inc.,% . 1 • 1.22xl0 6 16.1 1865 0.15 3063 0.25 3 1.34xl0 6 20 . 3 3970 •0.30 5370 0.40 5 1.78xi0 6 2 3 . 5 5340 0 . 3 0 7040 0 .39 10 2.82xl0 6 37.1 5035 ' 0.18 6600 0.24 Table 3- Uptake of U- C-L-phenylalanine and i t s i n c o r p o r a t i o n i n t o Compounds A -and B i n the leaves of western r e d cedar. / 7 2 . sent the average values of three runs with no s t a t i s t i c a l v a r i a n c e b e i n g c a l c u l a t e d . 7 3 . DISCUSSION S t r u c t u r a l S t u d i e s of the D i l i g n o l s  Compound A Compound A was obtained as an amorphous s o l i d from the column chromatographic s e p a r a t i o n of the e t h y l a c e t a t e e x t r a c t as p r e v i o u s l y d e s c r i b e d . Attempts to c r y s t a l l i z e the compound from o r g a n i c s o l v e n t s were u n _ . s u c c e s s f u l and p r e p a r a t i o n of a d e r i v a t i v e was i n i t i a t e d . The p r e p a r a t i o n of an a c e t a t e d e r i v a t i v e also yielded an amorphous s o l i d which f a i l e d to c r y s t a l l i z e from o r g a n i c s o l v e n t s . A t r i t u r a t e d s o l i d was obtained by s c r a p i n g under petroleum e t h e r ( 6 5 ° - 1 1 0°C) f o l l o w e d by c o o l i n g at 5°C. The petroleum e t h e r was allowed t o evaporate i n a vacuum d e s s i c a t o r l e a v i n g the t r i t u r a t e d a c e t a t e d e r i v a -t i v e w i t h a m e l t i n g p o i n t of 3 5 ° - 3 8°C ( u n c o r r e c t e d ) . At-tempts at r e c r y s t a l l i z a t i o n of t h i s d e r i v a t i v e were un-s u c c e s s f u l . Compound A was l a t e r shown, by NMR, to be a mixture of e r y t h r o - and threo-isomers which accounts f o r the low m e l t i n g p o i n t range of i t s a c e t a t e d e r i v a t i v e . There was no q u e s t i o n of the p u r i t y of Compound A s i n c e t h i n l a y e r chromatography ( s i l i c a and c e l l u l o s e ) , i n s e v e r -a l s o l v e n t systems, of Compound A and i t s a c e t a t e d e r i -v a t i v e showed a s i n g l e spot. 7 4 . Mass s p e c t r a l d e t e r m i n a t i o n of the ac e t a t e d e r i -v a t i v e of Compound A r e v e a l e d a molecular weight of 804 with a c a l c u l a t e d e m p i r i c a l formula (from M+l/M r a t i o ) of CggH^Ois* supported by carbon and hydrogen a n a l y s e s . S i x t e e n degrees of u n s a t u r a t i o n , i n d i c a t i v e of at l e a s t two s u b s t i t u t e d benzene r i n g s , are c a l c u l a t e d from the e m p i r i c a l formula. An u l t r a v i o l e t Ae value of 8.04 x 10 f o r Compound A showed the e x i s t e n c e of two p h e n o l i c h y -d r o x y l groups. I n f r a r e d data s u b s t a n t i a t e d the e x i s t e n c e of aromatic n u c l e i , absence of c a r b o n y l , and a l a r g e num-ber of h y d r o x y l groups. The 18 oxygen atoms of the f o r -mula f o r Compound A a c e t a t e were assign e d (on the b a s i s of NMR and mass s p e c t r a l data) t o two p h e n o l i c a c e t a t e s , one b e n z y l i c a c e t a t e , f o u r a l i p h a t i c a c e t a t e s , one methoxyl group, and three ether or h e m i a c e t a l oxygens. On the b a s i s of the f o l l o w i n g r e s u l t s , Compound A was con s i d e r e d to be l-( 3'-methoxy - 4'-hydroxyphenyl ) - 2 - 0 -1"- [ 2"-hydroxy - 4"-(propane - 3 "'-O-a-L-rhamnoside)phenyl]-propane-1,3, d i o l (XLI). CH," CH0OR £__5* - Q. -)y,.l-/t _ HCOR 2 OR 3 R0 OR XLI, R=H XLIa, R=Ac 75. Chemical c h a r a c t e r i z a t i o n . Compound A r e a c t s to produce a b r i g h t orange c o l o r when sprayed w i t h DSA, which i s c h a r a c t e r i s t i c of an a -h y d r o x y g u a i a c y l compound (46). A p o s i t i v e t e s t with G i e r e r ' s reagent (44) f u r t h e r s u b s t a n t i a t e s the e x i s t e n c e of a b e n z y l h y d r o x y l group. Schroeder's r e a c t i o n ' ( 8 2 ) , f o r the d e t e c t i o n of o-dihydroxy groups, was n e g a t i v e . Since a l k a l i n e nitrobenzene o x i d a t i o n of Compound A (to determine the aromatic s u b s t i t u t i o n p a t t e r n ) showed the presence of v a n i l l i n and protocatechualdehyde ( e s t a b l i s h -i n g the e x i s t e n c e of g u a i a c y l and c a t e c h o l n u c l e i ) ,the t e s t with Schroeder's reagent showed that one of the hy-d r o x y l groups on the c a t e c h o l nucleus must be e t h e r i f i e d . H y d r o l y s i s of the compound with 2% aqueous o x a l i c a c i d showed s e v e r a l p h e n o l i c p r o d u c t s . The presence of L-rhamnose i n the molecule was c o n c l u s i v e l y e s t a b l i s h e d by t h i n l a y e r c e l l u l o s e chromatography of the h y d r o l y s a t e . The occurrence of t h i s uncommon sugar bound through an unusual a l i p h a t i c g l y c o s i d e . l i n k a g e w i l l be f u r t h e r sub-s t a n t i a t e d l a t e r i n t h i s d i s c u s s i o n . An e m p i r i c a l formula of ^25^34^11 ^s i n d - i c a t e d f o r Compound A from the formula obtained f o r the a c e t a t e d e r i -v a t i v e . T h i s d i s c u s s i o n has accounted f o r ten oxygen atoms (f o u r on the benzene r i n g s , one b e n z y l i c h y d r o x y l , 76. f i v e from the L-rhamnose). There remains the placement of one a l i p h a t i c h y d r o x y l group and a d e s c r i p t i o n o f i t s r e -l a t i o n s h i p to the other a l i p h a t i c carbon and oxygen atoms. i These f e a t u r e s were d e l i n e a t e d i n the f o l l o w i n g two ex-periments. The f i r s t experiment s u b s t a n t i a t e d Compound A as a g u a i a c y l g l y c e r o l - 3 - a r y l ether through e t h a n o l y s i s . Com-p a r i s o n of the products of e t h a n o l y s i s w i t h standard H i b b e r t 1 s ketones showed the presence of 1-guaiacyl-2-ethoxypropan-l-one and l-guaiacylpropan-2-one. These pro-ducts were c o n s i d e r e d p r o o f of the g u a i a c y l g l y c e r o l - 3 -a r y l ether nucleus f o r Compound A. The g l y c e r o l s i d e chain c o n t a i n s two i s o m e r i c carbons and i s r e s p o n s i b l e f o r the mixture of erythro- and threo-isomers p r e v i o u s l y mentioned. Another e t h a n o l y s i s product showed a c o l o r r e a c t i o n and Rf values i n d i c a t i v e of c a t e c h o l d e r i v a t i v e s . The second experiment was an e t h a n o l y s i s of the i f u l l y methylated [(CH^^SOjj/CH^I.] d e r i v a t i v e of Compound A. T h i s e t h a n o l y s i s y i e l d e d d i h y d r o c o n i f e r y l a l c o h o l (XLII) as a major product. The f r e e aromatic h y d r o x y l group of . d i h y d r o c o n i f e r y l a l c o h o l e s t a b l i s h e d the unusual V - n -propan-3"'-ol sidechaln and the 2-4" alkyl-aryl ether linkage i n Compound A (XLI). The free aliphatic hydroxyl group of XLII es-tablished the point of attachment of the L-rhamnose i n the mol-ecule. The n-propyl side chain accounted for the remaining aliphatic 77. CHJOH XLII carbon atoms. Another product from t h i s e t h a n o l y s i s showed a c o l o r r e a c t i o n and Rf values i n d i c a t i v e of v e r a t r y l - d e r i v a t i v e s . Proof that the arrangement of c a t e c h o l and L-rhamnose groups were as shown (X L I ) , and not i n t e r c h a n g e d , or t h a t the s u b s t i t u t i o n was as a 3 - a r y l e t h e r r a t h e r than y- e t c . was obtained from the f o l l o w i n g experiment. P e r i o d a t e o x i d a t i o n of diazomethane methylated (aromatic h y d r o x y l s ) Compound A showed an uptake of e x a c t l y two moles. T h i s two mole consumption of p e r i o d a t e i s s o l e l y from the rhamnose moiety and f u r t h e r e s t a b l i s h e s , the absence of any other v i c i n a l d i o l s i n Compound A. Lack of a bathochromic s h i f t of the u l t r a v i o l e t maxima of the o x i d i z e d product i n base was evidence that the g l y c o s i d i c bond d i d not occur through an aromatic h y d r o x y l . Only the s t r u c t u r e shown f o