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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 U n i v e r s i t y , 1962 B.S. (Chern.), Oregon State U n i v e r s i t y , 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  i n the Department of Forestry  We accept t h i s thesis as conforming to the required standard.  THE UNIVERSITY OF BRITISH COLUMBIA December, 1970  In p r e s e n t i n g t h i s  thesis  an advanced degree at the L i b r a r y s h a l l I  f u r t h e r agree  in p a r t i a l  fulfilment of  the U n i v e r s i t y of  British  make i t f r e e l y a v a i l a b l e  that permission  for  the requirements f o r  Columbia,  I agree  r e f e r e n c e and  for extensive copying o f  this  that  study. thesis  f o r s c h o l a r l y purposes may be g r a n t e d by the Head o f my Department o r by h i s of  this  representatives. thesis  It  is understood that  f o r f i n a n c i a l gain shall  written permission.  Department The U n i v e r s i t y o f B r i t i s h Vancouver 8, Canada  Columbia  copying o r p u b l i c a t i o n  not be allowed without my  i.  ABSTRACT  Three d i l i g n o l rhamnosides were i s o l a t e d i n 0.15 t o 0.^0$ y i e l d from the ethyl acetate solubles of a methyl alcohol extract of western red cedar (Thuja p l i c a t a 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"-(propane-3"'-a-L--rhamnoside)phenyl]-propane-lj3 d i o l , using NMR and chemi c a l degradation i n conjunction with mass.spectral techniques on the compound and i t s d e r i v a t i v e s .  The other d i l i g n o l rhamnosides were  not completely characterized, but were shown to be chemically r e l a t e d to the d i l i g n o l i d e n t i f i e d .  Based upon NMR, chemical and_  mass spectral data, the uncharacterized d i l i g n o l rhamnosides are speculated t o 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 d i l i g n o l glycoside i n plant tissues.  The unusual a-L-rhamnose moiety of the d i l i g n o l occurs i n  a previously unreported linkage to the n-propyl hydroxyl group uncommon i n l i g n i n .  The rhamnoside also displays the previously  unreported guaiacylglycerol~catechol--8-aryl ether structure rather than the commonly observed guaiacylglycerol-guaiacyl~3a r y l ether structure.  i i .  A new combustion-absorption technique was  developed  and validated which allows high e f f i c i e n c y evaluation of low act i v i t y radioactive products separated on t h i n layer c e l l u l o s e chromatography p l a t e s .  The technique was applied to an analysis  of the anabolic products of an i n f u s i o n feeding of Ualanine to western red cedar leaves.  C--L-phenyl-  F a c i l e imbibition o f U--  14 C-L-phenylalahine occurs within ten hours. ation of 0.30%. and 0.^0%  Maximum incorpor-  of the a v a i l a b l e r a d i o a c t i v i t y occurs  i n the characterized d i l i g n o l rhamnoside, and i t s suspected phenylcoumaran homolog r e s p e c t i v e l y , at the three to f i v e hour period of the i n f u s i o n feeding.  The incorporation r e s u l t s indicate the  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 the leaves of western red cedar.  i n aromatic metabolism  This feeding experiment i s  preliminary to future d e t a i l e d biosynthetic studies i n the l e a f tissue. The combustion-absorption technique i s l i m i t e d to combustible sample weights of 7 nig.  iii .  TABLE OF CONTENTS PAGE  ABSTRACT  '.  i  TABLE OF CONTENTS  .  :  .  i i i  LIST OF TABLES.  ..... v i  LIST OF FIGURES............  ..  vii  ACKNOlttEDGEMENT  ix  INTRODUCTION  1  LITERATURE REVIEW  4  Definitions.. The  4  Status o f L i g n i n  /  .......  5  The .Location of L i g n i n  6  The I s o l a t i o n o f L i g n i n  8  The Characterization o f L i g n i n  9  The Biosynthesis  o f Lignin..  Aromatic Constituents  Related to L i g n i n  16 26  Aromatics o f Western Red Cedar  30  Metabolism and L i g n i n Formation  33  L i t e r a t u r e Summary and Observations  35  MATERIALS AND METHODS  37  C o l l e c t i o n o f Leaves.  37-  Leaf Extraction.  37  Chromatography  38  Thin l a y e r c e l l u l o s e chromatography  38  Thin l a y e r s i l i c a g e l 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)  iv  Periodate oxidation  46 •.. 47  Hydrolysis 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 Chromatograohic Samoles.  ....  47  ..  51  ill  , Leaf  C Feeding  ....  Spectral Techniques...  54  •  54  U l t r a v i o l e t and i n f r a r e d Nuclear magnetic resonance  . 55  .  55  Mass spectroscopy ' RESULTS  '  I s o l a t i o n o f Compounds Compounds A B 3  3  56  .  56  •  57  and C - I s o l a t i o n  Compounds A B, and C - Properties  60  Compounds A B, and C - Derivatives....  62  3  3  Methylation  62  ,  63  Acetylation Hydrogen.olysis  '  64  Compounds A ,B, and C - Degradative Studies  64  Hydrolysis  64  Ethanolysis  65  ....  A l k a l i n e nitrobenzene  66  oxidation..  66  Periodate oxidation  67  L i q u i d S c i n t i l l a t i o n Technique l2|  C Feeding  '  DISCUSSION  '.  S t r u c t u r a l Studies o f the D i l i g n o l s .  70 73 73 73  Compound A Chemical characterization.  75  NMR spectra o f Compound A and i t s acetate....  78  Mass spectrum o f Compound A acetate.  84 89  Compound B NMR spectra of Compound B and i t s acetate  91  Mass spectrum o f Compound B acetate  95  Compound C  •  NMR spectra o f Compound C and i t s derivatives  98 99  v.  DISCUSSION (Cont'd)  105  Mass spectrum of Compound C acetate L i g n i n Biosynthesis  •  '  113  S c i n t i l l a t i o n - Chromatography Technique Precursor Feeding Study CONCLUSION Future Research L i t e r a t u r e Cited  110  •  119 126 128 130  vi.  LIST OF TABLES TABLE 1.  2.  •  , PAGE  Sample preparation and r e s u l t s o f l i q u i d s c i n t i l l a t i o n counting comparison o f scraping and combustion methods applied t o samples from t h i n ' l a y e r c e l l u l o s e chromatographic plates ............ Properties o f Compounds A,B, and C  49 ........ 6 l  14 3. - Uptake o f U- C-L-phenylalanine and i t s inccporation i n t o Compounds A and B i n the leaves o f v;estern red cedar ..............  71  vii. LIST  FIGURE 1.  2. 3.  4.  OF FIGURES  '  PAGE  S h i k i m i c a c i d pathway t o t h e f o r m a t i o n o f a r o m a t i c amino a c i d s p h e n y l a l a n i n e and tyrosine  18 •  R e s o n a n c e forms o f t h e f r e e r a d i c a l 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  Scheme f o r t h e e x t r a c t i o n components f r o m w e s t e r n  derived  and s e p a r a t i o n o f r e d cedar l e a v e s . . . .  39  A schematic r e p r e s e n t a t i o n of the pressure column c h r o m a t o g r a p h y s y s t e m  42  A s c h e m a t i c chromatogram o f t h e 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 c e d a r l e a v e s ( s p r a y , DSA)  51  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 l e a v e s as r u n on LH-20 [CHCl^ :EtOH(4 :1)]. .  58  7.  The NMR  spectrum  o f Compound  79  8.  The NMR  spectrum  of the acetate  5.  6.  of 9.  10.  Compound  A derivative  A  The mass s p e c t r u m of Compound A  80. of the a c e t a t e d e r i v a t i v e 85  The p r o p o s e d mass s p e c t r a l  fragmentation of  rhamnose t r i a c e t a t e 11.  The NMR  12.  The NMR s p e c t r u m o f t h e a c e t a t e d e r i v a t i v e o f Compound B The mass s p e c t r u m o f t h e a c e t a t e d e r i v a t i v e  13.  of  spectrum  o f Compound  87 9  B  93... 96  Compound B  14.  The NMR  spectrum  15.  The NMR s p e c t r u m o f Compound C  2  o f Compound C  100  of the a c e t a t e d e r i v a t i v e •.  101"  LIST  OF FIGURES  (cont'd)_  viii.  FIGURE 16.  17.  18.  19.  20.  21.  22.  PAGE The NMR s p e c t r u m o f t h e a g l y c o n e o f Compound .C... .  .103  The NMR s p e c t r u m o f t h e a c e t a t e o f t h e a g l y c o n e o f Compound C  104  The mass s p e c t r u m o f Compound C  derivative  of the a c e t a t e d e r i v a t i v e '  106  The e f f e c t o f an e x t e r n a l q u e n c h i n g compound on t h e d e t e r m i n a t i o n o f a c t i v i t y o f s c r a p e d c h r o m a t o g r a p h i c samples......  114  The e f f e c t o f an e x t e r n a l q u e n c h i n g compound on t h e d e t e r m i n a t i o n . o f a c t i v i t y o f comb u s t e d c h r o m a t o g r a p h i c samples  115  The e f f e c t o f c h r o m a t o g r a p h i c s p o t 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 u n t i n g o f comb u s t i o n samples  116  Autoradiogram o f the gross e t h y l a c e t a t e s o l u b l e s f r o m U- 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  4  1  23. 24.  4  .  Uptake o f U-C-r-L-pheny l a l a n i n e r e d c e d a r leave's  by w e s t e r n  Incorporation of U-^C-L-pheny l a l a n i n e Compounds A a n d B i n ffhe l e a v e s o f western r e d cedar  124 into 124  ACKNOWLEDGEMENT  The E.P.  author.wishes  to gratefully  Swan,'part-time A s s o c i a t e  Professor,  Forestry, University of B r i t i s h guidance  i n the planning  acknowledge Dr.  Columbia  and e x p e r i m e n t a l  s t u d y , and f o r h i s a s s i s t a n c e  Faculty of f o r his, p a t i e n t stages  of this  i n the p r e p a r a t i o n  of this  thesis. Particular Vancouver F o r e s t Fisheries this be  r e c o g n i t i o n must  Products Laboratory,  and F o r e s t r y  Forest  phases o f  S p e c i a l acknowledgement  t o t h e wood c h e m i s t r y  P r o d u c t s Lab. f o r t h e i r  assistance  the i n t e r p r e t a t i o n of the included  resonance  throughout the  nuclear  Professor,  author Faculty  i s further indebted  magnetic  phases  t o D r . J.W.  Wilson,  of Forestry, University of B r i t i s h  f o r h i s suggestions,  preparatory  of this  criticisms  thesis.  go t o t h e U n i v e r s i t y o f B r i t i s h  .financial  support  extended  and a i d i n the  Acknowledgement as  w e l l must  program.  for h i s aid  spectra.  The  Columbia  must  s e c t i o n o f the Vancouver  s t u d y , and i n p a r t i c u l a r t o D r . J . F . M a n v i l l e in  of the  Department o f  where a l l e x p e r i m e n t a l  s t u d y were p e r f o r m e d .  extended  go t o t h e s t a f f  Columbia  f o r the duration  f o r the  o f the academic  X;.  F i n a l - acknowledgement without  whom t h e l a t t e r  been p o s s i b l e .  stages  must  go t o my  wife,  Carole,  o f t h e work w o u l d n o t have  INTRODUCTION  Lignin mensional tissues nature  of t h i s  tissues.  The  lignin  initiated  lignin  polymeric  i t s multi-stage  develop-  metabolizing 30%  r e p r e s e n t s as much as  of  the  stems, and t h i s high content  many i n v e s t i g a t i o n s  into  q u e s t i o n o f where l i g n i n micro  the  "where"  and  have p r o d u c e d lignin  analyses  o f amount and  cambium, c e l l  e v i d e n c e , but  polymer  quantitative  results  where l i g n i n  i s f o r m e d may  tion,  i s f o r m e d has  quantitative  i n t h e wood, b a r k ,  where t h e  i s formed.  suggests  that  the  i n woody  location  wall,  no  resulted  and  clear The  of  cell  answers,"as  variation  determination  d e p e n d upon t h e  lignin  in of  defini-  J The  tion  The  of phenylpropanoid  These i n v e s t i g a t i o n s  cytoplasm  its  tridi-  i n plant  support.  suggests  c o n i f e r o u s plan.t  many macro and  stems.  to  existing  a  of i t s f o r m a t i o n . The  in  substance  of  polymer  for structural  a wide r a n g e  constituents  "how"  g e n e r a l l y b e e n d e f i n e d as  phenylpropanoid  primarily  ment o v e r  has  has  definition  polymeric  form.  to i n c l u d e those  of l i g n i n  need not  I t Is p o s s i b l e  be  t o expand  monomeric, d i m e r i c  and  confined to this  defini-  oligomeric  phenylpropanoid lignin.  The  definition formed  inclusion  allows  t o be  where t h e ferred  precursors of  lignols  that the  first  with  first  appear.  initial the  phenylpropanoid  true l i g n i n  (36)  and  cursors  are  lignin  o f where  lignin  is  to that p o i n t  I t can be  further i n -  formation i s  of the m o n o l i g n o l s .  How-  serve as precursors t o other compounds.  character i s dimeric of the  have b e e n  chemical  Therefore,  the  (dilignol)  i n the  c h a r a c t e r of  l o c a t e d i n the  c o n s i d e r e d t o be  originating  transformation  from  appears  formed  t o be  lig-  of  further  highly  tissue. active  I t may tissues  aromatic  be  region pre-  (78).  This  a localized biosynthetic  the  the  from  cambial  t r a n s l o c a t e d sugars  mechanism a s s o c i a t e d w i t h  display  vitality  and  specialization  suggested  similar  that  localized  other biosynthe-  mechanisms.This  the  as  i . e . , a g u a i a c y l - g - a r y l e t h e r . i n most c o n i f e r s . Dilignols  tic  the  p o i n t of l i g n i n  appearance  present, d e f i n e d nature nin,  into  determination  e v e r , these same monolignols may non-lignin  (lignols)  a s s o c i a t e d more s p e c i f i c a l l y  lignols  coincident  the  of  lignin  investigation  leaves of western  serve  as  a metabolic  dilignols.  red  examines the cedar  tissue  (Thuja p l i c a t a  important  Such a h y p o t h e s i s  hypothesis  i n the  necessarily  that  Donn)  may  formation  requires  the  of  development for  of techniques  characterization.  participation  capable  of I s o l a t i n g d i l i g n o l s  V e r i f i c a t i o n .of t h e  i n the. m e t a b o l i s m radioactive  of the a c t i v e  be a c h i e v e d  through  anticipated  low l e v e l s o f i n c o r p o r a t e d  associated  with these  techniques  for their  tracer  dilignols'  tracer  studies  measurement.  tissue  studies.  The  radioactivity  will  require  new  may  4.  LITERATURE  Definitions  " .  Three in  general  classes  o f compounds  this dissertation; lignin,  close  may  be g e n e r a l l y  units.  neutral  All  The  This  solvents  walls  compounds  discussion.  L i g n i n has n e v e r b e e n s p e c i f i c a l l y defined  dimensional biopolymer  cell  considered  and l i g n a n .  t h e i r d e f i n i t i o n p r i o r t o the ensuing  Lignin:  noid  lignol  are  r e l a t i o n s h i p i n n o m e n c l a t u r e between t h e s e  requires  It  REVIEW  primarily  biopolymer  occurring  composed  of phenylpropa-  purpose  and b o n d e d t o  of s t r u c t u r a l support.  i n this dissertation will  n a t u r a l l y occurring l i g n i n .  S y n t h e t i c a l l y produced  be I n d i c a t e d Lignols:  Freudenberg in  Lignol  synthetic  alcohol  is a specific  lignin.  dissertation  lignin  intermediates  from c o n i f e r y l  " monolignols", dimeric  include  those  by  of c o n i f e r y l a l c o h o l to  "trilignols",  trilignol...oligolignol  will  introduced  Monomeric p r o d u c t s  t r i m e r i c products  terms d i l i g n o l ,  term  the "trapped"  dehydrogenation  are designated  "dilignols",  be t o  such.  (35) t o d e s c r i b e  the enzymatic  form  as  in  plant  to l i g n i n  will  tri-  Is e s s e n t i a l l y i n s o l u b l e  and e x i s t s w i t h i n  f o r the primary  reference  as a w i d e l y  defined.  dimeric,  products etc.  as u s e d  The  i n this  trimeric...  5.  oligomeric phenylpropanol d e r i v a t i v e s , both synthetic and natural, which exhibit a l k y l - a r y l ether and/or carbon-carbon bonds between the propanol chain of one unit and the aromatic nucleus of the next u n i t .  Such derivatives are  products of c o n i f e r y l alcohol. are  not r e s t r i c t e d to dehydrogenation These l i g n o l s , as Freudenberg's,  optically inactive. .Lignan: This term defines a natural dimeric compound which  is  obtained through  units  in a  carbons is  carbon-carbon  of t h e i r  formed  Since  propyl side  Its f i r s t  has  cal  investigations.  has  been a c l e a r e r  nin  been the  version zable This  chains.  The  combination  an o p t i c a l l y  active  2-L,' 3 - " T J ~ c b n f i g u r a t i o n .  the  of f o r e s t  r e c o g n i t i o n by  net  result  ( i f incomplete)  Payen i n 1833,  To  those  adding  comprises  as w a s t e , i n e x c e s s  investigations  understanding  involved  i n the  of  chemical pulping i n s t a l l a t i o n s  in atlig-  chemical  con-  t o n s , from  i n 1970  (48).  nonutili-  pollution.  22-34% o f most woods and  o f 50 m i l l i o n  lignin  of  represents a  to ever e n l a r g i n g  lig-  biochemi-  economic e x p l o i t a t i o n  products, l i g n i n  waste p r o d u c t  of these  coupled with f r u s t r a t i o n  significant  products.  lignin  as  Such a  s u b j e c t o f e x t e n s i v e c h e m i c a l and  structure,  to achieve  based  (3-3')  Lignin  nin  f o r m a t i o n and  phenylpropanoid  bond b e t w e e n t h e m i d d l e  commonly has  Status of  tempts  t o g e t h e r two  stereo-specifically  l i g n a n which The  joining  will  the  occur  world's,  Harkin  (48)  6. feels  that the l i m i t s  o f accomplishment  as r e g u l a t e d by p r e s e n t suggests  i n lignin  m e t h o d s , h a s been  t h a t t h e wood b a s e d i n d u s t r i e s  research,  approached.  must  He  improve, t h e i r  lignin  p e r f o r m a n c e t h r o u g h t h e . a p p l i c a t i o n o f t h e knowledge  gained  i n structural  The  elucidation  of l i g n i n  to date.  Location of L i g n i n Lignin  stones,  i n the f i b e r  roots, bast, pith,  o f members of p l a n t nature  i s found  and c o r k  life  (i.e.,  of l i g n i n  division  aromatic  fungi).  has e n a b l e d  and m o n o c o t y l e d o n o u s  (hardwoods)  lignin  .  well.  exhibit  lignina.  character of  typical guaiacylpropane  monomers, w h i l e t h e contain  The m o n o c o t y l e d o n s  syringylpropane  monomers and  (annual  p l a n t s and  t h e 4 - h y d r o x y p h e n y l p r o p a n e monomers as  Coniferous  o f a t t e n t i o n owing .history  The  contains  (3,5-dimethoxy-4-hydroxyphenylpropane) guaiacylpropane.  chemical  i n t h e p h e n y l p r o p a n e monomers  (coniferous) l i g n i n  dicotyledonous  basic  forms  a further d i v i s i o n as:  (3-methoxy-4-hydroxyphenylpropane)  grasses)  some l o w e r  The d i f f e r i n g  to the r e s p e c t i v e l i g n i n  gymnospermous  fruits,  of the majority  i s b a s e d upon t h e p r e d o m i n a n t  substitution  polymerized  cells  o f t h e p l a n t kingdom,, e x c e p t  gymnospermous,dicotyledonous This  cell walls,  lignin  has r e c e i v e d t h e g r e a t e s t  amount  t o i t s g r e a t e r homogeneity and l o n g e r  as a r e s i d u e  of the chemical  Distributional  pulping industry.  studies of l i g n i f i c a t i o n  i n woody  7. stems o f J a p a n e s e r e d p i n e (12)  have r e v e a l e d  content the  vertically  wood and  from the  half  i n the  layer.  r e g n a n s T.  mono- and  ment.  The  there  polysaccharide  and  xylarly Don)  differentiation  (101).  initially ners  along  study  deposited  during  during  the the  results  the  and  the  of  determined primary of the  of the  l a y e r and  S^  in  is  agreethan Is  laid  deposited.  during  (Pinus lignin  layer.  These r e s u l t s i n the  S^  ously  deposited  i n the  intercellular  l a y e r while  Fergus  lignin  in  includ  radiata  cell  cor-  Subsequently, is laid  down  and  Is  outer  the  or  continulayers  e_t §JL. (25)  across  the  i n d i c a t e that  layer during lignin  D.  appears  then s u c c e s s i v e l y In  of the  R e c e n t work by  are  w a l l near the  formation  of  a l . (91),  layer, lignin  radial walls.  distribution  et  lignin  that  after  amined the  distance--  hemicellulose  i n Monterey pine  appears  wall.  with  lignification  lignin  cell  content  l a t e r substantiated i n an  of  the  majority  study  formation  formation  lignin  i n d i c a t e t h a t more  most o f t h e  i n the  intercellular  tangential the  This  In  Zucc.  lignin  determinations,  The sequence of deposition was microscopic  the  Stewart  secondary w a l l before  ultraviolet  increase  centripetally  Muell.) by  cellulose  down i n t h e  i s an  et  S i m i l a r i n v e s t i g a t i o n s i n eucalyptus  polysaccharide  of the  d e n s i f l o r a Sieb.  tree while  bark i n c r e a s e s  cambial  (Eucalyptus ing  that  (Pinus  cell  has  of ex-  walls  of  early--and  Mill.)  using  analysis... wall, wall the  72%  Their of  The  results  the  total  to  latewood, 18$  28%  82%  I s o l a t i o n of  the  leaving  the  a protic  Both  of  of  i n the  to  the  densitometric  i n the  lignin  i n the occur  middle  eariywcod  i s i n the  middle i n the  cell  secondary  lamella,  In  secondary  wall  lamella.  Lignin  the  i s o l a t i o n of  lignin  carbohydrate with  insoluble solvent  lignin,  and  original lignin.  the. l i g n i n  and  or  involves  severe  solution  acid  of  either treatment  the  lignin (73)•  i t s subsequent p r e c i p i t a t i o n  t h e s e methods p r o d u c e  resemble ture  volume o f  found  chemical  removal of  show t h a t  occurring  was  occurred  The  i n b l a c k - s p r u c e ,(Pice.a m a r i a n a .  