UBC Theses and Dissertations

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UBC Theses and Dissertations

A high performance liquid chromatographic study of post-stress isoflavonoid accumulation in Phaseolus… Carlson, Robert Eric 1979

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A HIGH PERFORMANCE LIQUID CHROMATOGRAPHIC STUDY OF POST-STRESS ISOFLAVONOID ACCUMULATION IN PHASEOLUS VULGARIS AND PISUM SATIVUM by ROBERT ERIC CARLSON B . A . , U n i v e r s i t y o f M i n n e s o t a - D u l u t h , 1972 M . S c , U n i v e r s i t y o f B r i t i s h C o l u m b i a , 1976 A T H E S I S SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF a c c e p t t h i s t h e s i s a s c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d DOCTOR OF PHILOSOPHY 9 i n THE FACULTY OF GRADUATE STUDIES D e p a r t m e n t o f C h e m i s t r y THE UNIVERSITY OF B R I T I S H COLUMBIA J u l y , 1979 R o b e r t E r i c C a r l s o n , 1979 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e Head o f my D e p a r t m e n t o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p a r t m e n t o f The U n i v e r s i t y o f B r i t i s h C o l u m b i a 2075 W e s b r o o k P l a c e V a n c o u v e r , C a n a d a V6T 1W5 D a t e >E-6 B P 75-51 1 E ii ABSTRACT It has been observed in some plants that their post-infectional response to a potential pathogen leads to the synthesis and accumulation of compounds which may be significant in the processes of disease resistance. Consequently, a high performance liquid chromatographic procedure has been developed which will separate complex mixtures of Phaseolus vulgaris (green bean) and Pi sum sativum (garden pea) iso-flavonoids to give a chromatographic fingerprint of the post-stress metabolic response of these plants. This system has been used to determine the time course of the accumulation of the major de novo fungitoxic metabolites (phytoalexins) of abiotically stressed plants. These accumulation rate studies represent one of the most significant applications of the HPLC procedure because the quantification of a selected number of components of a mixture by other chromatographic methods can be, at best, tedious. The HPLC procedure has also been applied to the characterization of a number of other post-stress metabolites from P_. sativum. This study led to the identification of a previously unreported 2'-methoxychalcone (4,4'-dihydroxy-2'-methoxychalcone) and its benzylstyrene analogue (4-hydroxy-(4'-hydroxy-2'-methoxybenzyl)styrene) as well as a number of compounds which have been observed from other plant sources. The characterization of benzylstyrenes from P_. sativum is particularly interesting because until now these compounds have only been described from heartwoods. Consequently, their identification as de novo metabolites will allow an in vivo investigation into their biosynthesis and potential role in disease resistance. To facilitate i i i further studies on the chemistry and biochemistry of these compounds their M S , NMR and UV properties, as they pertain to the identification of structure, have been determined. A final aspect of this study was the qualitative evaluation of the response of P_. sativum to a pathogenic and a non-pathogenic fungus. These results illustrated that the plant's net response to the two fungi was similar. However, a more important aspect of the study was its demonstration that the HPLC procedure could be used to determine the effects of in vivo fungal metabolism on the plant's accumulation of isoflavonoids. i v TO j o a n d e r i c f o r f a i t h a n d e r i n e l i z a b e t h f o r h o p e V CONTENTS SECTION PAGE Abstract •. . ii Dedication iv Contents v List of Tables vii List of Figures viii List of Schemes xi Abbreviations and Definitions xii Acknowledgements xiv I. INTRODUCTION 1 Post-infectional metabolism in plants and the phytoalexin theory 1 Rationale for the choice of a phytoalexinic model system 5 Flavonoid structure and distribution 6 Flavonoid biosynthesis 10 II. DEVELOPMENT OF A GENERAL PROCEDURE FOR THE CHROMATOGRAPHIC RESOLUTION OF THE P. SATIVUM AND P. VULGARIS ISOFLAVONOIDS . 13 HPLC background 14 Isoflavone chromatography 15 Additional model studies 18 Application of the chromatographic procedure to an isoflavone extract 26 III. ANALYSIS OF THE RESPONSE OF P. SATIVUM AND P. VULGARIS TO ABIOTIC AND BIOTIC FORMS OF STRESS 31 A. The phytoalexinic response 31 Phytoalexin chromatography 31 Analysis of stressed and unstressed plants ... 38 vi R a t e o f a c c u m u l a t i o n s t u d i e s 4 6 EL I d e n t i f i c a t i o n o f t h e P , s a t i v u m m e t a b o l i t e s . . . . 51 1 , M e t a b o l i t e i d e n t i f i c a t i o n a n d s y n t h e s i s 51 I s o l a t i o n a n d i d e n t i f i c a t i o n . . . 51 S y n t h e s i s 55 2 , A d d i t i o n a l d a t a f o r t h e s t r u c t u r a l a n a l y s i s o f t h e b e n z y l s t y r e n e s 60 NMR a n a l y s i s 60 M a s s s p e c t r a l a n a l y s i s 6 5 UV s p e c t r o s c o p y a n d t h e l o c a t i o n o f t h e d o u b l e b o n d 73 C , The r e s p o n s e o f P i sum s a t i v u m t o p a t h o g e n i c a n d n o n - p a t h o g e n i c f u n g a l s t r e s s 81 D, A d i s c u s s i o n o f P i sum s a t i v u m s t r e s s m e t a b o l i s m . . . 87 1 , P h y t o c h e m i c a , ! a s p e c t s 87 B i o s y n t h e s i s 87 Chemotaxonomy 9 6 2 , P h y t o p a t h o l o g i c a l a s p e c t s , , 99 I V , CONCLUSION 102 V , EXPERIMENTAL , , , . , 104 A , G e n e r a l m e t h o d s 104 B , G r o w t h , s t r e s s a n d e x t r a c t i o n o f p l a n t s 106 G r o w i n g c o n d i t i o n s . 106 S t r e s s p r o c e d u r e s 107 E x t r a c t i o n o f p l a n t m a t e r i a l s 108 C , C h r o m a t o g r a p h y 110 D, S y n t h e s i s 116 BIBLIOGRAPHY , , , 128 APPENDIX , 136 v i i L I S T OF TABLES TABLE PAGE I S e l e c t e d members o f t h e L o t o i d e a e 9 I I The i d e n t i f i e d m e t a b o l i t e s o f s t r e s s e d P i s u m s a t i v u m . . 53 I I I The b e n z y l s t y r e n e s e r i e s 62 IV B e n z y l s t y r e n e NMR d a t a 63 V B e n z y l s t y r e n e mass s p e c t r a l d a t a 67 V I The r e l a t i v e i n t e n s i t i e s o f t h e b e n z y l s t y r e n e Cy f r a g m e n t . 69 V I I B e n z y l s t y r e n e UV s p e c t r a l d a t a 78 V I I I UV d a t a o f a s e l e c t e d s e r i e s o f t r a n s - p r o p e n y l b e n z e n e s . 79 IX T y p i c a l l e v e l s o f m e t a b o l i t e a c c u m u l a t i o n i n c o p p e r ( I I ) c h l o r i d e s t r e s s e d P_. s a t i v u m 115 v i i i L I S T OF FIGURES FIGURE PAGE 1 . S e l e c t e d e x a m p l e s o f t h e f u n g i t o x i c p r o d u c t s o f n o r m a l b i o s y n t h e s i s 2 2 . S e l e c t e d e x a m p l e s o f t h e f u n g i t o x i c p r o d u c t s o f de n o v o b i o s y n t h e s i s 3 3 . S k e l e t a l s t r u c t u r e s a n d n u m b e r i n g s c h e m e s o f t h e m a j o r f l a v o n o i d p h e n o l i c s w h i c h a r e r e l a t e d t o p t e r o c a r p a n b i o s y n t h e s i s 8 4 . C h a l c o n e / f l a v a n o n e b i o s y n t h e s i s f r o m p h e n y l a l a n i n e . . 11 5 . The b i o s y n t h e t i c r e l a t i o n s h i p s o f t h e p t e r o c a r p a n r e l a t e d i s o f l a v o n o i d s 12 6 . S e p a r a t i o n o f 4 ' , 6 , 7 - t r i h y d r o x y i s o f l a v o n e a n d 4 ' , 5 , 7 -t r i h y d r o x y i s o f l a v o n e on a r e v e r s e p h a s e c o l u m n . . . . 17 7 . G r a d i e n t c h r o m a t o g r a m o f s e l e c t e d i s o f l a v o n e s 19 8 a . G r a d i e n t c h r o m a t o g r a m o f 7 - h y d r o x y - 4 ' - m e t h o x y - a n d 7 - h y d r o x y - 3 ' , 4 ' - m e t h y l e n e d i o x y i s o f l a v o n e 21 8 b . I s o c r a t i c c h r o m a t o g r a m o f 4 ' , 7 - d i m e t h o x y - a n d 7 - m e t h o x y - 3 ' , 4 ' - m e t h y l e n e d i o x y i s o f l a v o n e 22 9 . G r a d i e n t c h r o m a t o g r a m o f 2 ' , 4 , 4 ' - t r i h y d r o x y c h a l c o n e . . 24 1 0 . G r a d i e n t c h r o m a t o g r a m o f s e l e c t e d c h a l c o n e s 25 1 1 . G r a d i e n t c h r o m a t o g r a m o f a G_. max e x t r a c t 28 1 2 . I s o c r a t i c c h r o m a t o g r a m o f a G. max h y d r o l y z a t e . . . . 30 1 3 . The m a j o r p h y t o a l e x i n s o f P_. s a t i v u m a n d P_. v u l g a r i s . 32 1 4 a . G r a d i e n t c h r o m a t o g r a m o f 4 - h y d r o x y - 2 , 3 , 9 - t r i m e t h o x y - , 3 - h y d r o x y - 2 , 9 - d i m e t h o x y - a n d 2 , 3 , 9 - t r i m e t h o x y p t e r o c a r p a n . 35 1 4 b . I s o c r a t i c c h r o m a t o g r a m o f 4 - h y d r o x y - 2 , 3 , 9 - t r i m e t h o x y - , 3 - h y d r o x y - 2 , 9 - d i m e t h o x y a n d 2 , 3 , 9 - t r i m e t h o x y -p t e r o c a r p a n 36 1 5 . G r a d i e n t c h r o m a t o g r a m o f p h a s e o l l i n , p h a s e o l l i n i s o -f l a v a n a n d k i e v i t o n e 37 1 6 . G r a d i e n t c h r o m a t o g r a m o f t h e h y d r o l y t i c e x t r a c t o f u n s t r e s s e d P_. s a t i v u m 39 1 7 . G r a d i e n t c h r o m a t o g r a m o f t h e e t h a n o l i c e x t r a c t o f u n s t r e s s e d ( A ) a n d s t r e s s e d ( B ) P . s a t i v u m 41 i x 1 8 . G r a d i e n t c h r o m a t o g r a m o f t h e e t h a n o l i c e x t r a c t o f u n s t r e s s e d ( A ) a n d s t r e s s e d ( B ) P_. v u l g a r i s 4 3 1 9 . I s o c r a t i c c h r o m a t o g r a m o f t h e p t e r o c a r p a n r e g i o n o f a s t r e s s e d P_. s a t i v u m s a m p l e 4 5 2 0 . T i m e c o u r s e s e q u e n c e i s o c r a t i c c h r o m a t o g r a m s o f c o p p e r ( I I ) c h l o r i d e s t r e s s e d P_. v u l g a r i s 4 8 2 1 . The r a t e o f p h a s e d 1 i n a n d p h a s e d 1 i n i s o f l a v a n a c c u m u l a t i o n i n P.. v u l g a r i s 49 2 2 . The r a t e o f p i s a t i n a c c u m u l a t i o n i n P_. s a t i v u m . . . . 50 2 3 . A r e p r e s e n t a t i v e g r a d i e n t c h r o m a t o g r a m o f t h e i d e n t -i f i e d m e t a b o l i t e s o f P_. s a t i v u m 52 2 4 . G r a d i e n t c h r o m a t o g r a m o f 2 ' , 4 ' - d i h y d r o x y b e n z y l s t y r e n e a n d i t s m e t h y l e t h e r d e r i v a t i v e s 61 2 5 . 270 MHz NMR s p e c t r u m o f 4 - m e t h o x y - ( 2 1 , 4 ' - d i m e t h o x y -b e n z y l ) s t y r e n e 64 2 6 . The mass s p e c t r u m o f 4 1 - h y d r o x y b e n z y l s t y r e n e 68 2 7 . The m e c h a n i s m s o f r a d i c a l s i t e m i g r a t i o n / h y d r o g e n r e a r r a n g e m e n t 70 2 8 . The mass s p e c t r a l f r a g m e n t a t i o n o f 4 ' - m e t h o x y b e n z y l -s t y r e n e 71 2 9 a . G r a d i e n t c h r o m a t o g r a m o f t h e r u t h e n i u m t e t r o x i d e r e a c t a n t s a n d p o t e n t i a l a l d e h y d e p r o d u c t s 74 2 9 b . G r a d i e n t c h r o m a t o g r a m o f t h e r u t h e n i u m t e t r o x i d e c l e a v a g e p r o d u c t o f s a t i v a s t y r e n e 75 3 0 . The UV a b s o r p t i o n s p e c t r a o f 2 ' , 4 1 - d i h y d r o x y b e n z y l -s t y r e n e a n d 4 - h y d r o x y - ( 2 ' , 4 ' - d i h y d r o x y b e n z y l ) s t y r e n e . 76 3 1 . G r a d i e n t c h r o m a t o g r a m s o f t h e e t h a n o l i c e x t r a c t o f M. f r u c t i c o l a a n d F_. s o l a n i p i s i s t r e s s e d P_. s a t i v u m 4 8 h o u r s a f t e r i n o c u l a t i o n 82 3 2 . G r a d i e n t c h r o m a t o g r a m s o f t h e e t h a n o l i c e x t r a c t o f M . f r u c t i c o l a a n d F_. s o l a n i p i s i s t r e s s e d P_. s a t i v u m 96 h o u r s a f t e r i n o c u l a t i o n 8 3 3 3 . Q u a n t i t a t i v e a s s e s s m e n t o f M. f r u c t i c o l a a n d F_. s o l a n i p i s i s t r e s s e d P_. s a t i v u m 8 4 3 4 . A p a r t i a l c h r o m a t o g r a m o f FSP s t r e s s e d P_. s a t i v u m 10 d a y s a f t e r i n o c u l a t i o n , 8 6 3 5 . The r a t e s o f m e t a b o l i t e a c c u m u l a t i o n i n c o p p e r ( I I ) X c h l o r i d e s t r e s s e d P_. s a t i v u m ; . . . . 8 8 3 6 . B i o g e n e t i c r e l a t i o n s h i p s o f t h e o b s e r v e d i s o f l a v o n o i d m e t a b o l i t e s o f P i sum s a t i v u m 8 9 3 7 . A l t e r n a t e p a t h w a y s o f b e n z y l s t y r e n e b i o s y n t h e s i s . . . 92 3 8 . P o s s i b l e b i o g e n e t i c r e l a t i o n s h i p s o f t h e IP. s a t i v u m s t y r e n e a n d 2 ' - m e t h o x y c h a l c o n e m e t a b o l i t e s 97 3 9 . G r a d i e n t c h r o m a t o g r a m o f p o s s i b l e P_. s a t i v u m s t y r e n e a n d 2 ' - m e t h o x y c h a l c o n e b i o s y n t h e t i c p r e c u r s o r s . . . . 98 4 0 . P a r t i a l c h r o m a t o g r a m s o f M. f r u c t i c o l a (A)! a n d F. s o l a n i p i s i ( B ) s t r e s s e d P_. s a t i v u m 6 d a y s a f t e r T n n o c u l a t i o n 100 4 1 . T h e s t a n d a r d g r a d i e n t I l l 4 2 . The g r a d i e n t m i x e r 112 x i L I S T OF SCHEMES SCHEME PAGE 1 . O u t l i n e o f i s o f l a v o n e s y n t h e s i s 56 2 . S y n t h e s i s o f 4 , 4 ' - d i h y d r o x y - 2 ' - m e t h o x y c h a l c o n e . . . . 58 3 . B e n z y l s t y r e n e s y n t h e s i s 59 4 . R u t h e n i u m t e t r o x i d e c l e a v a g e o f s a t i v a s t y r e n e a n d i s o -s a t i v a s t y r e n e 77 5 . C i n n a m y l a l c o h o l / p h e n o l c o n d e n s a t i o n p r o d u c t s 94 6 . F o r m a t i o n o f a q u i n o n e - t y p e p r o d u c t f r o m s a t i v a s t y r e n e . 