r Compound A (XLI) e x p l a i n s the forementioned data. F u r t h e r v e r i f i c a t i o n was achieved by NMR and mass s p e c t r a l a n a l y s e s . V 78. NMR s p e c t r a of Compound A and i t s a c e t a t e . NMR s p e c t r o s c o p i c techniques confirmed the b a s i c s t r u c t u r e of Compound A as determined i n de g r a d a t i v e s t u -d i e s . The NMR spectrum of Compound A was obtained i n deuteroacetone and i s presented i n F i g u r e 7. The NMR of the a c e t a t e d e r i v a t i v e of Compound A was o b t a i n e d i n deute-r o c h l o r o f o r m and i s shown i n F i g u r e 8 . NMR s p e c t r a l a n a l y s i s was p r i m a r i l y done on the ac e t a t e d e r i v a t i v e of Compound A ( F i g u r e 8 ) . The ensuing d i s c u s s i o n w i l l t h e r e -f o r e - r e f e r to t h i s spectrum and the NMR r e s u l t s o f the u n a c e t y l a t e d compound w i l l be t r e a t e d s e p a r a t e l y . The t o t a l p r o t o n i n t e g r a l of F i g u r e 8 i s 47 to 49 p r o t o n s . S i x of these protons are seen to resonate at T = 3.04- 3-30 and are r e p r e s e n t a t i v e of the aromatic p r o -tons of Compound A. These protons must be r e p r e s e n t a t i v e of two aromatic r i n g s s i n c e the s p e c t r a a l s o i n d i c a t e s a thr e e p r o t o n methoxyl resonance (T = 6 . 2 2 ) . Two aromatic h y d r o x y l s are c o n s i d e r e d t o occur i n Compound A, based upon the two non-equivalent aromatic a c e t a t e resonances at x = 7 . 7 3 and 7 - 77 . This slightly different shielding of these acetate resonances show the phenolic hydroxyls of Compound A to be non-equivalent. r F i g u r e 8. The NMR spectrum of the acetate d e r i v a t i v e of Compound A. 81. F i v e a l i p h a t i c h y d r o x y l s are i n d i c a t e d f o r Com-pound A by the f i v e a c e t a t e resonances o c c u r r i n g i n the T = 7-9 - 8 . 4 'region of F i g u r e 8 . The anomeric p r o t o n of rhamnose i s seen to occur as a s i n g l e t t o low f i e l d ( T = 5 . 3 6 ) as a r e s u l t of i t s a s s o c i a t i o n to the ex - L -hem i a c e t a l bonded ( 6 l ) . The hig h f i e l d three proton doublet at f = 8 . 8 3 o r i g i n a t e s from the methyl group of the rhamnose c o u p l i n g - w i t h the C^ proton (J = 6 . 5 H z ) . The C 2-Cjj protons are 4 s h i f t e d to lower f i e l d ( T = 4 . 6 - 5-2) i n the a c e t a t e d e r i v a t i v e of Compound A and are s p e c i f i -c a l l y l o c a t e d and d e s c r i b e d as: H 2, x = 4 . 7 9 (J" 2 ^= (ca.) 1Hz); H 3, T. = 4 . 7 7 ( J 2 3 = 1.5 Hz, J^ ^ = (ca.) 10 Hz); Hjj, T = 5.0 ( J ^ 5 = 4 . 5 Hz). The proton i s not s h i f t e d through a c e t y l a t i o n and i t s chemical s h i f t was determined to be T = 6 . 2 1 by n u c l e a r magnetic double resonance ex-periments (NMDR). A p p l i c a t i o n of NMDR t o the H,_ resonance r e s u l t e d i n a c o l l a p s e of the hig h f i e l d doublet t o a s i n g l e t . Two resonances at T = 4.00 ( i n t e g r a t e d v a l u e , one proton) can be as s i g n e d to the b e n z y l proton of a g l y c e r o l grouping. These doublets (T = 3 . 9 7 , J S f t X. = 7.0 Hz; T = 4 . 0 5 , J a « 3 » = ^ - 0 H z ) c a n b e a t t r i b u t e d to the oc-currence of a d i a s t e r e o m e r i c p a i r of 3 - g u a i a c y l g l y c e r o l • isomers of Compound A ( 6 0 ) . Since the 3 proton i s asso-c i a t e d with an et h e r bonded carbon, i t should appear i n 8 2 . the r e g i o n " T ' = 4 . 5 - 6 . 0 (5 4 ) . Using NMDR te c h n i q u e s , the x = 4 . 5 - 6 . 0 r e g i o n was scanned while m o n i t o r i n g the low f i e l d doublet f o r any changes. In t h i s way, the 3 proton resonance was l o c a t e d at T = 5 - 3 8 , hidden beneath the anomeric p r o t o n resonance. NMDR of the 3 proton caused the a prot o n doublets to c o l l a p s e to two s i n g l e t s ( T = 3 . 9 7 , 4 . 0 5 ) . T h i s i r r a d i a t i o n of the 3 p r o t o n a l s o r e s u l t e d i n c l a r i f i c a t i o n of the m u l t i p l e t s i n the r e g i o n T = 5 .6 - 6 . 1 i n d i c a t i n g the y protons resonance a s s o c i a t e d w i t h the m u l t i p l e t i n t h i s range. The chemical s h i f t i n t h i s resonance i s c h a r a c t e r i s t i c of an e s t e r i f i e d hydroxy-methylene (54) . The occurrence of the two proton t r i p l e t at x = 7-37 (J = 3 . 7 H z ) ( F i g u r e s 7 and 8) and the two proton m u l t i p l e t at x = 8 . 1 8 ( F i g u r e 7) are c h a r a c t e r i s t i c of the a'and 3' protons of an n-p r o p y l c h a i n . When the resonance o c c u r r i n g under the a l i p h a t i c a c e t a t e envelope at x = 8.18 was i r r a d i a t e d i n an NMDR experiment, the t r i p l e t at T = 7-37 c o l l a p s e d to a s i n g l e t thereby e s t a b l i s h i n g i t as the a'proton resonance and the x = 8 .18 resonance as the 3 'resonance. I r r a d i a t i o n of the 3'resonance c l a r i f i e d the' m u l t i p l e t i n the range 6 . 3 0 x to 7 .75 T to a d i s t i n c t AB qu a r t e t c e n t e r e d at T = 6 . 5 w i t h X^ = 6 . 3 5 , xfi = 6 . 6 5 ( J ( g e = 1 0 . 0 Hz). T h i s resonance i s r e p r e s e n t a t i v e of the y.' pro t o n s , and the chemical s h i f t of the AB qu a r t e t suggests 8 3 . that they are methylene protons a s s o c i a t e d with an et h e r bond r a t h e r than an e s t e r bond (occurrence i n T = 5 . 5 -6 . 0 r a n g e ) . The AB nature of the s p i n - s p i n c o u p l i n g of the geminal protons o f - t h e methylene group i n d i c a t e s non-equivalence and suggests r e s t r i c t e d r o t a t i o n due to the bulky IL-rhamnose moiety at the 3 ' - y ' carbon-carbon bond. Examination of the NMR spectrum of u n a c e t y l a t e d Compound A r e v e a l s s e v e r a l f e a t u r e s i n c l u d i n g : a t o t a l i n t e g r a l of 30 to 35 p r o t o n s , a more d e f i n i t i v e s i x p r o -ton aromatic resonance ( T = 2 .9 - 3 . 6 l ) , two aromatic hy-droxy resonances (x = 1 . 3 0 , 2 . 5 5 ) , a twelve to f o u r t e e n pro-ton m u l t i p l e t i n the x = 6 . 1 - 6 . 8 r e g i o n which i n c l u d e s the rhamnose h y d r o x y l groups. Other s p e c i f i c f e a t u r e s i n - , elude: b e n z y l p r o t o n resonances x = 5 , 0 7 , the methine pro-ton resonance (as determined by NMDR) x = 6 . 0 0 , and the two proton resonance m u l t i p l e t of the 3 ' protons at x= 8.18 without i n t e r f e r i n g a c e t a t e resonances. M e t h y l a t i o n of Compound A produces a nine proton resonance at x= 6 . 2 1 r e p r e s e n t a t i v e of three methoxyl groups or two f r e e aromatic h y d r o x y l groups i n Compound A. - The chemical ' s h i f t data and nature of the NMR spectrum of the a c e t a t e d e r i v a t i v e of Compound A i s i n agreement with s i m i l a r data recorded f o r 1 - ( 3 , 4-dimethoxy) 84 -2-(2'- methoxyphenoxy)-propane-l,3 d i o l a c e t a t e (XLIII) . r e p o r t e d by Ludwig et a l . (60). -OCH 3 X L I I I The d i s c u s s i o n of the NMR spectrum of compound XL I I I r e -l a t e s the c h a r a c t e r of the a, 3 3 and y resonances i n r e -l a t i o n t o the e x i s t e n c e of two d i a s t e r e o i s o m e r i c forms. These c h a r a c t e r i s t i c s are i n c l o s e agreement f o r resonan-ces r e c o r d e d f o r the a c e t a t e of Compound A. On.the b a s i s of t h i s comparison and on the degr a d a t i v e and NMR data compiled f o r Compound A, the compound i s c o n s i d e r e d t o be c h a r a c t e r i z e d as an i s o m e r i c mixture of 1-(3'-methoxy-4'-hydroxyphenyl)-2-0-1"-[2"-hydroxy-4"-(-propane-3"-0-a-L-rhamnoside ) phenyl]^-propane-l .,3 d i o l (XLI).. Mass spectrum of Compound A acetate. The mass spectrum of the acetate of Compound A i s shown i n Figure 9- The parent ion i s shown to occur at m/e=80k. Ex-85. i111 .1-' [11 III I . D.O «.D W.C in, i"i 100. D IZI.O ;?l).C J10.D tftf.O POO. D TO.O MOO WO.0 MOD «o.a 52-«a.i .