u l t r a v i o l e t m i c r o s c r o p y and  compared  while  in  latew.ocd c e l l s  lignin  w h i c h may  However, t h e  i t s environment  or  may  not  polymeric  seriously  na-  restrict  other a l t e r n a t i v e s . o f i s o l a t i o n . P h y s i c a l , and also  available  matrix. in  a ball  oxygen rotting lignin ted  The  (10,  to  b i o l o g i c a l methods o f  separate  physical  mill  with  16,  17).  fungi.:  to  lignin  methods employ  or without  the  carbohydrate  grinding  nonswelling  to  c e l l u l a s e s have a l s o  plant  polysaccharides  solvent been.used  extraction to  give  of  solvents  B i o l o g i c a l methods u t i l i z e  digest  more a c c e s s i b l e  from the  i s o l a t i o n are  wood and  brown-  leaving (83).  high  the  Isola-  y i e l d s , of  lignin  w h i c h has  been s l i g h t l y  altered  i n polar organic solvents (77). are  limited  by  may  be  The  Characterization  understood  vestigations  formed  tions  used. Mild  and  obtained  75,  i n these  benzaldehyde), of  and  c o n i f e r o u s bark  aldehydes dehyde)  the  nature upon t h e  oxides  applied  have  mercury,  yields  major aromatic  has  (3,5  produced  c o n t a i n s the  pro-  of  (alkaor aromatic  aldehydes  are  (3-methoxy-4-hydimethoxy-4-hydroxy-  p-hydroxybenzaldehyde. has  (e .g_. ,  of t h e . c o n d i -  to l i g n i n  of copper,  s yrInga ldehyde  Vanillin  lignin  which  severity  substantial  Three  lignin  of  o f the d e g r a d a t i o n  plus protocatechualdehyde  (5Q) .  synthetic i n -  breakdown o f l i g n i n  degradations: v a n i l l i n  droxybenzaldehyde),  lignin.  to o t h e r n a t u r a l polymers Partial  or the  98).  which  i t is presently  d i v e r s e nature  o x i d a t i o n techniques  (9,  as  d e g r a d a t i v e and  i n a i r ) have p r o d u c e d  aldehydes  techniques  lignin  degradation techniques  i s dependent  nitrobenzene  silver  of l i g n i n  The  of p r o t e i n s ) .  ducts  line  from  applied  been a c c o m p l i s h e d ,  dissolve  Lignin  of l i g n o l s .  been s u c c e s s f u l l y hydrolysis  of  evolved  severely limited  A l l isolation  r e p r e s e n t a t i v e of n a t u r a l  characterization has  will  p r o p e n s i t y to produce  only p a r t i a l l y  The  has  their  and  Mild oxidation the  forementioned  (3,4-dihydroxybenzalg u a i a c y l nucleus  which  10. is  predominant  i n the coniferous species , while  vanillin  and s y r i n g a l d e h y d e  are found  lignin.  The m o n o c o t y l e d o n o u s  lignin  p-hydroxybenzaldehyde nucleus.  CHO  both  i n dicotyledonous c o n t a i n mainly the  The d i m e r d e h y d r o d i v a n i l l i n ' ' ( . I )  CHO  I has  been o b t a i n e d  as a m i n o r p r o d u c t  of this oxidation  s u g g e s t i n g t h e e x i s t e n c e o f some a r y l - a r y l Stronger  o x i d a t i v e t e c h n i q u e s , u s i n g p e r m a n g a n a t e on  methylated and  wood  ( 3 0 , 59) y i e l d  methoxyl-substituted  lignin. ganate tural  aromatic  o x i d a t i o n technique i n f o r m a t i o n from  number o f a l i p h a t i c  a c i d s from-the o x i d i z e d  to obtain valuable l i g n i n  the.radioactively  labelled  o f wood c o n t a i n i n g r a d i o a c t i v e l y Reductive  and s o l v o l y s i s  derivatives  with  alkali  metals  74), which  of l i g n i n  indicate  oxidation  labelled  hydrogenation  i n liquid  of propylcyqlohexanol  catalyzed-ethanolysis  struc-  lignin.  methods a p p l i e d t o l i g n i n  ( s u c h as r e d u c t i o n by c a t a l y t i c  solvolysis  (58,  a larger  . Freudenberg et a l . (37, 39, 40, 4l) used the perman^-  products  (79)  bonds i n l i g n i n .  ammonia)  or yield  or propylphenol.  produces  the occurrence  Hibbert's o f a a nd/or L  Acid ketones 3-ether  11. linkages lignin (63)  The  o f Hondo s p r u c e  resulted  dimer as  in lignin.  from  r e c e n t h y d r o g e n o l y s i s of the  [Picea  jezoensis  i n the i s o l a t i o n  (Sieb.  and  of a carbon-carbon  the h y d r o g e n o l y s i s products which  was  Zucc.)] bonded  identified  1-(3'-methoxy-4'-hydroxypheny1)-2(2"-hydroxy-3"-methoxy-  5"-n-propyl)-n-propane soeugenol  ( I I I ) under  (II).  H y d r o g e n o l y s i s of  t h e same c o n d i t i o n s  cle.avage o f t h e p h e n y l c o u m a r a n  ring  II Nimz  (68),  bromide  has  or a c e t i c  thioacetic lignol  successfully  acid  quantitative  to y i e l d I I .  III l i g n i n using  anhydride with boron t r i f l u o r i d e  w i t h boron  fragments.  cleavage  degraded  gave  dehydrodii-  These  trifluoride  (72)  to  acetyl and  liberate  f r a g m e n t s were shown t o u n d e r g o  of the phenylcoumaran  ring  during i s o l a t i o n  (68).  I A trimeric-lignol coumaran s t r u c t u r e lignin acetone  based  was  (IV) i n c o r p o r a t i n g  considered to occur i n "spruce"  upon t h e i d e n t i f i c a t i o n  ( V ) , the  a phenyl-  substituted  of  w-hydroxyguaiacyl-  phenylcoumarone  ( V I ) , and  •  12.  the  s u b s t i t u t e d stilb'ene  the  acidolysis  acidolysis  (VII) i n the r e a c t i o n products  o f Bjorkman  products  "spruce"  lignin  and  of  related  f r o m m o d e l compounds ( 1 ) .  CHJOH  OH  IV  oca.  V  o  CH C CH OH 2  2  CH C CH OH 2  OCH^  2  OCH,  OH  VI  The  products  compound  ( V I I and V I ) were  considered  IV v i a a coumaran r i n g  opening  to arise  and l o s s  from  of formal-  dehyde t o f o r m V I I j and t h e r e a c t i o n o f t h e i n t e r m e d i a t e in VI.  a reverse  P r i n z r e a c t i o n to form the i s o l a t e d  A comparison  acylacetone  of the high  (V) w i t h  the y i e l d  yield  product  of the oi-hydroxyguai-  o f t h e compounds  obtained  13. in  the  considered of  conclusive  evidence  a r y l g l y c e r o l 3 - a r y l ether The  desire  n i n has  led to  niques.  Nimz  eliminate order  the  results  concluded  that  of  the  l i g n i n went  that  aryl  mild  ether  the  dissolved  lignin  to  lignin  by  t h e i r ' s-tructures  T h e s e were D , L - p i n o r e s i n o l (XI).  The  lignin.  ether  trimeric  of  Nimz  the  elucidated of  (VIII  ex-  bound Monomeric,  degradation products  obtained  acid hydrolysis  These  would  to represent  dehydrogenation products  been p r e v i o u s l y  re-  and  conditions  i n "spruce"  phenolic  in  lignin.  bonds.  and  alcohol  of  benzyl-aryl  obtained  hydrochloric  very  probably  oligomeric  had  the  p o l y m e r w h i c h was  and  (XVI)  conditions,  o f F r e u d e n b e r g e_t a l .  lignin  dimeric,  to  into solution.  hydrolysis  groups  tech-  necessary  100°C,  10%  lig-  hydrolysis  " s p r u c e " wood i n w a t e r a t  of  dimeric  from  groups  posed p o r t i o n  Two  fragments  i t was  benzyl  existence  lignin.  alkaline hydrolytic  and  was  the  ether  considered  "core"  of  c o n d e n s a t i o n r e a c t i o n s ' of  observed  benzyl  the  lignol  were i n a g r e e m e n t w i t h t h o s e  (33),who cleave  i n support  bonds i n  obtain  benzyl, h y d r o x y l  observed that  m o d e l compounds  a p p l i c a t i o n of mild  (69)  prevent  percolated  (69)  to  a c i d i c and  to  active He  3-ary.l e t h e r  a c i d o l y s i s of  to.  were  XV).  coniferyl alcohol  i n a mild  "spruce"  methyl  lignin  alcohol-  (31).  (XIII)  and  dehydrodiconiferyl .  (XIV)  and  tetrameric  l i g n o l s , i s o l a t e d and characterized by Nimz  (69)  had  .  (XV)  •  1.4.  HO  XIV  XV  XVI  3 - a r y l ether  glycerol  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 . and t h e g u a i a c y l g l y c e r o l - 3 - 0 - 4 ' -  Guaiacylglycerol  (VIII)  coniferylalcohol  (IX) o c c u r r e d  pounds in  X and XIV have a l s o b e e n i s o l a t e d  and  (8l).  (XIV)  (70)  Nimz  and t e t r a m e r i c  products  of "pine" The m i l d ,  convincing realized  groups similar  i n the water p e r c o l a t i o n  h y d r o l y s i s products  h y d r o l y s i s products  The  core  and c o n s e q u e n t l y character  a l . ( 2 8 ) , who  repeating  unit  have  proposed  ether  not c l e a v e d .  a basic  for lignin.  weight  of the l i g n i n  can be f o u n d  This  It i s the  peripheral  should  contain  bonds a r e  i n t h e work o f  polymeric  Forss  guaiacylpropane  b a s i c u n i t was  bonds and externally by a l k y l - a r y l ether bonds only. external a l k y l ether bonds which were s e l e c t i v e l y  less  Substantiation  be i n t e r n a l l y bound by a l k y l - a r y l ether and a r y l - a r y l  under mild a c i d i c conditions.  furnished  represent  t h e low m o l e c u l a r  g r o u p i n g s whose h y d r o l y z a b l e  lignin  the t r i m e r i c  on t h e s t r u c t u r e o f l i g n i n .  of only  of l i g n i n .  accessible of t h i s  acidic  these  identification  wood  wood.  evidence  that  I s o l a t e d and i d e n t i f i e d (XV) l i g n o l s  Com-  identified  t h e d i o x a n e : w a t e r h y d r o l y s i s o f a Hondo s p r u c e  meal  et  i n racemic mixtures.  3  thought to  alkyl I t was the  hydrolyzed  Assurance of the r e l i a b i l i t y  of these mild a c i d i c hydrolysis r e s u l t s was strengthened  by the  characterization of s i m i l a r products i n studies of a l k a l i n e hydrol y s i s of l i g n i n  (100).  16. The  Biosynthesis The  simulated  s t r u c t u r e of l i g n i n  b i o s y n t h e s i s , and (35).  degradation chemical The  of L i g n i n  verified  of the  lignin,  upon o r d e r l y d e g r a d a t i v e fostered  the  thesized  lignin  concept  of  difficulties  analysis.  and  biosynthetic The  enzymatic  extensive  chromatographic i n the  (13,  lignin  of  specific  These  the  prior  isolation  of  and  en-  difficulties biosyn-  growth o f  the  However,  clarification and  reverse.  i t imposes  natural conditions.  intermediates  use  of  aro-  identification necessary  to  the  lignin. of r a d i o a c t i v e t r a c e r s ,  techniques  have been t h e biosynthesis  enzymatic  primary  fac-  of  aromatic recent  atmospheric  carbon d i o x i d e .  Two  examine t h e  role  biosynthesis  formation.  The  during  e l u c i d a t i o n of the  35)  in  the  production  compounds f r o m  via  normal  nature  "trapping" a r t i f i c i a l l y ,  intermediates  b i o s y n t h e s i s and  chemical  views  to  and  the  by  chemical  from the  such an.approach r e q u i r e d the  tors  through  results  biopolymer under s i m u l a t e d  and  clarified  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  vironment  of  been  This, approach i s contrary  unorthodox procedure  matic  has  interest  These reviews are  conversion  photosynthesis  of aromatic  of  summarized  and  more r e c e n t  re-  work  below.  carbon d i o x i d e  i s w e l l understood,  to  and  carbohydrate is  presented  17. in  a text examining plant biochemistry  drate precursors of aromatic (EMP  COg.  carbohy-  biosynthetic  formation  or pentose phosphate shunt) o r The  o f the  conversion  (phosphoenol-pyruvate aromatic  The  compounds o r i g i n a t e from c a r b o h y d r a t e  pathway  sized  known t o i n i t i a t e  (8).  and  photosynthe-  carbohydrate  precursors  D-erythrose-4-phosphate;  amino a c i d s p h e n y l a l a n i n e  well understood.  metabolism  and  to  the  tyrosine i s also  This pathway (Figure 1) was elucidated i n n u t r i -  t i o n a l studies with the b a c t e r i a E. c o l i and has been reviewed(88). Chorismic (42,  43)  a c i d was and  of prephenic  the  serves acid  last  as  the branch  or a n t h r a n i l i c  sequence.  Although  this  subsequent  studies  ( 3 , 4,  applicable  to higher  Brown and and, to  to a l i m i t e d  lignin  intermediate  pathway was  Neish extent  (14,  15)  noted  K o u k o l and  characterize catalyzed  the  The  the  Conn  be  deamination  phenylalanine  (55)  the  conversion  isolation  of  lignin.  were a b l e t o i s o l a t e  and  ammonia-lyasewhich  of phenylalanine  e s t a b l i s h e d the  precursors  amino a c i d s i n t o  enzyme, p h e n y l a l a n i n e  concurrent  (66)  in bacteria,  pathway t o  that  i n numerous p l a n t s i n d i c a t i n g  I n 1961,  grass  determined  shown the  formation  aromatization  t y r o s i n e , were good  a c i d - d e r i v e d aromatic  in  i n the  identified  p l a n t s as w e l l .  shikimic  acid.  p o i n t to the acid  5) have  t o be  to  cinnamic  of t y r o s i n e ammonia-lyase  similar  deamination  of  18.  COOH I  -  COPO3H,  COOH I  c=o  CH,  Phosphoenol pyruvic acid CHO I H-C-OH' I H-C-OH I CH 0P0 H 2  3  HC>j—ADH  I CH, I HO-C-H • I H-C-OH I H - C - C H  Quinic acid V^COOH KOH J  HoV-^OH 5-Dehydro quinic acid  CH 0P0 H 2  3  COOH  3-Deoxy-D-arabino heptulosinic acid -7-phosphate  A  D-Erythrose-4phosphate  HC^  5-Dehydroshikimic acid  COOH  COOH  CB,  H^PO  ^  2  HjOjPCT Y  'COOH  6H  OH  OH  3-(Enolpyruvate ether) of phosphoshikimic acid  5-Phosphoshikimic acid  Shikimic acid  :HJ-CO-COOH HOOC  CHJ-CH-COO  CH,-CO-COOH  *"NH,  Phenylpyruvic acid L-Phenylalanine OH  CH3-CO-COOH  Anthraniiic acxd .  Figure  1.  tyrosine of  these to  p-Hydroxyphenylpyruvic acid  2  OH L-Tyrosine  S h i k i m i c a c i d pathway t o t h e f o r m a t i o n o f a r o m a t i c 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 ) .  to.p-hydroxycinnamic  the transformation  amic a c i d  CH -CH-COO"  ' <^"'  Prephenic acid  of the aromatic  derivative,  cinnamic  eventually  acid  acid.  With the establishment amino a c i d  i t became n e c e s s a r y  derivatives  form'lignin.  could  to cinn-  t o prove  be f u r t h e r  that  metabolized  Smith and Neish (87) claimed to  19. .demonstrate t h e • i r r e v e r s i b i l i t y of cinnamic  a c i d formation  when  • they  carbon  labelled  cinnamic  "aspen"  showed t h a t acid  acid  periments  precursors.  utilizing  and  sinapic  acids  all  of these  by N e i s h acid  to  l i g n i n , with sinapic  in  those  utilized  these  "spruce"  In a t a b u l a t i o n  (35) , i t was derivatives  acid  cinnamic ring  and  than being i n c o r p o r a t e d i n t o of t r a c e r  ex-  ferulic,  demonstrated  that  were good p r e c u r s o r s  b e i n g an e f f e c t i v e p r e c u r s o r  s p e c i e s (angiosperms) of the  i  of  cinnamic, p-coumaric,. c a f f e i c ,  cinnamic  fication  atoms  were i n c o r p o r a t e d i n t o  twig l i g n i n rather  cinnamic  a l l of the  '  acid  with s y r i n g y l  lignin.  metabolism.route  substituted  cinnamic  acid  yet  t h e mechanism o f r i n g h y d r o x y l a t i o n a n d / o r  had  o n l y been i n f e r r e d  because  of the  limited  Veri-  to l i g n i n derivatives, methoxylation data  available  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 methionine tion, and 1965,  the  been demonstrated  s e r v e s as a m e t h y l  group  o r i g i n of the r e q u i r e d  m e t h y l a t i n g enzyme were n o t cinnamic  acetone  acid  powders  cinnamic  acid  phenolase production ing  i t had  from  from  cinnamic  of c a f f e i c a c i d  enzymes have now  from  donor i n l i g n i n  determined  until  that  forma-  later.  In  discovered i n spinach  capable of forming  acid.  s p i n a c h l e a f has  (18)  hydroxylated substrate  h y d r o x y l a s e was  (64) w h i c h was  i n 1954  Recently  (99),  p-hydroxya purified  b e e n shown t o c a t a l y z e p-coumaric  been f o u n d w h i c h  are  acid. capable  the  Methylatof  20.  methylating methionine et  a l . (51)  caffeic  a c i d i n the presence  of S-adenosyl-  (26,  i n grasses  and woody  shrubs  have a l s o  described  an enzyme  27).  Higuchi,  preparation  (S-adenosylmethionine:catechol-O-methyltransferase)  which  selectively  and  methylated  5-hydroxyferulic is  the meta-hydroxyl  acids  i n bamboo  available regarding  tion  of the'guaiacyl  sequences  previously  nucleus  acid  in  conifers  carboxyl  way  however, t h a t cinnamic  of  meta-  acids  to lignin.  Such  -* c i n n a m i c  a c i d -* f e r u l i c  (4-0-coniferyl alcohol  (38)  suggesting  a c i d ->-  3-D-glucoside)  precursor  reduction  leaves  (89)  have e s t a b l i s h e d  acid  (chlorogenic  well  that  of the q u i n l c  acid)  from  the chlorogenic  or l i g n i n  24) d e m o n s t r a t e d  tive  intermediates  the b i o s y n t h e t i c  (acetone  of  El-Basyouni  part  path-  caffeic  acid, concluding  a c i d does n o t t a k e  the occurrence  acid  Experiments i n  acid ester  cinnamic  biosynthesis.  (23,  to l i g n i n  of a cinnamic  function prior t o . l i g n i f i c a t i o n .  to the formation  phenol  and m e t h y l a -  as: phenylalanine  a c i d -* c a f f e i c  evidence  t o the formation  as good p r e c u r s o r s  b e e n shown t o be an e x c e l l e n t  tobacco  Little  acid. Coniferin  has  I t i s evident,  be c o n s i d e r e d  -»• p - c o u m a r i c  sinapic  leading  lead t o the s u b s t i t u t e d  considered  a s e q u e n c e may  shoots.  further hydroxylation  the. s y r i n g y l n u c l e u s . bolic  of c a f f e i c  as  i n poly-  and .co-workers  of m e t a b o l i c a l l y ac-  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 , the  lignin  w h i c h were r e a d i l y i n c o r p o r a t e d  o f wheat and. b a r l e y  were b e t t e r  lignin  precursors  produced hydroxycinnamic Logan spp.  (11)  to  lignin.  acids  soluble  and  concluded  to  lignin  that  (49),  formed  feraldehyde  and  analogs  formation,  on  were  not  an  d i l u t e the. acid., and  and  coninto  wheat p l a n t  b a s e d upon t h e  from f e r u l i c  Bland  incorporation  studying  could  and  Eucalyptus  acids  their  c o n i f e r i n was  (a)'coniferyl alcohol  intermediate  cinnamic  p r i o r to  Brown  intermediates  upon h y d r o l y s i s .  hydroxylated  glucose * esters  nif ication,  that:  that  Hlguchi  precursor  than  These  found, i n l i g n i f i c a t l o n . studies  shoots,  verted  plants .  into  lig-  obligatory observations  lignification  (b)  c o n i f e r y l a l c o h o l were o b t a i n e d  that  coni-  in  ferulic  14 acidthe in  C feedings  importance lignin  rather  of e s t e r i f i e d  formation.  to  than  In  coniferin. cinnamic  summary, 'the  acid  These d a t a  suggest  intermediates  biosynthetic  pathway  l i g n i n f r o m C 0 may be c o n s i d e r e d t o be: C 0 P y ^ , , , , . 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, hydroxylation, methylation , , ., . , , . —— 2 ,—£ substituted cmh  2  o  t  o  s  n  t  h  e  s  2  2  esterifIcation namic a c i d s > i n s o l u b l e e s t e r s of cinnamic . , reduction ,, , „ . . .' -, , , acid c- a l k y l e t h e r s o f c i n n a m i c a l c o h o l s polymerization ,. - — 1* l i g n i n . The the  basis  concluding  for formation  step  i n the  studies  on  above scheme has artificial  been  lignin.  This  s  22. concept  of determining  lignin  synonymous w i t h F r e u d e n b e r g lignin  research.  alcohol  Freudenberg,  from  a common f i e l d  resembled  work on t h i s  lignin  after  K l a s o n , used  tified and  thereby  the  cent  structure  this  formulae  alcohols phenoxide  process  to l i g n i n  fied  the  lignin.  based  which on  the  mesomers  form'stabilized  at  least:  years  compiled  (Figure 2 ) .  related with  the  work . schematic  results. p-hydroxycinnamyl  free  v i a the  radicals  Harkin  free  iden-  most r e -  c o n s i d e r e d to procede  forms by  of  lignification  culminated  r e a c t i o n w i t h which these  to  which  Freudenberg's  of p o l y m e r i z a t i o n of i s now  campestris  i s o l a t e d 'and  o f h i s work and  (35)  20  pre-  polymer, i n t e r m e d i a t e s  forms which g i v e m e t a s t a b l e  four p r i n c i p a l  last  t h e mechanism o f  review  evolution  for lignin The  In the  coniferyl  enzyme  substance  r e a c t i o n s have been  of t h i s  comprehensive  traces  (34).  revealing  an  mushroom P s a l l i o t a  e n z y m a t i c a l l y produced  the .condensation  almost  the H e i d e l b e r g s c h o o l of  p r o d u c e ,at n e u t r a l pH ,a p o l y m e r i z e d  closely  of  and  i s now  as a s u b s t r a t e i n c o n j u n c t i o n w i t h  paration to  structure  (48)  radicals  following  five  1.  M o l e c u l a r g r o w t h by f r e e - r a d i c a l 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 r e a r r a n g e m e n t s o f some methides t o form phenoxides (phenols) may u n d e r g o renewed o x i d a t i o n t o f r e e  has  with classi-  combine  mechanisms  pairing  to  quinone that radicals.  Figure  2.  R e s o n a n c e forms o f t h e 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 alcohol.  3.  S t a b i l i z a t i o n o f some q u i n o n e m e t h i d e s 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) also 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 t o f r e e - r a d i c a l t r a n s f e r s and s u b s e q u e n t d i s proportionations.  5.  S t a b i l i z a t i o n o f some q u i n o n e m e t h i d e s 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 portionation. h'arkin (48) i l l u s t r a t e s the formation of " l i g n i n " pro-  ducts v i a the f i v e processes, through combinations of r a d i c a l s (Figure 2).  Process 1 (e.g_. 'R + P^, R + R &  b  + F^, R + R ) Q  followed by Process 2 w i l l produce the d i l i g n o l structures such as guaiacylglycerol-3-0-4'-coniferyl alcohol (IX) and p i n o r e s i n o l (XIII)..  24.  