101 x i i ABBREVIATIONS AND DEFINITIONS b r E E t FSP FT HPLC IR i s o c r a t i c Me MDC m. p . MF MS NMR OAc OMe p a t h o g e n PE p h y t o a l e x i n r a c e s f s o l u t e s t r e s s b r o a d d i e t h y l e t h e r e t h y l F u s a r i u m s o l a n i f . s p . p i s i ; a p e a p a t h o g e n F o u r i e r - t r a n s f o r m h i g h p e r f o r m a n c e l i q u i d c h r o m a t o g r a p h y i n f r a r e d a s o l v e n t s y s t e m o f c o n s t a n t c o m p o s i t i o n m e t h y l m e t h y l e n e d i c h l o r i d e m e l t i n g p o i n t M o n i l i n i a f r u c t i c o l a ; a n o n - p a t h o g e n o f p e a mass s p e c t r a n u c l e a r m a g n e t i c r e s o n a n c e a c e t y l m e t h o x y l a s p e c i f i c c a u s e o f d i s e a s e 3 0 - 5 0 ° C p e t r o l e u m e t h e r p h y t o - p l a n t , a l e x i n - p r o t e c t o r ; c f . s e c t i o n I . A a s u b s p e c i e s s o l v e n t f r o n t t h e c h r o m a t o g r a p h i c s a m p l e a p h y s i c a l , c h e m i c a l o r b i o l o g i c a l p e r t u r b a t i o n w h i c h r e s u l t s i n an a l t e r a t i o n o f t h e p l a n t ' s n o r m a l b i o l o g i c a l p r o c e s s e s TMS - t e t r a m e t h y l s i l a n e u l t r a v i o l e t v o l u m e v i s i b l e x i v THIS THESIS WOULD BE INCOMPLETE WITHOUT AN EXPRESSION OF THANKS TO: D r . D a v i d D o l p h i n f o r f r e e d o m a n d g u i d a n c e - a r a r e c o m b i n a t i o n . D r . James B e r g e r f o r h i s a c c u r a t e a d v i c e . The UBC MS s t a f f w i t h o u t w h o s e h e l p t h e unknowns w o u l d h a v e r e m a i n e d s o . D r s . Hans V a n E t t e n a n d James Rahe f o r t h e p h y t o a l e x i n s a m p l e s . D r s . D a v i d H a l k o a n d Gene J o h n s o n f o r t h e i r w i l l i n g n e s s t o l i s t e n . D r . C a r l A l l e y n e f o r s u f f e r i n g t h r o u g h i t a l l - t w i c e . 1 I . INTRODUCTION P o s t - I n f e c t i o n a l M e t a b o l i s m i n P l a n t s arid t h e P h y t o a l e x i n T h e o r y T h e s u r v i v a l o f a n o r g a n i s m d e p e n d s i n p a r t o n i t s a b i l i t y t o r e s i s t m o s t o f t h e p o t e n t i a l p a t h o g e n s w h i c h e n t e r i t s e n v i r o n m e n t . I n t h e p l a n t k i n g d o m t h e s e m e c h a n i s m s o f d i s e a s e r e s i s t a n c e c a n t a k e a v a r i e t y o f f o r m s J I n p a r t i c u l a r , i t h a s b e e n o b s e r v e d i n some p l a n t s t h a t t h e i r p o s t - i n f e c t i o n a l r e s p o n s e l e a d s t o t h e s y n t h e s i s a n d a c c u m u l a t i o n o f 2-4 c o m p o u n d s w h i c h e x h i b i t i n v i t r o f u n g i t o x i c i t y . 5 T h e s e c o m p o u n d s , a t l e a s t c o n c e p t u a l l y , c o u l d be t h e b a s i s o f d i s e a s e r e s i s t a n c e t h r o u g h t h e e l i m i n a t i o n o f p o t e n t i a l p a t h o g e n s w h i c h do n o t p o s s e s s m e c h a n i s m s f o r n o n - i n d u c t i o n , i n s e n s i t i v i t y o r d e t o x i f i c a t i o n . ^ T h e y c a n b e d i v i d e d i n t o c o m p o u n d s w h i c h a r e d e r i v e d f r o m n o r m a l b i o s y n t h e -s i s a n d c o m p o u n d s w h i c h a r e s y n t h e s i z e d s p e c i f i c a l l y i n r e s p o n s e t o ? 9 i n f e c t i o n / " 5 ' * A f e w o f t h e f u n g i t o x i c c o m p o u n d s o f n o r m a l b i o g e n e s i s a r e shown i n F i g u r e 1. T h e s e c o m p o u n d s a r e t y p i c a l l y f o r m e d b y t h e h y d r o l y s i s o r o x i d a -q t i o n o f p r e - e x i s t i n g n o n - t o x i c s u b s t r a t e s o n c e l l b r e a k d o w n . A c o m p l e t e l i s t w o u l d i n c l u d e a l a r g e number o f common p h e n o l s , a r o m a t i c h y d r o x y a c i d s , f l a v o n o i d s , a n t h o c y a n i n s and c o u m a r i n s a s w e l l a s c o m p o u n d s w h i c h a r e n o t 2 3 9 d e r i v e d f r o m a r o m a t i c b i o s y n t h e s i s . ' ' F i g u r e 2 i l l u s t r a t e s some o f t h e f u n g i t o x i c c o m p o u n d s o f de n o v o b i o s y n t h e s i s . T h e s e c o m p o u n d s , w h i c h h a v e b e e n named p h y t o a l e x i n s ( p h y t o - p l a n t ; a l e x i n - p r o t e c t o r ) J ° w e r e d e f i n e d i n 1956 by M u l l e r a s : ^ 1 " . . . a n t i b i o t i c s w h i c h a r e p r o d u c e d a s a r e s u l t o f t h e i n t e r a c t i o n o f t w o d i f f e r e n t m e t a b o l i c s y s t e m s , t h e h o s t a n d t h e p a r a s i t e , a n d w h i c h i n h i b i t t h e g r o w t h o f m i c r o -o r g a n i s m s p a t h o g e n i c t o p l a n t s . " 2 0 - £ - D - G l u c OH A rbutin (Pear) P i n o i y i v i n (Red Pine) HO HO OR N H 2 3 - H y d r o x y - t y r o m i n e (Sugar Beet) C H , = CH - C H , — S — S - C H , - C H = - C H -II ' c 0 A11 i c I n (Garlic) C H 2 Tul ipol in (Tulip) C H 2 = C H - C H 2 - N = C = S A l l y l i j o t h i o c y o n o t e (Crucifers) HO co - C H 2 — C H 2 — OH OH P h l o r i d l i n : R = ^ - 0- glucosyI P h l o r e t i n : R = H (Apple) R = O"°"TL>0"f) HO ; HO OH /9 - D - G l u c - O (Tomato) HO FIGURE 1. SELECTED EXAMPLES OF THE FUNGITOXIC PRODUCTS OF NORMAL BIOSYNTHESIS.2'3,9 3 ( F r e n c h B e a n ) ( S w e e t P o t a t o ) G o s s y p o l C a p s i d i o l ( C o t t o n ) ( G r e e n P e p p e r ) CH2OH - CHOH - CH = CH - [C • C] 3 - CH = CH - CH, S A F Y N O L ( S a f f l o w e r ) O r c h i n o l ( O r c h i d ) FIGURE 2. SELECTED EXAMPLES OF THE FUNGITOXIC PRODUCTS OF DE NOVO B I O S Y N T H E S I S . 2 , 3 ' 9 , 1 2 ' 1 5 4 M o r e r e c e n t l y t h e t e r m p h y t o a l e x i n h a s b e e n b r o a d e n e d t o c o v e r i n h i b i t o r y 12 c o m p o u n d s w h i c h a r e : " f o r m e d i n r e s p o n s e t o i n j u r y , p h y s i o l o g i c a l s t i m u l i , t h e p r e s e n c e o f i n f e c t i o u s a g e n t s o r t h e p r o d u c t s o f s u c h a g e n t s . " The p r o d u c t i o n o f p h y t o a l e x i n s h a s b e e n d e m o n s t a t e d i n many p l a n t t i s s u e s i n r e s p o n s e t o i n f e c t i o n , c h e m i c a l s t r e s s a n d a v a r i e t y o f o t h e r s t i m u l i . 1 2 ' ^ ' 1 7 H o w e v e r , t h e i r p r e c i s e f u n c t i o n a n d r o l e i n d i s e a s e r e s i s t a n c e h a s n o t b e e n p r o v e n . I n f a c t , a f t e r n e a r l y t w o d e c a d e s o f r e s e a r c h t h e v i e w s p r e s e n t e d i n t h e l i t e r a t u r e r a n g e f r o m t h e i r p o s s i b l e i m p l i c a t i o n i n r e s i s t a n c e ; ^ " . . . p r o d u c t i o n o f t h e a n t i f u n g a l p h y t o a l e x i n ( g l y c e o l l i n ) a p p e a r s t o be t h e b a s i s f o r r e s i s t a n c e o f c e r t a i n s o y b e a n c u l t i v a r s t o i n c o m p a t i b l e r a c e s o f . . . " t o a d i s m i s s a l o f t h e i r r o l e i n t h e d e t e r m i n a t i o n o f r e s i s t a n c e o r 5 s u s c e p t i b i l i t y : " P h y t o a l e x i n p r o d u c t i o n i s r e g a r d e d a s ' c o n s i d e r a b l y more r e m o t e a n d c e r t a i n l y w i t h o u t d i r e c t r e l a t i o n t o s p e c i -f i c i t y ( r e s i s t a n c e o r s u s c e p t i b i l i t y ) . I n f a c t , t h e r e i s e v i d e n c e t o s u p p o r t an e a r l y s u g g e s t i o n t h a t i t r e p r e s e n t s o n l y p a r t o f a m a j o r s t i m u l a t i o n o f s e c o n d a r y m e t a b o l i c a c t i v i t y t h a t f o l l o w s a h y p e r s e n s i t i v e r e s p o n s e , a p a r t t h a t h a s b e e n r e c o g n i z e d b e c a u s e i t s p r o d u c t s a r e f u n g i t o x i c . " A p p a r e n t l y , t h e c o n c e p t u a l l y a t t r a c t i v e o b s e r v a t i o n o f i n v i t r o f u n g i t o x i c i t y i s n o t s u f f i c i e n t t o p r o v e t h e r o l e o f t h e p h y t o a l e x i n s i n t h e d e t e r m i n a t i o n o f d i s e a s e r e s i s t a n c e o r s u s c e p t i b i l i t y . E v i d e n t l y one m u s t a s k : What a d d i t i o n a l i n f o r m a t i o n i s n e e d e d a n d how c a n i t be o b t a i n e d ? 5 The a n s w e r t o t h e f i r s t p a r t o f t h i s q u e s t i o n l i e s i n two a s p e c t s o f t h e s t u d y o f t h e p h y t o a l e x i n s . I n t h e f i r s t c a s e an i m p o r t a n t f a c t o r i n t h e d e t e r m i n a t i o n o f r e s i s t a n c e o r s u s c e p t i b i l i t y by p h y t o a l e x i n s i s t h e c i p r a t e a t w h i c h t h e f u n g i t o x i c c o m p o u n d s a c c u m u l a t e i n v i v o . * P r e s u m a b l y , i f t h e c o n c e n t r a t i o n a t t a i n e d a t a n e a r l y s t a g e i n i n f e c t i o n i s n o t s u f f i c i e n t t o e x e r t a n e f f e c t on t h e i n v a d i n g f u n g u s i t s c o l o n i z a t i o n w i l l n o t be a r r e s t e d . 5 The s e c o n d c a s e was a l l u d e d t o by Ward a n d S t o e s s l when t h e y s u g g e s t e d t h a t p h y t o a l e x i n p r o d u c t i o n " h a s b e e n r e c o g n i z e d b e c a u s e i t s p r o d u c t s a r e f u n g i t o x i c . " T h i s i m p l i e s t h a t a v a r i e t y o f p o t e n t i a l l y i n t e r e s t i n g m e t a b o l i t e s may h a v e b e e n i g n o r e d . T h e s e two a s p e c t s o f t h e s t u d y o f p o s t - s t r e s s m e t a b o l i s m a r e r e l a t e d b e c a u s e t h e i r s o l u t i o n l i e s i n t h e d e v e l o p m e n t o f an a n a l y t i c a l s y s t e m w h i c h c a n s i m u l t a n e o u s l y r e s o l v e a n d q u a n t i f y t h e m a j o r c o m p o n e n t s o f t h e d e s i r e d c l a s s o f compounds o f t h e s t r e s s m e t a b o l i t e m i x t u r e . C o n s e q u e n t l y , t h e f o c u s o f t h i s p r o j e c t h a s b e e n t h e d e v e l o p m e n t o f an a n a l y t i c a l s y s t e m b a s e d on t h e r e s o l u t i o n a n d q u a n t i f i c a t i o n c a p a c i t i e s o f a h i g h p e r f o r m a n c e l i q u i d c h r o m a t o g r a p h i c s y s t e m w h i c h i s c o u p l e d w i t h an o n - l i n e UV a b s o r p t i o n d e t e c t o r . R a t i o n a l f o r t h e C h o i c e o f a P h y t o a l e x i n i c M o d e l S y s t e m The d e v e l o p m e n t o f an a n a l y t i c a l s y s t e m w h i c h w o u l d p e r f o r m s a t i s f a c t o r i l y f o r a l l o f t h e d i f f e r e n t t y p e s o f p h y t o a l e x i n i c s t r u c t u r e s i l l u s t r a t e d i n F i g u r e 2 w o u l d be an i m p r a c t i c a l , i f n o t i m p o s s i b l e , t a s k . 2-4 H o w e v e r , t w o o f t h e m o s t e x t e n s i v e l y i n v e s t i g a t e d o f t h e p h y t o a l e x i n s , t h e p t e r o c a r p a n s p h a s e d 1 i n ( I ) a n d p i s a t i n ( I I ) , a r e members o f t h e i s o f l a v o n o i d c l a s s o f p l a n t p o l y p h e n o l s . T h u s t h e c h o i c e o f 6 PHASEOLLIN P I S A T I N P h a s e o l u s v u l g a r i s L. P i s u m s a t i v u m L. G r e e n b e a n G a r d e n p e a t h e L e g u m i n o s a e s p e c i e s w h i c h p r o d u c e t h e s e c o m p o u n d s , P h a s e o l u s v u l g a r i s a n d P i s u m s a t i v u m r e s p e c t i v e l y , a s t h e m o d e l s y s t e m s u s e d i n t h i s s t u d y i s b a s e d on t h e a v a i l a b i l i t y o f i n f o r m a t i o n on t h e c o n d i t i o n s r e q u i r e d f o r a c c u m u l a t i o n , c h r o m a t o g r a p h i c a n d p h y s i c a l p r o p e r t i e s , e t c . w h i c h a r e g e r m a n e t o t h e d e v e l o p m e n t o f a new a n a l y t i c a l s y s t e m . F l a v o n o i d S t r u c t u r e a n d D i s t r i b u t i o n P i s a t i n a n d p h a s e d ! i n b e l o n g t o a d i v e r s e g r o u p o f p o l y p h e n o l i c 19 n a t u r a l p r o d u c t s w h i c h a r e c h a r a c t e r i z e d by a C 5 - C 3 - C 5 s k e l e t o n , T h e s e c o m p o u n d s , w h i c h a s a c l a s s a r e c a l l e d f l a v o n o i d s , h a v e b e e n d i v i d e d i n t o 20 t h r e e s u b - g r o u p s on t h e b a s i s o f i s o m e r i z a t i o n o f t h e Cg l i n k . The t h r e e s u b - g r o u p s w h i c h a r e f o r m e d a s a r e s u l t o f t h i s i s o m e r i z a t i o n a r e t h e f l a v o n o i d s ( I I I ) , t h e i s o f l a v o n o i d s ( I V ) a n d t h e n e o f l a v o n o i d s ( V ) . 7 The p t e r o c a r p a n s , a s c a n be s e e n f r o m a c o m p a r i s o n o f t h e s k e l e t a l s t r u c -t u r e s V I a n d V I I b e l o n g t o t h e i s o f l a v o n o i d s u b - c l a s s . The m a j o r members o f t h e f l a v o n o i d a n d i s o f l a v o n o i d s u b - c l a s s e s w h i c h a r e o f i m p o r t a n c e t o o u r s t u d y o f p o s t - i n f e c t i o n a l p t e r o c a r p a n ( i s o f l a v o n o i d ) m e t a b o l i s m a r e shown i n F i g u r e 3. A l t h o u g h t h e f l a v o n o i d s ( H I ) a r e n e a r l y u b i q u i t o u s i n h i g h e r p l a n t s t h e i s o f l a v o n o i d s ( I V ) a r e c o n f i n e d p r e d o m i n a n t l y t o t h e s u b - f a m i l y 21 22 L o t o i d e a e o f t h e L e g u m i n o s a e . ' T h i s s u b - f a m i l y ( T a b l e I ) c o n t a i n s many e c o n o m i c a l l y i m p o r t a n t a n d o t h e r w i s e i n t e r e s t i n g p l a n t s . 8 CHALCONE (FLAVONOID) ISOFLAVONE (ISOFLAVONOID) 4 PTEROCARPAN (ISOFLAVONOID) FIGURE 3. SKELETAL STRUCTURES AND NUMBERING SCHEMES OF THE MAJOR FLAVONOID PHENOLICS WHICH ARE OF IMPORTANCE TO THIS STUDY. 