«».» . *«.B «o.e - «O.B WO.D va.fl .S4).i .SW.B .MO.D uo I KO.B -. 6«.B t&ve uu.g maj TO a i w.i w . i eoi.o ,<uo.i> F i g u r e 9. The mass spectrum of the acetate d e r i v a t i v e of Compound A. 86. amination of the hig h mass end of the spectrum r e v e a l s - s i x t r a n s i t i o n s which may be a s s o c i a t e d with a c e t a t e decompo-s i t i o n . Two of the t r a n s i t i o n s are accompanied by metas-t a b l e i o n s : 804 m*=722.2 e > ? 6 2 + C H c = 0 a n d 762 m * = 6 2 1 „ 0 ' . d , 702+CH^C-OH. The other t r a n s i t i o n s f o r which metastables 9 are not present a r e : 702 ^642+CH- C-OH, 6 4 2 ^ 600+ CH2=C = 0 , 702, ^ 660 + CH 2 = C = 0 and 6 6 0 - > 600+CH -C-OH. These a c e t a t e degradations i n d i c a t e the occurrence of at l e a s t two aromatic a c e t a t e s and two a l i p h a t i c a c e t a t e s . V a l i d a t i o n of the occurrence of Compound A as a rhamnoside can be seen i n the occurrence of an m/e = 273 i o n f o r the rhamnose t r i a c e t a t e fragment. T h i s fragment can i n t u r n decompose along a pathway s i m i l a r to that pro-posed by Pearl and Darling (74) for glucoside acetates. A proposed decomposition path of the rhamnose t r i a c e t a t e i s d e p i c t e d . i n F i g u r e 1 0 . The prominent i o n s 2 7 3 , 2 1 3 , 1 5 3 , 1 1 1 , and 109 are i n agreement with the proposed pathway. 1 8 7 . OAc m/e 109 CHjC-O m/e 111 F i g u r e 10. Proposed mass s p e c t r a l f r a g m e n t a t i o n pathway f o r rhamnose t r i a c e t a t e . S e v e r a l other f e a t u r e s of Compound A can be i l -l u s t r a t e d through probable formulae r e l a t e d to major Ions of the mass spectrum. Some p o s s i b i l i t i e s are i l l u s t r a t e d on the following page. S e v e r a l c h a r a c t e r i s t i c s of Compound A are n o v e l and r e q u i r e comment. F i r s t , t h i s compound c o n s t i t u t e s the f i r s t i s o l a t i o n and c h a r a c t e r i z a t i o n of a f r e e d i l i g n o l g l y c o s i d e . Second, the compound e x i s t s as an unusual 8 8 . CH„OAc I 2 HC+ I HCOAc Q OAc m/e 323 CH2OAc HC+ HCOAc OCH, OH m/e 281 H CH0OAc I 2 C+ II CH, OH m/e 221 HC+ HC I HCOAc o OCH-)H m/e 221 CH2OH C+ CH, OH m4 179 rhamnoside r a t h e r than much more commonly observed g l u -c o s i d e s . T h i r d , the rhamnoside i s connected t o the com-pound through a p r e v i o u s l y unreported a l i p h a t i c h y d r o x y l p o s i t i o n r a t h e r than the expected aromatic h y d r o x y l p o s i -t i o n . F o u r t h , the e x i s t e n c e of a s a t u r a t e d p r o p y l s i d e c h a i n i s unreported among d i l i g n o l s i s o l a t e d u s i n g hydro-l y t i c c o n d i t i o n s and i s b i o s y n t h e t i c a l l y unusual. F i n a l l y , the e x i s t e n c e of a c a t e c h o l group i s unreported among d i l i g n o l s p r e v i o u s l y i s o l a t e d and c h a r a c t e r i z e d . 89. The complete a n a l y s i s of the a n a l y t i c a l and spec- -t r a l data a v a i l a b l e . f o r Compound A has enabled i t s p o s i t i v e c h a r a c t e r i z a t i o n as d e s c r i b e d . Such data are incomplete for Compounds B and C which therefore cannot be as completely characterized. However, speculative conclusions may be made regarding the structures of these compounds based upon the available data and their observed chemical relationship to Com-pound A. The following discussions include such speculations. Compound B Compound B was i s o l a t e d , from the column chroma-t o g r a p h i c s e p a r a t i o n s p r e v i o u s l y d e s c r i b e d , as an amor-phous s o l i d which f a i l e d t o c r y s t a l l i z e from o r g a n i c s o l v -ents . A t r i t u r a t e d a c e t a t e d e r i v a t i v e was obtained by s c r a t c h i n g under petroleum ether (65°-110°C). The m e l t i n g p o i n t of the ace t a t e d e r i v a t i v e of Compound B was 34° -43°C ( u n c o r r e c t e d ) . Mass s p e c t r a l d e t e r m i n a t i o n of mo l e c u l a r weight was not c l e a r l y d e f i n e d because of the i n a b i l i t y to count mass u n i t s at the high mass end of the spectrum. However, a molec u l a r weight of 744 or 786 was i n d i c a t e d by i n t e r p o l a -t i o n . No meaningful e m p i r i c a l formula c o u l d be c a l c u l a t e d . D i f f e r e n c e s p e c t r a were not obtained f o r t h i s compound be-cause of i t s apparent e x i s t e n c e as a mixture. The i n f r a r e d spectrum of Compound B was e s s e n t i a l l y I d e n t i c a l with 90. t h a t of Compound A, showing an aromatic n a t u r e , no c a r -b o n y l , and a h i g h degree of h y d r o x y l a t i o n . Compound B shows an orange r e a c t i o n w i t h DSA while d i s p l a y i n g n e g a t i v e r e a c t i o n s w i t h G i e r e r ' s (44) and Schroeder's reagent (82). The compound t h e r e f o r e c o n t a i n s n e i t h e r an o-dihydroxyphenyl group nor a b e n z y l h y d r o x y l group. The low y i e l d of Compound B prevented i t s a n a l y s i s u s i n g e t h a n o l y s i s or a l k a l i n e n i t r o b e n z e n e d e g r a d a t i o n t e c h n i q u e s . Chromatography of the 2% o x a l i c a c i d h y d r o l y s i s products showed a mixture of p h e n o l i c compounds ( i n c l u d i n g Compound C) and a major occurrence of L-rhamnose. Compound B was a l s o shown to be formed i n 50% y i e l d under c o n d i t i o n s of c a t a l y t i c r e d u c t i o n of Compound A. Other low y i e l d p r o -d u c t s , i n a d d i t i o n t o unreacted Compound A, were noted i n the chromatographic examination o f the r e a c t i o n p r o d u c t s . Examination of the NMR and mass s p e c t r a l data of Compound B i n comparison t o t h a t o f Compound A, and the ob-served chemical r e l a t i o n s h i p between the compounds, l e d to the s p e c u l a t i v e phenylcoumaran s t r u c t u r e XLIV f o r Com-pound B. - " •' * XLIV XLV 9 1 . NMR s p e c t r a of Compound B and i t s a c e t a t e . The NMR s p e c t r a of Compound B and i t s a c e t a t e d e r i v a t i v e were ob t a i n e d as wit h Compound A and are d e p i c -ted i n F i g u r e s 11 and 1 2 . The s i m i l a r i t y of these s p e c t r a to those f o r Compound "A i s ev i d e n t and only the d i s t i n g u i s h -i n g d i f f e r e n c e s w i l l be d i s c u s s e d . The t o t a l i n t e g r a l s of 28 to 32 and 45 to 47 pro-tons f o r F i g u r e s 11 and 1 2 , r e s p e c t i v e l y , can only be con-s i d e r e d as approximations because of the known occurrence of Compound B as a mixture. The aromatic proton r e g i o n of these s p e c t r a i n t e g r a t e s f o r f i v e t o s i x pr o t o n s . The• high f i e l d rhamnose methyl resonance shows a f i v e p roton m u l t i p l e t i n F i g u r e 11 and c l a r i f i e s somewhat i n F i g u r e 12 to show the expected d o u b l e t . The methoxyl resonance at T = 6 . 2 0 i n t e g r a t e s to three protons but shows three d i s -t i n c t peaks i n d i c a t i n g the e x i s t e n c e of a methoxyl v a r y i n g between three aromatic h y d r o x y l s i t e s . The doublet asso-c i a t e d w i t h the a pro t o n i s centered at 5 . 2 1 T and i n t e -g r a t e s to l e s s than one p r o t o n , while the t o t a l i n t e g r a l ( 5 . 