HO  Process hols  and  3 i n t r o d u c e s water t o form  probably  i s i n v o l v e d i n the  carbohydrate  bond by  This  a l s o may  by  process  the  result  with u n o x i d i z e d phenoxides.  ethers, esters,  such  isolated  components  gradation products free  radical  nols sence  from  the  of the  of  ethers  can be and  e t §JL.  lignin after  Process  4 gives  chains, while  lignin,  of  and  acid  the the  rearrangements.  formed t h r e e  dilig-  d i m e r i z a t i o n o f c o n i f e r a l d e h y d e i n the  of peroxidase  enzyme and  de-  e x p l a i n e d i n terms  subsequent (19)  diols,  arylglyceric  T h e r e f o r e , most  of.lignin,  Connors  lignin-  as d i a r y l p r o p a n e  synthetic  combinations  Recently,  side  d l o x e p l n s , and  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 .  alco-  carbohydrates.  benzyl-aryl  5 leads to s t r u c t u r e s  diphenyl  of  with  to carbonyl functions i n a l k y l  Process  covalent  i n the branching  formation of n o n c y c l i c  condensation rise  condensation  benzyl  hydrogen peroxide in.  pre- .  25. The products were 2 , 3 - d i f o r m y l - l , 4 - d i - 5 - g u a i a -  aqueous solution. cylbuta-1 3--diene  (XVTI),'a-(4-3-fonnylvinyl~2~methoxyphenoxy)  3  coniferylaldehyde. (XVIII). a-.(5-3-foiiuylviriyI-2-hydroxy-3-methoxy- • v  phenyl) coniferylaldehyde (XIX).  • '  OH  XVII  These dimers 2.  Figure  aldehydes, at  the  aldehydic side chains  quinone methide the  loss  the a, stage of the  products.  what  free  At  precursors  radicals  of  coniferyl-  3-enone s y s t e m  rearranges  t o form acidic  phenoxyl  this  free o f two  carbonyl, subsequently  p r o t o n a t i o n of the  dimeric sider  e x p l a i n e d v i a the  comprising  a t o the  final  can., be  The  which through bon  XIX  XVIII  a stable proton  phenol,  from  rearomatizes  anion to y i e l d  point i t i s important  the  car-  with  the  the to  con-  (monomeric, d i m e r i c , e t c . )  have  26.  been i s o l a t e d  unchanged from  substantiate  the  biosynthetic  results.  Aromatic  sap  amounts o f  known s u b s t a n c e s  coniferyl  sulfanilic  acid,  IX  has  4-0-sinapyl  of  alcohol, quinic acid,  (36).  treatmentwas  The  sugars,  and  un-  w e i g h t w h i c h gave  "spruce"  a l s o shown t o  protocatechuic  guaiacylglycerol coniferyl  formaldehyde,  a c i d , have a l s o b e e n shown t o be  formaldehyde  The  yel-  color reactions with d i a z o t i z e d  d e a c t i v a t e d " spruce", sap  shikimic  metabolizing  C o n i f e r i n , various  of high molecular  or o r a n g e - y e l l o w  and  constituents.  3-D-glucosides  4-0-p-coumaryl a l c o h o l (32).  small  low  aromatic  deactivated with  c o n t a i n the  to f u r t h e r  to L i g n i n  i n v e s t i g a t i o n s of a c t i v e l y  of"spruce"  b e e n shown t o  systems  drawn f r o m d e g r a d a t i v e  t i s s u e s have . c o n s i d e r e d  cambial  and  conclusions  C o n s t i t u e n t s .Related Several  plant  lignifying  acid,  3 - c o n i f e r y l ether  present sap,  in  without  contain:  D,L-pinoresinol ( I X ) , and  dehydrodi-  alcohol (XI).  XI  (XIII),  XIII  27.  In  an e x a m i n a t i o n o f t h e cambium and sapwood o f  w e s t e r n hemlock precursors,  ( T s u g a h e t e r o p h y 11a  Goldschmid  and H e r g e r t  pounds i n c l u d i n g s e v e r a l structures tected  were n o t e l u c i d a t e d .  (XIII),  (XXI),  lignan  .  Among t h e compounds d e -  (6) has v e r y  7-methoxy a unique  (XX),  (XXII),  alcohol  pinoresinol sus-  recently  XXII  studied  the occurrence  w e s t e r n hemlock s a p -  of l i o v i l  ( X X I I I ) and a  2-(a-hydroxy-vanillyl)-5-oj-hydroxypropyl)-  coumaran  (XXIV).  This  compound  3 - y - l i n k a g e , and was a l s o ;  hydroxy-  ( I X ) , and s e v e r a l  XXI  dilignol  whose  glycosides.  wood and r e p o r t e d novel  lignan glycosides  oxomatairesinol  dehydrodiconiferyl  XX  Barton  (46) f o u n d many com-  i n t h e sapwood w e r e : c o n i d e n d r i n  matairesinol  pected  suspected  (Raf.) Sarg.) f o r l i g n i n  thought  (XXIV) e x h i b i t s to occur  as an  28.  aliphatic  glycoside  CH CH CH OH 2  XXIII Studies with  2  2  XXIV on- l e a f  carbohydrate  t i s s u e have m a i n l y  content.  been  However, i n a s t u d y  concerned o f low  molecular weight aromatic compounds i n the leaves of Scots pine "(Pinus s y l v e s t r i s L.) (97), two glycosides of g u a i a c y l g l y c e r o l . were  i d e n t i f i e d . • The two g l y c o s i d e s were  glucoguaiacylglycerol glycerol  a-threo-3-D-  (XXV) a n d 1 3 - t h r e o - 3 - D - g l u c o g u a i a c y l -  (XXVI).  GHpH  HG< HCOGIucose  CHpH HCOGIucose  I  HCOH  OCHOH  OH  XXV  XXVI  29.  In a study  of the  e x t r a c t i v e s of the  tamarack (Larix l a r i c i n a (Du Roi) K. Koch), found and  3 - g l u c o s i d e s .of v a n i l l i c  the  the  a~glucoside  et  a l . (96)  of  one  of  flavonpids.  the  hundred  leaves  and  conifers  p-coumaric acid.  polyphenolics  noting primarily  A recent  of Douglas  the  acids  Takahashi  i n the  the  leaves  occurrence (80)  i n c o r p o r a t i o n study  f i r (Pseudotsuga  menzesii  using (Mirb.)  F r a n c o ) showed i n c o r p o r a t i o n o f t h e T - g l u c o v a n i l l i n lignin  whereas T - f e r u l i c  recent  examination  (52)  acid  was  not  of  (67)  Niemann  of p-hydroxybenzoic  have e x a m i n e d  leaves  into  incorporated. . A  of phenylalanine  ammonia-lyase  (PAL) a c t i v i t y i n eucalyptus (Eucalyptus" s i e b e r i L. Johnson\ and _(E.  slderoxylori  cose in  may  the  A. Cunn et Wools) leaves indicated-that g l u -  serve  as  formation  flavanoids.  of polyphenols  acid  products  c a t e c h i n and  and  t o be the  as PAL  lignification  When p h e n y l a l a n i n e  p-coumarylquinic  vonoids  to  than  such  speculated that  related  biosynthesis.  duced  a better precursor  I t was  more d i r e c t l y  metabolic  "  was  the  stilbenes  activity than  may  and be  polyphenol  f e d to the  chlorogenic acid formed w h i l e  phenylalanine  leaves,  were t h e  glucose  first  feeding  g l y c o s i d e s of s t i l b e n e s  pro-  and f l a -  first. The  dimerization  formation  of the  lignans i s another  r e a c t i o n o f monomeric  phenylpropane  important  30.  precursors. the  No s p e c i f i c  biosynthesis  that  the' l i g n a n s  coupling  (65) has  Neish  are probably  formed  side  t o be s p e c i f i c a l l y  chain.  regarding  postulated  through a  reductive  through the 3 carbon Such a c o u p l i n g  enzyme c o n t r o l l e d , t h e r e b y  2 - L , 3-D c o n f i g u r a t i o n  common  i s available  of lignans.  of cinnamyl a l c o h o l s  of the propylene  the  evidence  Is suggested producing  o f the l i g n a n s .  s p e c i f i c configuration and the lack o f triphenylpropenoid cludes of  t h e q u i n o n e m e t h i d e pathway  coniferyl alcohol  optically lignans  synthetically coupling  since  the  The r o u t e  The" lignans,•pre-  polymerization  v i a free r a d i c a l reactions,  inactive products.  and l i g n i n ,  since  atoms  gives  of formation  h o w e v e r , may be c l o s e l y r e l a t e d they b o t h a r i s e from  cinnamyl  of bio-  alcohol  reactions.  AROMATICS OF WESTERN RED CEDAR The  ..  -  study of lignans and tropolones has been extensive  i n western red cedar (Thuja p l i c a t a Donn).  Characterization o f  these components has played a primary r o l e i n the u t i l i z a t i o n o f the species (62).  The tropolones  (2-hydroxy-2,4,6-cycloheptatrl-  en-l-ones) are non-benzenoid aromatics which are steam  volitile.  Those which.have been i s o l a t e d and characterized from western red cedar wood,according to Barton and MacDonald ( 7 ) , are:  a-thuja-  p l i c i n (XXVTI), 3 - t h u j a p l i c i n (XXVTII), y- t h u j a p l i c i n (XXIX), . 3- t h u j a p l i c i n o l  (XXX), 0- dolabrin (XXXI), together  with  31.  XXVII'  XXXVa, R = H XXXVb, R = O H  XXVIII  XXIX  XXX  XXXVIa, R , R = H XXXVIb, R = 0 H ; R - H XXXVIc, R = R = O H 1  2  1  1  2  2  XXXI  XXXVII  32. similar  compounds  such- as n e z u k o n e  ('.XXXIV.), and t h u j i c  acid  (XXXII) / methyl  (XXXIII).  The l i g n a n s  have b e e n i s o l a t e d and c h a r a c t e r i z e d are  either derivatives  ether  i n western r e d cedar  (XXXVb), t h u j . a p l i c a t i n  (XXXVIa), h y d r o x y t h u j a p l i c a t i n  methyl  (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 of  which  of 3,Y-dibenzylbutane: t h u j a p l i c a t i n  (XXXVa), d i h y d r o x y t h u j a p l i c a t i n methyl  thujate  tetrahydronapthalene:plicatic  acid  ether  (XXVIc), or  (XXXVII),  plicatin  ( X X X V I I I ) , p l i c a t i n a p t h o l ( X X I X ) , and p l i c a t i n a p t h a l e n e ( X L ) . The has  large  generated  formation.  number o f l i g n a n s . I n w e s t e r n r e d c e d a r  interest into their  In a chromatographic  biosynthesis study  and s i t e o f  o f the l i g n a n s o f  w e s t e r n r e d c e d a r r e l a t e d t o the sapwood-heartwood formation,  Swan e_t a l . ( 9 5 )  concluded  of  the lignans  at  t h e sapwood-heartwood boundary.  that  through h y d r o x y l a t i o n  was c o n s i d e r e d  dihydroxythuj a p l i c a t i n  ->- p l i c a t i n  later  proposal,  sidered  that  to this  extending- w e l l lation  -> p l i c a t i c  i n t o t h e newly  o f the tropone  i n t o the  acid.  Swan and J i a n g  and h y d r o x y l a t i o n  iri s i t u  t h u j a p l i c a t i n ->  p l i c a t i n a p t h o l was d e r i v e d  through dehydration  portion  transformation  to continue  heartwood- f o r many y e a r s by t h e r o u t e :  addition  the major  ( i n w e s t e r n r e d c e d a r ) a r e formed The  trans-  from  with  In a  (94)  con-  plicatin  the  formed heartwood.  transformation Hydroxy-  nezukone t o form t h e t h u j . a p l i c i n s  was  observed  boundary. ether  to occur  wholly  Mono- and  to the  considered  formation  • intermediates  which  the  dihydroxylated  d e r i v a t i v e s were  similar  at  sapwood-heartwood thujaplicatin  t o be  of p l i c a t i n .  c o u l d d e f i n e the  f o r m e d i n a manner No  recognized  transformation  phenylpropane  t o a l i g n a n have b e e n i s o l a t e d .  suggests  the  must in  that  extend  vitro  to the  f o r s u c h unknown  sapwood and  cambial  of (95)  Swan  intermediates  areas'by  utilizing  or i n v i v o r a d i o a c t i v e t r a c e r e x p e r i m e n t s .  Metabolism  and  This examination trients  search  methyl  L i g n i n Formation  d i s c u s s i o n w o u l d be of the  leading to  incomplete  without  total"consecutive utilization lignin  formation  i n the  an  of  living  nu-  plant.  Ik Coniferous  leaf  photosynthesis  shown r a d i o a c t i v i t y D - g l u c o s e , and occurring  incorporation into  D-fructose  of  86).  "^CO^  i n the  (78)  revealed  resinosa  Ait*);,/  synthate  predominantly  growth r e g i o n .  with  (85,  i n sucrose  assimilation  The  up  the  Autoradiography  acid,  the  concentration  quinic acid)  the  majority  of  study  raffinose, activity of  photo-  of red pine  (Pinus  distribution  main stem t o t h e  of  photo-  apical  a s s i m i l a t i n g from  stem and  c o u l d be  precursors  differentiating  the  detected  of i n t e r n o d a l sections  of l i g n i n  i n newly  CO ^ have  sucrose,  A recent  radioactivity  moved down t h e  vealed  the  branches  the  lower worls roots.  studies using  in  re-  (shikimic  tissue.  34. Preferential that  labeling  of l i g n i n  cellulose,  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  photos-ynthate  d u r i n g growth.  translocation  of carbohydrates  from  Stewart  (90),  (particularly tissues  that  t h e cambium  saccharides  cell.  verted  i s incorporated into  initiated  The b u l k  The s h i k i m i c a c i d  at t h i s  During  mic  acid  m e t a b o l i s m by  of the a v a i l a b l e  acids. sors  the primary  cell  and l i g n i n ,  p r e c u r s o r s may  vacuole, excreted into  to e s t e r i f i e d  the a c t i o n  and  precur-  as p r e v i o u s l y d e s c r i b e d .  t h e n be e x c r e t e d i n t o t h e  the c e l l  wall.  i n s t a n c e , t h e g l y c o s i d e s a r e h y d r o l y z e d , and through  shiki-  the'hydroxyaromatic  the t r a n s l o c a t o r y  r a y s , or i n c o r p o r a t e d i n t o  lignin  by t h e  by t h e  i n aminotransferase  These i n t u r n are c o n v e r t e d  esterified  wall  of p o l y s a c c h a r i d e  amino a c i d s p r o d u c e d  r e a c t i o n s t o produce  of the polyphenols  These  con-  pathway i s p r o b a b l y  the cytoplasmic stages  pathway, p a r t i c i p a t e  aminohydrolase  oligo-  stage.  s y n t h e s i s , the aromatic  to  i n trees,  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  protoplast.  the  tissues  for further  meta-  relating  c o n v e r t s d i s a c c h a r i d e s and  the d i f f e r e n t i a t i n g  in  the  sucrose)  i n a summary o f e v i d e n c e  to.monosaccharides  carbohydrate  f o r new  for further  the s e q u e n t i a l f o r m a t i o n of secondary  notes  suggested  The d a t a i n d i c a t e s  the leaves t o the cambial  bolism. to  over  systems o f In the  last  converted  o f an o x i d o r e d u c t a s e .  As t h e  fiber  enters  drates  senescence,  decreases  membrane, and with  those  the  causing  a breakdown o f t h e  a consequent  of the  c o n c e n t r a t i o n of  general  of vacuole  cytoplasm.  During  phenolic excretory  through, t h e  cell  activity  f o r more t h a n  sited  most  Literature It cess  of the  Summary  i s apparent  of l i g n i n  50%  of the  cellulose  and  from  the  has  s y n t h e t i c , b i o s y n t h e t i c , and  to  aromatic  lignin  their  t r a n s f o r m a t i o n to  formation  nature  characterized.  pro-  viewpoints.  involves a  which  locational  of intermediates  efficient  carbohydrate  t r a n s p o r t and  at  corre-  sequence a l l early  eventual  lignin.  have b e e n c o n c e r n e d  oligolignols  t h a t the  The  s t u d i e s of l i g n i n  important  down.  degradative  The  several  been l a i d  depo-  from  in  allows  probably being  monomer t o p o l y m e r r e l a t i o n s h i p  woody s t e m s .  biological  been e x t e n s i v e l y s t u d i e d  t o cambium t o c e l l  Most  in  literature  sponds t o a l e a f  stages  diffuse  lignin  has  stage  Observations  formation  c o m p o s i t e view, o f  change  components  this  senescence which i s  responsible after  constituents  It i s this  associated with  cytoplasmic  blending  much o f t h e  wall.  carbohy-  with  formation  cambial  monomeric  Considerably  i n woody  o r sapwood r e g i o n s  intermediates fewer f r e e  have been l o c a t e d o r  stems  located  dilignols  with  and to  characterized in a l l  36. . plant  systems, although  effective  precursors to  dilignols lignin  have b e e n shown  formation  as  i n synthetic  studies. The area the  f o r the  tage  of s t a b l e  metabolizing leaf  enzymatic  analysis  of t e s t  experimental of the  results  data  tissue.  leaves.  ticular tains  techniques are  the  The  potential  aromatic  average  promising  The  the  study  added  of  advan-  De-  of a c c e l e r a t e d  i n order  the m e t a b o l i c t h a t so  to  in radioactive  capable  little  to o b t a i n capability research  c o n s t i t u e n t s of c o n i f e r o u s ,  leaves of western for lignin  a  t o woody t i s s u e s .  necessary,  consistent with  a higher than  lignans.  offers  manipulation  I t i s remarkable  b e e n done on  plant  compounds.  s t u d i e s i n comparison  of a n a l y t i c a l  provides  intermediates related  tissues  of ease of e x p e r i m e n t a l  velopment  has  location  b i o s y n t h e s i s of polyaromatic  actively  and  leaf-branch relationship  red  cedar  s t u d i e s , s i n c e the amount  of both  offer  par-  s p e c i e s con-  lignin  and  37-  . MATERIALS  Collection  five  samples were o b t a i n e d  o r more  (Thuja p l i c a t a Columbia  randomly  Collections  first  three  lation  collection  of i s o l a t i o n  dates  1 9 6 9 , and J u n e  provided  techniques.  f o r feeding experiments  cosides.  The f i n a l  procedures  collections  and t o d e v e l o p  cones o r f l o w e r s .  1969,  samples  1970.  The t h i r d  served  The  f o r t h e de- . collection  and t h e i s o l a t i o n to repeat  improvements.  were c u t f r o m t h e t r e e s t a k i n g c a r e  a plastic  branches  were made i n J a n u a r y  1 9 6 9 , June 1 9 6 9 , October  used  low l e v e l  s e l e c t e d western r e d cedar  April  was  from  Donn) t r e e s on t h e U n i v e r s i t y o f B r i t i s h  campus.  velopment  ;  o f Leaves  Leaf of  AND METHODS  of g l y -  the i s o The l e a v e s  t o a v o i d branch  The s a m p l e s were i m m e d i a t e l y  material,  placed i n  b a g , s e a l e d , and t r a n s p o r t e d t o t h e l a b o r a t o r y .  Leaf E x t r a c t i o n 14 Following ing  experiments  the removal of l e a f  and m o i s t u r e - d e t e r m i n a t i o n ,  l e a v e s were chopped ent .  The c h o p p e d  large  Soxhlet  over  samples  for  the remaining  i n a l a r g e Waring b l e n d e r  without  l e a v e s were w e i g h e d and p l a c e d  extractor for.extraction with  a 72 h o u r p e r i o d .  A single  C feed-  solv-  ina  methyl a l c o h o l  e x t r a c t i o n was d e c i d e d  38. upon b e c a u s e prevented  true  The worked  the h i g h  up  percentage of moisture  i n the  sequential extraction i n a pilot  m e t h y l a l c o h o l e x t r a c t w h i c h was  according  to Figure  3.  The  leaves  attempt.  obtained  ethyl acetate  obtained contained l e a f phenolic glycosides without  was  extract  interfering  chlorophylls. c  Chromatography Thin  layer cellulose  Thin  chromatography  layer cellulose  p l a t e s were p r e p a r e d  grams o f 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 of  American V i s c o s e  homogenization resultant  C o . ) , i n 85 ml o f w a t e r  of f i f t e e n  slurry  seconds  ( t h i c k n e s s 0.25  p l a t e s were a l l o w e d Four  lulose acid:  acid The  of  acid:  (2%); first  system  (4:1:1:1)  nim) was  water  and two  applied to  layer cellulose  acetic  acetic  (20:1:2)  as t h e  solvent.  (IAW).  reference  chromatography  used e t h y l a c e t a t e : p r y i d i n e : water  as t h e d e v e l o p i n g  cel-  chloroform:  (3) 2% aqueous  s o l v e n t s were c o n s i d e r e d  sugar residues  five  layer  chloroform:  (2) l o w e r l a y e r (CAW);  The  layer applicator.  (4) i s o p r o p a n o l : a m m o n i a : w a t e r  Thin  by  overnight.  (1) n - b u t a n o l :  (2:3:1.5)  f o r the study.  (12:5:4)  to air-dry  (BCAW);  Corp. Div.  followed  s y s t e m s \\rere u s e d i n t h i n  chromatography: water  acetic  solvent  25  i n a Waring b l e n d e r .  8" x 8" g l a s s p l a t e s i n a Shandon t h i n The  (EMC  using  39. "  W e s t e r n Red C e d a r -Waring Chopped  Leaves blender  leaves  L e a f samples f o r 14 C f e e d i n g study - E x t r a c t i o n with 72 h o u r s Discard  alcohol  leaves Methyl  alcohol  solubles  -Filter Methyl a l c o h o l  through  insolubles  celite.  filtrate  1) E v a p o r a t e 2) Wash w i t h  Chloroform  methyl  to dryness 1 l i t e r chloroform  Chloroform discarded  washings-  1) Add 1.5 l i t e r s and celite to form thick slurry 2) F i l t e r t h r o u g h c e l i t e Water  insolubles-discarded  Water  solubles  1) C o n c e n t r a t e on evaporator 2) E x t r a c t w i t h e t h y l ether E t h y l ether solubles 0.8% - n o t a n a l y z e d  Water  solubles  Extract acetate Water s o l u b l e s discarded Figure  3..  with  ethyl  Ethyl acetate s o l u b l e s 3.9%  Scheme f o r t h e e x t r a c t i o n and s e p a r a t i o n o f components f r o m w e s t e r n r e d c e d a r l e a v e s .  40. Detection primarily  of  utilized  the  compounds on  the  diazotized sulfanilic  cellulose plates acid..  sprays,  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  reagent  (FeCl^  while  Other Barton's  'Fel^IN^ ) ) were used, t o d e t e c t  sugar r e s i d u e s  phenolics,  were, l o c a t e d w i t h p - a n i s i d i n e  hydro-  chloride . • Thin  layer s i l i c a  gel  chromatography  S i l i c a , gel. p l a t e s ' w e r e of  35  g o f Merck S i l i c a  slurry  was  applied  preparative using in  a  at  Gel  G In  prepared using 70  a thickness  c h r o m a t o g r a p h y ) on  a Shandon a p p l i c a t o r .  u s e d - i m m e d i a t e l y -were s t o r e d  of  of water.  0.25 8"  five  The  130°.C o v e n f o r !•£ h o u r s  ml  mm 8"  x  plates  This ' 0.50  or  mm  were t h e n  placed-  Plates  70°C o v e n t o  not  prevent  deactivation. Two (1)  ic  1  solvents  were u s e d  chloroform:methyl alcohol  ethyl alcohol acid,  (9:1)  (BE).  concentrated  i n g was  used  tively,  diazotized  detection  to  detect  for thin layer s i l i c a (4:1)  (CM),  A spray  nitric  acid  (2)  benzene:  of  concentrated  sulfur-  (1:1)  followed  by  heat-  plates.  Alterna-  compounds on  sulfanilic  and  plates:  the  a c i d was  a l s o u s e d -as  a  reagent.- .  • Column  chromatography  Column c h r o m a t o g r a p h y was of  (for  glass' p l a t e s  for activation. in'a  a. s l u r r y  isolating  the  phenolic  the  glycosides  most In  important  this  means  investigation.  41. acid  the  major c h r o m a t o g r a p h i c media used.  two  of LH-20  as  ( F i s h e r ) and  Sephadex L H - 2 0  Silicic  a gel-filtration  chromatographic on  the  experiments  ethyl acetate  (79)  extract.  and  with  Earlier  G-25,  separation  the  t h e n unknown d i l i g n o l  mono- and  eluted  with  cosides dex  were r e c l a i m e d  elutions.  most e f f e c t i v e (4:1)  (CE).  amined and the  on  The  although  solvent  silicic  water  a c i d was  coincident with  gel  the  separation  p l a t e s and  reproducible  on  the  phenolic  organic the  also  o f the  and;, a g a i n  required  u s e d as o r 3E  the as  other  eluants.  t h o s e u s e d on  silicic  a c i d columns.  final  plates  silica  the  glycosides.  ex-  glycosides,  s e e n on  u s e d p r i m a r i l y i n the  Sepha-  eluant.  e i t h e r CM  are  gly-  ethyl alcohol  definitive  e x t r a c t i o n of the  were  extraction.  separation  These s o l v e n t s  o f the  desired  system which proved  chloroform:  much l e s s  c h r o m a t o g r a p h i c medium w i t h  tion  the  the  from  These g e l s  comparable r e s u l t s u s i n g  i t provided  Deactivated  medium was  using. •  C e l l u l o s e column c h r o m a t o g r a p h y was  acetate  generally  studies  glycosides  glycosides.  ethyl, acetate  L H - 2 0 was  s e p a r a t i o n was  ethyl  by  prior  excellent results in  o r w a t e r - m e t h a n o l and  LH-20"was u s e d w i t h  solvent  provided  polyphenolic  water  selection  pilot  trials,  and  other  The  medium r e s u l t e d f r o m  Sephadex G-10 of  ( P h a r m a c i a ) were  stages  of  was This  separa-  42.  In borious  an e f f o r t  to mitigate.the  t a s k o f column  pressure  Recorder  ( 4 7 ) and u l t r a v i o l e t  The s c h e m a t i c d i a g r a m  chromatographic  l o n g and l a -  c h r o m a t o g r a p h y , newer methods o f  chromatography  were u s e d .  often  (Figure  monitoring  4) d e p i c t s t h e  apparatus used i n the i n v e s t i g a t i o n . .  UltraviolMonifor Column 2 Fraction Coll. Column 1  Figure  The  4.  196-47 c a p a b l e o f d e -  240 m l / h r a t a d i s c h a r g e  pressure  pump was u s e d t o a p p l y p r e s s u r e  tographic  Fraction Coll.  A schematic r e p r e s e n t a t i o n of the pressure column c h r o m a t o g r a p h y s y s t e m .  pump was a M i l t o n - R o y M o d e l  livering This  Sample Inj.  columns  a t 50 t o 10.0. p s i .  c o r d i n g m e t e r was a G i l s o n M o d e l Leeds Northrup s t r i p r e c o r d e r .  o f 1000 p s i .  f l o w t o t h e chromaThe u l t r a v i o l e t r e -  UV 280 I F c o u p l e d  to a  Column No. 1 h a d an LH-20  43. bed  dimension  of  Column No.  2 had  and  CM  either  adjustable silicic  on  LH-20  had  Derivative  cm  acid  same  with  CE  as  eluants .  times  cm  eluant. x 1  cm)  Flow r a t e s were,  ml/min, w h i l e  the  r a t e o f 1 m l / m i n due  of i t s connection. 10  the  (60  packing  column f o r 0-4  a maximum f l o w  as much as  the  x 2.5  o r BE were u s e d as  limitations  represent feed  cm  a silicic  i n the  acid  pressure  76  These  that available  flow  with  to rates  gravity  columns.  Preparations  Acetylation Acetic acetylate and  free  anhydride:  hydroxyl  groups.  compound were a l l o w e d  perature  and  steam b a t h  the  p y r i d i n e (1:1)  to  The  stand  removed f r o m t h e m i x t u r e  The  used  acetylation  overnight  f o l l o w i n g morning they  (100°C) f o r 1 nour.  was  to  mixture  a t room tem-  were warmed on  excess  reagent  a  was  u n d e r vacuum.  0-methylation Methylation excess  about  added. The  hydroxyl  diazomethane i n methyl a l c o h o l .  methylated to  of p h e n o l i c  groups The  utilized  compound t o  was  d i s s o l v e d i n minimal methyl  alcohol,  -10°C  f o r 1 h o u r a f t e r which' c o l d  diazomethane  The  mixture  was  then  f o l l o w i n g morning the  removed u n d e r  vacuum.  returned  to -10°C  residual methylating  be  cooled was  overnight. agents  were  44. Dimethyl  s u l f a t e - p o t a s s i u m c a r b o n a t e was  conjunction with methyl to  fully  methylate  Dimethyl  (50%  sulfate  e x c e s s ) v;ere added solution  nitrogen  air.  The  inorganic  (3  and  tive  p e r OH  form.  with acetone.  to dryness  g r o u p ) and  overnight.  from  The  tions  the t o t a l  utilized  cedure sequent  silver  and  t h e aqueous  Methyl oxide  morning  the m e t h y l a t e d  deriva-  solution with  chloro-  were r e q u i r e d  methylation procedure.  to that  of methyl  mentioned  iodide/silver  (2  temperature  These  dimethyl sulfate-sodium hydroxide  identical use  rotary  following  Stronger methylation conditions  complete  in a  The  The  i n dimethyIformamide.  added t o the m i x t u r e extracted  of moist  filtered.  g r o u p ) were added a t room  left  refluxed  exclusion  c o o l e d and  evaporated  e q u i v a l e n t s p e r OH  was was  was  redissolved  the mixture  water  m i x t u r e was  acetone  s o l u t i o n was  w i t h the  (50%  carbonate  t o an a n h y d r o u s  This  attempt  glycosides.  potassium  were washed t h r i c e  solution  equivalents and  f o r 5 hours  salts  evaporator iodide  e x c e s s ) and  i n portions  resultant  o x i d e I n an  of the i s o l a t e d  of the g l y c o s i d e .  under  acetone  one  iodide-silver  used i n  above w i t h o u t  to  condii n a prothe' s u b -  oxide.  Hydrogenolysis It benzyl  was  alcohol  necessary to c a t a l y t i c a l l y group  o f one  of the  hydrogenate  isolated  the  glycosides.  45. '.This hydrogenolysis was done with a 20 mg samole i n approximately of  5 ml  of e t h y l  catalyst  The  test  then  [(PdCl^-BaSO^) A d l e r and  tube  genation  a l c o h o l i n a test, tube  was  then  apparatus.  h y d r o g e n was  mixture  sample was  low  first  min  and  the  mg  was  added  pressure  hydro  evacuated  i n t r o d u c e d to a pressure  s h a k e n f o r 15  was  (2)]  Mar-ton  placed i n a.Parr  The  to which 50-70  o f 40  and  psi.  sample removed  The and  filtered.  Degradative  Techniques  Alkaline  nitrobenzene  Alkaline  nitrobenzene  mine t h e b a s i c a r o m a t i c of phenylpropane u n i t s The  procedure  and  and  used  t h e manner o f l i n k a g e  Blundell (76)  Siddequeullah hours at  (92)  with  was  The  the  used.  l 6 0 ° C i n the  sodium h y d r o x i d e .  resultant  extracted with  ether, a c i d i f i e d  and  extracted with  e t h e r t o o b t a i n the a r o m a t i c  final the  products  thin  layer  thin  layer  u s i n g CAW.  The  cellulose  improveA 10  of  mixture  with h y d r o c h l o r i c acid  cellulose  was  run  aldehydes. on  the  compounds were l o c a t e d on  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).  mg  presence  was  Qualitative  to d e t e r -  i n the major o c c u r i n g g l y c o s i d e .  o x i d i z e d 2.5  nitrobenzene  o x i d a t i o n was  nuclei  o f S t o n e and  ments o f P e p p e r and sample was  oxidation  46. Ethanolysis Ethanolysis Compound A of  2%  was  run  of  was  sealed  The  ethanolysis  under n i t r o g e n  tive  (BE)  standard  leaves. of  was  dissolved  ded  to  25  ethyl  Dyer  i n 1 ml  100°C.  with  cellulose in on  thin  n-  layer  Compara-  chromatogram  acetate  The  to  reaction.  5 ml.  sodium a r s e n i t e  and  stored  and  titrated  with  methylated'  of the of  compounds  the  western  according to  alcohol, This  solution at  2 hours.  starch  run  a  0°C.  and  (39.7  w a t e r was  s o l u t i o n was and  the  added  A 5 nil a l i q u o t  Sodium b i c a r b o n a t e ,  0.0534 M  iodine  rng) ad-  solution  i n d i c a t o r were added  red  the  methylated d e r i v a t i v e  volume o f  and  one  to  extract  was  ethyl  ml  applied • of  of  1  was  was  oxidation  (21).  withdrawn a f t e r  lution  tube  neutralized  layer  ml  hour at  methylated d e r i v a t i v e .  0.0693 M periodate to  and  thin  sample  f o r one  aqueous ammonia o r  oxidation  This  a total  brought  on  The  i n one  oxidation  from the  of  heated  (aromatic hydroxyls)  procedure  10.ml  a l c o h o l •.  cooled  methylated  contained  f r a g m e n t s were f o r m e d i n t h e  Periodate  cedar  with  f o r the  Periodate  isolated  run  fully  compounds were r u n . o n t h e  d e t e r m i n e what  derivative  and  • or  sample  and  m i x t u r e was  butanol saturated gel  mg  acid-ethyl  sodium b i c a r b o n a t e  silica  a 10  on  hydrochloric  Conpound A'  and  solution  .  to was  was excess the  so-  to  the  based the  s t a r c h end on  a prior  point.  Determination  determination  arsenite available  of molar uptake  of i o d i n e r e q u i r e d to  was  titrate  i n solution..  Hydrolysis Hydrolysis tempted w i t h l u t i o n was  2%  of the  aqueous  added t o an  the mixture  refluxed  the mixture  was  The  chloroform  tographed the  aqueous  c o o l e d and e x t r a c t was the  added t o t h e  cooled the  degree  of the  extracted with to dryness  of h y d r o l y s i s .  to hydrolyze  and  c o n d i t i o n s used  acid  so-  glycoside  F o l l o w i n g the  of the m e t h y l a t e d i n methanol.  g l y c o s i d e and  s o l u t i o n was chloroform  f o r the  with  solution  oxalic  at-  reflux  and time,  chloroform. and  chroma-  Several  of  required ref o r the  methy-  derivatives.  hydrochloric acid  and  The  evaporated  or h y d r o l y s i s under  Hydrolysis  was  acid.  thrice  compounds were d i f f i c u l t  lated  ly  oxalic  f o r 2 hours.  to determine  hydrolysis  6%  n o n - m e t h y l a t e d g l y c o s i d e s was  then  Liquid  The  aglycone,  refluxed  extracted with  while  the  run  in  f o r 2 hours. chloroform:  The water  chromatographical-  w a t e r s o l u b l e s were d e i o n i z e d  amine exchange r e s i n  compared t o s t a n d a r d  and  chromato-  sugars.  S c i n t i l l a t i o n C o u n t i n g of- Low C h r o m a t o g r a p h i c Samples A new  was  hydrolysis solution  s o l u b l e s were a n a l y z e d  a strong quaternary  graphically  derivatives  method f o r d e t e r m i n i n g  Activity  levels  of  radioactivity  •4-8.. of  c h r o m a t o g r a p h i c a l l y s e p a r a t e d compounds  in  this  was  study.  necessary  present proven study  to test  i t svalidity  technique  c o u l d t h e n be a p p l i e d  i n the leaves of western 1 denotes  were t a k e n  from  as p r e v i o u s l y  diameter  circles  applying the  described.  The r a d i o a c t i v e  1 cm c i r c l e s  l i j  (five  samples.  Those  from  cellulose ether  ly  nitrate  nitrate  ink. from  vials  to just also  without  for  1 cm  samples were p r e p a r e d  leaving stands  combustion.  by  solution to  to the were  s o l u t i o n of  circle.  alcohol:  The  c o n t a i n e d 3 ml/1 b l a c k they  layer  Other n o n - r a d i o a c t i v e  cover each  dried,  pre-  the c e l l u l o s e  by a p p l y i n g a 3% w/v  As t h e c i r c l e s  ready  through  s p r a y s were t h e n a p p l i e d  were t h e n mounted on w i r e tion  plate  w h i c h were t o be combusted  solution  the glass  experiment  (Parlodion-Mallinkrodt) i n ethyl  (1:1) d r o p w i s e  lulose ing  samples  the p l a t e s  feeding  On t h e s e p l a t e s ,  replicates).  and c h r o m o g e n i c  f o r the  of a - f i v e  C-L-phenylalanine  compounds  lifted  plates  were i n s c r i b e d  10 u l o f U -  in a pilot  The  r e d cedar.  The samples  two c e l l u l o s e  paration  replace.  t h e sample p r e p a r a t i o n s u s e d i n  o f t h e method.  the g l a s s .  and t o compare i t t o  i t would p o t e n t i a l l y  validation  to  developed  B e c a u s e t h e method i s a.new t e c h n i q u e , i t  methods w h i c h  Table  was  c o u l d be l i f t e d  a residue.  The  and p l a c e d i n  celmarkclean-  samples  scintilla-  49. TABLE 1 Sample p r e p a r a t i o n and r e s u l t s o f 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 c o m p a r i s o n o f s c r a p i n g and c o m b u s t i o n methods a p p l i e d t o samples f r o m 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 Standard Standard phe.n y i alari i n e  r quer cet i n  Amount added (Jug) 2 6 12 2 6 12 2 5 10 15 2 5 10  15  2  pinosylvin  blank  i  5 10 15 2 5 10 15 2 5 10 15 2 5 10 15 10 2 5 10 2 5 10  -  Spray — — Ninh ydrin  Combustion, dpm  2781  Scrape, Activity ratio dpm obs. r e s u l t / standard — — — 1941 2332 1. 20  2255 2463 . 2492 2423 no  '  Bisdiazo. benz .  2781 2748 2753 2773  2570 2255 2337 i Barton's  2433  2288  .886  .896 2036 1923 1675 1533  1530  1327  .837 2053  .853 1.06  1791 1652 1388  .851 .715  1  2424  Ninhydrin  1577  2736 196 4  1  1  2717  .966  .862  2169  Barton s  .923  .982 .975 .934  2381  D.B.  .923 .813  2294 2418  r  .788 .716  2480  '  • 79 1.00 .99 .99 .99  .608 .684  2695  Bis  .99 .86  1389  2711  2746 2685 2 762  .871  1.05  1181  2657 2730 DSA no  1.16 1.17  1.18 1.24 1.20 .987 .97 .99 .812  .872 1.11 .854 1.01 .977  50. Just sorbant  prior  1 ml o f c h i l l e d  t o combustion,  [ethanolamine:  ethylene  glycol  monomethyl  ( 1 : 2 ) ] was p l a c e d i n t h e b o t t o m o f e a c h v i a l . grease vial  was s p r e a d  was t h e n  thinly  about  flushed with  ly  -10°C  [5.5  (3:1)]  samples  p l a t e as c l e a n l y  scraped  was t h e n  Immediate-  (ethylene  requiring  s c r a p i n g were removed  as p o s s i b l e u s i n g a s c a l p e l .  (New E n g l a n d  ( 2 x 5  tion  a calibrated  with  of t h i s  glycol  counting.  p l e s were c o u n t e d counter  cock-  added and t h e sample was  to counting.  min) i n a P a c k a r d  experiment  vial  from  The t o which  N u c l e a r Corp.) was added. The  were s h a k e n v i g o r o u s l y p r i o r  results  light.  sample was p l a c e d i n a s c i n t i l l a t i o n  14 ml o f L i q u i f l u o r vials  infrared  T h i r t e e n ml o f s c i n t i l l a t i o n  forscintillation Those  the  The cap  and t h e sample was com-  g PPO i n t o l u e n e : C e l l o s o l v e  monomethyl-ether) ready  and t h e  c o m b u s t i o n t h e sample was s h a k e n and c o o l e d a t  f o r 10 m i n u t e s .  tail  Stopcock  oxygen f o r 5 seconds.  u s i n g an e x t e r n a l f o c u s e d  after  ether  the l i p of the v i a l  was q u i c k l y t i g h t e n e d on t h e v i a l busted  CO^ a b -  A l l sam-  1200 s c i n t i l l a -  external standard.  are included i n Table  The 1.  A separate experiment was run to determine the l i m i t s of spot s i z e upon combustion e f f i c i e n c y .  •  51.  14 Leaf  C  Feeding A p o r t i o n of the leaves  collected  i n the f i e l d  were  14 immediately purpose  of this  stantiate tion  prepared  kinetic  were  e x p e r i m e n t was  and  studies  leaves 1/8  about  inspected  of the leaves  were i m m e d i a t e l y hours  petri  containing  2 g portions approximately  u l samples  withdrawn from each p e t r i tion  o f the samples.  vials and  amount the  step to  These cut l e a v e s  and p l a c e d  o v e n f o r 18  of the f e e d i n g  leaves  in.four 14  2 u c / m l o f U-  C-L-  water. s o l u t i o n were after  T h e s e samplesv.were p l a c e d (New E n g l a n d N u c l e a r  to s c i n t i l l a t i o n  A  experiment  The r e m a i n i n g  o f the f e e d i n g  of labeled phenylalanine  beginning  anabolic  cedar.  i n a 105°C  d i s h immediately  containing Liquifluor  were s u b j e c t e d  combus-  t o e l i m i n a t e woody m a t e r i a l .  i n 6 ml o f s t e r i l i z e d 10  sub-  scissors into  inch i n length.  oven-dry weight.  into four  Three  the r e l a t i v e  a v a i l a b l e f o r the feeding  were w e i g h e d  phenylalanine  developed  were c u t w i t h  The  further  as a p r e l i m i n a r y  w e i g h e d and p l a c e d  to determine  dishes  (l).to  i n the leaves.of western r e d  averaging  portion  o f t h e newly  dilignols  collected  carefully  experiment.  twofold:  (2) t o d e t e r m i n e  of i s o l a t e d  The pieces  C feeding  the a p p l i c a b i l i t y  technique  activity  for a  counting  introduci n counting Corp.)  t o determine the  a v a i l a b l e to the leaves at  period.  The d i s h e s  were  then  52. and i l l u m i n a t e d by two 250 w a t t  covered at  3 feet.  a distance of approximately  represented end  5 and 10 h o u r s .  p e r i o d , 3 more 10  y l samples were  drawn f r o m t h e i n d i v i d u a l determine time.  the percent  water.  tion  of the l a b e l l e d  o f each  from the l a b e l l e d  solution  f o r e x t r a c t i o n with  and workup a c c o r d i n g  3 y i e l d e d a gross The  ethyl  ethyl  was  flasks.  replicates time  point  first  direction  acetate  second d i r e c t i o n  and  sprayed  designated  with  CAW..  diazotized  evidence, i t  of i n t e r e s t  extract.  thin  two d i m e n s i o n a l l y w i t h  with  under  Three  60 u'l  ( r e p r e s e n t i n g the f o u r  f o l l o w e d by a i r - d r y i n g  the  to dryness  chromatographic  feeding solution  and d e v e l o p e d  Extrac-  a l c o h o l t o f o u r 2 ml v o l u m e -  p e r i o d s ) were a p p l i e d t o s e p a r a t e  plates  taken  t h a t t h e compounds  i n the e t h y l  o f each  i n individual-  methyl a l c o h o l .  e x t r a c t was  B a s e d upon e a r l i e r  w o u l d be p r e s e n t  distilled  acetate extract.  acetate  assumed a t t h i s  with  were  and washed w i t h placed  to  t o t h e scheme d e p i c t e d i n F i g u r e  vacuum and t r a n s f e r e d i n e t h y l tric  with-  solution  feeding, the leaves  The washed l e a v e s were t h e n  micro-soxhlets  At t h e  f e e d i n g s o l u t i o n s and c o u n t e d  uptake  Upon c o m p l e t i o n  filtered  o f 1,  bulbs  The f o u r ' d i s h e s ,  3,  f e e d i n g times  o f each time  incandescent  layer  cellulose  BCAW i n t h e  and d e v e l o p m e n t i n  The p l a t e s were t h e n  sulfanilic  as A and B on t h e s c h e m a t i c  acid.  The  dried,  compounds  chromatogram ( F i g u r e s )  53. were to  then  s t r i p p e d .from t h e p l a t e  t h e method p r e v i o u s l y  with  irregular.shape  with  a razor  This  subdivision insured  the  count  obtain  blade  rates  described.  and s i z e  Chromatographic  required  total  combustion  count  was  rate  being  rates  with  summed t o  spot.  a Packard  count  surface.  of the spots,  f o r each  counted using  c o u n t e r and t h e o b s e r v e d  spots  careful subdivision,  o f the i n d i v i d u a l s e c t i o n s  radioactivity  tillation  according  f o l l o w i n g r e m o v a l from the p l a t e  the r e p r e s e n t a t i v e  absorbed  and c o u n t e d  The 1200  scin-  were r e l a t e d  14 to the o r i g i n a l tion  i n each  Spectral  C uptake  compound  tographically  run  s p e c t r a were o b t a i n e d  purified  scraped  from  samples  or methyl  " '  appropriate  s a m p l e s ..-.were d e t e r m i n e d  blanks.  Ultraviolet  a n a l y s i s was  termination  of phenolic  hydroxyl  (45).  difference  spectra  The method obtained  recording  were  spectro-  Spectral shifts  of  o f 0.1 N s o d i u m  a l s o used  according  involves  eluants  The s p e c t r a  i n the presence  methoxide.  Goldschmid  f o r column chromaalcohol  cellulose plates.  i n m e t h y l a l c o h o l i n a Beckman DK-2  photometer u s i n g the  time.  and i n f a r e d  Ultraviolet  spots  r e l a t e d to feeding  incorpora-  Techniques  Ultraviolet  of  t o determine p e r cent  i n the de-  t o t h e method o f .  the d e t e r m i n a t i o n  f o r the s h i f t  of  of the phenoxide  54.  Figure  5:  A s c h e m a t i c chromatogram o f t h e 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 w e s t e r n r e d c e d a r l e a v e s . ( s p r a y -DSA),  ion  formed  extent  with the a d d i t i o n  o f 0 . 1 N sodium  o f change o f maxima was compared t o a e u g e n o l  dard t o determine  free phenolic  hydroxyl content.  s p e c t r a were o b t a i n e d f r o m samples  Nuclear magnetic All  samples  acetonei  was  n u c l e a r magnetic  nance  Infrared'  bromide  spectrophotometer.•  resonance 100  (NMR) s p e c t r a were,  mHz NMR  spectrophotometer.  T e t r a m e t h y l s i l a n e was added signal,  a t t = 10.  as an i n t e r n a l  N o r m a l sample  a p p r o x i m a t e l y 10 mg/ 300 y l o f s o l v e n t .  o f 1-5  stan-  were p r e p a r e d i n d e u t e r o c h l o r o f o r m o r d e u t e r a t e d  and l o c k  of l e s s  The  resonance  on a V a r i a n H A - 1 0 0 ,  obtained  dard  i n potassium  on a P e r k i n - E l m e r 521 i n f r a r e d  pellets  The  methoxide.  stan-  dilution  F o r samples  t h a n 5 mg a m i c r o - c e l l was u s e d w i t h . the. d i l u t i o n mg/30 u-1 o f s o l v e n t .  (NMDR) e x p e r i m e n t s  determine  coupled  Nuclear magnetic  were a p p l i e d  double  reso-  to derivatives to  signals.  Mass s p e c t r o s c o p y Mass s p e c t r a , were o b t a i n e d f o r p u r i f i e d a c e t a t e derivatives  of d i l i g n o l  r u n by M o r g a n - S c h a f f e r High  amplification  ments were  rhamnosides.  spectra  were  C o r p o r a t i o n , M o n t r e a l , Quebec.  