9 T A B L E . I SELECTED MEMBERS Of THE LOTOIDEAE Glycine max ( L . ) M e r r Phaseolus aureus R o x b . Phaseolus vulgaris L . Pisum sativum L . Mediaago sativa L . Trifolium re-pens L . Dalbergia latifola R o x b . Pterooarpus santalinus L . Genista tinatoria L . Ononis spinosa L . Derris elliptiaa ( W a l l . ) Thumb. Cytisus sooparius ( L . ) L i n k . Lathyrus odoratus L . - S o y b e a n ( F o o d ) ^ Mung B e a n ( F o o d ) - F r e n c h b e a n ( F o o d ) - G a r d e n p e a ( F o o d ) - L u c e r n e ( F o d d e r J - W h i t e c l o v e r ( F o d d e r ) ^ I n d i a n r o s e w o o d ( T i m b e r ) Red s a n d a l w o o d ( T i m b e r ) - D y e r s broom ( D y e ) - R e s t h a r r o w ( M e d i c i n a l ) - D e r r i s ( I n s e c t i c i d a l ) - Broom ( O r n a m e n t a l ) - S w e e t p e a ( O r n a m e n t a l ) 10 Flavonoid Biosynthesis The flavonoids are products of the secondary metabolic processes of plants. The first specific reaction in flavonoid biosynthesis is the enzyme-mediated condensation of an activated cinnamic acid derivative with three molecules of malonyl CoA to give the isomeric chalcone/ flavanone intermediate ' (Figure 4). The metabolic link between the plant's primary metabolism and this pathway is the formation of trans-24 25 cinnamic acid from L-phenylalanine by phenylalanine ammonia-lyase ' (Figure 4). In most cases it is not known whether the chalcone or the flavanone 24 is the more direct precursor of the particular flavonoid in question. In the case of isoflavonoid formation, however, the experimental evidence 2fi favors the chalcone form of the precursor. The biosynthetic relation-ships of the different classes of the isoflavonoids which are of interest to this study are given in Figure 5. Note that although pterocarpan 21 27 biosynthesis is well defined through the 2'-hydroxyisoflavanone stage ' the relationships between the isoflavonols and the pterocarpans and isoflavans are not completely understood. 11 PHENYLALANINE PHENYLALANINE AMMONIA-LYASE C 0 0 H -HO OJ C O o H CINNAMIC ACID CINNAMIC A C I D 4-HYDROXYLASE 4-HYDROXYCINNAMIC ACID CoA LIGASE C O - S C o A 4-HYDROXYCINNAMYL CoA MALONYL CoA CHALCONE/FLAVANONE SYNTHASE(S) OH ISOMERASES FLAVANONE •H o r -OH FIGURE 4. CHALCONE/FLAVANONE BIOSYNTHESIS FROM P H E N Y L A L A N I N E . 2 4 ' 2 5 12 FIGURE 5 . THE BIOSYNTHETIC RELATIONSHIPS OF THE PTEROCARPAN RELATED I S 0 F L A V 0 N 0 I D S . 2 1 ' 2 7 ~ 2 9 P r o b a b l e p a t h w a y s ( ) . P o s t u l a t e d p a t h w a y s ( — ) . 13 I I . DEVELOPMENT OF A GENERAL PROCEDURE FOR THE CHROMATOGRAPHIC RESOLUTION OF THE P . SATIVUM AND P . VULGARIS ISOFLAVONOIDS The c u r r e n t l y e m p l o y e d m e t h o d s o f i s o f l a v o n o i d s e p a r a t i o n r e l y 30 p r e d o m i n a n t l y on t h i n - l a y e r ( T L C ) o r c o l u m n c h r o m a t o g r a p h i c p r o c e d u r e s . A l t h o u g h p a p e r a n d g a s c h r o m a t o g r a p h y h a v e f o u n d o c c a s i o n a l u s e " 5 ^ f n t h e s e p a r a t i o n o f i s o f l a v o n o i d s t h e p o o r r e s o l u t i o n o b s e r v e d f o r t h e f o r m e r 31 p r o c e d u r e a n d t h e n e e d f o r d e r i v a t i z a t i o n a n d t h e p o s s i b i l i t y o f a r t i f a c t 32 f o r m a t i o n i n t h e l a t t e r c a s e do n o t make t h e s e a l t e r n a t i v e m e t h o d s p a r t i c u l a r l y a t t r a c t i v e . U n f o r t u n a t e l y , e v e n t h o u g h TLC a n d r e l a t e d p r o c e d u r e s h a v e u s u a l l y b e e n t h e m e t h o d s o f c h o i c e , t h e a v a i l a b l e r e f e r e n c e s t o t h e i r u s e i n t h e r e s o l u t i o n o f c o m p l e x i s o f l a v o n o i d m i x t u r e s a r e r e p l e t e w i t h e x a m p l e s o f t h e i r i n e f f i c i e n c y . A t y p i c a l TLC p r o c e d u r e w h i c h was u s e d f o r t h e r e s o l u t i o n o f t h e i s o f l a v o n o i d c o m p o n e n t s i s o l a t e d f r o m s t r e s s e d g r e e n b e a n s [P_. v u l g a r i s ) r e q u i r e d 3 t o 5 s u c c e s s i v e d e v e l o p m e n t s i n a s many d i f f e r e n t s o l v e n t s y s t e m s f o r e a c h o f t h e 4 3 3 m e t a b o l i t e s s t u d i e d . T h e s e r e s u l t s i n d i c a t e t h a t an i n h e r e n t a s p e c t o f t h e u s e o f TLC f o r p o s t - s t r e s s s t u d i e s i s i t s i n a b i l i t y t o s u f f i c i e n t l y r e s o l v e t h e m a j o r c o m p o n e n t s i n a s a m p l e s o t h a t t h e b a s i c p a t t e r n ( " f i n g e r p r i n t " ) a n d t h e a c c u m u l a t i o n r a t e o f t h e s t r e s s r e s p o n s e c a n be e a s i l y d e t e r m i n e d . A l t h o u g h h i g h p e r f o r m a n c e l i q u i d c h r o m a t o g r a p h y ( H P L C ) h a d n o t b e e n a p p l i e d t o t h e r e s o l u t i o n o f i s o f l a v o n o i d s b e f o r e t h i s w o r k was i n i t i a t e d i t s s u p e r i o r r e s o l u t i o n , d e t e c t i o n , q u a n t i f i c a t i o n a n d 3 4 p r e p a r a t i v e p o t e n t i a l ^ i m m e d i a t e l y s u g g e s t e d t h a t i t w o u l d be t h e c h r o m a t o g r a p h i c m e t h o d o f c h o i c e . C o n s e q u e n t l y , t h e i n i t i a l p h a s e o f t h i s 14 p r o j e c t c o n s i s t e d o f t h e d e v e l o p m e n t o f an HPLC s y s t e m w h i c h w o u l d be s u i t a b l e f o r t h e s e p a r a t i o n o f t h e IP. v u l g a r i s a n d P_. s a t i v u m i s o f l a v o n o i d s . HPLC B a c k g r o u n d R e s o l u t i o n i s a m e a s u r e o f t h e d e g r e e o f s e p a r a t i o n o f t h e c o m p o n e n t s i n a m i x t u r e . C o n s e q u e n t l y , i t i s t h e c r i t e r i o n u s e d t o d e t e r m i n e 34 34 t h e s u c c e s s o f a s p e c i f i c c h r o m a t o g r a p h i c p r o c e d u r e . I t i s d e p e n d e n t o n : S e l e c t i v i t y - a m e a s u r e o f r e l a t i v e compound r e t e n t i o n . C a p a c i t y - a m e a s u r e o f a n a l y s i s t i m e ( e . g . i n j e c t i o n t o e l u t i o n t i m e ) . Random D i s p e r s i o n - a m e a s u r e o f t h e e f f i c i e n c y o f t h e c o l u m n w h i c h i s o b s e r v e d a s p e a k b a n d w i d t h a n d i s d e f i n e d i n t e r m s o f t h e h e i g h t e q u i v a l e n t o f t h e t h e o r e t i c a l p l a t e ( H ) . T h e t h e o r e t i c a l p l a t e i s d e f i n e d a s t h e c o l u m n v o l u m e e l e m e n t o f m i n i m u m d i m e n s i o n s u c h t h a t t h e p a r t i t i o n i n g o f t h e s o l u t e b e t w e e n t h e m o b i l e a n d s t a t i o n a r y p h a s e s r e a c h e s e q u i l i b r i u m b e f o r e m o v i n g on t o t h e n e x t v o l u m e e l e m e n t . I t i s a m e a s u r e o f e d d y d i f f u s i o n o r n o n - h o m o g e n e o u s f l o w a n d i s d e p e n d e n t on p a r t i c l e s i z e a n d t h e n o n - e q u i l i b r i u m w h i c h r e s u l t s f r o m r e s i s t a n c e t o mass t r a n s f e r i n t h e s t a t i o n a r y a n d m o b i l e p h a s e s . The s m a l l e r t h e v a l u e o f H t h e m o r e p l a t e s / m e t e r g e n e r a t e d a n d t h e g r e a t e r t h e e f f i c i e n c y o f r e s o l u t i o n . T h e i n c r e a s e d r e s o l u t i o n w h i c h r e s u l t s f r o m d e c r e a s e d p a r t i c l e s i z e was t h e p r i n c i p l e f a c t o r w h i c h l e d t o t h e d e v e l o p m e n t o f h i g h p e r f o r -mance ( p r e s s u r e , s p e e d ) l i q u i d c h r o m a t o g r a p h y . 3 4 A s t h e a v e r a g e p a r t i c l e s i z e o f t h e p a c k i n g d e c r e a s e s a n a l y s i s t i m e i n c r e a s e s u n l e s s t h e s o l v e n t i s pumped t h r o u g h t h e c o l u m n . M o d e r n HPLC s y s t e m s a r e c a p a b l e o f 10 t o 20 m l / m i n . a t up t o 8 0 0 0 P S I . The d e s i r e t o i m p r o v e r e s o l u t i o n w h i c h l e d t o 15 the development o f smal l p a r t i c l e packings and t h e i r a s s o c i a t e d hardware has been achieved i n p r a c t i c e w i t h the a t ta inment o f up t o 45,000 p l a t e s / 34c meter f o r some s o l u t e / s o l v e n t / p a c k i n g c o m b i n a t i o n s . I s o f l a v o n e Chromatography The development o f an HPLC system f o r the s e p a r a t i o n and a n a l y s i s of complex i s o f l a v o n o i d mi xt u re s must begin w i t h the s e l e c t i o n o f a s o l v e n t and adsorbent system which i s s u i t a b l e f o r the r e s o l u t i o n of t e s t m i x t u r e s . This procedure can then be e v a l u a t e d by i t s a p p l i c a t i o n to the r e s o l u t i o n of p l a n t e x t r a c t s . Test mi xt u re s o f i s o f l a v o n e s ( V I I I ) were chosen f o r V I I I the pr imary development o f the s o l v e n t and adsorbent system because they are c e n t r a l t o the b i o s y n t h e s i s o f a l l o f the o t h e r i s o f l a v o n o i d s and are r e a d i l y a v a i l a b l e through i s o l a t i o n o r s y n t h e s i s . The i s o f l a v o n e s are r e l a t i v e l y a p o l a r compounds which have, as would be e x p e c t e d , poor s o l u b i l i t y i n predominant ly aqueous s o l v e n t systems. Consequent ly , t h e i r chromatography on a r e v e r s e phase adsorbent ( e . g . octadecane permanently bonded to a s i l i c a s u p p o r t j / a q u e o u s s o l v e n t system would be expected t o show the e f f e c t s of poor s o l u t e t r a n s f e r between the s t a t i o n a r y and moving phases. T h i s i s p r e c i s e l y what i s observed i n the 35 r e c e n t l y r e p o r t e d r e s u l t s of West, et_al_. T h e i r p r o c e d u r e , which was developed f o r the r e s o l u t i o n o f the i s o m e r i c i s o f l a v o n e s g e n i s t e i n 16 (IX) and 4',5,7-tnhydroxyisoflavone (X) used a reverse phase adsorbent in combination with an acetonitrile/water solvent. The results obtained from this system, which are shown in Figure 6, illustrate that the severe band spreading which is observed would make the adequate resolution of multi-component mixtures impossible. Additionally, the application of this system to the more lipophilic pterocarpan isoflavonoids could result in unacceptably long retention times. A variety of HPLC adsorbents are available. However, the primary alternative to the reverse phase packing-is a si l ica adsorbent which would be used with an apolar organic solvent system. Although the development of the precise solvent ratios which would be used with the si l ica adsorbent is somewhat empirical, the choice of the solvents can be based on the properties of the isoflavone solutes. Specifically, the isoflavones can exhibit a range of polarities based predominantly on their degree of hydroxylation and hence a range of adsorption potentials for the si l ica adsorbent. In addition, the phenolic hydroxyls would be expected to show some degree of ionization in non-acidic media. The probable chromatographic result of ionization would be peak tailing which would beexpected to have a deleterious effect on 17 1 O 5 10 15 20 Time, min FIGURE 6 . SEPARATION OF 4 ' , 6 , 7 - T R I H Y D R O X Y I S O F L A V O N E ( 1 ) AND 4 ' , 5 , 7 - T R I H Y D R O X Y I S O F L A V O N E ( 2 , G E N I S T E I N ) ON A REVERSE PHASE C O L U M N 3 5 18 b o t h r e s o l u t i o n a n d q u a n t i f i c a t i o n . The s o l u t i o n t o t h e p r o b l e m p r e s e n t e d by t h e s p a n o f i s o f l a v o n e p o l a r i t y r e q u i r e d t h a t t h e s o l v e n t s y s t e m be f o r m u l a t e d t o c o n t a i n a n o n - p o l a r s o l v e n t t o i n c r e a s e r e t e n t i o n o f t h e m o r e a p o l a r s o l u t e s , a c a r r i e r s o l v e n t i n w h i c h t h e i s o f l a v o n e s o l u t e s w e r e r e a s o n a b l y s o l u b l e a n d a p o l a r s o l v e n t f o r t h e e l u t i o n o f t h e m o r e p o l a r s o l u t e s w i t h a m i n i m u m o f b a n d s p r e a d i n g ( t a i l i n g ) . A t e r n a r y s y s t e m o f t h i s t y p e was c h o s e n b e c a u s e i t w o u l d a l l o w f o r b o t h g r o s s a n d f i n e v a r i a t i o n i n r e t e n t i o n o f d i f f e r e n t s o l u t e p o l a r i t i e s by a l t e r a t i o n i n t h e c o m p o n e n t s o l v e n t r a t i o s t h r o u g h t h e u s e o f a g r a d i e n t m i x e r . The s o l u t i o n t o t h e p r o b l e m o f m e d i a a c i d i t y s i m p l y r e q u i r e d t h e a d d i t i o n o f a s m a l l amount o f o r g a n i c a c i d t o i n h i b i t i o n i z a t i o n . The s y s t e m w h i c h was d e v e l o p e d t o m e e t t h e s e c r i t e r i a c o n t a i n s p e t r o l e u m e t h e r , d i c n l o r o m e t h a n e , e t h a n o l a n d a c e t i c a c i d . F i g u r e 7 i l l u s t r a t e s t h a t t h i s s o l v e n t s y s t e m s a t i s f a c t o r i l y r e s o l v e s a n i s o f l a v o n e t e s t m i x t u r e w i t h a m i n i m u m o f t a i l i n g . A d d i t i o n a l M o d e l S t u d i e s A l t h o u g h t h e c h r o m a t o g r a p h i c s y s t e m w h i c h was d e v e l o p e d f o r i s o f l a v o n e r e s o l u t i o n a p p e a r s t o f u n c t i o n s a t i s f a c t o r i l y , s e v e r a l a d d i t i o n a l s t u d i e s on t e s t s o l u t e s w e r e p e r f o r m e d t o h e l p d e f i n e t h e c a p a b i l i t i e s a n d l i m i t a t i o n s o f t h e p r o c e d u r e . To d e t e r m i n e i f c l o s e l y r e l a t e d c o m p o u n d s c o u l d be r e s o l v e d , t h e i s o f l a v o n e p a i r s i l l u s t r a t e d by t h e s t r u c t u r e s X I , X I I a n d X I I I , XIV w e r e c h r o m a t o g r a p h e d . The r e s u l t s w h i c h a r e g i v e n i n F i g u r e 8 a r e p a r t i c u l a r l y s t r i k i n g when o n e c o n s i d e r s t h a t t h e 7 - h y d r o x y - 4 ' - m e t h o x y ( X I ) FIGURE 7, GRADIENT CHROMATOGRAM OF SELECTED ISOFLAVONES, S o l v e n t s y s t e m A (A c o m p l e t e d e s c r i p t i o n o f t h e HPLC p a r a m e t e r s i s g i v e n i n t h e e x p e r i m e n t a l s e c t i o n ) , s f r S o l v e n t f r o n t , P r - / * , ^ - d i m e t h y l a l l y l - . 20 XIV a n d t h e 7 - h y d r o x y - 3 ' , 4 ' - m e t h y l e n e d i o x y ( . X I I ) p a i r o f i . s o f l a v o n e s h a v e n o t p r e v i o u s l y b e e n s u c c e s s f u l l y r e s o l v e d by a l i q u i d c h r o m a t o g r a p h i c A 36 p r o c e d u r e . The c h r o m a t o g r a p h i c c o n d i t i o n s o f F i g u r e 8 a w e r e a l s o u s e d i n an a t t e m p t t o s e p a r a t e a m i x t u r e o f t h e 7 - h y d r o x y - 4 K - m e t h o x y i s o f l a v o n e ( X I ) a n d i t s 4 ' - h y d r o x y - 7 - m e t h o x y i s o f l a v o n e i s o m e r . No r e s o l u t i o n was o b s e r v e d . O b v i o u s l y , t h i s c h r o m a t o g r a p h i c s y s t e m d o e s h a v e l i m i t a t i o n s . 