0 0 - 5-40T'), i n c l u d i n g the anomeric s i n g l e t , i n t e g r a t e s to two pr o t o n s . These o b s e r v a t i o n s are i n agreement with the occurrence of t h i s compound as a mole.cular and/or i s o -meric mixture even though i t appears c h r o m a t o g r a p h i c a l l y pure. The NMR spectrum of the diazomethane methylated d e r i v a t i v e of t h i s compound i n d i c a t e d s i x methoxyl protons or at l e a s t one f r e e aromatic h y d r o x y l . The methylated Acetone-d 94. product a l s o shows secondary s p l i t t i n g of the low f i e l d b e n z y l proton d o u b l e t , . f u r t h e r s u g g e s t i n g an i s o m e r i c mixture of e r y t h r o - and t h r e o - c o n f i g u r a t i o n . The NMR of the- a c e t a t e d e r i v a t i v e shows one a r o -matic a c e t a t e ( t = 1.12) and a low f i e l d resonance (x = 2.25) which may be a f r e e h y d r o x y l group. Such a h y d r o x y l resonance i n the a c e t a t e d e r i v a t i v e suggests a s t r o n g l y hydrogen bonded or s t e r i c a l l y h i n d e r e d h y d r o x y l which i s not e f f i c i e n t l y a c e t y l a t e d . I t i s however, more probable t h a t the low f i e l d resonance i s the r e s u l t of an i m p u r i t y . The a c e t a t e d e r i v a t i v e ( F i g u r e 12) a l s o r e v e a l e d no down-f i e l d s h i f t of the b e n z y l proton doublet on a c e t y l a t i o n ( T = 5.05). The spectrum pre s e n t s only f o u r a l i p h a t i c a c e t a t e resonances ( x = 7.8 - 8.2) s u g g e s t i n g t h a t the b e n z y l carbon atom i s i n v o l v e d i n an ether l i n k a g e causing the s h i f t of the a proton resonance to lower f i e l d . NMDR experiments on the a c e t a t e e s t a b l i s h e d the appearance of the g and y proton resonances at 5.83 x and 5.48T , r e s p e c t i v e l y . These resonance l o c a t i o n s r e p r e s e n t opposite chemical s h i f t values to those observed f o r the a c e t a t e of Compound A. Such changes i n chemical s h i f t are- noted for dehydrodiconiferylalcohol triacetate (XLV) (see page 90) i n comparison to l-(3,4-dimethoxyphenyl)~2-(2'-methoxyphenoxy)-pro-pane-1,3 d i o l d i a c e t a t e (XLII) as d e s c r i b e d by Ludwig et_ a l . (60). 95. Mass spectrum of Compound B acetate. ' The mass spectrum of the acetate d e r i v a t i v e of Compound B is-shown i n F i g u r e 13. The occurrence of p o t e n t i a l parent ions at m/e = 702, 744 and 786 i n d i c a t e s the e x i s t e n c e of the compound i n a mol e c u l a r mixture. Such a mixture s e v e r e l y r e s t r i c t s the a n a l y s i s of the spectrum f o r meaningful i o n s , however, the m/e = 744 i o n would be. i n agreement wi t h the mol e c u l a r weight of the s p e c u l a t e d s t r u c t u r e f o r Compound B. The p r e v i o u s l y mentioned ions a s s o c i a t e d with rhamnose t r i a c e t a t e . d e c o m -p o s i t i o n are a l s o present i n the spectrum. The g e n e r a l c h a r a c t e r of the spectrum Is i n agreement with the spec-trum of the phenylcoumaran d e h y d r o d i c o n i f e r y l a l c o h o l as r e p o r t e d by Kovacik and Skamla (56). The probable e x i s t e n c e of more than one compound (and t h r e o - and erythro- -isomers) makes exact a n a l y s i s of Compound B d i f f i c u l t . However, i t would appear that the b e n z y l carbon atom i s e i t h e r e ther or a l k y l - a r y l l i n k e d to a second phenyl n u c l e u s , thereby e l i m i n a t i n g the a h y d r o x y l and the a s s o c i a t e d downfield s h i f t of the be n z y l proton a f t e r a c e t y l a t i o n . A l s o , t h e 3 carbon i s bonded i n such a manner as to cause an u p f i e l d s h i f t of i t s resonance i n comparison to the same resonance i n Compound A. The nature of the . substituent bonded to the 96. ' Pi .1 .1.1,;. l ib i J U L i I MMhA 0.0 M.O «.fl 60.0 BO.O .100.0 120.D i«.0 o . teo.o ?tf>.0 240.0 £60.0 f^lfl.O M3.0 32D.0 MD.O 3E0.0 aoo.o «o.a a.a .. «a.0 . «a.o SJO.B • xs.o 9<a.o sua.a a* . cva.9 . MO.a tni.a coa.o TM.O TJO.I T*.O TCO.B a.a aaa.a aa.% F i g u r e 13. The mass spectrum of the acetate d e r i v a t i v e of Compound B. 94. 3 carbon atom may cause the downfield shift of the y proton i n comparison to the same resonance i n Compound A. . Based upon the nuclear magnetic resonance data i n comparison to that of Ludwig et a l . (60) and Compound A, and the observed chemical relationship between Compound B and Compound A, a phenylcoumaran (XLIV) i s considered as the most probable structure. The a c c e p t a b i l i t y of s t r u c t u r e XLIV f o r Compound an a, 3-phenoxy m i g r a t i o n can occur f o r a-hydroxy-g u a i a c y l - 3 - a r y l e t h e r compounds under m i l d a c i d i c c o n d i -t i o n s . Nimz showed the c o n v e r s i o n of XLVI to XLVII i n water (100°C)'in seven days. The c a t a l y t i c hydrogenolysis conditions - '- -under which Compound A was formed at 50% y i e l d from Com-pound. B may be comparative to Nimz' c o n d i t i o n s l e a d i n g t o the f o r m a t i o n of a quinone methide i o n capable of r e a r -rangement to y i e l d compound XLIV. B i o s y n t h e t i c f o r m a t i o n B may be strengthened by the o b s e r v a t i o n of Nimz (71) that XL VI XLVII 98. of the n a t u r a l l y o c c u r r i n g Compound B may r e s u l t from Compound A by a s i m i l a r b i o s y n t h e t i c reaction,, or i t may be formed as a separate e n t i t y p r i o r or concurrent to the forma t i o n of Compound A v i a the quinone methide pathway. Compound C Compound C, as i s o l a t e d from the chromatographic column, was an amorphous s o l i d which f a i l e d to c r y s t a l l i z e ' from o r g a n i c s o l v e n t s . A t r i t u r a t e d a c e t a t e d e r i v a t i v e was ob t a i n e d through s c r a p i n g under petroleum ether (65° -110°C), m e l t i n g p o i n t 39° - 43°C ( u n c o r r e c t e d ) . The mole-c u l a r weight of the acetate d e r i v a t i v e was determined to be 702 from the mol e c u l a r i o n of i t s mass spectrum. A c a l c u l a t e d e m p i r i c a l formula of Cnr-H,,„0-, r was obtained from 3b> 15 the M + 1/M r a t i o and 15 degrees of u n s a t u r a t i o n were c a l -c u l a t e d from the e m p i r i c a l formula. The compound r e a c t e d • with DSA to produce a red c o l o r and t e s t s f o r o-dihydroxy and b e n z y l h y d r o x y l groups were n e g a t i v e . No a l k a l i n e u l t r a v i o l e t spectrum was obtained because of the low y i e l d of t h i s compound. H y d r o l y s i s of Compound C was s u c c e s s f u l i n producing a low y i e l d of a c h r o m a t o g r a p h i c a l l y pure aglycone and L-rhamnose was e s t a b l i s h e d as the g l y c o s i d e . Low y i e l d s of the compound prevented the a p p l i c a t i o n of f u r t h e r degra-d a t i v e t e c h n i q u e s . The i n f r a r e d spectrum of t h i s compound, 9 9 . was. again s i m i l a r to Compounds A and B. Based on the f o l l o w i n g examination of NMR and mass s p e c t r a l data i n r e l a t i o n to the chemical r e l a t i o n s h i p of Compound C to Compounds A and B, Compound C i s c o n s i d e r e d to be a guaiacyl benzdioxane d i l i g n o l possessing either structure XLVIII or XLIX. mnose XLVIII ' XLIX NMR s p e c t r a of Compound C and i t s d e r i v a t i v e s . The NMR s p e c t r a of Compound C and i t s a c e t a t e d e r i v a t i v e were ob t a i n e d as f o r Compounds A and B and d i s p l a y e d the same'general c h a r a c t e r i s t i c s ( F i g u r e s 14 and 15). The t o t a l approximate p r o t o n i n t e g r a l s f o r F i g u r e s 14 and 15 r e s p e c t i v e l y , are 29-33 and 40-43. Important f e a t u r e s i n c l u d e : s i x aromatic p r o t o n s , no s h i f t of the b e n z y l proton ( x - 5-14) i n the a c e t a t e d e r i v a t i v e , s i m i l a r i t y of chemical s h i f t s of the 3 and Acetone-d F i g u r e 14. The NMR spectrum of Compound C. 102. Y resonances ( T = 5.91, 6.37, F i g u r e 15)to those of the acetate of Compound B (F i g u r e 13) r a t h e r than the ac e t a t e of Compound A, one aromatic a c e t a t e resonance ( T= 7.72), and f o u r a l i p h a t i c a c e t a t e resonances (T = 7.82 - 8.30). These c h a r a c t e r i s t i c s show Compound C to be s i m i l a r to Compound B with an eth e r or an a l k y l - a r y l bond to the a carbon of the g u a i a c y l g l y c e r o l group. H o w e v e r t h i s com-pound c o n t a i n s one l e s s aromatic h y d r o x y l group. Examination of the NMR s p e c t r a o f the. aglycone of Compound C and i t s a c e t a t e d e r