spectra  included.  These  o f h i g h m o l e c u l a r weight  frag-  56.  RESULTS  Isolation  o f Compounds  Yields 23.8 g o f e t h y l  o f 1.47 g o f e t h y l acetate  solubles  ether solubles were o b t a i n e d  (58.6% moisture) of f r e s h western r e d cedar according  to Figure  3.. T h e s e  yields  o f 0.76%  weight  of the oven-dry  ing  extract  revealed  flavonoids.  red-orange  near the solvent  tic  acid  (46).  examination  a wide r a n g e  front  on t h i n l a y e r  acetate).by  of the gross  dimensional  ethyl.ace-  of polyphenolics  sulfanilic  when d e v e l o p e d  includ-  cellulose plates.  acid  (DSA)  i n 2% aqueous This  i s i n d i c a t i v e o f an a l p h a h y d r o x y  Two  represent  compounds w h i c h gave an o r a n g e ,  with diazotized  ran  w i t h DSA  weights  treated  leaves.  Several  reaction  f r o m 1.5 k g  leaves  ( e t h y l e t h e r ) and 3.86$ ( e t h y l  Chromatographic tate  extract  and  reaction  guaiacyl  chromatography u s i n g  nucleus  BCAW i n t h e  first  d i r e c t i o n and CAW  i n the second d i r e c t i o n , w i t h  layer  cellulose plates,  resulted  these  compounds  5, compounds A t o I ) w i t h  range  o f Rf v a l u e s  versely the  related  (Figure  i n CAW.  i n good s e p a r a t i o n  The R f v a l u e s  t o t h e number o f f r e e  chromatographed  compounds ( 2 2 ) .  ace-  i n CAW  hydroxyl  thin  of  a wide are i n -  groups o f  5(7.  Consideration s p r a y e d w i t h DSA highest  yield  and  gave t h e  Several  reaction  (B^EjI^J)  other  but  dimensional  were not  very  distinct  occurred  gave a r e d  chromatography-  Compound A o c c u r r e d  compounds a l s o  but  D,K,F,G,H,L). . O t h e r DSA  two  i n d i c a t e d that  action.  pounds a l s o  of the  orange  gave an  in  color  orange  i n lower y i e l d .  the re-  color  Some com-  o r r e d - o r a n g e r e a c t i o n w i t h DSA (0  compounds  investigated  t o T)  beyond  also  reacted  (C, with  chromatographic  detec-  tion. Compounds.A, B and The tionate the  on  gross  C-  Isolation  ethyl  LH-20 w i t h  prior  to  LH-20  extract  divided  i n t o two  applied  separately  Fifty  ml  ly  thin layer  on  Pilot  separation  was  tion and  schematic now  the  i n the  of  almost  r e l a t e d compounds.  form: methyl a l c o h o l  i n the  (3.5  a l l of  (CM).  5)  the  was  as that  advisable ethyl  solvent,  25  ml  cm  x 40 one  The ml  reveals  each  and  cm).  dimensional-  compounds of  eluant.  the  undesirable  column was  , reswollen  500  frac-  (4:1)  crude  eluting  first  (Figure  The  (3:7)  the  monitored  gel plates  chromatogram  devoid  extract  column  f r a c t i o n s were t a k e n and silica  found to  determined  (approximately  to. a L H - 2 0  u n d e r s t u d y were o b t a i n e d The  of  also  Therefore,  dissolved  fractions  was  ethyl alcohol  studies  a quantitative, run.  acetate  extract  chloroform:  eluting solvent.  a preparative  acetate  stripped  subfracflavonoid  using  to n e a r the  chloro-  original  bed  level,  and  run,  and  The The  the  the  second  elution clarified  quantitative  LH-20 column..  rate  o f 2 m l / m i n u t e , and  plates.  monitored  The  elution  e x t r a c t was  thin  layer  now  ready  I t was  silica  for  application  to  15  pressure  g),  fractions  were t a k e n .  u s i n g the  G i l s o n UV  monitor  of a t y p i c a l  run  and  in  divided  at a  ml  curve  gel  concentrated  10  applied,under 10  applied  constant.  (estimated weight  4 e q u a l p o r t i o n s and  e l u a n t was  by  e x t r a c t was  into  flow The silica  i s shown i n  6.  0  100  200  300  •Elution Figure  o f the  volume r e m a i n e d  vacuum e v a p o r a t o r  Figure  half  again being monitored  plates.  to  the  6.  400  volume,  500  ml  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 l e a v e s as r u n - o n LH-20 (CHCl^:Et0H(4:1)).  600  Thin  layer  fraction while 420  chromatography  contained  fraction.  a p p e a r e d t o be vestigated separated  to  applied  x  and  up  to  to  ml  any  high  either the  solvent.  failing  and  A and  quantitative The trated  to  (5 t o  CM.  500  on  and  evaporator  gel  The  two  column i n t h e  35  to  50  ml in-  runs  were  and  These f r a c t i o n s column  (3-5  cm  taken  desired  com-  approximately  main p u r p o s e s :  separation  eliminated  running  of the the  first,  near  compounds  semi-micro  achieved. the  and  of  The range.  thin layer s i l i c a  compounds were  applied The  a rate  ml  the  100 not  four  column o r by  2 under p r e s s u r e . at  was  compounds were  the  column was  pressure  of  f r a c t i o n s were ml.  ml  constituents  ml).  column w i t h  f r a c t i o n s were c o l l e c t e d .  d e t e r m i n e d by  ml  column s e r v e d  run  10  silica  Ten  fractions containing  under constant  280  constituents  J p r i o r t o . a p p l i c a t i o n to  in a rotary  to  major e l u t i o n peaks  volume  from t h i s  silica  t o c o l u m n No.  the  two  molecular non-phenolic  by  major  a preparative  The  major  a cyclitol  f r o n t ; second,.some p r i m a r y  B,C,D  5 ml  or  e l u t i o n volume o f  pounds were e l u t e d  220  f r a c t i o n s from the  the  eluted with  a total  250  to  a small  were now cm)  The  according  as  the  A f r a c t i o n appearing near  carbohydrate  further.  concentrated  that  Compounds B,C,D  Compounds A a n d . J were t h e  to.480 ml  40  revealed  gel  i n 2 ml  c o l u m n was 0.75  concen-  portions  eluted  ml/min and  compounds e l u t e d Pure  4  to  from  f r a c t i o n s were  chromatography  and  set  60. aside.  Those f r a c t i o n s  n o t 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 t h e r e m a i n i n g for  other runs.By  these  fractions  procedures,  were r e c o m b i n e d  chromatographically pure  fractions o f Compounds A, B and C were obtained. The  yield  o f Compound A ( b a s e d  o f l e a v e s ) was 0.15%. all  compounds g i v i n g  was a p p r o x i m a t e l y Compounds  in  It  distinctive  0.5%  that the y i e l d o f  c o l o r r e a c t i o n s w i t h DSA  o f t h e weight  No. 1 p r e s e n t s  o f oven-dry  leaves.  the chromatographic,  and p h y s i c a l p r o p e r t i e s o f Compounds  t a b u l a r form.  both  I t was e s t i m a t e d  weight  A, B and C - P r o p e r t i e s  Table spectral  on t h e o v e n - d r y  Rf values  s h o u l d be n o t e d  BCAW was u s e d  A, B, and C  In c o n s i d e r i n g chromatographic  and chromogenic  spray  o f t h e more u s u a l BAW  a c i d , water  (4:1:5)] because  duplicating  t h e Rf v a l u e s  i t gave  less  of the c l a s s i c  In t h e d e t e r m i n a t i o n  properties,  reactions are included.  that the chromatographic  i n lieu  chromogenic,  solvent  designated  [butanol,  acetic  streaking while solvent.  o f an a l k a l i n e  difference  curve,  a eugenol standard was run to check the procedure and r e a f f i r m 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 f o r the compounds and t h e i r derivatives are represented i n Figures 7 t o 9 and Figures 11 to 18.  Figure 10 i s a s p e c i f i c rep-  resentation of mass spectral fragmentation associated with the acetate d e r i v a t i v e of Compound A.  TABLE PROPERTIES OF CHROMATOGRAPHIC PROPERTIES ON CELLUL05E SILICA BCAW CAW 2* IAW CM BE COMPOUND A  0.63  SPECTRAL PROPERTIES (EXCLUDING NMR. MASS SPECTRAL) INFHARED BANDS c m Pi Tl TTA ULTRAVIOLET MAX ..... BAND Str. Str BAND ACID DSA JNMeOH MBOH+BASE DOO . it* D o n . U.V. 0 1207 m 3400 B 1123 a (- FIT Y-0 279nm 289.5nn._ B04 292B W 1009 1770 a 1605 1042 B a 10 W 1505 978 a 1449 m 909 . w 1429 w m 877 1370 w m 805 1270 w B 781 1230 m  CHROMOGENIC PROPERTIES ON CELLULOSE B DSA pNA 5  ( + ) R-0  Q ,14 0.39  0.50 0.76  2 i COMPOUNDS A, B AND C  _ x  PHYSICAL PROPERTIES ACETATE DERIVATIVE MalWt, C.HANAL m.p. " CALC. Emp.Form %C MH/M %H |(unc o n . ) 804  - -39 4B°1B C  H  57.98 5.84 48-50*  J  COMPOUND B  0.73  0.95  0.B7 0.23 0.51  P  ( + ),  q  Y  PAT  2B8nro 248nn  280.5nn  *  •  COMPOUND C  0.73  0.84 0.70  «  0.48  R,  It)  f  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 , w a t e r ( 4 : l i l d ) CAU « c h l o r o f o r m , a c e t i c a c i d , w a t e r ( 2 i 3 : 1 . 5 ) IAW - i s o p r o p o n o l , ammonia, w a t e r ( 2 0 i l t 2 ) 2% = 2% aqueoua a c e t i c a c i d BE " b e n z e n e , 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)  H  nbs,  Q  i  2Blnm 251nn  281nn  I  •  i.  s  a  w B B  m  m m  a  w a  B  W  a a w  H  w a w DSA » d i a z o t i z e d s u l f o n i l i c B • Barton'o reagent „ Schrooder'o reagent pNA m p - n i t r o o n o l i n e B  '  3400 292B 1700 1600 1502 1442 1436 1370 1268 1231 , 3420 2925 1470 1600 1503 1447 1434 1375 126B 1205  •  n  o  t dotorainad  1120 100B 1040 977 902 871 805 ' 7B1 1230 1121 1085 1040 974 909 B71 B05 790 , acid  m  a a  «  70677447  58.08 6,32  w  w w  w w w m a a w w w w w 0 Y R {•1  702  C  35 42°15-  ».Orange - yellow « red m poaitiva reaction  H  br = b r i g h t  fl - fluoreBcant ab8« aboarbo (-)-  negativo reaction  •62.  Figures of  t h e mass  Compounds  9, 13, 18 a r e b a r g r a p h r e p r e s e n t a t i o n s  spectral  A, B and C.  been d i v i d e d i n t o the  spectrum  composition  spectra.  so t h a t  i s i n magnified  the f i g u r e s  t h e h i g h mass  end o f  as p r i m a r y  have b e e n l a b e l e d w i t h  Metastable  their  s p e c t r a t h e m/e  de-  molecular  p e a k s have n o t b e e n n o t e d  I n a l l t h r e e mass  have  proportion.  fragments which appear  products  number.  of the a c e t a t e s of  For convenience,  two p a r t s  T h o s e mass  ion  fragmentation  on t h e s e ,  43 i o n was t h e  100% i o n .  Compounds  A, B and C - D e r i v a t i v e s  Methylation Aromatic-hydroxyl diazomethane i n methyl chromatography  R f compound  reagent  alcohol.  Thin  o f two compounds  o f an a r o m a t i c  Rf compound  showed no r e a c t i o n .  tion  mixture  separated  was  collected  thane. reaction  was  CM as t h e e l u a n t .  Thin  and s u b j e c t e d layer  silica gel  reaction  (Rf  0.45,  0.55).  The  Barton's  The m e t h y l a t e d  reac-  g e l pressure  The s e p a r a t e d  low R f  to re-methylation  showed f u r t h e r f o r m a t i o n  mixture  reaction.to  on t h e s i l i c a  chromatography  A used  h y d r o x y l ) , while the  high  using  layer  displayed a positive  (indicative  umn  o f Compound  (CM).of t h e m e t h y l a t i o n  showed t h e p r e s e n c e lower  methylation  compound  with  of the products o f t h e Rf 0.55  col-  diazomefrom  this  compound.  6 3 .  B a s e d upon t h e c h r o m a t o g r a p h i c t h e Rf . 0 . 5 5  tions,  compound  tic  hydroxyl methylated  the  lower  tially  Rf ( 0 . 4 5 )  methylated Complete  behaviour  was  derivative  was  first  using dimethyl  These  tive  benzyl hydroxyl  chromatography these  occur  group  to apply  partially  methylated  was  product sorbance  was  with  carbo-  oxide  i n Compound A.  i n dimethyl  Thin  re-  layer  t o be i n c o m p l e t e f o r  two a t t e m p t s .  product  I t was t h e r e f o r e c o n d i t i o n s on  using dimethyl  Verification  reagent  sodium  because' o f t h e h i g h l y r e a c -  o b t a i n e d by a n e g a t i v e  to Barton's  o f Compound  Methylation  the s t r o n g e r m e t h y l a t i o n  sodium h y d r o x i d e .  lation  •  chosen t o minimize  showed m e t h y l a t i o n  c o n d i t i o n s even a f t e r  necessary  with  sulfate  c o n d i t i o n s were  arrangements which might  the  conditions.  f o l l o w e d by m e t h y l i o d i d e / s i l v e r  formamide.  while  c o n s i d e r e d t o be a p a r -  ( a l lhydroxyls) methylation stringent  nate  o f Compound A,  aroma-  o f Compound A.  A r e q u i r e d more attempted  reac-  considered the f u l l y  derivative  compound  and c o l o r  sulfate  of the complete  reaction  o f the  methylated  and t h e l a c k o f i n f r a r e d  i n t h e 2700 .nm t o 2900 nm  methy-  ab-  range.  Acetylation Acetate formed  derivatives  of these  according t o the procedure  Triturated  three  compounds  were  previously outlined.  a c e t a t e s were o b t a i n e d _ t h r o u g h  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  ether  ( 6 5 - 1 1 0 ° C ) and  at 5°C.  cooling  Hydrogenolysis Catalytic A using a catalyst  ' hydrogenolyis (2)  was a t t e m p t e d  highly reactive  b e n z y l h y d r o x y l groups.  on Compound  i n the reduction of  The r e a c t i o n was r u n w i t h  t h e 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 m i n u t e s and t h e product  was c h r o m a t o g r a p h i c a l l y  degree  of  sulted  i n the apparent (Rf 0 . 5 1  compound yield. test  hydrogenolysis .  examined t o d e t e r m i n e  The 10 m i n u t e  the  hydrogenolysis  re-  c o n v e r s i o n o f Compound A t o a  second  i n C H C l ^ M e O H ) , Compound B, i n about  When i s o l a t e d ,  this  f o r the benzyl hydroxyl  compound gave a n e g a t i v e  50%  Gierer  group.  • #  Compounds  A, B and C - D e g r a d a t i v e  Studies  .  Hydrolysis Hydrolysis using  o f Compounds  2% aqueous o x a l i c  methanol.  acid  A, B and C was  o r 6% h y d r o c h l o r i c a c i d i n  H y d r o l y s i s o f Compounds  A and B was  c o n s i d e r e d u n s u c c e s s f u l because o f the f o r m a t i o n by-products, a low y i e l d the parent products  however.hydrolysis of a s i n g l e  glycoside.  o f Compound  attempted  product,  o f Compound  generally of several  C resulted i n  the p u t a t i v e aglycone  An e x a m i n a t i o n  of  of the h y d r o l y s i s  B r e v e a l e d the presence  o f Compound  C,  65. indicating two  a chemical  compounds u n d e r The  of L~arabinose of  the  matic  presence  D-xylose  h y d r o l y s i s was r e v e a l e d the  existing  between  were d e t e r m i n e d  also  by  separated  minor  enzyme h y d r o l y s i s b u t  formation  in,yield  amounts  chromatography  compounds.  a p p l i e d t o Compound A.  probable  the  hydrolysis.  o f L-rhamnose and  hydrolysate of i n i t i a l l y  graphy the  c o n d i t i o n s of  primary and  relationship  Enzy-  Chromato-  o f some a g l y c o n e  insufficient  for  in  effective  study.  Ethanolysis Compound A was described.  The  resulting  sodium b i c a r b o n a t e alcohol on t h e  s u b j e c t e d t o e t h a n o l y s i s as p r e v i o u s l y  and  run  saturated with plate  products on  the  one  a t Rf  second  as  spot  h i g h Rf  Rf  •  reaction  typical  W  h  e  sprayed  n  color,  w h i c h d i s p l a y e d a lemon y e l l o w  compound e x h i b i t e d e s s e n t i a l l y  with a color reaction  similar  of the  of p-hydroxyl  placed  spot  to  with  o f Compound A showed 2  ' l-ethoxy-l-guaiacylpropan-2-one  color  compounds  which d i s p l a y e d a red-orange 0.20  in butyl  l-guaiacyl-2--ethoxypropan-l-one,  e t h a n o l y s i s products 0.92  cellulose  Standard  l-guaiacylpropan-2-ohe.  DSA,  The  layer  with,  l~guaiacylpropan-l,2--one, ..  l-ethoxy-l-gualacylpropan-2-one, and  thin  ammonia.  included:  were n e u t r a l i z e d  the  or  the  spots,  and  a  color.  same Rf  value  l-guaiacylpropan-2-one, former. • The  o c c u r r i n g a t Rf  b e n z y l groups  (46).  DSA  0.20  i s considered  66. The also  subjected  product  was  tographed layer  value  methylated  to  n e u t r a l i z e d with  plates  ethanolysis as  the  identical  to  i n BE. was  of t h i s  tographed gave an  the  to  cream).  comparison to  the  (phenolic tion  partially  according  hours.  thin  constituents  exactly  the  same  Rf  standard  and  displayed  to  a l k a l i n e nitrobenzene  and  of  major  0.50).  Rf  described..'  spots  The  the  when chroma-  high  Rf  w i t h DSA"while the color reaction  The  compound lower  ( o f f .white  a l k a l i n e nitrobenzene  standards  vanillin  and  color reactions  pro-  protocate-  and  Rf  values.  oxidation  hydroxyls  m i x t u r e was  at  method p r e v i o u s l y  c h u a l d e h y d e , showed i d e n t i c a l Periodate  major  on  oxidation  a less distinct  Chromatography  chroma-  DSA.  c o l o r when s p r a y e d  compound gave  The  the  run  subjected  0.95  (Rf  Rf  duct.,in  of  o x i d a t i o n ' showed two  i n CAW  orange  to  and  dihydroconiferyl alcohol One  was  ethanolysis  sodium b i c a r b o n a t e  color reaction with  according  Compound A  resulting  dihydroconiferyl alcohol  Compound A was  results  The  shown t o  Alkaline nitrobenzene  oxidation  d e r i v a t i v e of  ethanolysis.  i n comparison  silica  of the  an  fully  o n l y ) was  t o the  titrated  Consumption  methylated  d e r i v a t i v e of  subjected  procedure f o r excess  of p e r i o d a t e  o f Dyer arsenite  Compound  to periodate (21).  The  a f t e r one  showed 2 m o l e s  A  oxida-  reaction and  two  consumed  at  67. the  end  o f one  considered  and  two  complete.  extracted  with  centrated  to. d r y n e s s  ultraviolet nm  hours. The  r e a c t i o n mixture  chloroform. and  spectrum  w h i c h gave no  T h e r e f o r e , the  The  taken  was  was  acidified  c h l o r o f o r m e x t r a c t was up  i n methyl a l c o h o l .  of the p r o d u c t ,  shift  reaction  upon t h e  o f 0.1  N  con-  •  An  showed a peak a t  addition  and  280  sodium  methoxide.  Liquid  Scintillation This  study  Technique  includes a preliminary investigation  of  14 the  infusion  o f U-  western  red  cularly  with  floated  i n an  nine. the  The  cedar..  would govern  be  low  through  into  i n v e s t i g a t i o n was  formation  aqueous  of d i l i g n o l  solution  of the the  the  leaves  concerned  of  parti-  glycosides i n leaves radioactive  chemical  phenyala-  p r o p e r t i e s of  g l y c o s i d e s e s t a b l i s h e d t h a t two  primary  t h e measurement  radioactivity.  levels  expected.  activity  The  f e e d i n g method and  dilignol  First,  the  C-L-phenylalanine  of i n c o r p o r a t i o n (near  Second, those  c o u l d be  of i n c o r p o r a t e d  compounds  chromatographically  autoradiography  1 per  cent)  factors  could  incorporating radiolocated  or g e n e r a l l y with  specifically  chromogenic  spray  reagents. The corporation  experimental on  chromatographically  methods were a v a i l a b l e separated  design r e q u i r e d e v a l u a t i o n of i n -  radioactive  for analysis compounds:  separated of the  samples.  Four  chromatographically  (a) a u t o r a d i o g r a p h y  (densi-  68. tometric  a n a l y s i s ) (b) c h r o m a t o g r a p h i c  window G e i g e r - M e u l l e r  c o u n t e r ) , (c).  (liquid  analysis),  scintillation  scintillation The eliminated  anticipated  (d) s u s p e n s i o n  low l e v e l s  autoradiography  phenolic  nature  required  location  severely  limit, l i q u i d  activity  samples.  products  (from v a r i o u s  that  scraped  of a c t i v i t y  and s t r i p  effectively  counting.  Autoradio-  while' s t r i p  counting  o f t h e compounds t o be s t u d i e d , and with  chromogenic  Many  suspended  chemical  silica  cal  quenching  agent.present.  the a v a i l a b l e  would  spray  could  o f s u s p e n d e d low  reagents) a r e .  quenchers d u r i n g  g e l samples, a d v e r s e l y ratio  liquid (53)  affect  r e g a r d l e s s o f the chemi-  alternatives,  samples  effectively  their  I t has a l s o been shown by Houx  external standard  it  analysis  chromatographic  recorded  chromatographic  reagents,  The  p h e n o l i c compounds and t h e i r - c o l o r e d  analysis.  of  spray  scintillation  the  Of  (liquid  f o r m e a s u r i n g low a c t i v i t y .  t o be e f f e c t i v e  scintillation  (thin  analysis).  o f t o o low e f f i c i e n c y  recognized  counting  combustion-absorption  graphy would r e q u i r e e x c e s s i v e t i m e , was  strip  combustion-absorption  seemed t h e most p l a u s i b l e  eliminate chemical  and p h y s i c a l  because quench-  m mg,  while  available factor of  n e a r l y 100 p e r c e n t for liquid  of the  scintillation  t o a combustion technique  chromatographically  separated  C label  w o u l d be  analysis(£0). The  was  the e f f i c i e n t  samples.  The  limiting combustion  combustion  69. would  be most  layer  cellulose  because Thin  feasible  i t does  layer  chromatography. not burn  small  cellulose  of  the  plates  silica  the  the  plate  a  These  was  spots  suitable which  spot from the p l a t e  designed to e s t a b l i s h cellulose  i n g - s u s p e n s i o n method,  i n this  vial  study  spots are  i n the  nitrate  t h e t e c h n i q u e , an  : (1) t h e  comparative  combustion  (2) changes  v e r s u s the  in efficiency  due  combusted  (3) t h e e f f i c i e n c y  changes  of chromogenic  in  combustion.  then  experi-  f o r t h e s c r a p e d and  f o r t h e two  from  presence  chemical quenching  reactions  of  counted.  o r d e r to. s u b s t a n t i a t e  o f the  method  p r e p a r e d samples  i n the s c i n t i l l a t i o n then  scraping  Although the scraped  The method u s e d  These  thin  subsequently  of the chromatographic  spraying.  a b s o r b e n t , and  ment was  and  a c l e a n e r , more e f f i c i e n t  c l e a n removal  layer  those from  preparations involve  desirable.  