21 FIGURE 8 a . GRADIENT CHROMATOGRAM OF 7 - H Y D R 0 X Y - 4 ' - M E T H 0 X Y - AND 7-HYDROXY-3 1 . 4 ' - M E T H Y L E N E D I O X Y I S O F L A V O N E . S o l v e n t s y s t e m D. 0 - 2 5 m i n u t e s o m i t t e d f o r c l a r i t y . 22 FIGURE 8b. ISOCRATIC CHROMATOGRAM OF 4 ' ,7 - D I M E T H O X Y - , 7-METHOXY-3 1 ,4 1 - M E T H Y L E N E D I O X Y - AND 7 - P R E N Y L 0 X Y-4 ' - M E T H 0 X Y I S 0 F L A V 0 N E S o l v e n t s y s t e m 1. 2 3 An a d d i t i o n a l l i m i t a t i o n o f t h e s y s t e m was d i s c o v e r e d when 2 " , 4 , 4 ' - t r i h y d r o x y c h a l c o n e ( X V ) , w h i c h i s t h e c h a l c o n e p r e c u r s o r o f t h e L e g u m i n o s a e i s o f l a v o n o i d s , ^ ' was c h r o m a t o g r a p h e d . As i l l u s t r a t e d i n F i g u r e 9 , c h r o m a t o g r a p h y o f t h e c h a l c o n e on o u r u s u a l s o l v e n t s y s t e m r e s u l t e d i n t h e e l u t i o n o f t h e compound a s a s e v e r e l y t a i l e d b a n d . T h i s e f f e c t , w h i c h i s a n i n d i c a t i o n o f p o o r s o l u t e m a s s t r a n s f e r , i s p r o b a b l y t h e r e s u l t o f t h e d i f f e r e n t modes o f s o l u t e a d s o r p t i o n t o t h e s u p p o r t w h i c h c a n o c c u r b e c a u s e o f t h e p o t e n t i a l f o r a h y d r o g e n b o n d b e t w e e n t h e 2 ' - h y d r o x y l a n d t h e c a r b o n y l . T h i s a s s u m p t i o n i s s u p p o r t e d by t h e r e s u l t s shown i n F i g u r e 10 w h e r e a number o f c h a l c o n e s , a l l o f w h i c h , h a v e t h e 2 ' - h y d r o x y l e i t h e r b l o c k e d a s an e t h e r o r a b s e n t , a r e o b s e r v e d t o e l u t e s a t i s f a c t o r i l y . A l t h o u g h an i n c r e a s e i n t h e s o l v e n t a c e t i c a c i d c o n t e n t was f o u n d t o c o n s i d e r a b l y r e d u c e t h e o b s e r v e d t a i l i n g f o r t h e t r i h y d r o x y -c h a l c o n e ( F i g u r e 9 ) i t was n o t e l i m i n a t e d . P r e l i m i n a r y e x p e r i m e n t s i n d i c a t e t h a t t h e a d d i t i o n o f a s t r o n g e r 37 a c i d ( e . g . f o r m i c a c i d ) t o t h e s o l v e n t s y s t e m w o u l d a l l e v i a t e t h i s p r o b l e m . H o w e v e r , t h i s s o l u t i o n t o t h e p r o b l e m i s n o t p r a c t i c a l f o r t h i s p r o j e c t b e c a u s e o f t h e p o t e n t i a l f o r a c i d i c d e g r a d a t i o n o f t h e p l a n t 38 e x t r a c t s . An e x a m p l e o f t h i s e f f e c t i s t h e o b s e r v e d a b i l i t y o f f o r m i c a c i d t o c a t a l y z e t h e d e h y d r a t i o n o f p i s a t i n ( I I ) t o a n h y d r o p i s a t i n ( X V I ) . O xv 24 A / » / \ i i i i J i i_ 30 25 20 T IME.mln FIGURE 9 . GRADIENT CHROMATOGRAM OF 2 ' , 4 , 4 ' - T R I H Y D R O X Y C H A L C O N E N o r m a l S o l v e n t S y s t e m ( A ; - — - ) . I n c r e a s e d A c e t i c A c i d S o l v e n t S y s t e m ( B ; — - - ) . F I G U R E 10. G R A D I E N T C H R O M A T O G R A M O F S E L E C T E D C H A L C O N E S . Solvent system A . 26 x v i A p p l i c a t i o n of the Chromatographic Procedure to an IsOflaVone E x t r a c t With the development of a chromatographic system which c o u l d adequately r e s o l v e i s o f l a v o n e t e s t mixtures our a t t e n t i o n c o u l d be focused on the a p p l i c a b i l i t y of the system to the a n a l y s i s o f i s o l a t e d i s o f l a v o n e s . Soybean ( G l y c i n e max) seed c o n t a i n s d a i d z e i n (XVII) XVII as w e l l as g l y c o s i d e s o f d a i d z e i n and g e n i s t e i n ( I X ) , T h e r e f o r e , i t was chosen as the i s o f l a v o n e source f o r t h i s t e s t procedure . I t i s p o s s i b l e to analyze f o r both i s o f l a v o n e aglycones and g l y c o s i d e s on our chromatographic system by the s e l e c t i o n o f s u i t a b l e e x t r a c t i o n procedures . The aglycones a r e i s o l a t e d by a l c o h o l i c e x t r a c t i o n w h i l e the g l y c o s i d e s can be i s o l a t e d as t h e i r aglycones a f t e r h y d r o l y s i s .,, . . 40 w i t h aqueous a c i d . 27 The c o m b i n a t i o n o f t h e two p r o c e d u r e s w i l l p r o v i d e t h e r e q u i r e d s a m p l e s f o r a g l y c o n e / g l y c o s i d e a n a l y s i s . F i g u r e 11 i l l u s t r a t e s t h a t t h e HPLC m e t h o d i n c o m b i n a t i o n w i t h t h e e t h a n o l i c a n d h y d r o l y t i c e x t r a c t i o n p r o c e d u r e s d o e s p r o v i d e a s a t i s f a c t o r y m e t h o d f o r i s o f l a v o n e a n a l y s i s . 39 As was e x p e c t e d , t h e e t h a n o l i c e x t r a c t c o n t a i n s o n l y d a i d z e i n w h i l e t h e h y d r o l y t i c e x t r a c t was f o u n d t o c o n t a i n d a i d z e i n and g e n i s t e i n ( N o t e ) . The a d d i t i o n a l p e a k i n t h e h y d r o l y t i c e x t r a c t c h r o m a t o g r a m was i d e n t i f i e d a s 5 - h y d r o x y m e t h y l - 2 - f u r a l d e h y d e ( X V I I I ) ; R = - C H 9 0 H . T h i s c o m p o u n d , w h i c h may be r e l a t e d t o t h e o n l y n o n - i s o f l a v o n o i d p h y t o a l e x i n 2 3 i s o l a t e d f r o m a L e g u m i n o s a e g e n u s ( X I X ) , ' p r o v i d e d an i n t e r e s t i n g a s i d e R' = -CO-C=C-CH=CH-CH -CH 0 XVIII 2 3 WYREONE from V i c i a faba XIX N o t e : To a v o i d t h e r e p e t i t o u s p r e s e n t a t i o n o f a n c i l l a r y m a t e r i a l t h e d a t a u s e d t o i d e n t i f y t h e i s o l a t e s d i s c u s s e d i n t h i s t h e s i s h a s b e e n c o m p i l e d i n an a p p e n d i x . 28 FIGURE 1 1 . GRADIENT CHROMATOGRAM OF A G. MAX EXTRACT. S o l v e n t s y s t e m A . 50 u l o f a . 5 m l / g d i l u t i o n i n j e c t e d . A - E t h a n o l i c e x t r a c t . B - H y d r o l y t i c e x t r a c t . 29 t o o u r s t u d y o f i s o f l a v o n o i d m e t a b o l i t e s b e c a u s e i t i s a s s o c i a t e d w i t h 41 s c o r c h i n g a n d b i t t e r t a s t e i n some f o o d s . As a r e s u l t , i t s d e t e r m i n a t i o n i s o f i m p o r t a n c e t o t h e f o o d p r o c e s s i n g i n d u s t r y . H o w e v e r , t h e p r o c e d u r e s 41 c u r r e n t l y u s e d f o r i t s a n a l y s i s r e q u i r e t h e p r e p a r a t i o n o f d e r i v a t i v e s a n d i n t e r f e r e n c e by f u r f u r a l d e h y d e ( X V I I I ; R = - H ) c a n be t r o u b l e s o m e . 4 ^ A t e s t c h r o m a t o g r a m o f t h i s compound a n d f u r f u r a l d e h y d e showed t h a t f u r f u r a l d e h y d e e l u t e s o v e r 12 m i n u t e s b e f o r e t h e h y d r o x y c o m p o u n d . T h i s e x p e r i m e n t s u g g e s t s t h a t t h e d e v e l o p m e n t d f a n a n a l y t i c a l s y s t e m b a s e d on t h e c h r o m a t o g r a p h i c r e s o l u t i o n o f t h e s e two c o m p o u n d s w o u l d p r o b a b l y be a s i m p l e t a s k . I n a d d i t i o n t o i l l u s t r a t i n g t h e p r e s e n c e o f d a i d z e i n , g e n i s t e i n a n d 5 - h y d r o x y m e t h y l - 2 - f u r a l d e h y d e t h e c h r o m a t o g r a m o f t h e h y d r o l y t i c e x t r a c t o f 6 . max ( F i g u r e l i b ) shows t h a t t h e s e p a r a t i o n o b s e r v e d f o r t h e t h r e e m a j o r p e a k s i s b e t t e r t h a n t h a t r e q u i r e d f o r a c c e p t a b l e r e s o l u t i o n . A l s o , t h e 0 t o 17 m i n u t e z o n e d o e s n o t i n d i c a t e t h e p r e s e n c e o f a n y a d d i t i o n a l m a j o r m e t a b o l i t e s a t t h e l e v e l o f a n a l y t i c a l s e n s i t i v i t y u s e d . A s i l l u s t r a t e d i n F i g u r e 1 2 , t h e same i n f o r m a t i o n on d a i d z e i n , g e n i s t e i n a n d h y d r o x y m e t h y l f u r a l d e h y d e c o n c e n t r a t i o n c a n be o b t a i n e d b y t h e u s e o f a p r o c e d u r a l l y s u p e r i o r i s o c r a t i c s o l v e n t s y s t e m . N o t o n l y i s a n a l y s i s t i m e r e d u c e d f r o m 30 t o 10 m i n u t e s b u t b e c a u s e t h e i s o c r a t i c r u n d o e s n o t r e q u i r e s o l v e n t r e - e q u i l i b r a t i o n f o r e a c h s a m p l e t h e o v e r a l l a n a l y s i s r a t e i s i n c r e a s e d f r o m 1 s a m p l e p e r h o u r f o r t h e g r a d i e n t s y s t e m t o 5 s a m p l e s p e r h o u r when t h e i s o c r a t i c p r o c e d u r e i s u s e d . 30 FIGURE 1 2 . ISOCRATIC CHROMATOGRAM OF A G. MAX HYDROLYZATE. S o l v e n t s y s t e m 4 . D - d a i d z e i n , G - g e n i s t e i n a n d F - 5 - h y d r o x y m e t h y l - 2 - f u r a l d e h y d e . 31 I I I . A N A L Y S I S OF THE RESPONSE OF P . SATIVUM AND P. VULGARIS TO A B I O T I C AND B I O T I C FORMS OF STRESS A . The P h y t o a l e x i n i c R e s p o n s e P h y t o a l e x i n C h r o m a t o g r a p h y A l t h o u g h p h a s e d 1 i n ( I ) a n d p i s a t i n ( I I ) w e r e t h e f i r s t r e c o g n i z e d p h y t o a l e x i n i c c o m p o n e n t s t o be i s o l a t e d f r o m P_. v u l g a r i s and P.; s a t i v u m r e s p e c t i v e l y , a number o f a d d i t i o n a l f u n g i t o x i c c o m p o u n d s h a v e s u b s e q u e n t l y b e e n d e s c r i b e d f r o m t h e s e p l a n t s . F i g u r e 13 i d e n t i f i e s t h e m a j o r p h y t o -a l e x i n s o f P.. v u l g a r i s and P . s a t i v u m w h i l e F i g u r e s 14 a n d 15 i l l u s t r a t e t h e a p p l i c a t i o n o f o u r g r a d i e n t c h r o m a t o g r a p h y p r o c e d u r e t o t h e i r r e s o l u -t i o n . I t i s a p p a r e n t f r o m F i g u r e s 14 a n d 15 t h a t a l t h o u g h m o s t o f t h e compounds b e h a v e s a t i s f a c t o r i l y o n t h e HPLC s y s t e m t h e r e s u l t s o b t a i n e d f o r k i e v i t o n e ( X X ; F i g u r e 15) a r e p o o r . H o w e v e r , t h e d e g r e e o f t a i l i n g o b s e r v e d f o r k i e v i t o n e i s n o t u n e x p e c t e d b e c a u s e o u r p r e v i o u s c h a l c o n e r e s u l t s h a d shown t h a t t h e p r e s e n c e o f a s t r o n g l y h y d r o g e n b o n d e d h y d r o x y l i n t h e m o l e c u l e w o u l d r e s u l t i n t a i l i n g . An e x a m i n a t i o n o f t h e s t r u c t u r e o f k i e v i t o n e ( X X ) shows t h a t t h e m o l e c u l e d o e s i n f a c t c o n t a i n t h e p o t e n t i a l f o r t h e f o r m a t i o n o f a h y d r o g e n b o n d . H O O H xx 32 FIGURE 1 3 . THE MAJOR PHYTOALEXINS OF P . SATIVUM AND P . V U L G A R I S . 33 A . PISUM SATIVUM 34 KIEVITONE B. PHASEOLUS VULGARIS 35 FIGURE 1 4 a . GRADIENT CHROMATOGRAM OF 4 - H Y D R 0 X Y - 2 , 3 , 9 - T R I M f c T H 0 X Y - , 3-HYDROXY-2 , 9 - D I M E T H O X Y - AND 2 , 3 , 9 - T R I M E T H O X Y P T E R O C A R P A N . S o l v e n t s y s t e m A . 36 T I M E , m i n FIGURE 1 4 b . ISOCRATIC CHROMATOGRAM OF 4 - H Y D R 0 X Y - 2 . 3 . 9 - T R I M E T H 0 X Y - , 3-HYDROXY-2 , 9 - D I M E T H O X Y - AND 2 , 3 , 9 - T R I M E T H O X Y PTEROCARPAN. S o l v e n t s y s t e m 2 . The p o s i t i o n o f p i s a t i n h a s b e e n i n d i c a t e d on t h e c h r o m a t o g r a m . — I I 1 1 1 -L j -30 25 20 15 10 5 ', FIGURE 1 5 . GRADIENT CHROMATOGRAM OF P H A S E O L L I N , PHASEOLLINISOFLAVAN AND KIEVTTONE. S o l v e n t s y s t e m A . 38 The analysis of kievitone could be approached hy two methods. In the f irst case the peak could be collected for quantification by UV absorbance after minor impurities are removed by TLC. This procedure, although somewhat tedious, would s t i l l be superior to the 4 or 33 5 successive TLC developments which have been previously required. In the second case the results of preliminary experiments have shown that the observed tailing will be decreased by the addition of formic acid to the solvent system. This procedure, however, will require a thorough examina-tion of the effects of the formic acid on the stress extract sample. Analysis of Stressed and Unstressed Plants Analysis of the isoflavones of G. max has shown that variations in the method of extraction can lead to the differentiation of metabolite source. Specifically, we observed that an extraction with ethanol gave a measure of the isoflavone aglycones while an extraction with ethyl acetate after acidic hydrolysis gave a measure of the isoflavone glycosides (as their aglycones) as well as the aglycones. By definition, the observed phytoalexins of P. vulgaris and P_. sativum are produced by the plant de novo in response to infection. Consequently, one would not expect to observe these compounds in the unstressed plant either as aglycones or glycosides. This is what is observed. Figure 16 illustrates the results of a hydrolytic extraction of P. sativum for plants which were of a typical age for our experiments (ca. 10 days after germination). Interestingly, in plants which were ca. 17 days old both of the major components of the extract, 5-hydroxymethyl-2-furaldehyde and the unidentified compound(s) at ca_. 13 minutes had almost completely disappeared at an analytical sensitivity 0HC-1L0J1-CH20H PISATIN V ANHYDROPISATIN 2 5 2 0 . 15 T I M E , m i n 1 0 s'f FIGURE 1 6 . GRADIENT CHROMATOGRAM OF THE HYDROLYTIC EXTRACT OF UNSTRESSED f\ SATIVUM. S o l v e n t s y s t e m A . 50 y l o f a . 5 m l / g d i l u t i o n i n j e c t e d . 40 t w i c e t h a t o f F i g u r e 1 6 , T h e s e a n a l y s e s i n d i c a t e t h a t t h e u n s t r e s s e d p i a n t d o e s n o t c o n t a i n q u a n t i t i e s o f i s o f l a v o n o i d g l y c o s i d e s i n e x c e s s o f c a . 1 j i g / g ( w e t w e i g h t ) . The e t h a n o l i c e x t r a c t s o f u n s t r e s s e d P . v u l g a r i s a n d P . s a t i v u m w e r e a l s o a n a l y z e d t o d e t e r m i n e t h e i r a g l y c o n e c o n t e n t . The c h r o m a t o g r a m s o b t a i n e d ( F i g u r e s 17a a n d 1 8 a ) show t h a t t h e h e a l t h y p l a n t s do n o t c o n t a i n a p p r e c i a b l e q u a n t i t i e s o f m e t a b o l i t e s a t a maximum a n a l y t i c a l s e n s i t i v i t y o f c a . . 