i v a t i v e ( F i g u r e s 16,17) o f -f e r s f u r t h e r evidence f o r the s t r u c t u r e of Compound C. Both of these s p e c t r a show s i x aromatic protons i n the T = 3.00 - 3.40 range. The a proton i s shown to occur at T = 5.14 i n F i g u r e 16 and does not s h i f t a f t e r a c e t y l a t i o n ( F i g u r e 17). NMDR e s t a b l i s h e d the occurrence of the 3 and y protons at 5.92 T and 6.39 T r e s p e c t i v e l y . Such chemical s h i f t s are i n d i c a t i v e of the a s s o c i a t i o n of these protons with e l e c t r o n - w i t h d r a w i n g groups bonded to both the 3 and y carbon atoms. NMDR experiments on the a and 3 protons i n the ac e t a t e NMR spectrum r e v e a l e d no s h i f t s a f t e r a c e t y l a t i o n while the y protons have been s h i f t e d to lower f i e l d ( r = 5.65). NMDR experiments e s t a b l i s h e d the chemical s h i f t s , of the a 1 , and 3', and y' protons to occur at T. = 7-41, 8.15 and 6.37 r e s p e c t i v e l y . The a c e t y l a t i o n o f . t h e 105. aglycone produces a s h i f t of the y' proton to 6.08T while the a' and 3' protons show no change. T h i s evidence i s proof of the e x i s t e n c e of the n-propanol s i d e c h a i n i n the aglycone of Compound C. The e x i s t e n c e of only one aromatic a c e t a t e i n both the g l y c o s i d e a c e t a t e and the aglycone a c e t a t e s p e c t r a o f f e r s evidence of the e x i s t e n c e of the g l y c o s i d i c l i n k a g e through t h i s a l i p h a t i c h y d r o x y l . An AB qu a r t e t s i m i l a r t o t h a t r e p o r t e d f o r the ac e t a t e d e r i v a t i v e s of Compounds A and B, i s observed upon i r r a -d i a t i o n of the resonance at 8.18 ? i n the NMR s p e c t r a of the acetate of Compound C. These o b s e r v a t i o n s are con-s i d e r e d proof of the e x i s t e n c e of the rhamnosidic l i n k a g e through the n-propanol s i d e c h a i n as r e p o r t e d f o r Compounds A and B. Mass spectrum of Compound C acetate. F i g u r e 18 shows the mass spectrum of the ac e t a t e d e r i v a t i v e of Compound C. The molecular i o n (M = 702) and ace t a t e decomposition pathways are c o n s i s t e n t w i t h the s p e c u l a t e d s t r u c t u r e . M o l e c u l a r i o n t r a n s i t i o n s 702 ->-660, 660 •> 600, 642 -> 600 , and 702 -> 642, are observed and s u b s t a n t i a t e d by the e x i s t e n c e of metastable ions at 621, 546, 562, and 587, r e s p e c t i v e l y . T h i s a c e t a t e de-composition i n d i c a t e s one aromatic a c e t a t e and one 1 0 6 . • , u.*v . vii.* •- "*aj.u . W.H .. .. w.u .. vju.u 3UJ.V {^U.B W.U 1VJ 3CJU.V DbTU.v WJ.fl UWJ WJ.V F i g u r e 18 . /The mass spectrum of the acetate d e r i v a t i v e ** of Compound C. 107.. a l i p h a t i c a c e t a t e . The mass spectrum r e v e a l s the ions p r e v i o u s l y mentioned f o r the rhamnose t r i a c e t a t e and i t s decomposition p r o d u c t s . Prominent ions are a l s o seen at m/e = 149, 222. These ions may o r i g i n a t e from the cleavage of the dioxane r i n g of the s p e c u l a t e d s t r u c t u r e of Compound C, f o l l o w e d by l o s s of a c e t a t e . Two p o s s i b l e molecular i o n s t r u c t u r e s are pres e n t e d below which may be r e p r e s e n t a t i v e of the m/e = 222 and 1^9 i o n s . In c o n s i d e r i n g the t r a n s f o r m a t i o n of Compounds A and B to Compound C by h y d r o l y s i s and the r e l a t e d NMR da t a of Compounds A and B, Compound C i s s p e c u l a t e d to be a g u a i a c y l benzdioxane d e r i v a t i v e (XLVIII or XLIX). 108... OH OH X L V I I I X L I X These s t r u c t u r e s may be s p e c u l a t e d t o be c h e m i c a l l y d e r i v e d from Compound A from an i n t e r m e d i a r y quinone methide form of Compound B o r i g i n a t i n g a c c o r d i n g to the rearrangement proposed by Nimz ( 7 1 ) w h i c h may subsequently r e a c t along three d i s t i n c t pathways. F i r s t , i n the quinone methide stage, the f r e e c a t e c h o l h y d r o x y l group may r e a c t w i t h the r e s o n a n c e - s t a b i l i z e d b e n z y l carbon atom to form s t r u c t u r e XLIX. Second, the 1,2 phenoxy' s h i f t of Nimz (71) may occur, with the subsequent formation of a l k y l - a r y l r i n g c l o s u r e to form the phenylcoumaran"XLIV. F i n a l l y , i n s t e a d of phenylcoumaran f o r m a t i o n , r o t a t i o n may occur about the new-l y formed a et h e r bond to p l a c e the c a t e c h o l h y d r o x y l i n c l o s e p r o x i m i t y t o the g carbonium i o n (formed i n the phenoxy t r a n s f e r ) with subsequent fo r m a t i o n of s t r u c t u r e XLVTIIc The c h a r a c t e r i z a t i o n of Compound A and the s p e c u l a -t i o n of Compounds B and C i m p l i e s the pathway of Nimz 109. as p l a u s i b l e i n the A to B to C i n t e r r e l a t i o n s h i p both c h e m i c a l l y and b i o s y n t h e t i c a l l y . of s t r u c t u r a l formulae f o r Compounds B and C are s p e c u l a -t i v e and are based s o l e l y on s p e c t r a l data. However, the ge n e r a l r e l a t i o n s h i p of A to B to C can stren g t h e n the ob-s e r v a t i o n s based upon the known s t r u c t u r e of A. F u r t h e r r e s e a r c h w i l l e s t a b l i s h i f t h i s i s a probable l i n k of l i g n i n t o l i g n a n b i o s y n t h e s i s i n western r e d cedar. Fur-t h e r r e s e a r c h w i l l a l s o e s t a b l i s h the exact nature of the a and 3 bondings i n Compounds B and C. The I s o l a t i o n of other compounds g i v i n g the p o s i t i v e DSA r e a c t i o n i n the e t h y l acetate e x t r a c t w i l l add v a l u a b l e i n f o r m a t i o n t o the e l u c i d a t i o n of these s t r u c t u r e s . I t i s of i n t e r e s t to note t h a t the three d i l i g n o l rhamnosides e x h i b i t a "missing" methoxyl group, common among the western red cedar heartwood l i g n a n s (e.g., XXXVIII). I t should again be noted that the p r e s e n t a t i o n XXXVIII 110 . L i g n i n B i o s y n t h e s i s The r e s u l t s of t h i s i n v e s t i g a t i o n l e a d to specu-l a t i o n i n t o the r o l e of the i s o l a t e d compounds i n l i g n i n b i o s y n t h e s i s . The g l y c o s i d i c nature of the i s o l a t e d d i l i g n o l s suggests the p o s s i b i l i t y of t h e i r involvement i n a t r a n s l o c a t o r y process to a s i t e of a c t i v e l i g n i n f ormation w i t h t h e i r subsequent h y d r o l y s i s , and p o l y m e r i -z a t i o n i n t o the l i g n i n molecule. The noted occurrence of c a t e c h o l groupings i n bark l i g n i n s (50) f u r t h e r sug-gests one p o s s i b l e d e s t i n a t i o n of the d i l i g n o l g l y c o s i d e s . However, t r a n s l o c a t i o n much b e y o n d l l o c a l e n v i r o n s , of aromatic ( p a r t i c u l a r l y d i m e r i c ) l i g n i n p r e c u r s o r s i s u n l i k e l y . Thus, the i s o l a t e d " compound would be d e s t i n e d to i n c o r p o r a t i o n i n t o l e a f or branch l i g n i n . Assuming that t r a n s l o c a t i o n i s l i m i t e d , the d i -l i g n o l s may ma i n t a i n one of two p o s i t i o n s r e g a r d i n g t h e i r r o l e i n l i g n i n b i o s y n t h e s i s . F i r s t , the d i l i g n o l s i s o -l a t e d may be r e p r e s e n t a t i v e of a d e f i n e d c l a s s of ex-t r a c t i v e components which may undergo f u r t h e r minor s t r u c -t u r a l a l t e r a t i o n but w i l l e x i s t e v e n t u a l l y as f r e e d i l i g n o l s . Second, the d i l i g n o l s may serve a s . t r u e i n -termediates to l i g n i n f o r m a t i o n . The. occurrence of the d i l i g n o l rhamnosides as a d i s t i n c t c l a s s may f i n d some s u b s t a n t i a t i o n i n t h e i r high y i e l d i n l e a f t i s s u e s , and t h e i r unusual chemical 111. s t r u c t u r e s (compared to other d i l i g n o l s ) . The high y i e l d i n leaves may be compared to that of the f l a v o n o i d g l y c o s i d e s i n the same t i s s u e . Such an ana-logy would p l a c e the d i l i g n o l rhamnosides as end products of p h e n o l i c anabolism w i t h secondary p h y s i o l o g i c a l func-t i o n s . One such p h y s i o l o g i c a l r o l e might be the p a r t i c i -p a t i o n of the compounds i n p r o t e c t i o n mechanisms i n the l e a v e s . I t i s p o s s i b l e that i n j u r y or d i s e a s e r e a c t i o n s may occur i n the leaves which would c a t a l y z e the cleavage of the unusual rhamnosldic l i n k a g e of the d i l i g n o l rhamno-s i d e to render the aglycone a v a i l a b l e f o r p a r t i c i p a t i o n i n d e t o x i f i c a t i o n or s e a l i n g - o f f a c t i v i t i e s . The n - p r o p y l s i d e c h a i n of the d i l i g n o l rhamnosides i s unusual i n l i g n i n and phenylpropane b i o s y n t h e s i s . Ac-t i v e p r e c u r s o r s to l i g n i n and p o l y p h e n o l i c s are c o n s i d e r e d to r e q u i r e phenylprop^rie. ?:.