directly  paralleled  as a s u s p e n s i o n .  burned,  after  In  ciency  plates.  can be  combusted of  conditions.  t h e most  chromatographic  i n t h e p a s t had  t h e sample  preparation allows  sized  combustion  offered  thin  not c o n s i d e r e d  sample p r e p a r a t i o n s f r o m t h i n  chromatographic  counting sample  normal  or  combusted. Radioactive  layer  under  S i l i c a g e l was  c e l l u l o s e , chromatography  medium t o p r o d u c e c o u l d be  i f a s s o c i a t e d with paper,  as a r e s u l t  methods^and  (4) sample  size  effiscrapto  samples, spray  limitations  •  70. 1 denotes the  Table proposals  and  replications  the  counting  (3,5  quenching  agent  radiation  and  and  Barton's ferric  reagent  iron  Figures sults  and  of Barton's  sprays  was  a distinctive  chemical  reagent  primarily  blue  separate  graphically  determination  efficiency  However,  mechanisms. with  complex; b i s -  is graphically  21  because  compounds.  depends upon a z o - c o u p l i n g  Figure  agent  ultraviolet  reactions via different  of Table l  20.  counting  a  depends upon c h e l a t i o n o f p h e n o l s  data  o f the on  color  3',4'  availability.  c h o s e n as  to- a b s o r b  choice as  five  7,  color^and  i n detecting phenolic  benzidine  19  i t Is a b l e The  5,  the  a p h e n o l i c quenching  conjugation,  benzidine  to produce  compounds. The  as  to t e s t from  (3,  Quercetin  used  fluoresce.  cause  diazotized  size  because  common use  sprays  obtained  d i h y d r o x y s t i l b e n e ) was  bis-diazotized  of t h e i r the  was  of i t s extensive  Pinosylvin  results  of each sample.  pentahydroxyflavone) because  samples p r e p a r e d  to  phenolic  recorded  presents  in  the  of chromatographic  o f combusted  -  respot  samples.  14 C Feeding The the  leaf  :  a n a l y s i s of s c i n t i l l a t i o n  f e e d i n g experiment  are  results  summarized  obtained  i n Table  in  3.  Figure 22 represents the preliminary autoradiographic v e r i f i c a t i o n of U-^C-L-phenylananine incorporation into Compounds A and B i n western red cedar leaves. during the  The r a d i o a c t i v i t y uptake by the leaves  l a t e r i n f u s i o n study  i s shown i n Figure 23.  The  r e l a t i v e incorporation of r a d i o a c t i v e phenylalanine i n t o Compounds A and B  i s depicted g r a p h i c a l l y i n Figure 24.  The r e s u l t s repre-  iamnose  Compound A  Feeding time h r .  Activity i n leaves dpm  Inc.,%.  Activity i n Compound B dpm  Inc.,% .  1865  0.15  3063  0.25  6  20.3  3970  •0.30  5370  0.40  1.78xi0  6  23.5  5340  0.30  7040  0.39  2.82xl0  6  37.1  5035  ' 0.18  6600  0.24  1.22xl0  6  3  1.34xl0  5 10  3-  Activity i n Compound A dpm  16.1  1 •  Table  Inc.,%  Compound B (speculated)  Uptake o f UC - L - p h e n y l a l a n i n e and i t s i n c o r p o r a t i o n i n t o Compounds A a n d B i n t h e l e a v e s o f w e s t e r n r e d cedar. -  /  72.  sent  the  average  variance being  values  of three  calculated.  runs  with  no  statistical  73.  DISCUSSION Structural  Studies  Compound  of the D i l i g n o l s  A  Compound  A was  obtained  f r o m t h e column c h r o m a t o g r a p h i c acetate  successful  t h e compound  from o r g a n i c  and p r e p a r a t i o n  preparation  amorphous s o l i d solvents.  which f a i l e d  s o l v e n t s were  with  tempts  a melting  e t h e r was  mixture the  point  was  obtained  allowed  Compound  of erythro-  low m e l t i n g  A was  al  point  range  vative  initiated.  from by  organic  scraping  t o evaporate  later  (silica  spot.  in a  deriva-  (uncorrected).  At-  d e r i v a t i v e were un-  shown, by NMR,  t o be a  which accounts f o r  of i t s acetate d e r i v a t i v e .  s y s t e m s , o f Compound  showed a s i n g l e  acetate  o f t h e p u r i t y o f Compound  l a y e r chromatography  solvent  of this  and t h r e o - i s o m e r s  T h e r e was no q u e s t i o n thin  of 35°-38°C  at r e c r y s t a l l i z a t i o n  successful.  _ .  d e r i v a t i v e also y i e l d e d an  vacuum d e s s i c a t o r l e a v i n g t h e t r i t u r a t e d tive  u n  ( 6 5 ° - 1 1 0 ° C ) f o l l o w e d by c o o l i n g a t  ether  The p e t r o l e u m  Attempts t o  to c r y s t a l l i z e  solid  solid  of the e t h y l  o f a d e r i v a t i v e was  o f an a c e t a t e  A triturated  under petroleum 5°C.  separation  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 .  crystallize  The  as an amorphous  A since  and c e l l u l o s e ) ,  A and i t s a c e t a t e  i n severderi-  74.  Mass s p e c t r a l d e t e r m i n a t i o n vative with  o f Compound A r e v e a l e d  Sixteen  supported  degrees  Compound  of aromatic  An u l t r a v i o l e t  I n f r a r e d data  nuclei,  of hydroxyl  o f NMR  o f 804 ratio)  analyses.  of at least  Ae v a l u e  o f 8.04 x 10  o f two p h e n o l i c  s u b s t a n t i a t e d the existence  The 18 o x y g e n atoms o f t h e f o r -  groups.  A acetate  were a s s i g n e d  (on t h e b a s i s  and mass s p e c t r a l d a t a ) t o two p h e n o l i c  benzylic acetate,  On t h e b a s i s was c o n s i d e r e d  hy-  a b s e n c e o f c a r b o n y l , and a l a r g e num-  four a l i p h a t i c  m e t h o x y l g r o u p , and t h r e e  1"-  indicative  A showed t h e e x i s t e n c e  mula f o r Compound  one  M+l/M  by c a r b o n and h y d r o g e n  of unsaturation,  formula.  d r o x y l groups.  ber  (from  weight  deri-  s u b s t i t u t e d benzene r i n g s , a r e c a l c u l a t e d from t h e  empirical for  a molecular  a c a l c u l a t e d e m p i r i c a l formula  of C g g H ^ O i s *  two  of the acetate  t o be  ether  acetates,  a c e t a t e s , one  or hemiacetal  of the f o l l o w i n g r e s u l t s ,  oxygens. Compound A  l-(3'-methoxy-4'-hydroxyphenyl)-2-0-  [2"-hydroxy-4"-(propane-3"'-O-a-L-rhamnoside)phenyl]-  propane-1,3, d i o l  (XLI).  CH OR £__5* 0  - .  HCOR 2)y,. -/ OR  _  -  l  t  3  XLI, XLIa,  CH,"  Q  R=H R=Ac  R0 OR  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 when s p r a y e d w i t h hydroxyguaiacyl Gierer's  f o r the Since  detection  presence i n g the  droxyl  aromatic  on  L-rhamnose i n t h e  The  the  catechol  showed t h a t  products.  m o l e c u l e was  of t h i s  unusual a l i p h a t i c stantiated An  later  vative. atoms  ( f o u r on  2%  of the  hy-  etherified.  aqueous o x a l i c  The  presence  of the  will  of by  hydrolysate.  be  further  o f ^25^34^11 ^  s  formula obtained  d i s c u s s i o n has the  one  an sub-  discussion.  e m p i r i c a l formula  This  the  n u c l e i ) ,the  uncommon s u g a r bound t h r o u g h  in this  (to  conclusively established  glycoside.linkage  Compound A f r o m t h e  existence  showed  n u c l e u s must be  l a y e r c e l l u l o s e chromatography occurrence  with  o f Compound A  compound w i t h  showed s e v e r a l p h e n o l i c  thin  and  catechol  of  -  protocatechualdehyde ( e s t a b l i s h -  of g u a i a c y l  Hydrolysis  a  negative.  s u b s t i t u t i o n pattern)  and  the  the  g r o u p s , was  oxidation  Schroeder's reagent  groups  color  reaction'(82),  Schroeder's  of o-dihydroxy  existence  orange  A positive test  further substantiates group.  of v a n i l l i n  with  acid  (46).  a l k a l i n e nitrobenzene  determine the  test  (44)  hydroxyl  a bright  w h i c h i s c h a r a c t e r i s t i c o f an  compound  reagent  of a benzyl  DSA,  to produce  f o r the  i  -icated  acetate  accounted f o r ten  benzene r i n g s , one  n d  benzylic  for  deri-  oxygen hydroxyl,  76. five one  from the L-rhamnose). aliphatic  lationship These  hydroxyl  group  to the other  features  There remains the placement o f and a d e s c r i p t i o n o f i t s r e -  aliphatic  were d e l i n e a t e d  c a r b o n and o x y g e n i  atoms.  i n t h e f o l l o w i n g two e x -  periments. The  first  experiment  substantiated  a g u a i a c y l g l y c e r o l - 3 - a r y l ether parison Hibbert  of the products 1  s ketones  through  ethanolysis.  of ethanolysis with  showed t h e p r e s e n c e  Compound A as  of  standard 1-guaiacyl-  2-ethoxypropan-l-one and l-guaiacylpropan-2-one. d u c t s were  considered  aryl  nucleus  ether  contains mixture  Another e t h a n o l y s i s values  of the g u a i a c y l  f o r Compound A.  two i s o m e r i c of erythro-  proof  carbons  product  These  pro-  glycerol-3-  The g l y c e r o l s i d e  and i s r e s p o n s i b l e  and t h r e o - i s o m e r s  Com-  chain  f o r the  previously  mentioned.  showed a c o l o r r e a c t i o n and R f  i n d i c a t i v e of catechol d e r i v a t i v e s . The  s e c o n d e x p e r i m e n t was an e t h a n o l y s i s  of the  i  fully This as  m e t h y l a t e d [(CH^^SOjj/CH^I.] d e r i v a t i v e o f Compound ethanolysis  yielded dihydroconiferyl alcohol  a major p r o d u c t .  dihydroconiferyl  The f r e e a r o m a t i c h y d r o x y l  a l c o h o l e s t a b l i s h e d the unusual  A.  (XLII)  group  of .  V-n-  propan-3"'-ol sidechaln and the 2-4" a l k y l - a r y l ether linkage i n Compound A (XLI).  The free a l i p h a t i c hydroxyl group of XLII es-  tablished the point o f attachment o f the L-rhamnose i n the molecule. The n-propyl side chain accounted f o r the remaining a l i p h a t i c  77. CHJOH  XLII  carbon  atoms.  Another  showed a c o l o r  product  reaction  and  from  this  ethanolysis  Rf v a l u e s i n d i c a t i v e  of  veratryl -derivatives. Proof that rhamnose g r o u p s  arrangement  were as  or t h a t  the  t h a n y-  e t c . was  Periodate  the  shown  s u b s t i t u t i o n was  (XLI), as  o b t a i n e d from  oxidation  o f c a t e c h o l and and  a 3-aryl the  from  the  absence  T h i s two  mole  product  bond d i d n o t Only explains  the  achieved  by  and  other v i c i n a l  of a bathochromic oxidized  consumption  shift  of exactly  i n Compound A.  evidence  that  the  the  shown f o r Compound A  NMR  and  data.  mass s p e c t r a l  V  Lack the  glycosidic  an a r o m a t i c h y d r o x y l .  forementioned  solely  maxima o f  occur through structure  two  e s t a b l i s h e s , the  of the u l t r a v i o l e t  i n b a s e was  (aromatic  of p e r i o d a t e i s  further diols  rather  experiment.  of diazomethane m e t h y l a t e d  rhamnose m o i e t y o f any  interchanged,  ether  following  h y d r o x y l s ) Compound A showed an u p t a k e moles.  not  L-  (XLI)  Further v e r i f i c a t i o n analyses.  was  78.  NMR  s p e c t r a o f Compound A and i t s a c e t a t e . NMR  structure dies.  spectroscopic techniques  o f Compound A as d e t e r m i n e d  The NMR  deuteroacetone the  spectrum  rochloroform  fore -refer to t h i s  The protons. T  The e n s u i n g  spectrum  proton  S i x of these  NMR  of  spectral  discussion w i l l  and t h e NMR  results  there-  o f the  be t r e a t e d s e p a r a t e l y .  integral  protons  o f Compound A.  o f F i g u r e 8 i s 47 t o 49  are seen  two a r o m a t i c  These protons  rings  t o resonate at  hydroxyls  x=  (T = 6.22).  indicates Two  a r e c o n s i d e r e d t o o c c u r i n Compound A,  upon t h e two n o n - e q u i v a l e n t 7.73  and 7 - 77 .  pro-  must be r e p r e s e n t a t i v e  since the s p e c t r a also  three proton methoxyl resonance  at  The NMR  = 3.04- 3 - 3 0 and a r e r e p r e s e n t a t i v e o f t h e a r o m a t i c  tons of  i n F i g u r e 7.  done on t h e a c e t a t e d e r i v a t i v e o f  compound w i l l total  stu-  o f Compound A was o b t a i n e d i n d e u t e -  Compound A ( F i g u r e 8 ) .  unacetylated  i n degradative  and i s shown i n F i g u r e 8 .  was p r i m a r i l y  the basic  o f Compound A was o b t a i n e d i n  and i s p r e s e n t e d  acetate derivative  analysis  confirmed  aromatic  acetate  s h i e l d i n g o f these acetate resonances show the phenolic hydroxyls of Compound A to be non-equivalent.  aromatic based  resonances  This s l i g h t l y d i f f e r e n t  a  Figure  8.  The  NMR  r spectrum of the  acetate  derivative  o f Compound  A.  81. Five pound  A by t h e f i v e  T = 7-9  -  rhamnose  (  = 5.36)  as a r e s u l t  resonances 8.  rhamnose  The a n o m e r i c  are  2  t o low  The h i g h  field  the acetate  shifted  4  derivative  t h e C^ p r o t o n t o lower  field  ( T = 4.6  1Hz);  H ,  T. = 4 . 7 7  3  T = 5.0  ( J ^  5  (J  = 4.5  through  acetylation  to  = 6.21  be T  periments resulted  Hz).  Hz, J^  The  and i t s c h e m i c a l  grouping. J  a  These  «3» ^-0 =  H z  ciated with  2  i s not s h i f t e d was  determined  resonance ex-  )  c  a  A (60).  an e t h e r b o n d e d  n  b  e  doublet  to a  (integrated value,  t o the b e n z y l proton o f  doublets  of a diastereomeric o f Compound  field  a t T = 4.00  c a n be a s s i g n e d  = 4.05,  ( J " ^= ( c a . )  A p p l i c a t i o n o f NMDR t o t h e H,_ r e s o n a n c e  Two r e s o n a n c e s  a glycerol  shift  double  i n a c o l l a p s e of the high  one p r o t o n )  5-2)  ( c a . ) 10 H z ) ;  ^ =  proton  by n u c l e a r m a g n e t i c  (NMDR).  singlet.  currence  3 = 1.5  2  -  The  o f Compound A a n d a r e s p e c i f i 2  isomers  proton  (J = 6.5Hz).  l o c a t e d and d e s c r i b e d a s : H , x = 4 . 7 9  T  field  three  cally  Hz;  proton  o f i t s a s s o c i a t i o n t o t h e ex - L  coupling-with  C -Cjj protons  Hjj,  f o r Com-  o c c u r r i n g i n the  as a s i n g l e t  (6l).  bonded  are indicated  a t f = 8 . 8 3 o r i g i n a t e s from the methyl group o f  doublet  in  acetate  i s seen t o occur  -hemiacetal  the  hydroxyls  8.4 'region o f F i g u r e  of T  aliphatic  (T = 3 . 9 7 ,  attributed  pair  J  S f t X  . = 7.0  to the oc-  of 3 - g u a i a c y l g l y c e r o l •  Since carbon,  the 3 proton i t should  i s asso-  appear i n  82. the  region"T'= 4.5 -  the  x =4.5 -  low  f i e l d doublet  proton the  (  T  doublets  6.1 indicating  with  themultiplet  this  resonance  monitoring the way, t h e 3  In t h i s  to collapse  This i r r a d i a t i o n  i nclarification  techniques,  t o two s i n g l e t s  of the 3 proton  of them u l t i p l e t s the  i nthis  beneath  NMDR o f t h e 3 p r o t o n  resonance.  the a proton  = 5.6 -  NMDR  = 5 - 3 8 , hidden  was l o c a t e d a t  anomeric p r o t o n  resulted T  f o r any c h a n g e s .  = 3.97, 4.05).  T  Using  6 . 0 r e g i o n was s c a n n e d w h i l e  resonance  caused  6 . 0 (5 4 ) .  y protons  range.  i scharacteristic  also  i n the region  resonance  The c h e m i c a l  associated shift i n  o f an e s t e r i f i e d  hydroxy-  m e t h y l e n e (54) . The 7-37  =  multiplet  T  o f an n - p r o p y l  under t h e a l i p h a t i c  was i r r a d i a t e d  a'proton  3'resonance. multiplet quartet  chain.  acetate envelope  Irradiation  centered  at  T  thereby  at x =  the t r i p l e t at  establishing  and t h e x = 8 . 1 8 resonance  resonance  o f the 3'resonance  6.30 x t o 7.75  T  = 6.5 with  T h i s resonance  and t h e c h e m i c a l  of the  When t h e r e s o n a n c e  i n an NMDR e x p e r i m e n t ,  i n t h e range  = 1 0 . 0 Hz). protons,  at x =  7 and 8) a n d t h e two p r o t o n  = 7-37 c o l l a p s e d t o a s i n g l e t  the  triplet  a t x = 8 . 1 8 ( F i g u r e 7) a r e c h a r a c t e r i s t i c  3' p r o t o n s  occurring 8.18  o f t h e two p r o t o n  3.7 Hz)(Figures  (J  a'and  occurrence  as t h e  clarified  to a distinct  X^ = 6 . 3 5 , x  i t as  fi  the'  AB  = 6.65 (  J  (  g  e  i s r e p r e s e n t a t i v e o f t h e y.'  shift  o f t h e AB q u a r t e t  suggests  83. that  they  are methylene protons  bond r a t h e r t h a n 6.0  range).  an e s t e r bond  The AB n a t u r e  the geminal  protons  equivalence  and s u g g e s t s  bulky  a s s o c i a t e d w i t h an e t h e r (occurrence i n  o f the s p i n - s p i n  o f - t h e m e t h y l e n e group restricted at the 3 '  IL-rhamnose m o i e t y  = 5.5  T  -  coupling of i n d i c a t e s non-  r o t a t i o n due t o t h e  - y'  carbon-carbon  bond. Examination  o f t h e NMR  spectrum  of unacetylated  Compound  A reveals several  integral  o f 30 t o 35 p r o t o n s , a more d e f i n i t i v e  ton  aromatic  droxy  resonance  resonances  features including:  = 2.9  ( T  (x = 1 . 3 0 ,  2.55),  i n the x = 6.1  ton m u l t i p l e t  -  t h e rhamnose h y d r o x y l g r o u p s .  two 8.18  proton resonance without  o f Compound 6.21  (as d e t e r m i n e d  A produces  a twelve  6.8 r e g i o n which i n c l u d e s  x  specific  =5,07,  spectrum  of the 3'  i n Compound  the methine  protons  similar  pro-  x=  Methylation at  o r two  x= free  o f t h e NMR  o f Compound  data recorded  at  A.  d a t a and n a t u r e  o f the a c e t a t e d e r i v a t i v e  agreement w i t h  features i n - ,  acetate resonances.  - The c h e m i c a l ' s h i f t  hy-  to fourteen pro-  a nine proton resonance  h y d r o x y l groups  s i x pro-  two a r o m a t i c  r e p r e s e n t a t i v e of t h r e e methoxyl groups  aromatic  total  by NMDR) x = 6 . 0 0 , a n d t h e  multiplet  interfering  3.6l),  Other  elude: b e n z y l proton resonances ton resonance  -  a  A isin  f o r 1-(3,4-dimethoxy)  84 -2-(2'- methoxyphenoxy)-propane-l,3 reported  by L u d w i g  diol  acetate  (XLIII) .  et a l . (60). -  OCH  3  XLIII The  d i s c u s s i o n o f t h e NMR  lates  t h e c h a r a c t e r o f t h e a, 3  lation These ces of  spectrum  t o the e x i s t e n c e characteristics  recorded this  compiled  a r e i n c l o s e agreement o f Compound  and on t h e d e g r a d a t i v e  f o r Compound  characterized  and y r e s o n a n c e s  i n re-  o f two d i a s t e r e o i s o m e r i c f o r m s .  f o r the acetate  comparison  3  o f compound X L I I I r e -  A.  f o r resonanOn.the b a s i s  and NMR  data  A, t h e compound i s c o n s i d e r e d  t o be  as an i s o m e r i c m i x t u r e o f 1 - ( 3 ' - m e t h o x y - 4 ' -  hydroxyphenyl)-2-0-1"-[2"-hydroxy-4"-(-propane-3"-0-a-Lr h a m n o s i d e ) phenyl]^-propane-l .,3 d i o l (XLI).. Mass s p e c t r u m  o f Compound  A acetate.  The mass spectrum of the acetate o f Compound A i s shown i n Figure 9-  The parent i o n i s shown to occur at m/e=80k.  Ex-  85.  i11 1  . D.O  «.D [ W.C I .1-' 111 III I  100. D  IZI.O  ;?l).C  J10.D  tftf.O  POO. D  in, i"i TO.O  MOO  WO.0  MOD  «o.a  52-  «a.i  .«».» . *«.B Figure  «o.e  9.  - «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  The mass s p e c t r u m o f t h e a c e t a t e d e r i v a t i v e o f Compound A.  iw.i  w.i  eoi.o  ,<uo.i>  86.  amination  o f t h e h i g h mass end o f t h e s p e c t r u m  transitions sition. table  w h i c h may be a s s o c i a t e d w i t h  Two o f t h e t r a n s i t i o n s 804 m*=722.2  ions:  0  '  e  >  ?  6  2  acetate  decompo-  a r e a c c o m p a n i e d by m e t a s +  C  H  .  702+CH^C-OH.  reveals-six  c  =  0  a  n  762  d  m  *  =  6  The o t h e r t r a n s i t i o n s  2  „  1  ,  d  f o r which  metastables  9 are not p r e s e n t CH =C = 0 , 2  702,  a r e : 702  ^ 660 + C H = C = 0 and 6 6 0 -  acetate degradations  least  two a r o m a t i c Validation  indicate  642  the occurrence  a c e t a t e s and two a l i p h a t i c of the occurrence  c a n be s e e n  ^  600+  > 600+CH -C-OH.  2  These  rhamnoside  C-OH,  ^642+CH-  ofat  acetates.  o f Compound A as a  i n the occurrence  o f an m/e = 273  ion  f o r t h e rhamnose t r i a c e t a t e  fragment.  This  can  i n t u r n decompose a l o n g a pathway s i m i l a r  fragment  to that  pro-  posed by Pearl and Darling (74) f o r glucoside acetates. A proposed decomposition .in Figure 10. 109  path  o f t h e rhamnose t r i a c e t a t e  The p r o m i n e n t  a r e i n agreement w i t h  i s depicted  i o n s 2 7 3 , 2 1 3 , 1 5 3 , 1 1 1 , and  the proposed  pathway.  1  87.  OAc  CHjC-O  m  Figure  10.  of  m  /e 111  P r o p o s e d 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 .  Several lustrated  /e 109  other features  through  probable  t h e mass s p e c t r u m .  o f Compound A c a n be  formulae  i l -  r e l a t e d t o major  Some p o s s i b i l i t i e s  Ions  are i l l u s t r a t e d  on the following page. Several and  require  first  comment.  isolation  glycoside.  c h a r a c t e r i s t i c s o f Compound A a r e n o v e l First,  this  compound c o n s t i t u t e s t h e  and c h a r a c t e r i z a t i o n  Second,  of a free  t h e compound e x i s t s  dilignol  as an u n u s u a l  88.  CH OAc  CH„OAc I 2 HC+ I HCOAc  2  HCOAc  Q  m  m  CH OH  HC I HCOAc  C+  CH, m  4 179  t h a n much more  T h i r d , the rhamnoside  pound t h r o u g h a p r e v i o u s l y rather  Fourth,  lytic  conditions  among  dilignols  aliphatic  aromatic  dilignols  of a catechol  previously  isolated  propyl  isolated  using  posiside hydro-  unusual.  group and  hydroxyl  hydroxyl  of a saturated  and i s b i o s y n t h e t i c a l l y  the existence  observed g l u -  i s c o n n e c t e d t o t h e com-  unreported  the existence  i s unreported  among  commonly  than the expected  chain  Finally,  OH /e 221  OH  rhamnoside r a t h e r  position  m  2  OCH-  cosides.  CH,  OH /e 281  HC+  )H /e 221  tion.  H  OCH,  OAc m/e 323  o  CH OAc I C+ II 0 2  HC+  is  unreported  characterized.  