5 u g / g ( w e t w e i g h t ) . H o w e v e r , t h e a n a l o g o u s c h r o m a t o g r a m s f o r 42 h e a v y m e t a l ( c o p p e r ( I I ) c h l o r i d e ) s t r e s s e d p l a n t s ( F i g u r e s 17b a n d 1 8 b ) show t h e a c c u m u l a t i o n o f a number o f u n i d e n t i f i e d c o m p o n e n t s i n a d d i t i o n t o some o f t h e r e c o g n i z e d p h y t o a l e x i n s . The P.. v u l g a r i s r e s u l t s c o n f i r m t h e p r e s e n c e o f p h a s e o l l i n , p h a s e o l l i n i s o f l a v a n a n d k i e v i t o n e i n t h e c o p p e r s t r e s s e x t r a c t s . I n a d d i t i o n , c o u m e s t r o l ( X X I ) , w h i c h h a s b e e n p r e v i o u s l y o b s e r v e d i n s t r e s s e d b e a n , 4 7 was i d e n t i f i e d . T h e P . s a t i v u m r e s u l t s , p a r t i c u l a r l y a s XXI p r e s e n t e d i n F i g u r e 19, show t h a t p i s a t i n i s t h e o n l y o b s e r v e d p e a p h y t o a l e x i n . F i g u r e s 17 a n d 18 i l l u s t r a t e n o t o n l y t h e de n o v o p r o d u c t i o n o f a v a r i e t y o f c o m p o u n d s b y t h e s t r e s s e d p l a n t b u t t h e y a l s o show t h a t 41 FIGURE 17. GRADIENT CHROMATOGRAM OF THE ETHANOLIC EXTRACT OF UNSTRESSED ( A ) AND STRESSED ( B ) P. S A T I V U M . S o l v e n t s y s t e m A . 50 u l o f a .1 m l / g d i l u t i o n i n j e c t e d . 43 FIGURE 1 8 . GRADIENT CHROMATOGRAM OF THE ETHANOLIC EXTRACT OF UNSTRESSED ( A ) AND STRESSED ( B ) P . VULGARIS S o l v e n t s y s t e m A . 50 y l o f a . 2 m l / g d i l u t i o n i n j e c t e d . 45 T I M E , m i n FIGURE 1 9 . ISOCRATIC CHROMATOGRAM OF THE PTEROCARPAN REGION OF A STRESSED P . SATIVUM SAMPLE. S o l v e n t s y s t e m 2 . The p o s i t i o n s o f 3 - h y d r o x y - 2 , 9 - d i m e t h o x y - ( a ) , 4 - h y d r o x y - 2 , 3 , 9 - t r i m e t h o x y - ( b ) a n d 2 , 3 , 9 - t r i m e t h o x y p t e r o c a r p a n ( c ) h a v e b e e n i n d i c a t e d on t h e c h r o m a t o g r a m . 46 t h e HPLC s y s t e m c a n s a t i s f a c t o r i l y r e s o l v e many o f t h e c o m p o n e n t s o f t h e s t r e s s m i x t u r e w h i c h f a l l w i t h i n t h e r a n g e o f o u r c h o s e n c h r o m a t o g r a p h i c z o n e . Two q u e s t i o n s , h o w e v e r , r e m a i n u n a n s w e r e d . I n t h e f i r s t c a s e , a l t h o u g h p r a c t i c a l c o n s i d e r a t i o n s r e q u i r e t h a t we n o t a t t e m p t t o r e s o l v e a l l o f t h e new m e t a b o l i t e s w h i c h t h e s t r e s s e d p l a n t i s l i k e l y t o p r o d u c e , i t s h o u l d be d e t e r m i n e d t h a t t h e b o u n d a r i e s o f t h e c h r o m a t o g r a p h i c z o n e do n o t i n a d v e r t a n t l y " j u s t m i s s " w h a t may be i n t e r e s t i n g c o m p o n e n t s o f t h e s t r e s s m i x t u r e . Our t e s t s , h o w e v e r , w h i c h i n v o l v e d i n c r e a s i n g a n d d e c r e a s i n g s o l v e n t p o l a r i t y f r o m a m i n i m u m o f n e a t h e x a n e s t o a maximum o f 10% e t h a n o l i n m e t h y l e n e d i c h l o r i d e d i d n o t r e v e a l a n y a d d i t i o n a l a p o l a r o r p o l a r c o m p o n e n t s . I n t h e s e c o n d c a s e , many o f t h e p r e v i o u s s t u d i e s on s t r e s s m e t a b o l i t e a c c u m u l a t i o n h a v e r e l i e d on t h e e x t r a c t i o n o f a q u e o u s w a s h i n g s o f t h e s t r e s s e d p l a n t ' s s u r f a c e . ' T h e s e s a m p l e s , w h i c h a r e t e r m e d e x u d a t e e x t r a c t s , w e r e a p p r o x i m a t e d by r i n s i n g a c o p p e r ( I I ) c h l o r i d e s t r e s s e d s a m p l e w i t h w a t e r f o l l o w e d by e x t r a c t i o n a n d c h r o m a t o g r a p h y o f t h i s a q u e o u s w a s h . T h e r e s u l t s o f t h i s e x p e r i m e n t show t h a t t h e r e i s a s l i g h t ( c a . 10%) r e l a t i v e c o n c e n t r a t i o n i n c r e a s e o f t h e more p o l a r c o m p o n e n t s o f t h e s t r e s s m i x t u r e c o m p a r e d t o t h e e t h a n o l i c e x t r a c t . The r e s u l t s o f t h e s e t e s t s c o n f i r m t h a t t h e e t h a n o l i c e x t r a c t s o f t h e s t r e s s e d p l a n t a r e r e p r e s e n t a t i v e o f i t s de n o v o a c c u m u l a t i o n o f i s o f l a v o n o i d m e t a b o l i t e s . R a t e o f A c c u m u l a t i o n S t u d i e s C o n s i d e r a b l e i n f o r m a t i o n i s a v a i l a b l e c o n c e r n i n g p h y t o a l e x i n o c c u r r e n c e , c h a r a c t e r i z a t i o n a n d i n v i t r o f u n g i t o x i c i t y . H o w e v e r , b e c a u s e o f t h e t e c h n i c a l d i f f i c u l t i e s w h i c h a r e a s s o c i a t e d w i t h TLC b a s e d a n a l y s e s 47 only a limited number of studies have dealt with the rate of phytoalexin accumulation. The chromatograms of phased!in and phaseollinisoflavan in P. vulgaris and pisatin in 'P. sativum illustrate that the HPLC procedure gives adequate resolution of these components of the stress metabolite mixture. Consequently, i t should be possible to determine the rate at which these phytoalexins accumulate by a time course analysis of a stressed sample. The chromatograms obtained from a time course study of phaseollin and phaseollinisoflavan accumulation are given in Figure 20. Note that because the gradient procedure gave excess resolution a more time effective isocratic system was used for these analyses. The results of this trial illustrate that the chromatograms obtained can provide not only the data for a quantitative analysis of phytoalexin accumulation, but also a comparison of the chromatograms can give a visual impression of the overall pattern of metabolite accumulation and decline irrespective of the identif-ication of all of the observed peaks. The results of the P.. vulgaris test and a similar trial on P.. sativum are also presented in a more usual graphical format in Figures 21a and 22a respectively. The accumulation curves obtained from these studies, which compare favorably to the results of similar TLC based studies (Figures 21b and 22b), show that the rate of metabolite accumula-tion can be readily determined using the HPLC technique. TIHt.iala FIGURE 20. TIME COURSE SEQUENCE ISOCRATIC CHROMATOGRAMS OF COPPER(II) CHLORIDE STRESSED P. VULGARIS. 0(a), 12(b), 24(c), 36(d), 48(e), 60(f), 72(g), 84(h) and 96(i) hours after stress. Solvent system 3. 50ul of a .2 ml/gm dilution injected. For 3-11 minutes the vertical scale has been expanded by a factor of two. 49 FIGURE 21. THE RATE OF PHASEOLLIN (X) AND PHASEOLLINISOFLAVAN (•) ACCUMULATION IN P. VULGARIS. A - This study. B - A TLC based study on excised pods. X axis - Hours after stress. Y axis - Concentration. 50 \ ' i 1 1 24 48 72 FIGURE 22. THE RATE OF P I S A T I N ACCUMULATION IN P . SATIVUM. A - T h i s s t u d y . B - A TLC b a s e d s t u d y o n e x c i s e d p o d s . X a x i s - H o u r s a f t e r s t r e s s . Y a x i s - C o n c e n t r a t i o n . 51 B. Identification of the P. Sativum Metabolites 1. Metabblite Identification and Synthesis In the last section we observed that the HPLC procedure could be used to resolve most of the known phytoalexins of P. vulgaris and P_. sativum. However, inspection of the chromatograms in Figures 17 and 18 indicates that there are a number of additional compounds from each plant which have not been previously identified. The structural determination of these compounds may provide additional potentially useful information on isoflavonoid stress metabolism. Consequently, all of the major metabolites which were observed to accumu-late i n s a t i v u m have been isolated and identified. Isolation and Identification The tedious extraction and separation steps which are normally necessary for the isolation of minor metabolites from plant extracts are not needed with HPLC because it simply requires a "scaling up" of sample size with collection of the appropriate fractions as they leave the detector. This fractionation procedure, when combined with a subsequent thin-layer chromatography step to ensure sample homogeneity, provides an easy method for the preparation of isolate samples for spectroscopic analysis. The structures of the components which were identified from P_. sativum are presented in Figure 23. Note that many of the compounds, as indicated in Table II, have been previously isolated from other plant sources. Most, however, have not been reported from P. sativum. Two of the metabolites which were identified during the course of this study have not been previously identified from any other source. Their identification is outlined here. FIGURE 2 3 . A REPRESENTATIVE GRADIENT CHROMATOGRAM OF THE IDENTIFIED ISOLATES OF P . SATIVUM The p e a k n u m b e r s r e f e r t o T a b l e I I . 53 TABLE II THE IDENTIFIED METABOLITES OF STRESSED PISUM SATIVUM Peak No.a Compound Previous 'Isolation 1 Sativone None 49a 2 Liquiritigenin Many sources 49b 3 Isoliquiritigenin Many sources 4 Sativastyrene None 21 5 Formononetin Many Leguminosae genera 21 6 Afromosin Many Leguminosae genera 7 Unidentified isoflavone 43 8 Pisatin Pisum sativum 9 Obtustyrene Dalbergia retusa 5 0 (Leguminosae) 10 4-Hydroxy-2,3,9-trimethoxy-, 3-Hydroxy-2,9-dimethoxy- and 2,3,9-Trimethoxypterocarpan Pisum sativum 51 11 Anhydropisatin (Flemichapparin B) Flemingia chappar (Leguminosae) a - see Figure 23 54 The characteristic UV spectrumdt of the f irst new metabolite (Figure 23, peak number 1) in combination with its molecular formula of ^16^14^4' w n ^ c n w a s derived from high resolution mass spectral measurements, suggested that the compound was a dihydroxymethoxychalcone. The mass 53 spectrum, which had the diagnostic fragments LsH7^3 a n ( ^ ^ 9^ 7^ 2 ^ e and 151 respectively), was used in combination with the observed NMR aromatic couplings to limit the potential isomeric possibilities for the isolate to either 4,4,-dihydroxy-2,-methoxychalcone (XXII) or 2',4-dihydroxy-4'-methoxy-chalcone (XXIII). Chromatographic comparison of the isolate with synthetic samples of these compounds proved that tt was the 2'Hnethoxy isomer. This isolate is a new natural product chalcone with an unusual methoxylation pattern. To reflect its isolation from P. sativum we have named it sativone. The second isolate (Figure 23, peak number 4) gave a high resolu-tion mass spectral molecular formula of C 7 6 H f 6 * ^ 3 * T h ^ s i | r f ° n T , a t i ° n > i n combination with an A B X 2 coupling pattern in the NMR spectrum and an increase in isolate mass of 28 after reaction with methyl iodide/potassium carbonate, indicated that the compound did not have a carbonyl oxygen and was therefore a dihydroxymethoxybenzylstyrene. Analysis of the mass 55 spectrum, which had the tropyl iuro fragments C^ HyQ and CgHgO^  (ro/e 107 and 137 respectively; see section IV.B), in combination with the similarity of the NMR aromatic couplings of this isolate and the previously identified chalcone indicated that the isolate might be 4-hydroxy-(4'-hydroxy-2'-methoxybenzyl)styrene (XXIV) or its 2'-hydroxy-4*-methoxy (XXV) isomer. Chromatographic comparison of the isolate with synthetic samples of these XXIV , XXV compounds proved that it was the 4'-rhydroxy-2'>methoxy compound. This isolate is a new natural product styrene. To reflect its structural relationship to the P_. sativum chalcone sativone and in accord 50 with literature precedent , we have named it sativastyrene. Synthesis Many of the components described in the last section have been previously isolated and/or synthesized. Consequently, confirmation of isolate structure for these compounds relied on recognized isolation procedures or synthesis by the route outlined in Scheme 1. However, two of the compounds, sativone (XXII) and sativastyrene (XXIV), had not been previously prepared. Additionally, the preparation of 4'-rhydroxy-2'-methoxybenzylstyrene (XXVI; obtustyrene) has been included in this discus-56 57 XXVI sion because the literature data on this compound and its precursors was, 50 until recently , largely incomplete. Synthesis of sativone (XXII) by the aldol condensation of £-hydroxybenzaldehyde and 4'-hydroxy-2'-methoxyacetophenone gave a low yield (21%) even after reaction times of up to six weeks. More importantly, the product obtained by this procedure was diff icult to purify. As a result, the synthesis of this compound was effected via the methylation and debenzylation of the readily available aldol product 4,4'-bis(benzyloxy)-2'-hydroxychalcone56 (Scheme 2). This procedure, which capitalized on the inability of the 2'-methoxychalcone intermediate to isomerize in acid to a 57 flavanone during the debenzylation step, gave a pure product. The synthesis of sativastyrene (XXIV) and obtustyrene (XXVI) required two separate routes for complete confirmation of structure (Scheme 3). Synthesis via acidic condensation5**'5 9 of the appropriate cinnamyl alcohol^ and m-methoxyphenol gave, as would be expected, the 2'-hydroxy-4'-methoxybenzyl isomer as well as the required 4'-hydroxy^'-methoxybenzyl-styrene. Although it was not diff icult to isolate and separate the two compounds the unambiguous assignment of the correct structure to each isolated fraction required a TLC and HPLC comparison of the isomers with SCHEME 2. SYNTHESIS OF 4,4'-DIHYDROXY-2 '-METHOXYCHALCONE (SATIVONE) 59 SCHEME 3. BENZYLSTYRENE SYNTHESIS Synthesis of 4-hydroxy-(4'-hydroxy-2'-methoxybenzyl)styrene (Sativastyrene) and 4-hydroxy-(2'-hydroxy-4'-methoxybenzyl)-styrene (Isosativastyrene). 60 the products of the aluminum hydride0 1 reduction of the corresponding chalcones. This reaction, By i tsel f , was not sufficient for the synthesis of the desired benzylstyrene because of the possibility of double bond isomerization.0^ An additional route for the synthesis of oBtustyrene also served to illustrate the use of the HPLC system for the resolution of Benzylstyrene mixtures, The chromatogram in Figure 24 shows that the reaction of 2 \ 4 ' v dihydroxybenzylstyrene with methyl iodide and potassium carbonate proceeds normally to produce, as intermediates to the final product 2',4'vdimethoxy-benzylstyrene, the 4'-hydroxy^'^methoxyBenzylstyrene (oBtustyrene) and its 2'-hydroxy<-4'HTiethoxy isomer. 