- c h a r a c t e r to allow the o x i -d a t i v e or r e d u c t i v e c o u p l i n g r e a c t i o n e s s e n t i a l i n the formation of l i g n i n and p o l y p h e n o l i c s . P a r t i c i p a t i o n of the d i l i g n o l rhamnosides i n wood l i g n i f i c a t i o n would r e -q u i r e m e t h y l a t i o n of the f r e e c a t e c h o l h y d r o x y l grouping and o x i d a t i o n of the p r o p y l s i d e chain p r i o r to f u r t h e r p o l y m e r i z a t i o n t o l i g n i n i n the accepted sense. While m e t h y l a t i n g enzymes are a v a i l a b l e , the o x i d a t i v e step i s h i g h l y improbable. 112. The f o r e g o i n g d i s c u s s i o n would seem to p r e c l u d e the p a r t i c i p a t i o n of the observed d i l i g n o l rhamnosides i n wood l i g n i n f o r m a t i o n . However, the assumed non-trans-l o c a t o r y nature of the compounds and t h e i r unusual s t r u c -t u r a l c h a r a c t e r suggest t h e i r p o s s i b l e p a r t i c i p a t i o n i n the l e s s i n t e n s i v e l y s t u d i e d f o r m a t i o n of bark or l e a f l i g n i n . Degradative examination of bark l i g n i n . h a s es-t a b l i s h e d the occurrence of c a t e c h o l groupings i n the l i g n i n s t r u c t u r e ( 5 0 ) - A s p e c i f i c study of western red cedar bark u s i n g NMR (93) has shown a lower methoxyl con-ten t and more a l i p h a t i c c h a r a c t e r than wood l i g n i n . Such evidence leads to s p e c u l a t i o n t h a t ' t h e d i l i g n o l rhamnosides of t h i s study may act a s . p r e c u r s o r s to l i g n i n d i f f e r e n t i n c h a r a c t e r than t h a t observed i n the wood. Such l i g n i n may c o n t a i n a r y l - a r y l bonded l i g n i n polymers, which o r i g i n a t e from c o u p l i n g ortho to the n - p r o p y l - s i d e c h a i n of d i l i g n o l s a c t i v a t e d by the f r e e c a t e c h o l h y d r o x y l grouping. I f the d i l i g n o l rhamnosides do p a r t i c i p a t e i n bark l i g n i n f o r m a t i o n , they c o u l d o r i g i n a t e i n the i n n e r phloem, i n c o n t r a s t to the o r i g i n of wood l i g n i n p r e c u r s o r s i n the cambium. T h i s d i s c u s s i o n i s h i g h l y s p e c u l a t i v e . The bark t i s s u e s must b'e s p e c i f i c a l l y examined f o r the p a r t i -c i p a t i o n of the d i l i g n o l s i n bark l i g n i n f o r m a t i o n . Evidence r e g a r d i n g the nature and f o r m a t i o n of l e a f l i g n i n i s wanting. I t i s t h e r e f o r e necessary to thoroughly i n v e s t i g a t e t h i s t i s s u e b e f o r e any s i g n i f i c a n c e can be a t t r i b u t e d t o the d i l i g n o l rhamnosides. S c i n t i l l a t i o n - C h r o m a t o g r a p h y Technique The v a l i d a t i o n procedure i n e s t a b l i s h i n g combus-t i o n as a s u p e r i o r a n a l y t i c a l technique i n the measurement of low a c t i v i t y , c h r o m a t o g r a p h i c a l l y separated compounds i s d e s c r i b e d i n a p r e v i o u s s e c t i o n . The r e s u l t s are tabu-l a t e d i n Table 1 and are g r a p h i c a l l y d e p i c t e d i n F i g u r e s 19, 20 and 21. Examination of the data y i e l d e d the f o l l o w -i n g r e s u l t s . A comparison of Figures 19 and 20 reveals a much smaller e f f e c t of e x t e r n a l component upon a c t i v i t y r a t i o i n the combusted samples. The scraped-suspended samples show s i g n i f i c a n t e f f e c t s of both e x t e r n a l component and spray reagent upon the r e c o r d e d a c t i v i t y r a t i o . The l a r g e s t e f f e c t on the a c t i v i t y r a t i o appears when the chromogenic spray b i s - d i a z o t i z e d b e n z i d i n e was used as a d e t e c t i n g reagent. When only 2'ug of q u e r c e t i n were present on the chromatographic s p o t s , subsequent d e t e c t i o n with t h i s spray, f o l l o w e d by c a r e f u l s c r a p i n g from the chromatographic p l a t e , produced a r e d u c t i o n of over 20 per cent i n d e t e c t a b l e a c t i v i t y . In c o n t r a s t , combustion and s c i n t i l l a t i o n c o u n t i n g of t h a t same sample . Amount of e x t e r n a l compound, yg - t F i g u r e 19. The e f f e c t of an e x t e r n a l quenching compound on the d e t e r m i n a t i o n of a c t i v i t y of scraped chromatographic samples. 1.11 1.00, o •H -P aj U $090 •H > •H -P O < 0.8$ 0.70 B - H Compound added Q u e r c e t i n P i n o s y l v i n Q u e r c e t i n P h e n y l a l a n i n e Q u e r c e t i n Spray Barton's Reagent No b i s - d i a z o . b e n z . N i n h y d r i n No 5 10 15 amount of e x t e r n a l compoundj ug The e f f e c t of. an external- quenching compound' on the d e t e r m i n a t i o n of a c t i v i t y of combusted chromatographic samples. F i g u r e 20 116. o C CD •H O •H CM ^ cd •H -P o o o •H -P cd -p co c •H c cd cd cd H H >3 •H -p •H • O CO Xi 0 > CD co o 0.90 0.80 0.70 1.0 •2.0 Area of chromatographic spot, cm' .Figure 21 The e f f e c t of chromatographic spot s i z e on l i q u i d s c i n t i l l a t i o n count-i n g of combustion samples. 117. shows a l o s s of l e s s than 10 per cent i n the d e t e c t a b l e a c t i v i t y . When q u e r c e t i n was d e t e c t e d w i t h Barton's r e a -gent, f o l l o w e d by s c r a p i n g and suspension i n the s c i n t i l l a -t i o n s o l u t i o n ^ the r e d u c t i o n i n a c t i v i t y ranged from 7 to 28 per cent. Comparative r e s u l t s f o r the combustion t e c h -nique show l o s s e s of l e s s than 5 per cent. F i g u r e s 19 and 20 show a v i v i d _ c o m p a r i s o n between the scraped-suspension and combustion r e s u l t s f o r p i n o s y l v i n without s p r a y i n g . The sample shows an i n c r e a s e i n a c t i v i t y of 15-20 per cent when analyzed as a^scraped sample.This i n c r e a s e must be the r e s u l t of chemolumi'nescence, which the s c i n t i l l a t i o n counter cannot d i s t i n g u i s h from t r u e r a d i o a c t i v i t y . Analy-s i s of the r e s u l t s of combustion f o r t h i s , same sample shows r e d u c t i o n s i n a c t i v i t y r a t i o of l e s s than 5%. Chemo-luminescence may a l s o e x p l a i n the i n c r e a s e i n a c t i v i t y noted f o r 2 ug of q u e r c e t i n . In g e n e r a l , the combustion technique minimizes the l o s s of d e t e c t a b l e r a d i o a c t i v i t y due to the e x t e r n a l i n -f l u e n c e s of chemical and p h y s i c a l quenching. F i g u r e 21 i s a summary of data r e l a t e d to the e f -f e c t of spot s i z e upon counting e f f i c i e n c y f o r the combus-t i o n t e c h nique. I t can be seen from the f i g u r e t hat an 2 2 i n c r e a s e i n spot s i z e from 0.6 cm to 2 cm r e s u l t e d i n a r e d u c t i o n of counting e f f i c i