89. The tral  data  complete  a n a l y s i s of the a n a l y t i c a l  available.for  characterization  Compound A has e n a b l e d  as d e s c r i b e d .  Such d a t a  and s p e c - i t s positive  are incomplete  f o r Compounds B and C which therefore cannot be as completely characterized.  However, speculative conclusions may be made  regarding the structures o f these compounds based upon the a v a i l a b l e data and t h e i r observed chemical r e l a t i o n s h i p t o Compound A.  The following discussions include such speculations.  Compound B Compound B was i s o l a t e d , tographic  separations  phous s o l i d ents .  43°C  under petroleum  from o r g a n i c  solv-  d e r i v a t i v e was o b t a i n e d by ether  (65°-110°C).  The m e l t i n g  d e r i v a t i v e o f Compound B was 34° -  (uncorrected). Mass s p e c t r a l  not  to crystallize  acetate  of the acetate  chroma-  p r e v i o u s l y d e s c r i b e d , as an amor-  failed  A triturated  scratching point  which  f r o m t h e column  clearly  units  tion.  weight  of molecular  of the i n a b i l i t y  mass e n d o f t h e s p e c t r u m .  s p e c t r a were n o t o b t a i n e d  of i t s apparent  spectrum  existence  mass  However, a interpola-  c o u l d be c a l c u l a t e d .  forthis  as a m i x t u r e .  o f Compound B was e s s e n t i a l l y  w e i g h t was  t o count  o f 744 o r 786 was i n d i c a t e d by  No m e a n i n g f u l e m p i r i c a l f o r m u l a  Difference cause  d e f i n e d because  at the high  molecular  determination  compound b e The i n f r a r e d  Identical  with  90. that  o f Compound A, s h o w i n g an a r o m a t i c  b o n y l , and a h i g h d e g r e e  n a t u r e , no c a r -  of hydroxylation.  Compound B shows an o r a n g e r e a c t i o n w i t h DSA displaying  negative reactions with  Schroeder's an  reagent  low y i e l d  ethanolysis  group n o r a b e n z y l h y d r o x y l  o f Compound B p r e v e n t e d  or a l k a l i n e  Chromatography  nitrobenzene  o f t h e 2% o x a l i c  showed a m i x t u r e  ducts,  r e d u c t i o n o f Compound A.  i n addition  chromatographic Examination  t o unreacted  to  o f t h e NMR  products  Compound B was  under Other  Compound  conditions of low y i e l d  pro-  products.  and mass s p e c t r a l  data of  o f Compound A, and t h e ob-  b e t w e e n t h e compounds, l e d  the s p e c u l a t i v e phenylcoumaran  XLIV  hydrolysis  techniques.  o f the r e a c t i o n  to that  chemical r e l a t i o n s h i p  pound B. -  degradation  using  Compound A, were n o t e d i n  examination  Compound B i n c o m p a r i s o n served  i t s analysis  o f L-rhamnose.  shown t o be f o r m e d i n 50% y i e l d  catalytic  the  acid  group.  o f p h e n o l i c compounds ( i n c l u d i n g  C) and a m a j o r o c c u r r e n c e also  and  ( 8 2 ) . 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  o-dihydroxyphenyl  The  G i e r e r ' s (44)  while  structure  X L I V f o r Com-  " •'  *  XLV  91. NMR  s p e c t r a o f Compound B and i t s a c e t a t e . The  derivative ted  ing  11  differences  tons  high  total  and 1 2 ,  tinct  peaks  with  grates  to less -  aromatic  occurrence mixture The NMR  derivative or at l e a s t  of t h i s  of t h i s  region of The• proton  The m e t h o x y l r e s o n a n c e  12  at  b u t shows t h r e e  hydroxyl  sites.  The d o u b l e t  at 5.21  i s centered  dis-  observations  integral  singlet, integrates  a r e i n agreement  compound as a mole.cular  with  and/or  iso-  chromatographically  o f the diazomethane  aromatic  asso-  T and i n t e -  the t o t a l  the anomeric  compound i n d i c a t e d  one f r e e  proton  somewhat i n F i g u r e  even though i t appears spectrum  occurrence  shows a f i v e  one p r o t o n , w h i l e  These  con-  the existence o f a methoxyl v a r y i n g  5-40T'), i n c l u d i n g  t o two p r o t o n s .  pro-  c a n o n l y be  to s i x protons.  and c l a r i f i e s  the a proton than  the d i s t i n g u i s h -  o f t h e known  The a r o m a t i c  doublet.  indicating  ciated  pure.  because  i n t e g r a t e s to three protons  between t h r e e  meric  respectively,  rhamnose m e t h y l r e s o n a n c e  show t h e e x p e c t e d  T = 6.20  and o n l y  spectra  o f 28 t o 32 and 45 t o 47  B as a m i x t u r e .  i n F i g u r e 11  multiplet  the  integrals 11  of these  be d i s c u s s e d .  spectra integrates f o r five field  (5.00  Compound A and a r e d e p i c -  The s i m i l a r i t y  as a p p r o x i m a t i o n s  Compound  these  and 1 2 .  will  f o r Figures  sidered  as w i t h  f o r Compound "A i s e v i d e n t  The  to  s p e c t r a o f Compound B and i t s a c e t a t e  were o b t a i n e d  i n Figures  to those  of  NMR  methylated  s i x methoxyl  hydroxyl.  The  protons  methylated  Acetone-d  94. product benzyl  a l s o shows s e c o n d a r y proton  mixture  NMR  acetate  2.25)  w h i c h may  ( t = 1.12) be  i n the  and  efficiently  that The  the  acetate  field (  T  shift  benzyl  carbon  NMDR e x p e r i m e n t s of  the  g and  on  the  y proton  hydroxyl  a strongly  hydroxyl  which i s  i s the  impurity.  benzyl proton  a proton  Such a  result  a l s o r e v e a l e d no  doublet only  - 8.2)  acetylation  four  aliphatic  ether  that  linkage  to lower  at  5.83  x and  5.48T ,  locations represent  chemical  values  observed  of  Compound A.  Such c h a n g e s i n c h e m i c a l  causing  appearance  These resonance to those  the  field.  e s t a b l i s h e d the  resonances  down-  on  suggesting  resonance  acetate  o f an  respectively. shift  (x =  probable  ( F i g u r e 12)  ( x = 7.8  resonance  aro-  I t i s h o w e v e r , more  atom i s i n v o l v e d i n an  o f the  isomeric  suggests  hindered  spectrum presents  resonances  shift  resonance  derivative  The  field  shows one  group.  derivative  acetylated.  o f the  field  a free hydroxyl  acetate  field  = 5.05).  acetate  the  low  an  derivative  a low  hydrogen bonded or s t e r i c a l l y not  low  and t h r e o - c o n f i g u r a t i o n .  o f the- a c e t a t e  matic  resonance  o f the  doublet, . further suggesting  of e r y t h r o The  splitting  opposite  f o r the  acetate  shift  are- n o t e d  f o r dehydrodiconiferylalcohol t r i a c e t a t e (XLV) (see page 90) i n comparison to l-(3,4-dimethoxyphenyl)~2-(2'-methoxyphenoxy)-propane-1,3 d i o l a l . (60).  diacetate  (XLII)  as  d e s c r i b e d by  L u d w i g et_  95. Mass s p e c t r u m The  o f Compound B acetate.  mass s p e c t r u m  Compound B i s - s h o w n potential the  parent  '  of the acetate d e r i v a t i v e of  i n F i g u r e 13.  i o n s a t m/e  The o c c u r r e n c e o f  = 702, 744 and 786  e x i s t e n c e o f t h e compound i n a m o l e c u l a r  Such a m i x t u r e spectrum  severely restricts  f o r meaningful  speculated mentioned position  structure  character  the a n a l y s i s  the m o l e c u l a r weight  f o r Compound B.  ions associated with are also  mixture.  i o n s , h o w e v e r , t h e m/e  w o u l d be. i n a g r e e m e n t w i t h  present  o f the spectrum  indicates  o f the = 744 i o n of the  The p r e v i o u s l y  rhamnose t r i a c e t a t e . d e c o m -  i n the spectrum.  The g e n e r a l  I s i n agreement w i t h t h e s p e c -  t r u m o f t h e p h e n y l c o u m a r a n 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 reported  by K o v a c i k The  probable  (and t h r e o - and of  proton  after  bonded i n such resonance  Compound  A.  phenyl  Also,the  a manner as t o c a u s e i n comparison  eliminating shift  o f the  3 carbon i s  an u p f i e l d  shift  t o t h e same r e s o n a n c e  of the .  that  ether or a l k y l - a r y l  nucleus, thereby  acetylation.  The n a t u r e  analysis  However, i t w o u l d a p p e a r  atom i s e i t h e r  t o a second  one compound  makes e x a c t  a h y d r o x y l and t h e a s s o c i a t e d d o w n f i e l d  benzyl  its  e x i s t e n c e o f more t h a n  Compound B d i f f i c u l t .  linked  (56).  erythro- -isomers)  the b e n z y l carbon  the  and Skamla  of  in  substituent bonded to the  96.  0.0  '  M.O  Pi .1 «.fl  1 ..1,;. l i b i J U L i I 60.0  BO.O  .100.0  MMhA 120.D i«.0  o  . teo.o  ?tf>.0  a.a  ..  «a.0  Figure  . «a.o 13.  SJO.B • x s . o The  9<a.o  sua.a  a*  .  cva.9  240.0 £60.0  . MO.a  tni.a  ^flfl.O  coa.o  M3.0 32D.0 MD.O 3E0.0  TM.O  mass s p e c t r u m o f t h e a c e t a t e o f Compound B.  TJO.I T*.O TCO.B  aoo.o  a.a  derivative  «o.a  aaa.a  aa.%  94. 3  carbon  proton  atom  may  cause  the downfield  shift  of  the  i n comparison to the same resonance i n Compound  y  A. .  Based upon the nuclear magnetic resonance data i n comparison to that of Ludwig et a l . (60) and Compound chemical r e l a t i o n s h i p phenylcoumaran  (XLIV)  between Compound  A, and the observed  B and Compound  A, a  i s considered as the most probable  structure. The B may an  be  acceptability  o f s t r u c t u r e XLIV  s t r e n g t h e n e d by t h e o b s e r v a t i o n  a , 3-phenoxy m i g r a t i o n  guaiacyl-3-aryl tions. water  ether  compounds u n d e r m i l d  The  catalytic  that  acidic  condi-  to XLVII i n hydrogenolysis  -'--  XL VI  XLVII  under which  Compound A was  pound. B may  be  formation  rangement  (71)  can o c c u r f o r a - h y d r o x y -  seven days.  conditions  the  o f Nimz  Nimz showed t h e c o n v e r s i o n o f XLVI (100°C)'in  f o r Compound  formed  a t 50%  yield  c o m p a r a t i v e t o Nimz' c o n d i t i o n s of a quinone methide  to y i e l d  compound X L I V .  f r o m Comleading  i o n capable of Biosynthetic  to  rear-  formation  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 r e a c t i o n , , o r i t may  be  entity  formed  formation  as a s e p a r a t e  prior  or concurrent  o f Compound A v i a t h e q u i n o n e m e t h i d e  to the  pathway.  Compound C Compound C, as i s o l a t e d c o l u m n , was  an amorphous s o l i d  from o r g a n i c  solvents.  was o b t a i n e d  through  110°C), m e l t i n g c u l a r weight be  point  culated with and  derivative  nr  and 15 d e g r e e s  ultraviolet of t h i s  hydroxyl  ether  (65° -  The m o l e -  determined  a red color  r  to A  was o b t a i n e d  from  15  o f u n s a t u r a t i o n were  cal-  The compound r e a c t e d •  and t e s t s  g r o u p s were n e g a t i v e .  s p e c t r u m was o b t a i n e d  because  f o r o-dihydroxy No  alkaline  o f t h e low y i e l d  compound. Hydrolysis  a low y i e l d  o f Compound  C was  of a chromatographically  L-rhamnose was  s u c c e s s f u l i n producing pure  aglycone  e s t a b l i s h e d as t h e g l y c o s i d e .  o f t h e compound p r e v e n t e d dative  was  o f C -H,,„0-,  from t h e e m p i r i c a l f o r m u l a .  benzyl  derivative  i o n o f i t s mass s p e c t r u m .  e m p i r i c a l formula  DSA t o p r o d u c e  acetate  39° - 43°C ( u n c o r r e c t e d ) .  of the acetate  ratio  to c r y s t a l l i z e '  s c r a p i n g under petroleum  3b> t h e M + 1/M  chromatographic  which f a i l e d  A triturated  702 f r o m t h e m o l e c u l a r  calculated  from the  techniques.  the a p p l i c a t i o n  The i n f r a r e d  spectrum  Low  and yields  of further of this  degra-  compound,  99. was. a g a i n  similar  following  examination  relation  t o Compounds A and B. o f NMR  to the chemical  Compounds  B a s e d on t h e  and mass s p e c t r a l  relationship  A and B, Compound  data i n  o f Compound  C to  C i s c o n s i d e r e d t o be a  guaiacyl benzdioxane d i l i g n o l possessing e i t h e r structure XLVIII or XLIX.  mnose  XLVIII NMR  '  XLIX  s p e c t r a o f Compound The  derivative  NMR  s p e c t r a o f Compound  were o b t a i n e d  C and i t s a c e t a t e  as f o r Compounds A and B and  displayed  the same'general  and  The t o t a l  15).  C and i t s d e r i v a t i v e s .  characteristics  approximate proton  ( F i g u r e s 14  integrals for  F i g u r e s 14 and 15 r e s p e c t i v e l y , a r e 29-33 and 40-43. Important shift  f e a t u r e s i n c l u d e : s i x aromatic  o f the b e n z y l proton  derivative,  similarity  p r o t o n s , no  ( x - 5-14) i n t h e a c e t a t e  of chemical  shifts  o f t h e 3 and  Acetone-d  Figure  14.  The NMR s p e c t r u m  o f Compound  C.  102.  Y resonances acetate of  ( T = 5.91, 6.37, F i g u r e 1 5 ) t o t h o s e  o f Compound B ( F i g u r e  Compound A, one a r o m a t i c  and  four  These  aliphatic  carbon pound  Examination  Both  o f these  chemical protons  show s i x a r o m a t i c  protons  T  T  and  3 protons  field  of the 3 Such o f these  bonded t o b o t h  NMDR e x p e r i m e n t s  a c e t y l a t i o n while  acetylation  o f the a s s o c i a t i o n  i n t h e a c e t a t e NMR s p e c t r u m  t o lower  i n the  respectively.  w i t h e l e c t r o n - w i t h d r a w i n g groups atoms.  after  the occurrence  a n d 6.39  are i n d i c a t i v e  3 and y carbon  shifted  16 and does n o t s h i f t  a t 5.92  of-  The a p r o t o n i s shown t o o c c u r a t  NMDR e s t a b l i s h e d  shifts  after  16,17)  spectra  the  shifts  (Figures  o f Compound C.  = 5.14 i n F i g u r e  y protons  group.  f o r the structure  T  and  aromatic hydroxyl  com-  evidence  = 3.00 - 3.40 r a n g e .  17).  Howeverthis  o f t h e NMR s p e c t r a o f the. a g l y c o n e o f  T  (Figure  ( T = 7.82 - 8.30).  group.  C and i t s a c e t a t e d e r i v a t i v e  further  T  an e t h e r o r an a l k y l - a r y l b o n d t o t h e a  c o n t a i n s one l e s s  fers  ( = 7.72),  show Compound C t o be s i m i l a r t o  o f the g u a i a c y l g l y c e r o l  Compound  than the acetate  acetate resonance  acetate resonances  characteristics  Compound B w i t h  13) r a t h e r  of the  on t h e a  r e v e a l e d no  the y protons  have  been  ( r = 5.65).  NMDR e x p e r i m e n t s  established  o f t h e a , and 3', a n d y ' p r o t o n s 1  8.15 a n d 6.37 r e s p e c t i v e l y .  the chemical  shifts,  t o o c c u r a t T. = 7-41,  The a c e t y l a t i o n  of.the  105.  aglycone while is  a shift  o f the y' proton  t h e a ' and 3' p r o t o n s  proof  the  produces  of the existence  aglycone  show no c h a n g e .  The e x i s t e n c e  aromatic  acetate  i n both  aglycone  acetate  spectra offers  An  AB q u a r t e t  derivatives diation the  acetate  evidence  to that reported  sidered proof  These  of the existence  through the n-propanol  side  and t h e  aliphatic  hydroxyl.  f o r the acetate  a t 8.18 ? i n t h e NMR  o f Compound C.  observations  upon  as r e p o r t e d  irra-  spectra of are con-  of the rhamnosidic  chain  one  of the existence  o f Compounds A and B, i s o b s e r v e d  o f the resonance  evidence  of only  the g l y c o s i d e acetate  linkage through t h i s  similar  This  of the n-propanol side chain i n  o f Compound C.  of the g l y c o s i d i c  t o 6.08T  linkage  f o r Compounds  A a n d B.  Mass s p e c t r u m Figure derivative acetate  and 621,  18 shows t h e mass s p e c t r u m  o f Compound C.  decomposition  speculated 660,  o f Compound C acetate.  structure.  The m o l e c u l a r  of the acetate i o n (M = 702) and  pathways a r e c o n s i s t e n t w i t h t h e Molecular  ion transitions  660 •> 600, 642 -> 600 , and 702 -> 642, a r e s u b s t a n t i a t e d by t h e e x i s t e n c e  composition  i n d i c a t e s one a r o m a t i c  observed  of metastable  546, 562, and 587, r e s p e c t i v e l y .  This  acetate  702 ->-  ions at  acetate deand one  106.  •,  u.*v  Figure  . vii.* •-  18.  "*aj.u  . W.H  .. .. w.u  .. vju.u  3UJ.V  ^{U.B  W.U  1VJ  3CJUV .  DbTUv .  /The mass s p e c t r u m o f t h e a c e t a t e ** o f Compound C.  WJ.fl  UWJ  WJ.V  derivative  107..  aliphatic  acetate.  The mass s p e c t r u m r e v e a l s  the ions  p r e v i o u s l y m e n t i o n e d f o r t h e rhamnose t r i a c e t a t e and i t s decomposition m/e  = 149, 222.  cleavage  molecular  These i o n s  C, f o l l o w e d  In  o f t h e m/e  considering  B t o Compound  may  ions  seen a t  o r i g i n a t e from t h e  of the speculated  are presented  structure  Two  possible  b e l o w w h i c h may be  = 222 and 1^9  the transformation  ions.  o f Compounds A  C by h y d r o l y s i s and t h e r e l a t e d  o f Compounds A and B, Compound  a guaiacyl  are also  by l o s s o f a c e t a t e .  ion structures  representative  data  Prominent  of the dioxane r i n g  o f Compound  and  products.  benzdioxane  derivative  NMR  C i s speculated  (XLVIII  or XLIX).  t o be  108...  OH  OH  X L V I I I  XLIX  T h e s e 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 f r o m Compound A f r o m an i n t e r m e d i a r y o f Compound B o r i g i n a t i n g  three d i s t i n c t stage,  pathways.  XLIX. with to  benzyl  S e c o n d , t h e 1,2 the subsequent  carbon  formation  form  along  methide  g r o u p may r e a c t w i t h t h e atom t o f o r m s t r u c t u r e o f Nimz  of a l k y l - a r y l  form the phenylcoumaran"XLIV.  react  i n the quinone  phenoxy' s h i f t  phenylcoumaran f o r m a t i o n , ly  First,  subsequently  the f r e e c a t e c h o l hydroxyl  resonance-stabilized  quinone methide  a c c o r d i n g t o the rearrangement  ( 7 1 ) w h i c h may  p r o p o s e d by Nimz  derived  Finally,  (71) ring  may  occur,  closure  instead of  r o t a t i o n may o c c u r  a b o u t t h e new-  f o r m e d a e t h e r bond t o p l a c e t h e c a t e c h o l h y d r o x y l i n  close proximity  t o the g carbonium i o n (formed  phenoxy t r a n s f e r ) w i t h  subsequent f o r m a t i o n  XLVTIIc The c h a r a c t e r i z a t i o n o f Compound tion  o f Compounds  i n the  of structure  A and t h e s p e c u l a -  B and C i m p l i e s t h e pathway o f Nimz  109. as  plausible  chemically It of  i n the A t o B to C i n t e r r e l a t i o n s h i p  and b i o s y n t h e t i c a l l y . should  a g a i n be n o t e d  s t r u c t u r a l formulae  tive  and a r e b a s e d  ther  to lignan  B and C a r e s p e c u l a -  on s p e c t r a l  also  data.  compounds g i v i n g  ethyl  acetate  to note t h a t  However, t h e  extract  of these  the three  Further  l i n k of  i n western r e d cedar. the exact  B and C.  the p o s i t i v e will  o f A.  i s a probable  establish  i n Compounds  other  elucidation  i f this  biosynthesis  research w i l l  presentation  o f A t o B t o C c a n s t r e n g t h e n t h e ob-  establish  a and 3 b o n d i n g s  the  the  b a s e d upon t h e known s t r u c t u r e  research w i l l lignin  that  f o r Compounds  solely  general relationship servations  both  dilignol  of the  The I s o l a t i o n o f  DSA r e a c t i o n  add v a l u a b l e  structures.  nature  Fur-  i n the  information to  It i s of interest  rhamnosides  exhibit  a  " m i s s i n g " m e t h o x y l g r o u p , common among t h e w e s t e r n r e d cedar heartwood  lignans  (e.g.,  XXXVIII  XXXVIII).  110 .  Lignin  Biosynthesis The  lation  into  results the r o l e  biosynthesis. dilignols in  of this investigation  lead  t o specu-  o f t h e i s o l a t e d compounds  i n lignin  The g l y c o s i d i c n a t u r e  suggests  a translocatory  the p o s s i b i l i t y process  of  i n t o the l i g n i n  catechol  gests  groupings  one p o s s i b l e  i n bark  aromatic  to  much b e y o n d l l o c a l lignin  T h u s , t h e i s o l a t e d " compound  incorporation  into  l e a f or branch  Assuming  that  translocation  lignols role  may m a i n t a i n  i n lignin  l a t e d may be r e p r e s e n t a t i v e tractive tural  a l t e r a t i o n but w i l l  destined  regarding  the d i l i g n o l s class  eventually  termediates  to lignin  serve  their iso-  o f exminor  struc-  as f r e e as.true i n -  formation.  The. o c c u r r e n c e  high y i e l d  w o u l d be  undergo f u r t h e r  S e c o n d , t h e d i l i g n o l s may  c l a s s may  glycosides.  precursors i s  of a defined  exist  sug-  i s l i m i t e d , the d i -  dilignols.  distinct  further  lignin.  First,  components w h i c h may  occurrence  environs, of  one o f two p o s i t i o n s  biosynthesis.  and p o l y m e r i -  The n o t e d (50)  lignin  o f the d i l i g n o l  ( p a r t i c u l a r l y dimeric)  unlikely.  of active  lignins  destination  involvement  hydrolysis,  molecule.  However, t r a n s l o c a t i o n  of t h e i r  to a site  f o r m a t i o n w i t h t h e i r subsequent zation  o f the i s o l a t e d  find  of the d i l i g n o l  rhamnosides  some s u b s t a n t i a t i o n  as a  i n their  i n l e a f t i s s u e s , and t h e i r u n u s u a l  chemical  111. structures  (compared t o o t h e r  The  high  yield  i n leaves  of the f l a v o n o i d g l y c o s i d e s logy  would p l a c e  dilignols). may  the d i l i g n o l  rhamnosides  anabolism with  tions.  such p h y s i o l o g i c a l r o l e  pation  o f t h e compounds  leaves. may  secondary  i n the leaves  in  to render  The is  to  of l i g n i n  dilignol  quire  chain  methylating highly  or disease  i n the  reactions  c a t a l y z e the  cleavage  o f the d i l i g n o l  ?:.- c h a r a c t e r  rhamno-  rhamnosides  are  to allow  Ac-  considered the  oxi-  r e a c t i o n e s s e n t i a l i n the Participation  of  i n wood l i g n i f i c a t i o n w o u l d r e -  of the f r e e  enzymes  of the d i l i g n o l  and p o l y p h e n o l i c s .  