2 . Additional Data for the Structural Analysis of the Benzylstyrenes Structural identification of the P_. sativum Benzyl styrenes, sativastyrene and oBtustyrene, was ini t ia l ly hampered By a lack of adequate literature data on this class of compounds. Consequently, during the course of this project the UV, MS and NMR properties of a series of 10 Benzyl^ styrenes (TaBle III) were determined. NMR Analysis Benzylstyrene NMR spectra usually show only partial resolution of 62 the aromatic resonances and the ABX2 pattern of the propene. Our analysis of the Benzylstyrene series on a 270 MHz instrument (TaBle i v ) , as i l lus-trated by the partial spectrum in Figure 2 5 , gave sufficient resolution in some cases to obtain a first-order analysis of the spectrum. The resonances observed in Figure 2 5 , which do not include the methoxyl singlets (3 . 7 5 , 3 .76 and 3 . 8 0 ppm relative to TMS) or the Benzyl FIGURE 2 4 . GRADIENT CHROMATOGRAM OF 2 ' , 4 ' ^ D I H Y D R O X Y B E N Z Y L S T Y R E N E AND ITS METHYL ETHER D E R I V A T I V E S . S o l v e n t s y s t e m A . 62 TABLE III THE BENZYLSTYRENE SERIES TABLE IV BENZYLSTYRENE NMR DATA A s s i g n . 3' . -5' 6' Benzyl -0CH3 2,6 3,5 2',4'-Di-hydroxy " 4'-Hydroxy-2'-methoxy 21 -Hydroxy-41-methoxy 2',4'-Di-methoxy 2\4,4'^Tri-hydroxy 4,4*-Di-hydroxy-21-methoxy 4,2'-Di-hydroxy-41-methoxy 2K,4,4VTri-methoxy 6.30-6.443 (m)b 6.31-6.46 On) 6.28-6.47 On) 6.40-6.48 (m) 6.37 (d,2) 6.45 (d,2) 6.44 (a,2) 6.44 (d,2) 6.30-6.44 On). 6.31-6.46 On) 6.28-6.47 (m) 6.40-6.48 On)" 6.23-6.33 On) 6.38 (dxd,2,8) 6.30-6.39 On) 6.41 (dxd,2,8) 6.93 (d,8) 7.03 (d,8) 7.06 (d,8) 7.06 (d,8) 6.87 (d,8) 6.96 (d,8) 7.01 (d.8) 7.06 (d,8) 3.47 (d,6) 3.47 (d,6) 3.48 (d,6) 3.45 (d,6). 3.37 (d,6) 3.36 (d,6) 3.43 . (d,6) 3.43 (d,6) 6.30-6.44 On) 6.31-6.46 On) 6.28-6.47 On) 6.28-6.48 On)" 6.16 (dxt,6,16) 6.16 (dxt,6,16) 6.20 (dxt,6,16) 6.19 (dxt,6,16) 6.30-6.44 (m) 6.31-6.46 (m) 6.28-6.47 'On) 6.28-6.48 (m) 6.23-6.33 (m) 6.33 (d,16) 6.30-6.39 (m) 6.33 (d,16) 7.20-7.36 On) 7.19-7.39 (m) 7.17-7.37 (m) 7.22-7.36 (m) 3.83 3.77 3.79,3.81 3.82 3.70 3.75,3.76, 3.80 7.17 (d,9) 7.22 (d,9) 7.21 (d,9) 7.26 (d,9) 6.72 (d>9) 6.76 (d,9) 6.76 (d,9) 6.80 (d,9) co ppm; b - (d - doublet, t - triplet, m - multiplet); c - (d,8) - doublet, J = 8 Hz. 64 FIGURE 25. 270 MHZ NMR SPECTRUM OF 4-METH0XY-(21,4'-DIMETHOXYBENZYL)STYRENE. 65 doublet (.3.43 ppm, J = 6 'HzJ', were assigned on the following basis, The styrene aromatic protons would be expected to give an AB quartet with a coupling constant of ca^ . 10 Hz. Consequently, the peaks observed at 6.80 ppm and 7.26 ppm CJ = 9 Hz J have been assigned to protons g and f respec-tively. The benzyl aromatic protons would be expected to give an ortho coupled doublet (e), a meta coupled doublet (c) and a doublet of doublets (d). Additionally, c and d should be upfield (shielded) with respect to e. Thus e can be assigned the low field doublet at 7.06 ppm (J = 8 Hz) while c is observed at 6.44 ppm (J = 2 Hz) and d is observed at 6.41 ppm (J = 2, 8 Hz). The peak assignments for the ABX2 resonances are equally straight-forward with the %2 Benzyl doublet at 3.43 ppm as mentioned previously and the B doublet of triplets observed at 6.19 ppm CJ = 6, 16 Hz) with the A doublet at 6.33 ppm (J = 16 Hz). Unfortunately, as is apparent from an inspection of Table IV, the incomplete resolution observed for some compounds in combination with the relatively small observed differences (ca. .1 ppm) from spectrum to spectrum does not allow a positive identification of the position of the double bond or the relative positions of the -0H/-0CH3 in obtustyrene/ isoobtustyrene and sativastyrene/isosativastyrene based solely on their NMR data. Mass Spectral Analysis The mass spectral analysis of a benzylstyrene can potentially provide information not only on molecular formula and substituents but also on the division of the observed substituents between the phenyl groups and the relative position of the double bond. 66 The results obtained for all of the compounds used in this study are given in Table V while a typical mass spectrum is shown in Figure 26. The illustrated peak assignments have been confirmed By high resolution measurements. It is apparent from these results that fragmentation proceeds via isomerization of the propene to give an analagous series of Cg, Cg and fragments for Both of the original phenyl groups. This oBservation is not unexpected Because previous analyses of alkene isomers have shown that radical site migration accompanied By hydrogen rearrangement is a process which can occur within the expected lifetime of the ion. These results indicate that the molecular formula and substituents of the molecule can be determined from the parent ion and the presence of fragments which show the loss of m/e 15 (-CH3), 17 (-QH) and 31 (-0CH3). Additionally, the data presented in Table VI indicates that Both fragments of the Benzylic (tropylium) pair are always present in sufficient intensi-ties to allow the division of the identified suBstituents Between the two original phenyl groups. Unfortunately, the apparently random relationship of the Cj fragment intensities in TaBle VI precludes the positional assignment of the double bond. The mechanisms by which the radical site migration/hydrogen rearrangement process and the subsequent fragmentation pathways lead to the oBserved fragments are shown in Figures 27 and 28. In Figure 27 i t is illustrated that the migration/rearrangement step may take place By sequential 1,2 - sh i f ts 6 4 (Figure 27a) or By a 1,3 - s h i f t 6 5 (Figure 27B). Although the determination of the actual mechanism was not attempted it is apparent from the figure that either mechanism leads to an equivalent final result. 67 TABLE V BENZYLSTYRENE MASS SPECTRAL DATA 4'-0H 4'-0CH3 2',4'-OH 2'-OH 4'-0CH~ . 4'-0H 2<-0CH3 2i,4'-OCH3 2',4',4-OH 2'4-OH 4>-0CH3 4,4'-0H 2'-OCH3 2',4,4-+ m m - H m - CH~ m+ - 0R A 9 8 B 46 19 100 37 24 87 27 33 100 20 11 1 6 / l l d 100 22 19 35/26 100 25 20 34 100 10 17 100 18 6 7/10 100 20 14 18/20 100 13 11 58 C q H 7 + OR, OR CpH^ + OR, OR CgH^ + OR, OR 30 6 33 8 4 6 10 5 6 8 10 8 40 7 4 11 5 6 10 3 5 CpH,- + OR, OR C°H: + OR, OR CgHg + OR, OR 37 13 3 12 8 4 16 7 7 _ 10 9 7 E 14 6 6 15 10 12 8 7 C?-KQ + OR, OR 33 18 22 28 42 65 49 18 C Q H o + OR C q H ° + OR CgHg + OR 20 34 50 10 6 7 2 2 E 16 28 19 9 17 14 12 26 5 20 42 C p H , + OR ( M + OR C°HJ + OR 32 6 -C 4 29 8 48 4 9 4 4 8 C 7 H g + OR 45 23 69 25 92 39 CgHg CgHg 20 37 100 5 12 45 40 37 80 12 9 21 14 15 34 4 7 29 C 8 H ? 32 8 3 7 4 20 10 4 6 4 3 7 6 5 -C 7 H 7 60 19 100 16 19 20 a - OR is -OH and/or -OCH3 as appropriate b - Relative Intensity (%) c - Less than 3% Relative Intensity d - -OH/ - O C H 3 e - Division based on high resolution measurements FIGURE 2 6 . THE MASS SPECTRUM OF 4 ' - H Y D R O X Y B E N Z Y L S T Y R E N E . 69 TABLE VI THE RELATIVE INTENSITIES OF THE BENZYLSTYRENE C, FRAGMENT Relative Intensity (%) Benzylstyrene of the C7 Fragment 1' 2' 4_ Ring A Ring B -OH -H -H 45 60 -OCH3 -H -H 23 19 -OH -OH -H 33 100 -OCH3 -OH -H 18 16 -OH -OCH3 -H 22 19 -OCH3 -OCH3 -H 28 20 -OH -OH -OH 42 69 -OCH3 -OH -OH 65 25 -OH -OCH3 -OH 49 92 -OCH3 -OCH3 -OCH3 18 39 70 + • H + H • H + • y\ _ >ri ^ » < f> -c -c -c -< i> = z ± r < f > - c - c - c-4> * c p - c - c - c - o > i \ i ^ I I I ^ i I I H H H H H H H H H + H . H + H * + C - C - <j> ^ < j > - C - C - C - < j , * - <j> - c - c - c I I ^ I I I ^ I I I H H H H H H H H 1 , 2 - S h i f t 6 4 + / ^ \ H c - c - c 1 I I H H H - • C l H C l H C - $ H . ^ P \ H H + • C - C - C - <J> ^ (j>- C - C - C I I I ^ I I I H H H H H H B . 1 , 3 - S h i f t 6 5 FIGURE 2 7 . THE MECHANISMS OF RADICAL S I T E MIGRATION/HYDROGEN REARRANGEMENT 71 a - See Figure 27 for the mechanism of this migration/ rearrangement (Mechanism A Illustrated). b - The appropriate metastable peak has been observed. c - Homolytic cleavage (e.g. -CHg-CH^ CIir) . + Heterolytic cleavage (e.g. -CH?-CH-CH-) FIGURE 28. THE MASS SPECTRAL FRAGMENTATION OF 4'-METHOXYBENZYLSTYRENE (R = -0CH3). 73 These results have shown that MS analysis can provide molecular weight and substituent data on an isolated benzylstyrene. However, it appears that the determination of the position of the double bond will cc require additional procedures (e.g. chemical modification } or more exotic (e.g. f ield ionization 6 7) techniques, UV Spectroscopy and the Location of the Benzylstyrene Doubl e Bond As observed above, the NMR and MS data cannot be used to positively determine which phenyl is the styrene portion of the molecule. go Although Oil is e_t al_. have placed the double bond on new isolates "in conjugation with the least substituted aryl group, in analogy with the established position of the double bond in all other reported (benzyl -styrenes)" a more definitive analytical method seemed appropriate. One approach to the solution of this problem is the use of a 69 degradative reaction. Ollis et_ al_. have used osmium tetroxide/sodium metaperiodate in a two step procedure to produce diagnostic aldehyde fragments. As an alternative to this procedure we found during the course of this study that the addition of ruthenium tetroxide7^ to a benzylstyrene solution in acetone gave a reaction mixture which could be directly analyzed by HPLC for the production of aldehyde products (Figure 29). Our results, which showed that the cleavage of sativastyrene (XXIV) and isosativastyrene (XXV) gave £-hydroxybenzaldehyde as the only observed product (Scheme 4; Figure 29b), indicate that this procedure may be potentially useful in benzylstyrene degradations. Another potentially useful approach to the problem of styrene placement is the use of UV spectroscopy. Figure 30 shows the neutral and basic spectra of 2',4'-dihydroxybenzylstyrene and 4-hydroxy-(2',4'-dihydroxy-74 FIGURE 2 9 a . GRADIENT CHROMATOGRAM OF THE RUTHENIUM TETROXIDE REACTANTS AND POTENTIAL ALDEHYDE PRODUCTS. S o l v e n t s y s t e m C . 75 T I M E . m i n FIGURE 2 9 b . GRADIENT CHROMATOGRAM OF THE RUTHENIUM TETROXIDE CLEAVAGE PRODUCT OF SATIVASTYRENE. S o l v e n t s y s t e m C . 76 2 (nm) FIGURE 3 0 . THE UV ABSORPTION SPECTRA OF V , 4 ' - D I H Y D R 0 X Y B E N Z Y L S T Y R E N E AND 4 - H Y D R 0 X Y - ( 2 1 , 4 ' - D I H Y D R O X Y B E N Z Y L ) S T Y R E N E S o l v e n t m e t h a n o l o n l y ( ) . S o d i u m m e t h o x i d e a d d e d ( — ) . 77 OH C H O SCHEME 4. Ruthenium Tetroxide Cleavage of Sativastyrene (R-j = -OH, R2 = -0CH3) and Isosativastyrene (R1 = -0CH3, R2 = -OH) benzyl)styrene while Table VII presents the UV data for all of the benzyl-styrenes used in this study. An examination of the spectra in Figure 30 in combination with the tabular data indicate that the major absorption is observed at ca_. 253 nm when the styrene portion of the molecule does not have any oxy- substituents. This absorption is not greatly affected by changes in the pattern of hydroxylation or methoxylation of the benzyl portion of the molecule (Table VII; compounds 1-6). However, it shows an 8-10 nm bathochromic shift when the styrene is n-oxygenated (Table VII; compounds 7-10). In addition, i f the styrene is £-hydroxylated an additional 20 nm bathochromic shift is observed on addition of base (Table VII; compound pairs 3 and 7, 4 and 8, 5 and 9). These observed shifts are supported by the data from a series of trans-propenylbenzenes (XXVII; Table VIII) from which i t has been observed that the parent compound, t_-propenylbenzene (XXVII) has an absorption maximum at 250 nm. This absorption shows a 9 nm bathochromic shift on £-oxygenation of the ring with an additional 25 nm shift to 284 nm observed 78 TABLE VII BENZYLSTYRENE UV SPECTRAL DATA Compound UV Absorption (nm) No. R l R2 R3 Methanol Solvent NaOMe Added 1 -OH -H -H 253,a 282sh, 292sh 250, 283sh, 292 2 -OCH3 -H -H 255, 283, 292 NCb 3 -OH -OH -H 253, 282sh 249, 286, 292sh 4 -OCH3 -OH -H 254, 282sh 249, 286, 292sh 5 -OH -OCH3 -H 253, 281sh 246, 285, 292sh 6 -OCH3 -0CH3 -H 253, 281sh NC 7 -OH -OH -OH 262 283 8 -OCH3 -OH -OH 262 282 9 -OH -OCH3 -OH 261 281 10 -OCH3 -OCH3 -OCH3 262 NC a. Principal absorption underlined. b. No change 79 TABLE V I I I UV DATA OF A SELECTED S E R I E S OF TRANS-PROPENYLBENZENES Compound UV A b s o r p t i o n (nm) R M e t h a n o l S o l v e n t NaOMe A d d e d - H 2 5 0 -OH 259 2 8 4 - 0 C H o 259 80 on addition of base to the p_-hydroxylated derivative. In accord with these data and the observation of the insensitivity of the major absorption to changes in the substitution pattern of the benzyl ring of the benzylstyrene (e.g. Table VII; compounds 1 and 3) it has also been observed that the UV spectra of the analogous series of allyl phenols (XXVIII) "correspond 72 closely to those of the respective phenols and phenol ethers." The sum of these data indicate that, within the series studied, the position of the double bond and the presence or absence of a styrene n-hydroxyl can be determined from the UV spectrum. It should be noted, however, that these results have been observed for and may be restricted to the trans double bond and ^-oxygenation. XXVII XXVIII 81 C. The Response of Pi sum Sativum to Pathogen ic and Non-Pathogen i c  Fungal Stress As a preliminary step toward the metabolic evaluation of the post-infectional factors which determine disease resistance the chromatographic system was used to evaluate the response of P.. sativum to a pathogenic and a non-pathogenic fungus. The choice of Monilinia fructicola (MF, a pathogen of stone fruits) and Fusariuro solani f. sp. pi si (FSP) as, respectively, the non-pathogenic and the pathogenic stressors used in these studies was primarily based on their previous use in other investigations into the role of pisatin accumulation in disease response . 