e n c y from 82% to 75%. T o t a l 118. weight data f o r the spot.removed from the chromatographic p l a t e showed that a maximum of 7 mg per spot could be com-busted e f f i c i e n t l y and the optimum weight of chromatographic spots was. i n the range of 4 to 5 mg. Such a chromatographic 2 spot weight was r e p r e s e n t a t i v e of a spot approximately 1 cm i n c l u d i n g the a d d i t i o n of the c e l l u l o s e n i t r a t e s o l u t i o n . The combustion of the chromatographic spots r e -moved from t h i n l a y e r c e l l u l o s e p l a t e s o f f e r s a s u b s t a n t i a l advantage i n counting low a c t i v i t y samples compared t o the sc r a p i n g - s u s p e n s i o n method. The sample to be combusted i s removed from the p l a t e c l e a n l y , e a s i l y handled, and m i n i -mizes l o s s e s d u r i n g sample t r a n s f e r . In comparison, scraped samples i n v o l v e t e d i o u s i s o l a t i o n of a p a r t i c u l a t e sample which can e a s i l y be s c a t t e r e d d u r i n g t r a n s f e r . The scraped samples o f t e n e x h i b i t e l e c t r o s t a t i c p r o p e r t i e s which f u r -t h e r hamper h a n d l i n g . Thus, the combustion technique o f f e r s the a b i l i t y t o count p h e n o l i c compounds, which can only be d e t e c t e d u s i n g chromogenic spray r e a g e n t s , while l i m i t i n g experimen-t a l e r r o r a s s o c i a t e d with sample p r e p a r a t i o n , h a n d l i n g , and coun t i n g . T h i s combustion technique a l s o p r o v i d e s an advan-tage i n terms of experimental design.. I t enables r a d i o a c -t i v e p r e c u r s o r i n h i b i t i o n i n t o m e t a b o l i z i n g t i s s u e s , f o l -119. lowed by e x t r a c t i o n , chromatographic, i s o l a t i o n and count-i n g of l a b e l l e d chromatographic spots i n a matter of days. When autoradiography i s used i n a s i m i l a r sequence, the r e s u l t s would not be a v a i l a b l e f o r a month or two f o r low a c t i v i t y samples. Other t e c h n i q u e s , such as t h i n window counting of chromatographic s t r i p s , or sample i s o l a t i o n to constant s p e c i f i c a c t i v i t y , are e i t h e r l i m i t e d by the l e v e l of a c t i v i t y present or long experimental workups The low a c t i v i t y combustion technique was l i m i t e d to t h i n l a y e r c e l l u l o s e chromatographic p l a t e s and may have l i m i t a t i o n s a s s o c i a t e d with chromogenic sprays not i n v e s t i -gated. Although the method i s a p p l i e d only to c e l l u l o s e p l a t e s , i t i s p o s s i b l e t h a t i t may f i n d f u t u r e a p p l i c a t i o n s on other chromatographic supports which can be combusted (e.g., polyamide, d e x t r o s e ) . The combustion technique i s not a p p l i c a b l e to t h i n l a y e r s i l i c a g e l chromatography be-cause i t i s non-combustible. However, the c e l l u l o s e n i -t r a t e s o l u t i o n i s u s e f u l i n removing chromatographic spots from the s i l i c a g e l p l a t e priorjysuspension c o u n t i n g . P r e c u r s o r Feeding Study The m e t a b o l i c a c t i v i t y of leaves makes them a very, promising t i s s u e f o r the study of i n c o r p o r a t i o n of known aromatic p r e c u r s o r s . P h e n y l a l a n i n e r e p r e s e n t s a w e l l es-t a b l i s h e d aromatic p r e c u r s o r which, when a d m i n i s t e r e d to 120 . a c t i v e l y m e t a b o l i z i n g t i s s u e s , w i l l p a r t i c i p a t e i n the for m a t i o n of aromatic compounds with a minimum r e v e r s i o n to the g l y c o l y t i c pathway. I t i s f o r these reasons that p h e n y l a l a n i n e was chosen f o r aromatic I n c o r p o r a t i o n s t u d i e s i n the leaves of western r e d cedar. A d m i n i s t r a t i o n of l i g n i n p r e c u r s o r s i n t o a c t i v e l y m e t a b o l i z i n g t i s s u e s g e n e r a l l y f a l l s i n t o two separate c l a s s e s : (1) i n f u s i o n , and (2) i m p l a n t a t i o n . K r a t z l (57) has shown t h a t the i n f u s i o n of r a d i o a c t i v e l i g n i n p r e c u r -sors i n t o the stem produces h i g h e r r a d i o a c t i v e i n c o r p o r a -t i o n i n t o l i g n i n than the i m p l a n t a t i o n o f the same p r e -c u r s o r s i n t o the cambial area. I m p l a n t a t i o n techniques are c o n s i d e r e d to i n i t i a t e secondary wound r e a c t i o n s which subsequently a l t e r the pathway to the form a t i o n of aroma-t i c compounds. Freudenberg (29) has noted the fo r m a t i o n of r a d i o a c t i v e wood l i g n i n u s i n g the technique of i n f u s i o n of known l i g n i n p r e c u r s o r s through the leaves of c o n i f e r o u s s p e c i e s . I t i s evident t h e r e f o r e , that i n f u s i o n f e e d i n g methods allow the e f f i c i e n t uptake of l i g n i n p r e c u r s o r s which may be a l t e r e d i n the l e a f t i s s u e p r i o r to the u l t i -mate for m a t i o n of l i g n i n i n the wood. These o b s e r v a t i o n s suggested the c l o s e r examination of western r e d cedar leaves f o r . l i g n i n p r e c u r s o r s which would show a c t i v e me-t a b o l i s m of r a d i o a c t i v e l y l a b e l e d aromatic p r e c u r s o r s d u r i n g i n f u s i o n f e e d i n g . 121. Preliminary, i n f u s i o n f e e d i n g experiments e s t a b l i s h e d t h a t the leaves of western red cedar were able to a s s i m i -l a t e r a d i o a c t i v e p h e n y l a l a n i n e to form r a d i o a c t i v e p h e n o l i c compounds. Autoradiography (Figure 22) of the gross e t h y l a c e t a t e e x t r a c t of the p r e l i m i n a r y f e e d i n g experiment showed i n c o r p o r a t i o n of the l a b e l i n a f o u r hour f e e d i n g time. Four major spots o c c u r r i n g between Rf-i 0.1 and 0.45 on the chromatogram d i s p l a y e d an orange c o l o r r e a c t i o n when 14 sprayed with DSA. The i n c o r p o r a t i o n of U- C-L-phenylala-nine Into two of these compounds (designated A and B) was l a t e r examined i n a ten hour f e e d i n g experiment u t i l i z i n g the v a l i d a t e d t h i n l a y e r chromatographic combustion t e c h -nique. D i a z o t i z e d s u l f a n i l i c a c i d (DSA) was used as a de-t e c t i n g reagent i n p l a c e of b i s - d i a z o t i z e d b e n z i d i n e i n the l e a f f e e d i n g experiment s i n c e i t gave more d i s t i n c t c o l o r r e a c t i o n s and somewhat h i g h e r counting e f f i c i e n c y a f -t e r combustion (Table 1). The use of DSA d i d e n t a i l some problems i n the spot removal from the chromatographic p l a t e , making i . i t " necessary t h a t the c e l l u l o s e l a y e r be of c o n s i s t e n t .thickness. I t was a l s o necessary to allow ade-quate time f o r the DSA spray to dry p r i o r to removal of the chromatographic spots with c e l l u l o s e n i t r a t e . The spots r e p r e s e n t a t i v e of Compounds A and B i n the ten hour l e a f f e e d i n g experiment r e q u i r e d d i v i s i o n to s m a l l e r s i z e p r i o r to combustion. The r e s u l t s of the combusted samples i n the ten h o u r . f e e d i n g experiment are t a b u l a t e d i n Table 3 and 1 2 2 : F i g u r e 2 3 . Autoradiogram 'of the gross e t h y l a c etate s o l u b l e s from U - 1 4 C - L - p h e n y l a l a n i n e f e d western red cedar leaves ( s o l v e n t - B E ) . 