to lignin  improbable.  m i g h t be t h e p a r t i c i -  and p o l y p h e n o l i c s  o x i d a t i o n of the p r o p y l  polymerization  p h y s i o l o g i c a l func-  available for participation  coupling  rhamnosides  methylation  products  and p h e n y l p r o p a n e b i o s y n t h e s i s .  to l i g n i n  or r e d u c t i v e  formation  and  side  r e q u i r e phenylprop^rie.  dative  the  n-propyl  precursors  as end  or s e a l i n g - o f f a c t i v i t i e s .  unusual i n l i g n i n  tive  linkage  the aglycone  detoxification  injury  which would  of the unusual r h a m n o s l d i c side  Such an a n a -  i n p r o t e c t i o n mechanisms  I t i s possible that  occur  to that  i n t h e same t i s s u e .  of p h e n o l i c One  be compared  catechol hydroxyl  side  chain p r i o r  i n the accepted  grouping  to further  sense.  are a v a i l a b l e , the o x i d a t i v e  While step i s  112. The  foregoing discussion  the p a r t i c i p a t i o n i n wood l i g n i n locatory tural the  o f the  of the  intensively  dilignol  However, t h e  compounds and  c h a r a c t e r suggest  less  observed  formation.  nature  w o u l d seem t o p r e c l u d e  their  studied  rhamnosides  assumed  their  possible  non-trans-  unusual  struc-  participation  f o r m a t i o n of bark  or  in  leaf  lignin. Degradative tablished lignin  the  tent  and  (50)-  of t h i s  character contain  than that  aryl-aryl  study  i n the  shown a l o w e r  bonded  methoxyl  that'the d i l i g n o l  the  rhamnosides  dilignol  formation, contrast cambium.  they  to the  i n t h e wood.  lignin  Such l i g n i n  polymers,  participate  which  i s highly  must b'e s p e c i f i c a l l y  of the d i l i g n o l s Evidence  i n bark  in may  originate dilignols If  lignin  i n the i n n e r phloem, i n  o f wood l i g n i n  This discussion  tissues  cipation  do  Such  different  catechol hydroxyl grouping.  could originate origin  con-  rhamnosides  c o u p l i n g o r t h o t o the n - p r o p y l - s i d e c h a i n of free  red  c h a r a c t e r t h a n wood l i g n i n .  observed  the  es-  of western  act as.precursors to l i g n i n  a c t i v a t e d by  bark  has  leads to s p e c u l a t i o n s t u d y may  lignin.has  of c a t e c h o l groupings  (93)  u s i n g NMR  of bark  A specific  more a l i p h a t i c  evidence  from  occurrence  structure  cedar bark  examination  i n bark  p r e c u r s o r s i n the  speculative.  examined lignin  r e g a r d i n g the nature  and  The  f o r the  parti-  formation. formation of  leaf  lignin  thoroughly can  i s wanting.  It i s therefore  investigate this  t i s s u e before  be a t t r i b u t e d t o t h e d i l i g n o l  Scintillation-Chromatography The v a l i d a t i o n tion  low a c t i v i t y ,  is  described  lated  i n Table  any s i g n i f i c a n c e  rhamnosides.  i n e s t a b l i s h i n g combus-  technique  chromatographically  i n t h e measurement  separated  section.  Examination  compounds  The r e s u l t s  1 and a r e g r a p h i c a l l y d e p i c t e d  19, 20 and 21. ing  procedure  i n a previous  to  Technique  as a s u p e r i o r a n a l y t i c a l  of  necessary  of the data  are  tabu-  i n Figures  y i e l d e d the f o l l o w -  results. A comparison of Figures 19 and 20 reveals a much smaller  effect  of external  combusted  samples.  significant reagent  component  effects  The s c r a p e d - s u s p e n d e d of both  upon t h e r e c o r d e d The l a r g e s t e f f e c t  when t h e c h r o m o g e n i c  upon a c t i v i t y  spray  external  activity  this  spray,  20 p e r c e n t  combustion  show  and  When o n l y  followed  i n detectable  and s c i n t i l l a t i o n  spray  ratio. ratio  appears  subsequent  by c a r e f u l  activity.  counting  was  2'ug o f q u e r c e t i n  spots,  from the chromatographic p l a t e , produced over  samples  b i s - d i a z o t i z e d benzidine  on t h e c h r o m a t o g r a p h i c  d e t e c t i o n with  i n the  component  on t h e a c t i v i t y  u s e d as a d e t e c t i n g r e a g e n t . were p r e s e n t  ratio  scraping  a reduction of In c o n t r a s t ,  of that  same  sample  Figure  19.  . Amount o f e x t e r n a l compound, y g The e f f e c t o f an e x t e r n a l q u e n c h i n g compound the d e t e r m i n a t i o n o f a c t i v i t y o f s c r a p e d chromatographic samples.  - t on  1.11  o  1.00,  •H -P aj  U  $090 •H > •H -P O  <  0.8$  0.70  B  5  amount F i g u r e 20  The the  -H  Compound a d d e d Quercetin Pinosylvin Quercetin Phenylalanine Quercetin  Spray B a r t o n ' s Reagent No bis-diazo.benz. Ninhydrin No  10  o f e x t e r n a l compoundj ug  e f f e c t of. an e x t e r n a l - q u e n c h i n g compound' on d e t e r m i n a t i o n o f a c t i v i t y o f combusted chromatographic samples.  15  116.  o C  CD •H O •H CM  ^  0.90  cd •H cd -P -p co  o o c  •H  o  c  •H cd -P cd cd H H >3 •H -p •H • O  0.80  CO  Xi 0 >  CD co  o  0.70  1.0  Area .Figure  21  •2.0  of chromatographic  spot,  cm'  The e f f e c t o f c h r o m a t o g r a p h i c s p o t s i z e on l i q u i d s c i n t i l l a t i o n counti n g of combustion samples.  117. shows a l o s s activity. gent, tion  solution^  nique  than  10 p e r  When q u e r c e t i n was  f o l l o w e d by  28 p e r  20  of l e s s  cent.  cent i n the d e t e c t a b l e  detected with Barton's  s c r a p i n g and  s u s p e n s i o n i n the  the r e d u c t i o n i n a c t i v i t y Comparative  show l o s s e s  results  of l e s s  than  f o r the  5 per  combustion  The  sample  results  shows an  the r e s u l t counter sis  of the  distinguish  results  from  of combustion  luminescence  the  noted  f o r 2 ug  of  explain  o f c h e m i c a l and Figure  fect  of spot  tion  technique.  21  size  of less  combustion  reduction  i n spot  be  than  Analy-  sample 5%.  Chemo-  activity  due  physical  i s a summary  technique minimizes t o the  size  from  seen  0.6  external i n -  of data r e l a t e d  upon c o u n t i n g e f f i c i e n c y I t can be  from  cm  of counting e f f i c i e n c y  the  quenching.  the  t o the  f o r the  figure  2 increase  cent  scintillation  increase i n  of d e t e c t a b l e r a d i o a c t i v i t y  fluences  and  quercetin.  In g e n e r a l , the loss  i n c r e a s e must  f o r t h i s , same  ratio  also  per  true r a d i o a c t i v i t y .  shows r e d u c t i o n s i n a c t i v i t y may  tech-  spraying.  o f 15-20  o f chemolumi'nescence, w h i c h t h e  cannot  7 to  scraped-suspension  increase i n activity sample.This  from  F i g u r e s 19  f o r p i n o s y l v i n without  when a n a l y z e d as a ^ s c r a p e d  scintilla-  combustion  cent.  show a v i v i d _ c o m p a r i s o n between t h e  and  ranged  rea-  ef-  combus-  that  an  2 t o 2 cm  from  82%  resulted to  75%.  in a  Total  118.  weight plate  data  showed t h a t  busted spots  f o r the spot.removed a maximum  efficiently  from the  o f 7 mg p e r s p o t  and t h e optimum w e i g h t  was. i n t h e r a n g e  chromatographic  o f 4 t o 5 mg.  c o u l d be com-  of  chromatographic  Such a  chromatographic 2  spot  weight  including  was r e p r e s e n t a t i v e o f a s p o t  the a d d i t i o n of the c e l l u l o s e  The  combustion  moved f r o m t h i n advantage  layer  i n counting  scraping-suspension removed mizes  samples which  samples  cellulose  method.  sample  The sample  transfer.  isolation  count  using tal  compared t o t h e  t o be c o m b u s t e d i s handled,  and m i n i -  In c o m p a r i s o n ,  of a p a r t i c u l a t e  electrostatic  scraped  sample  The  p r o p e r t i e s which  scraped fur-  handling.  Thus, the combustion to  easily  solution.  a substantial  be s c a t t e r e d d u r i n g t r a n s f e r .  often exhibit  t h e r hamper  samples  1 cm  spots r e -  plates offers  low a c t i v i t y  involve tedious  can e a s i l y  nitrate  of the chromatographic  from t h e p l a t e c l e a n l y ,  losses during  approximately  phenolic  chromogenic  technique  compounds, w h i c h spray  reagents,  e r r o r associated with  offers  the a b i l i t y  c a n o n l y be d e t e c t e d while  limiting  experimen-  sample p r e p a r a t i o n , h a n d l i n g , and  counting. This  combustion  technique  tage  i n terms o f e x p e r i m e n t a l  tive  precursor  inhibition  into  also provides  design..  an a d v a n -  I t enables  metabolizing  radioac-  tissues, f o l -  119. lowed ing  by  extraction,  of l a b e l l e d  chromatographic, i s o l a t i o n  chromatographic  When a u t o r a d i o g r a p h y i s u s e d results  w o u l d n o t be samples.  counting  of chromatographic  strips,  constant  specific  are e i t h e r  activity The  to  thin  present or long low  layer  limitations  t e c h n i q u e s , s u c h as t h i n  activity,  activity  cellulose  o r sample  a s s o c i a t e d w i t h chromogenic  gated.  A l t h o u g h t h e method i s a p p l i e d  plates,  i t i s possible  (e.g., polyamide, not  applicable  aromatic tablished  the  level  plates  and may  s p r a y s not only  to  limited have  investi-  cellulose  combusted  The  combustion  silica  technique i s  g e l chromatography  However, t h e  i n removing  be-  cellulose n i -  chromatographic  spots  priorjysuspension counting.  Study  metabolic a c t i v i t y  promising tissue  by  c a n be  solution  The  limited  to  s u p p o r t s which  trate  Precursor Feeding  window  applications  i t i s non-combustible.  gel plate  f o r low  future  layer  i s useful  the  find  cause  from the s i l i c a  i t may  dextrose).  to t h i n  days.  isolation  t e c h n i q u e was  chromatographic  on o t h e r c h r o m a t o g r a p h i c  of  e x p e r i m e n t a l workups  combustion  that  count-  sequence,  f o r a month o r two  activity  of  Other  spots i n a matter  in a similar  available  and  f o r the study  precursors.  o f l e a v e s makes them a v e r y , of i n c o r p o r a t i o n  o f known  Phenylalanine represents a well  aromatic p r e c u r s o r which,  es-  when a d m i n i s t e r e d t o  120 .  actively  metabolizing tissues, w i l l  formation to  of aromatic  the g l y c o l y t i c  phenylalanine studies  compounds w i t h  pathway.  has  of l i g n i n  into  t h e stem p r o d u c e s h i g h e r  tion  into  lignin  are  subsequently tic  area.  to i n i t i a t e  alter  compounds.  separate  radioactive incorpora-  Implantation  secondary  wound r e a c t i o n s w h i c h  (29) has n o t e d  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  known l i g n i n  through  species.  I t i s evident  methods a l l o w  the e f f i c i e n t  w h i c h may be a l t e r e d mate f o r m a t i o n suggested leaves  for.lignin  tabolism during  precursors  feeding.  of infusion of coniferous  infusion  of l i g n i n  tissue  i n t h e wood.  examination  of radioactively  infusion  uptake  i n the l e a f  of lignin  the c l o s e r  the leaves  t h e r e f o r e , that  o f aroma-  the formation  of  precursors  pre-  techniques  t h e pathway t o t h e f o r m a t i o n  Freudenberg  (57)  precur-  t h e i m p l a n t a t i o n o f t h e same  the cambial  considered  two  actively  of r a d i o a c t i v e l i g n i n  sors  into  into  into  and (2) i m p l a n t a t i o n . K r a t z l  shown t h a t t h e i n f u s i o n  cursors  that  Incorporation  precursors  generally f a l l s  (1) i n f u s i o n ,  than  reasons  of western r e d cedar.  Administration metabolizing tissues  i n the  a minimum r e v e r s i o n  I t i s f o r these  was c h o s e n f o r a r o m a t i c  i n the leaves  classes:  participate  prior These  feeding  precursors to the u l t i observations  o f western r e d cedar  w h i c h would  show a c t i v e  l a b e l e d aromatic  precursors  me-  121.  Preliminary, i n f u s i o n feeding that  the  leaves  late  radioactive phenylalanine  compounds. acetate  of western red  Autoradiography  extract  of the  to  label  Four major spots  on  chromatogram d i s p l a y e d  established  to  assimi-  form r a d i o a c t i v e  preliminary  time. the  c e d a r were a b l e  (Figure  showed i n c o r p o r a t i o n o f t h e  experiments  22)  of the  feeding  gross  i n a f o u r hour  orange  ethyl  experiment feeding  o c c u r r i n g b e t w e e n Rf-i 0.1 an  phenolic  and  0.45  c o l o r r e a c t i o n when 14  sprayed with nine  Into  later the  DSA.  two  The  of these  validated thin Diazotized  tecting  reagent  leaf  color ter  C-L-phenylala-  compounds  A and  examined i n a t e n  nique.  the  i n c o r p o r a t i o n o f U-  reactions  making i . i t  sulfanilic  was  experiment  since  1).  The  removal  " necessary  that  quate  spray  the  chromatographic  representative feeding to ten  spots  use  from  I t was  DSA  combustion. hour.feeding  The  as  a  efficiency  to  cellulose nitrate.  the  plate,  B i n the to  combusted  tabulated  of spots  leaf  size  samples  i n Table  ade-  The  ten hour  smaller  of  allow  to removal  af-  some  chromatographic  prior  division  are  distinct  c e l l u l o s e l a y e r be  to dry  r e s u l t s of  experiment  the  de-  in  did entail  also necessary  with  required  counting o f DSA  the  o f Compounds A and  experiment  used  i t gave more  somewhat h i g h e r  spot  f o r the  (DSA)  tech-  of b i s - d i a z o t i z e d benzidine  c o n s i s t e n t .thickness. time  acid  was  utilizing  combustion  (Table  i n the  experiment  B)  l a y e r chromatographic  and  combustion  problems  hour f e e d i n g  i n place  feeding  (designated  3  prior in and  the  122:  Figure  2 3 .  A u t o r a d i o g r a m 'of t h e g r o s s e t h y l a c e t a t e s o l u b l e s from U - 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 ) . 1 4  123. graphically  depicted  Table cedar is  leaves  3 reveals  that  time  cent  i n h o u r 1, w i t h  This  precursor rate  truction might  cut western r e d .  i s shown i n F i g u r e  to a level  o f uptake  optimize  into  the metabolizing  2 3 which  continuous  effectively  precursor  i n the l e a v e s .  also  the aromatic  10 h o u r s .  minimize  the des-  by m i c r o - o r g a n i s m s  This  during  pre-  of the l a -  after  rapid rate  the i n t r o d u c t i o n of the aromatic areas  pre-  shows 16 p e r  uptake  o f 37 p e r c e n t  should  of the aromatic  be p r e s e n t  phenylalanine  i n c o r p o r a t i o n of the l a b e l e d  cursor with  beled  floating  method o f a d m i n i s t e r i n g  The p e r c e n t  uptake  23 and 24.  i n a r a d i o - a c t i v e s o l u t i o n of  an e f f i c i e n t  cursor.  i n Figures  a period  which  should  precursor  of normal  me-  tabolism. The  level  A and B d u r i n g depicted of  of a c t i v i t y  i n c o r p o r a t i o n Into  1, 3, 5, and 10 h o u r f e e d i n g  i n Table  3 and F i g u r e  0.15% t o 0.40% Were o b s e r v e d  nine  i n t o Coumpounds  cate  that  Compound  A and B.  than  pounds showed a r e d u c t i o n This  associated sequent  reduction with  Incorporation  Examination  in activity  a r e needed  phenylala-  of the data  does Compound A.  i n activity  levels  indi-  a l a r g e r p o r t i o n of the  after  f u r t h e r metabolism  experiments  periods i s  for radioactive  B incorporates  available.radioactivity  ing.  24.  Compounds  after  Both  5 hours  comfeed-  5 h o u r s may be  o f t h e compounds.  to c l a r i f y  this, p o i n t .  Sub-  t  10  124  90  080 70 60 50 40 30 20 10  3  1  5  7  9  11  Feeding  Figure  time, h r s . 14 U p t a k e o f UC - L - p h e n y l a l a n i n e by • w e s t e r n r e d cedar l e a v e s .  23.  * B  3  5  Feeding  Figure  24.  7  Compound A Compound B  9  11  time, h r s . 14 I n c o r p o r a t i o n o f UC-L-phenylalanlne i n t o compounds A and B i n t h e l e a v e s . of western r e d cedar.  125. The two  results  important  of t h i s  points.  First,  lanine  is actively  in  leaves of western  the  incorporation  into  kinetic.studies glycosides  and  may  f e e d i n g experiment i t i s apparent  that  incorporated into,dilignol red cedar.  subsequently  n i n p r e c u r s o r s i n the  relate  .explain-" t h e  leaves of western  that the  phenyla  rhamnosides  Second, the  these p r e c u r s o r s i s such biosynthetically  revealed  level future  dilignol  formation of red  of  cedar.  lig-  126.  CONCLUSION  The  ethyl acetate  hol  extracts  The  purpose o f the  terize  nosides sure  of f r e s h western  active  in lignin  red  cedar  of these  alcoexamined.  t o i s o l a t e and  charac-  serve  dilignol  techniques.  dilignol  as  rham-  the a p p l i c a t i o n of  gel f i l t r a t i o n  formation  l e a v e s was  Three such  have b e e n i s o l a t e d t h r o u g h  metabolic  of methyl  d i l i g n o l s , w h i c h may  formation.  c h r o m a t o g r a p h y and  rapid  portion  i n v e s t i g a t i o n was  metabolically  precursors  soluble  pres-.  The  rhamnosides . 14  was  shown t h r o u g h  lanine  their incorporation  i n a leaf infusion'feeding  o f U-  C-L-phenyla-  experiment.  Of the three d i l i g n o l s i s o l a t e d , the d i l i g n o l rhamnoside 1-(3'-methoxy-4'-hydroxyphenyl)-2-0-1'-[2'-hydroxy4'-(propane-3"'-O-a-L-rhamnoside)phenyl]-propane-l,3 obtained i n high y i e l d (0.15$), and was  diol  was  characterized success-  f u l l y by chemical degradation i n conjunction with NMR  and mass-  s p e c t r a l techniques. The lated  i n lower  structures accumulated the  other  two  yields  were n o t  dilignol  r h a m n o s i d e s have b e e n  ( l e s s than  completely  c h e m i c a l , NMR,  compounds were f o u n d  and t o be  0.05$ e a c h ) ,  characterized.  their Based  mass s p e c t r a l d a t a , closely related  iso-  upon  however,  homologs  127.  of the c h a r a c t e r i z e d ship  dilignol  rhamnoside.  a p p e a r e d t o be a s s o c i a t e d  active  benzylhydroxyl  fied  much o f t h e e v i d e n c e  Chromatographic other  similar  ponents  with reactions  and c a t e c h o l  p h e n y l c o u m a r a n and g u a i a c y l  evidence  compounds.  i n the leaves  This  groups.  benzdioxane  collected suggested A total  relation-  of ther e Speculative  structures  satis-  f o r t h e s e two compounds. the existence  yield  of several  f o r a l l such  com-  o f w e s t e r n r e d c e d a r may be as  high  as 0.5%. The  characterized  several novel previously reported  structural  dilignol  characteristics  reported  dilignols  including:  occurrence  of a free  dilignol  unusual occurrence  of the glycoside  (3)  unreported  the previously  nin precursor phatic  aryl in  unreported  rather ether  than  t h e common  i n a potential through  an  propyl  alias a  side  chain  character.  dilignol  infusion  study  revealed  r h a m n o s i d e , and i t s s u s -  pected' phenylcoumaran homolog, i n c o r p o r a t e d  0.3% and 0.4%  14 U-  lig-  guaiacylglycerol-guaiacyl-3-  radioactive  the c h a r a c t e r i z e d  (2) t h e  guaiacylglycerol-catechol-3-aryl  a compound o f l i g n i n  that  (1) t h e f i r s t  of the d i l i g n o l  and, (5) t h e u n u s u a l s a t u r a t e d  A specific  i n r e l a t i o n to  as an a - L - r h a m n o s i d e ,  linkage  (4) t h e e x i s t e n c e  possessed  glycoside,  existence  of the g l y c o s i d i c  hydroxyl,  previously ether  rhamnoside  C-L-phenylalanine within  a t e n hour f e e d i n g  period.  128.  The  degree  tained nique  of i n c o r p o r a t i o n i n t o  through f o r the  samples, lulose  the  application  efficient  separated  plates.  low  samples.  The  of the  role was  lignin  high  yield  leaf  tissue  the  leaf  Future  discussed.  of these  indicated roles  on  thin  of the  be  upon t h e i r i n the  t h a t t h e y may  tech-  layer  in  analysis,  lignin  that  important  the bark  and  structures.c.:'The  actively also  cel-  techniques  suggested  l e a v e s may  compounds  ob-  radioactivity  rhamnosides  I t was  p r e c u r s o r s , based  physiological in  validation  dilignol  g l y c o s i d e s of the  leaf  o f low  to- c o m p a r a b l e methods o f  activity  biosynthesis dilignol  counting  A separate  compounds was  o f a newly d e v e l o p e d  chromatographically  showed i t s s u p e r i o r i t y for  these  metabolizing  fulfill  important  (e.g. disease' p r o t e c t i o n , wound-sealing)  tissues.  Research The  observed  d i s c o v e r y of the  metabolic  dilignol  participation  future  investigations.  future  research with  rhamnosides  suggests  many  F i v e major p r o p o s a l s  these  compounds a r e  and  their  important  relating  to  as f o l l o w s :  The characterization of the two remaining d i l i 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 r e l a t i o n s h i p 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 o f o t h e r d i l i g n o l a n d / o r o l i g o l i g n o l s w h i c h may be p r e s e n t i n these l e a v e s .  Further b i o s y n t h e t i c l a b e l l i n g studies with more p r e c i s e p r e c u r s o r s t o a s c e r t a i n s p e c i f i c pathways i n t h e b i o g e n e s i s o f t h e d i l i g n o l s and l i g n i n . 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