7 3 , 7 4 The results obtained from these studies are illustrated in Figures 31 to 33. Note that the analytical time period has been extended over that used for the abiotic t r ia ls . This change in procedure reflects a shift from a quantitative assessment of response to a more qualitative determina-tion of the effect of the fungus on metabolite accumulation and degradation. Specifically, because it is frequently diff icult or impossible to detect the 75 formation of fungal metabolites in infected tissue, the goal of these analyses was to determine i f the fungi exhibited major differences in their stimulation of metabolite production or in their ability to degrade the plant's isoflavonoid products. In Figures 31 and 32, which give the chromatograms for both MF and FSP 48 and 96 hours after inoculation , the major observed differences are a differential accumulation of sativastyrene at 96 hours and the appar-ent lack of accumulation of obtustyrene in MF at both 48 and 96 hours. However, i t would appear from Figure 33, which gives the accumulation trend lines for the major observed metabolites over a 10 day period, that the 82 FIGURE 31. GRADIENT CHROMATOGRAMS OF THE ETHANOLIC EXTRACT OF M. FRUCTICOLA (UPPER CHROMATOGRAM) AND F. SOLANI PISI (LOWER CHROMATOGRAM) STRESSED P. SATIVUM 48 HOURS AFTER INOCULATION . Solvent system A. 50 yl of a .1 ml/g dilution injected. 83 FIGURE 32. GRADIENT CHROMATOGRAMS OF THE ETHANOLIC EXTRACT OF M. FRUCTICOLA (UPPER CHROMATOGRAM) AND F. SOLANI PISI (LOWER CHROMATOGRAM) STRESSED P. SATIVUM 96 HOURS AFTER INOCULATION . Solvent system A. 50 yl of a .1 ml/g dilution injected. 84 FIGURE 33. QUANTITATIVE ASSESSMENT OF M. FRUCTICOLA AND F. SOLANI PISI STRESSED P. SATIVUM. Compound Identification: A, Afromosin; B, Obtustyrene; P, Pi satin; S, Sativastyrene. The following compounds had reasonably constant concentrations (ug/g): Sativone (4-8), Formononetin (2-4) and Anhydropisatin (10-15). 85 determination of pathogenicity, at least for this pair of fungi, is not dependent on a simple on/off mechanism of metabolite accumulation. Consequ-ently, the interpretation of these data will require a much more extensive examination of the differential effect of these fungi on P. sativum. In addition to the above results, the chromatogram in Figure 34 shows a novel metabolite which was observed in the analysis of MF/FSP stress. This compound, which is only observed in the 8 and 10 day Fusarium innoculated samples, has been identified as 3,6a-dihydroxy-8,9-methylenedioxypterocarpan (XXXI). This compound has been previously °> XXXI 74 reported as a product of pisatin metabolism by FSP. Our observation of its accumulation at a late stage in infection correlates with the previous conclusion^4'^6 that the degradation of pisatin to this compound by Fusarium is probably not significant in the determination of successful colonization. The resolution of this component of the stress mixture does, however, illustrate the ability of the HPLC system to detect the products of in vivo fungal metabolism. 86 j i i _ S O 25 20 T I M E . m i n FIGURE 34. A PARTIAL CHROMATOGRAM OF FSP STRESSED P. SATIVUM 10 DAYS AFTER INOCULATION. Solvent system A. 50 ul of a .1 ml/g dilution injected. 0-18 minutes omitted for clarity. 87 D. A Discussion of Pisum Sativum Stress Metabolism The development of an analytical technique which would be suitable for the resolution of complex isoflavonoid mixtures was the major goal of this project. However, the structural information which was presented in Section III.B.l and the rate of accumulation data given in Figure 35 which is analagous to and an extension of the results of Section III.A can provide information on biosynthetic, chemotaxonomic and pathological aspects of stress metabolite accumulation in P_. sativum. Consequently, a discussion of these results is presented here. 1. Phytochemical Aspects  Biosynthesis The pterocarpans have been recognized as natural products for 21 many years; mainly as heartwood constituents of various leguminous plants. However, it was not until their observation as de novo metabolites produced by growing tissues in response to stress that a route was opened for studies 21 of their biosynthesis. The phytoalexins of f_. sativum are pterocarpans. Therefore, it is not surprising that our structural results have shown that many of the observed metabolites of P^. sativum are related to the known pathways of pterocarpan biosynthesis. As a result, their presence in P_. sativum is in agreement with the previous work which has been done on pterocarpan biosynthesis in other Lotoideae species. In general, the relationships of these components of the stress mixture can be reasonably well illustrated by the biosynthetic flow scheme shown in Figure 36. The uncertainty in the biosynthesis of the 6a-hydroxypterocarpan (pisatin) is illustrated by the presence of two possible pathways (Figure 36, pathways A and B) from the 8,9-methylenedioxyisoflavone through the observed 88 24 48 72 FIGURE 35. THE RATES OF METABOLITE ACCUMULATION IN COPPER(II) CHLORIDE STRESSED P. SATIVUM. X axis - Hours after stress. Y axis - Concentration (ug/g). 89 mslonate /clnnamate BIOGENETIC RELATIONSHIPS^ 1*L'~C^ OF THE OBSERVED ISOFLAVONOID METABOLITES OF PISUM SATIVUM. Minor components (-•—). (x) - Observed in previous44'74 studies. Routes A and B reflect alternate possibilities. 90 pterocarpan and pterocarpene intermediates. The available rate data (Figure 35) indicates that the isoflavonoid metabolites can be divided into two accumulation types. In the first case, both pisatin, which is the apparent major end product of isoflavonoid stress metabolism, and formononetin, (XXXII) which is a key 21 27 isoflavone intermediate in pterocarpan biosynthesis, ' show similar accumulation curves which begin soon after stress and level off at ca_. 72 hours after stress. The isoflavone afromosin, (XXXIII), however, which is a branch point isoflavone to three minor component pterocarpans, only begins to accumulate at ca_. 48 hours after stress. These data support the observation that pisatin is a rapidly formed and potentially significant 43 metabolite of stress while the afromosin derived pterocarpans are part of a minor, latent pathway. XXXII 91 There are three additional metabolites which do not appear to fit the scheme of isoflavonoid biosynthesis. These compounds are the 2'-methoxychalcone sativone (XXII) and the benzylstyrenes sativastyrene (XXIV) and obtustyrene (XXVI). The biosynthetic pathways which may lead O XXII XXVI to the formation of these metabolites are not as well documented as is the 7 8 case in isoflavonoid synthesis. In particular, the currently favored scheme of benzylstyrene biosynthesis, which is given in Figure 37, is predominantly based on their co-occurrence in some of the Lotoideae (Leguminosae) with the neoflavonoid 3,3-diarylpropenes (XXIV) in combination with the in vitro observation of the formation of diarylpropenes as well as 93 XXXIV benzylstyrenes in the acidic condensation of cinnamyl alcohols ' or 79 cinnamyl pyrophosphate with phenols (Scheme 5). Until now the in vivo study of benzylstyrene biosynthesis has been difficult because these compounds are characteristically isolated 80 from heartwoods. Consequently, tracer studies on their synthesis have been virtually impossible. Our identification of the benzylstyrenes sativastyrene and obtustyrene as induced metabolites of f_. sativum should provide a system for the study of the mechanism of their biosynthesis with the aid of labelled precursors. Five points must be considered in the development of an acceptable biosynthetic scheme for the 2'-methoxychalcone and the benzylstyrene components of stressed f_. sativum. 1. The structural similarity of sativone and sativastyrene suggests that they may be biogenetically related. 2. In vivo reductive dehydroxylation of a phenol is a rarely observed 81 biosynthetic reaction. Consequently, the biosynthetic conversion of sativastyrene to obtustyrene is not favored. 94 SCHEME 5. CINNAMYL ALCOHOL/PHENOL CONDENSATION PRODUCTS . R and R' are -OH and -0CHo, 95 3. The partial methylation of a flavonoid by methyl transferase enzymes, as would be the case in the formation of the 2'-methoxychalcone from V ,4,4'-trihydroxychalcone, has not been observed. The evolutionary development of this route in the Lotoideae would be unusual because the 2'-hydroxyl is required for the utilization of the chalcone as an isofla-vonoid or flavonoid precursor. 4. The benzylstyrenes have characteristically been isolated with the 3,3-diarylpropene neoflavonoids.61 Although the possibility of widely differential accumulation cannot be excluded the absence of any major unidentified peaks in the f_. sativum chromatogram indicates that the parallel accumulation of approximately equivalent quantities of diarylpro-penes has not occurred. However, it should be noted that a preliminary analysis of the products of the acidic condensation of m-methoxyphenol and p_-hydroxycinnamyl alcohol by HPLC and MS indicated that none of the observed products were neoflanonoids. Consequently, the formation of sativastyrene by an activated cinnamyl alcohol/phenol condensation without the concomitant forma-tion of neoflavonoids cannot be ruled out. 5. The rate data (Figure 35) indicate that sativone and sativastyrene have similar accumulation curves. The accumulation pattern observed for obtustyrene is significantly different in that it begins much later. Unfortunately, these data do not determine whether obtustyrene is a precursor or a product of sativone or sativastyrene. In fact, the possibility of the late development of a second benzylstyrene pathway also cannot be excluded. This alternative would suggest the presence of parallel benzylstyrene pathways. 96 Figure 38 presents what appears to be the most favored biosyn-thetic scheme for the styrene and methoxychalcone metabolites along with several alternatives. Obviously, this scheme must be considered to be strictly hypothetical until additional studies have been performed. Further study of the biosynthesis of these compounds illustrates both a limitation and an additional application of HPLC to this type of problem. Figure 39 shows that the determination of possible biosynthetic intermediates which do not accumulate in the plant is nearly impossible using the chromatographic technique due to their low concentrations. However, the HPLC procedure will be useful in the next phase of this study which will require the isolation of labelled intermediates for the eluci-dation of the biogenetic relationships of sativone, sativastyrene and obtustyrene. Chemotaxonomy An additional phytochemical aspect of this study which is suggested by our results is the potential use of the structural data for chemotaxonomic studies. The pterocarpans have been shown to be of value 82 as taxonomic markers in the Lotoideae. Our studies on P.. sativum suggest that the benzylstyrenes, which have previously only been isolated from a number of Da1 bergia (Lotoideae) and Machaerium (Lotoideae) species, may be a new class of markers with taxonomic potential. Specifically, the 4'-hydroxy-2'-methoxybenzylstyrene, which was identified from stressed P. sativum (Lotoideae), has also been reported from Dal bergia retusa (Lotoideae).50 97 21-HYDROXYCHALCONES FIGURE 38. POSSIBLE BIOGENETIC RELATIONSHIPS OF THE P. SATIVUM STYRENE AND 2'-METHOXYCHALCONE METABOLITES. Favored pathway ( ). a - Not observed to accumulate b - The phenol precursors may not be of the form illustrated. FIGURE 39. GRADIENT CHROMATOGRAM OF POSSIBLE P. SATIVUM STYRENE AND 21-METHOXYCHALCONE BIOGENETIC PRECURSORS ( ). COPPER(II) CHLORIDE STRESSED SAMPLE AS REFERENCE (-—). Solvent system A. 99 2. Phytopathological Aspects Our analysis of the response of P. sativum to pathogenic and non-pathogenic stress has shown that the plant's accumulation of isofla-vonoid and related metabolites is qualitatively similar for the two fungi used in this study. This result suggests that the fungi do not exhibit gross differences in metabolite induction although a comparison of the chromatographic results obtained 6 days after innoculation (Figure 40) would seem to indicate that the Fusarium stressed plant is showing a greater commitment to benzylstyrene accumulation. It should be noted, however, that the differences observed are not particularly large and a variety of factors which may effect benzylstyrene accumulation (e.g. fungal polyphenol oxidases) have not been considered. An aspect of the presence of sativastyrene in stressed £_. sativum which is at present hypothetical but nonetheless interesting is the possibility that sativastyrene may function in the plant's disease response through its ability to form reactive quinone-methides. The reaction in Scheme 6, which is plausible on chemical grounds, could g furnish "quinones which may participate in the resistance process by condensation with proteins and enzymes or by polymerization to tannins and lignans which may act as protein precipitants or as physical barriers to parasite expansion." The proof of this possibility will require further investigation into the in vitro and in vivo chemistry and biochemistry of this class of compounds. 100 FIGURE 40. PARTIAL CHR0MAT0GRAMS OF M. FRUCTICOLA (A) AND F. SOLANI  PISI (B) STRESSED P. SATIVUM 6 DAYS AFTER INOCULATION . 101 SCHEME 64 FORMATION OF A QUINONE-TYPE PRODUCT FROM SATIVASTYRENE 102 IV. CONCLUSION A high performance liquid chromatographic procedure has been developed which will separate complex mixtures of P.. vulgaris (green bean) and P_. sativum (garden pea) isoflavonoids to give a chromatographic fingerprint of the post-stress metabolic response of these plants. This system has been used to determine the time course of metabolite accumula-tion in extracts from abiotically stressed plants. These accumulation rate studies represent one of the most significant applications of the HPLC procedure because the quantification of a selected number of components of a mixture by other chromatographic methods can be, at best, tedious. In addition to the study of the P_. vulgaris and P_. sativum phytoalexins the HPLC procedure has been used for the isolation and characterization of a number of other post-stress metabolites from P. sativum. This study led to the characterization of the previously unreported 2'-methoxychalcone sativone and the benzylstyrene sativasty-rene as well as a number of compounds which have been previously observed from other plant sources. The characterization of the benzylstyrenes sativastyrene and obtustyrene is particularly interesting because until now these compounds have only been described from heartwoods. Consequently, their identifica-tion as de novo metabolites of stressed P. sativum will allow ah in vivo investigation into their biosynthesis. To facilitate further studies on the chemistry and biochemistry of these compounds their MS, NMR and UV properties have been evaluated. 