123. g r a p h i c a l l y d e p i c t e d i n F i g u r e s 23 and 24. Table 3 r e v e a l s that f l o a t i n g cut western r e d . cedar leaves i n a r a d i o - a c t i v e s o l u t i o n of p h e n y l a l a n i n e i s an e f f i c i e n t method of a d m i n i s t e r i n g the aromatic p r e -c u r s o r . The per cent i n c o r p o r a t i o n of the l a b e l e d p r e -c u r s o r w i t h time i s shown i n F i g u r e 2 3 which shows 16 per cent uptake i n hour 1, with continuous uptake of the l a -b e l e d p r e c u r s o r t o a l e v e l of 37 per cent a f t e r 10 hours. T h i s r a t e of uptake should e f f e c t i v e l y minimize the des-t r u c t i o n of the aromatic p r e c u r s o r by micro-organisms which might be present i n the l e a v e s . T h i s r a p i d r a t e should a l s o optimize the i n t r o d u c t i o n of the aromatic p r e c u r s o r i n t o the m e t a b o l i z i n g areas d u r i n g a p e r i o d of normal me-t a b o l i s m . The l e v e l of a c t i v i t y i n c o r p o r a t i o n Into Compounds A and B d u r i n g 1, 3, 5, and 10 hour f e e d i n g p e r i o d s i s d e p i c t e d i n Table 3 and F i g u r e 24. I n c o r p o r a t i o n l e v e l s of 0.15% to 0.40% Were observed f o r r a d i o a c t i v e p h e n y l a l a -nine i n t o Coumpounds A and B. Examination of the data i n d i -cate that Compound B i n c o r p o r a t e s a l a r g e r p o r t i o n of the a v a i l a b l e . r a d i o a c t i v i t y than does Compound A. Both com-pounds showed a r e d u c t i o n i n a c t i v i t y a f t e r 5 hours f e e d -i n g . T h i s r e d u c t i o n i n a c t i v i t y a f t e r 5 hours may be a s s o c i a t e d with f u r t h e r metabolism of the compounds. Sub-sequent experiments are needed to c l a r i f y this, p o i n t . 10 90 80 t 0 -124 70 60 50 40 30 20 10 1 11 3 5 7 9 Feeding time, h r s . 14 Fi g u r e 2 3 . Uptake of U- C-L-phenylalanine by • w e s t e r n r e d cedar l e a v e s . F i g u r e 24. * Compound A B Compound B 3 5 7 9 11 Feeding time, h r s . 14 I n c o r p o r a t i o n of U- C-L-phenylalanlne i n t o compounds A and B i n the leaves . of western r e d cedar. 1 2 5 . The r e s u l t s of t h i s f e e d i n g experiment r e v e a l e d two important p o i n t s . F i r s t , i t i s apparent that phenyla l a n i n e i s a c t i v e l y i n c o r p o r a t e d i n t o , d i l i g n o l rhamnosides i n the leaves of western r e d cedar. Second, the l e v e l of i n c o r p o r a t i o n i n t o these p r e c u r s o r s i s such that f u t u r e k i n e t i c . s t u d i e s may b i o s y n t h e t i c a l l y r e l a t e the d i l i g n o l g l y c o s i d e s and subsequently .explain-" the f o r m a t i o n of l i g -n i n p r e c u r s o r s i n the leaves of western red cedar. 126. CONCLUSION The e t h y l a c e t a t e s o l u b l e p o r t i o n of methyl a l c o -h o l e x t r a c t s of f r e s h western red cedar leaves was examined. The purpose of the i n v e s t i g a t i o n was to i s o l a t e and charac-t e r i z e m e t a b o l i c a l l y a c t i v e d i l i g n o l s , which may serve as p r e c u r s o r s i n l i g n i n f o r m a t i o n . Three such d i l i g n o l rham-nosides have been i s o l a t e d through the a p p l i c a t i o n of pres-. sure chromatography and g e l f i l t r a t i o n t e c h n i q u e s . The r a p i d m e t a b o l i c f o r m a t i o n of these d i l i g n o l rhamnosides . 14 was shown through t h e i r i n c o r p o r a t i o n of U- C-L-phenyla-l a n i n e i n a l e a f i n f u s i o n ' f e e d i n g experiment. Of the three dilignols isolated, the d i l i g n o l rhamnoside 1-(3'-methoxy-4'-hydroxyphenyl)-2-0-1'-[2'-hydroxy-4'-(propane-3"'-O-a-L-rhamnoside)phenyl]-propane-l,3 d i o l was obtained i n high yi e l d (0.15$), and was characterized success-f u l l y by chemical degradation i n conjunction with NMR and mass-spectral techniques. The other two d i l i g n o l rhamnosides have been i s o -l a t e d i n lower y i e l d s ( l e s s than 0.05$ each), t h e i r s t r u c t u r e s were not completely c h a r a c t e r i z e d . Based upon accumulated c h e m i c a l , NMR, and mass s p e c t r a l d a t a , however, the compounds were found to be c l o s e l y r e l a t e d homologs 127. of the c h a r a c t e r i z e d d i l i g n o l rhamnoside. T h i s r e l a t i o n -s h i p appeared to be a s s o c i a t e d with r e a c t i o n s of the r e -a c t i v e b e n z y l h y d r o x y l and c a t e c h o l groups. S p e c u l a t i v e phenylcoumaran and g u a i a c y l benzdioxane s t r u c t u r e s s a t i s -f i e d much of the evidence c o l l e c t e d f o r these two compounds. Chromatographic evidence suggested the e x i s t e n c e of s e v e r a l other s i m i l a r compounds. A t o t a l y i e l d f o r a l l such com-ponents i n the leaves of western r e d cedar may be as hig h as 0.5%. The c h a r a c t e r i z e d d i l i g n o l rhamnoside possessed s e v e r a l n o v e l s t r u c t u r a l c h a r a c t e r i s t i c s i n r e l a t i o n to p r e v i o u s l y r e p o r t e d d i l i g n o l s i n c l u d i n g : (1) the f i r s t r e p o r t e d occurrence of a f r e e d i l i g n o l g l y c o s i d e , (2) the unusual occurrence of the g l y c o s i d e as an a-L-rhamnoside, (3) the p r e v i o u s l y unreported e x i s t e n c e i n a p o t e n t i a l l i g -n i n p r e c u r s o r of the g l y c o s i d i c l i n k a g e through an a l i -p h a t i c h y d r o x y l , (4) the e x i s t e n c e of the d i l i g n o l as a p r e v i o u s l y unreported g u a i a c y l g l y c e r o l - c a t e c h o l - 3 - a r y l ether r a t h e r than the common g u a i a c y l g l y c e r o l - g u a i a c y l - 3 -a r y l ether and, (5) the unusual s a t u r a t e d p r o p y l s i d e c h a i n i n a compound of l i g n i n c h a r a c t e r . A s p e c i f i c r a d i o a c t i v e i n f u s i o n study r e v e a l e d t h a t the c h a r a c t e r i z e d d i l i g n o l rhamnoside, and i t s sus-pected' phenylcoumaran homolog, i n c o r p o r a t e d 0.3% and 0.4% 14 U- C-L-phenylalanine w i t h i n a ten hour f e e d i n g p e r i o d . 128. The degree of i n c o r p o r a t i o n i n t o these compounds was ob-t a i n e d through the a p p l i c a t i o n of a newly developed t e c h -nique f o r the e f f i c i e n t c o u n t i n g of low r a d i o a c t i v i t y samples, se p a r a t e d c h r o m a t o g r a p h i c a l l y on t h i n l a y e r c e l -l u l o s e p l a t e s . A separate v a l i d a t i o n of the techniques showed i t s s u p e r i o r i t y to- comparable methods of a n a l y s i s , f o r low a c t i v i t y samples. The r o l e of the d i l i g n o l rhamnosides i n l i g n i n b i o s y n t h e s i s was d i s c u s s e d . I t was suggested t h a t the d i l i g n o l g l y c o s i d e s of the leaves may be important bark and l e a f l i g n i n p r e c u r s o r s , based upon t h e i r structures.c.:'The h i g h y i e l d of these compounds i n the a c t i v e l y m e t a b o l i z i n g l e a f t i s s u e i n d i c a t e d t h a t they may a l s o f u l f i l l important p h y s i o l o g i c a l r o l e s (e.g. disease' p r o t e c t i o n , wound-sealing) i n the l e a f t i s s u e s . Future Research The d i s c o v e r y of the d i l i g n o l rhamnosides and t h e i r observed m e t a b o l i c p a r t i c i p a t i o n suggests many important f u t u r e i n v e s t i g a t i o n s . F i v e major p r o p o s a l s r e l a t i n g to f u t u r e r e s e a r c h w i t h these compounds are as f o l l o w s : The characterization of the two remaining dili g n o l . rhamnosides found i n the leaves of western red cedar and the c l a r i f i c a t i o n of the chemical and biochemical relationship between them and Compound A. The i s o l a t i o n a n d . c h a r a c t e r i z a t i o n of other d i l i g n o l and/or o l i g o l i g n o l s which may be present i n these l e a v e s . F u r t h e r b i o s y n t h e t i c l a b e l l i n g s t u d i e s with more p r e c i s e p r e c u r s o r s to a s c e r t a i n s p e c i f i c pathways i n the b i o g e n e s i s of the d i l i g n o l s and l i g n i n . Radiochemical k i n e t i c s t u d i e s u t i l i z i n g the t h i n l a y e r c e l l u l o s e sample technique to a s s o c i a t e the order of f o r m a t i o n of the d i l i g n o l s and t h e i r subsequent c o n v e r s i o n s . 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