103 •'- A final aspect of this study was the qualitative evaluation of the response of f_. sativum to a pathogenic and a non-pathogenic fungus. These results illustrated that the plant's net response to the two fungi was similar. However, a more important aspect of the study was its demonstration that the HPLC procedure could be used to determine the effects of in vivo fungal metabolism on the plant's accumulation of isoflavonoids. 104 V. EXPERIMENTAL A. General Methods Methods Electronic Spectroscopy A Cary recording spectrophotometer (Model 17) was used to obtain ultra-violet and visible spectra. Solution spectra were obtained with matched silica glass cells of 10 mm path length. The addition of reagents 84 to the methanolic solutions follows the procedure of Mabry. Infrared Spectroscopy Infrared spectra were recorded on a Perkin-Elmer Model 457 grating spectrophotometer covering the frequency range 4000-600 cm-1. The cell windows used were NaCl. IR spectra were calibrated with polystyrene film at 1601.4 cm"1. Nuclear Magnetic Resonance Nuclear magnetic resonance spectra were obtained at 270 MHz (Fourier-transform) with a U.B.C. NMR Centre modified Brliker spectrometer and at 100 MHz with a Varian HA-100 spectrometer for continuous-wave spectra and a Varian XL-100 or Nicolet Model NIC-80 spectrometer for Fourier-transform spectra. The chemical shifts are recorded in the 6 (ppm) scale with tetramethylsilane (TMS) as an internal standard. Mass Spectrometry Mass spectra were recorded on an Atlas CH-4 spectrometer or an A.E.I. MS-902 spectrometer. High resolution measurements were obtained on 105 the latter instrument. In most cases only those fragments of greater than 10% relative intensity are reported. The form used is; m/e. (% Relative Intensity). Melting Point Determination Melting points were measured with a Thomas-Hoover capillary melting point apparatus and are uncorrected. Elemental Analysis Elemental analysis for carbon and hydrogen were performed by Mr. P. Borda of the Microanalytical Laboratory, U.B.C. Reagents and Sol vents All chemicals and solvents were reagent grade unless otherwise indicated. Spectral grade solvents were used in all solutions for ultra-violet and visible spectra. 106 B. Growth, Stress and Extraction of Plants Growing Conditions The pea and bean seed used in these studies was obtained from Buckerfields Ltd., Vancouver, B.C., Canada. The varieties used were: Garden pea -Pisum sativum cv. Melting sugar Green bean - Phaseolus vulgaris cv. Topcrop The soybean seed used was purchased in a grocery store. The variety was not determined. All plants, unless otherwise indicated, were grown in the dark at 23 +2°C. Bulk Growth 100 g samples of seed were grown and stressed in bulk for the extraction of large quantities of metabolites for isolation and identifi-cation. In a typical experiment the seed was placed in a liter beaker and rinsed twice with water before the addition of 700 ml of water. After standing overnight the excess water was decanted and the seed rinsed twice with water. The container was covered loosely with foil, and left in the dark. The water rinses were repeated twice daily for c£. 6 days. The seedlings were then ready for copper(II) chloride stress. Growth for Rate or Fungal Stress Studies In a typical experiment 40 g of seed in a suitable beaker was rinsed twice with water and the seed culled for cracked seed coats, etc. The seed was then placed in a 10 x 30 cm glass dish which had 1 cm of water soaked filter paper placed in the bottom. A sheet of filter paper was placed over the seed and the container was covered loosely with a glass 107 plate. The container was placed in a dark cabinet at room temperature. The seedlings were allowed to grow for ca_. 6 days (radical length 1-2 cm) with the addition of water to the paper mat as necessary. The seedlings were subsequently stressed for the required study. Stress Procedures  Chemical Stress In both the bulk and rate study experiments the seedlings were stressed by soaking for 30-45 minutes in copper(II) chloride (3 or 9x10 M ) . After decanting the solution the samples were left loosely covered in the dark and moistened with water as necessary. The bulk tests were typically extracted 4 days after the initial stress. The rate tests were extracted at appropriate time intervals as designated in the text. The results of these abiotic tests have been replicated at least 3 times. Fungal Stress The fungi used in these studies were sub-cultured from pure isolates on potato-dextrose agar (Difco) in petri plates and allowed to grow under ambient conditions (20-25°C; dark) for 30 days before storage at 2°C until needed. The pure cultures were obtained from: Monilinia fructicola - Dr. R. J. Copeman, Dept. of Plant Pathology, The University of British Columbia. Fusarium solani f. sp. pisi - Dr. B. MacNeill, Dept. of Environ-(Isolate No. 160) mental Biology, The University of Guelph, Ontario. 108 The innoculum used in the stress trials was prepared by irrigating the plates with 1Q ml of sterile distilled water for 15 minutes and straining the resulting suspension through 2 layers of cheese cloth. 4 85 This gave an innoculum density of approximately 3x10 spores/ml. This solution was used at a rate of 15-30 ml per dish of seedlings. The excess liquid from the inoculation solutions was usually taken up by the paper mat and as a consequence was not decanted. The Fusarium isolate used was periodically checked for virulence on P. sativum. This was done by irrigating 2 week old plants which were growing in autoclaved soil with the innoculum solution described above. After an additional 2 weeks the plants were usually wilted and showed the expected root necrosis.**6 The fungal stress results were replicated twice. Extraction of Plant Materials After the appropriate growth period the plants were weighed (wet weight; total sample for bulk extracts, 3-6 g for rate studies) and extracted as described below. Ethanolic Extracts Ethanolic extraction of samples followed the general procedure 44 of VanEtten. The sample was twice macerated with 95% ethanol. After filtration two volumes of water were added and the ethanol removed under reduced pressure on a rotary evaporator. The aqueous fraction was acidified with 5% aqueous HC1 (ca. 2 ml) and extracted with ether (2x1 volume). After drying over anhydrous sodium sulfate the sample was filtered and the solvent removed on a rotary evaporator. This procedure was not observed, 109 by UV analysis, to cause the dehydration of pisatin to anhydropisatin. The residue was dissolved in methylene dichloride for analysis. Typical dilutions ranged from .1 to .5 ml of methylene dichloride per gram of sample (wet weight). Extraction of Aqueous Washings As described in the text the bulk growth experiments were washed with water to determine if the stressed plant exudates were significantly different from the ethanolic extracts. To obtain these data the bulk stressed samples were soaked with 500 ml of water 4 days after the initial stress and the resultant aqueous solution extracted with 2x1 volume of ether after acidification with 25% aqueous NCI. After drying the ethereal solution over anhydrous sodium sulfate, filtration and removal of solvent on a rotary evaporator the residue was dissolved in methylene dichloride for analysis. The dilutions used (.1-.5 ml/g) were based on the wet weight of the sample which provided the original aqueous solution. Hydrolytic Extraction Hydrolytic extraction of samples followed the procedure 87 of Harborne. The samples (ca. 5 g) were added to 50 ml of 2N HC1 and hydrolyzed for 30 minutes at 100°C. After cooling and filtration through 2 layers of cheesecloth the solution was twice extracted with equal volumes of ethyl acetate. If necessary minimal quantities of methanol were added to break troublesome emulsions. After drying over anhydrous sodium sulfate and filtration the solvent was removed on.a rotary evaporator. The residue was dissolved in methylene dichloride/ethanol (19/1) using the usual dilutions. no C. Chromatography High Performance Liquid Chromatography The HPLC system used was a Waters Assoc. ALC 202 equipped with a 2 cm Corasil II (Waters) guard column, a y-Porasil (Waters) column and a 280 nm detector. The solvent gradient (Figure 41) was generated via a simple solvent displacement mixer (Figure 42) and was not varied throughout the study. Therefore, it is presented here and is not reproduced on each of the chromatograms. The pertinent standard conditions were: Solvent flow: 1.5 ml/min. Pressure: 300 - 800 psi. Detector: Range setting 32. Chart speed: .75 inch/min. The solvents used were purified as follows: Hexanes: Traces of aromatics were removed from reagent hexanes by absorption on a silica gel 93 column. Methylene dichloride (MDC): Reagent grade methylene dichloride was distilled once to remove non-volatile impurities. Ethanol: 100% ethanol was distilled once. Acetic acid (AcOH): Glacial. FIGURE 41. THE STANDARD GRADIENT 112 > > P u m p A B > F l o w FIGURE 42. THE GRADIENT MIXER. The initial solvent volume of B was 25 ml. A is the high polarity solvent. B is the low polarity solvent. 113 The solvent systems used were (all ratios are v/v): Gradient chromatography: System High Polarity Solvent Low Polarity Solvent A MDC/EtOH/AcOH (97/3/.2) 10% high polarity in hexanes B MDC/EtOH/ACOH (97/3/3) 10% high polarity in hexanes C MDC/EtOH/AcOH (97/3/.2) 30% high polarity in hexanes D MDC/EtOH/AcOH (99/1/.2) 10% high polarity in hexanes All gradients were generated from the low polarity solvent to the high polarity solvent. Isocratic chromatography: System Solvent 1 12% of high polarity solvent A (as above) in hexanes 2 15% of high polarity solvent A (as above) in hexanes 3 60% of high polarity solvent A (as above) in hexanes 4 80% of high polarity solvent A (as above) in hexanes 5 Low polarity solvent D (as above) HPLC quantification of most of the-uietabolites of this study was performed by the injection of standard solutions to give straight line correlations for peak height to ug sample. Note that although the peak which was quantified as 'afromosin' was a mixture since the UV absorptions of the two components were similar their detector response characteristics should be nearly equivalent. Additionally, the quantification of anhydropisatin was by collection and 51 measurement of absorbance at 357 nm. 114 Typical accumulation levels observed for bulk stressed P\ sativum are given in Table IX. Thin-layer Chromatography The appropriate TLC conditions are listed in the text in the form TLC (PE/E; 1/1: = .xx). This means: Thin-layer chromatography; Solvent system: 30-50°C petroleum ether/ether (1/1; v/v); Rf = .xx. The TLC plates used were: Silica gel GF precoated plates (Analtech uniplate, 250 j i ) . Column Chromatography The appropriate column chromatography conditions are listed in the synthetic section. The silica gel used was Woelm, activity I (ICN Pharmaceuticals). 115 TABLE IX TYPICAL LEVELS OF METABOLITE ACCUMULATION IN COPPER(II) CHLORIDE STRESSED P. SATIVUM Compound yg/g (Wet- Weight) 4,4'-Di hydroxy-21-methoxychalcone 20a Sativastyrene 33 (30-65)b Formononetin 2 'Afromosin' 1 Pisatin 100 (80-130) 4'-Hydroxy-2'-methoxybenzy1styrene 4 Anhydropisatin 13 a - 48 hours after the initial stress b - 96 hours after the initial stress 116 D. Synthesis The following compounds were prepared or isolated by known procedures: Chalcones p o 41-Benzyloxy-2'-hydroxychalcone EC 4,4'-Bis(benzyloxy)-2'-hydroxychalcone 89 4,4'-Dihydroxychal cone 90 2',4-Di hydroxy-41-methoxychalcone 91 4,4'-D imethoxychal cone 92 3',4'-Dimethoxy-4-hydroxychalcone 93 4'-Hydroxy-4-methoxychalcone q n 2' ,4,4'-Trihydroxychal cone Isoflavones 94 4',7-Dihydroxyisoflavone 94 4',7-Dimethoxyisoflavone 95 41,6-Dimethoxy-7-hydroxyi sof1avone 7-(Y,Y-Dimethylallyloxy)-4,-methoxyisoflavone 96 96 94 7-Hydroxy-4'-methoxyisoflavone96 41 -Hydroxy-7-methoxyisof1avone" 7-Hydroxy-3',41-methylenedioxyisof1avone96 97 7-Methoxy-31,41-methylenedioxyisof1avone 98 4',5,7-Trihydroxyisoflavone Pterocarpans 51 6a,7a-Dehydro-3-methoxy-8,9-methylenedioxypterocarpan 43 6a-Hydroxy-3-methoxy-8,9-methylenedioxypterocarpan 117 Miscellaneous 59 2',4'-Dihydroxybenzylstyrene 57 4',7-Dihydroxyflavanone CO 4'-Hydroxybenzylstyrene p_-Hydroxycinnaniyl alcohol60 55 41-Hydroxy-21-methoxyacetophenone 99 4-Hydroxy-2-methoxybenzaldehyde 50 41-Hydroxy-2 -methoxybenzylstyrene The following compounds were received as gifts: 2,3,9-Trimethoxy-, 3-hydroxy-2,9-dimethoxy- and 4-hydroxy-2-3,9-trimethoxy-pterocarpan were received from Dr. H. VanEtten. Phaseollin, phaseollinisoflavan and kievitone were received from Dr. J. Rahe. The following compounds were synthesized during the course of this study. Their syntheses are given in the order: 4,4'-B i s(benzyloxy)-2'-methoxychalcone 4,41-Di hydroxy-2'-methoxychalcone 4-Hydroxy-(2',4'-dihydroxybenzyl)styrene 4-Methoxy-(2'-4'dimethoxybenzyl)styrene 4-Hydroxy-(4'-hydroxy-2'-methoxybenzyl)styrene 4-Hydroxy-(21-hydroxy-41-methoxybenzyl)styrene 41-Benzyloxy-2'-methoxychalcone 4'-Hydroxy-2'-methoxychalcone 4'-Hydroxy-2'-methoxybenzylstyrene 2'-Hydroxy-41-methoxybenzylstyrene 2',4'-Dimethoxybenzylstyrene 4'-Methoxybenzylstyrene 118 4,4'-Bis(benzyloxy)-2'-methoxychalcone 4,4'Bis(benzyloxy)-2'-hydroxychalcone (2.10g, 4.79 mmoles) was refluxed for 2 hours with an excess of methyl iodide (3 ml) and potassium carbonate (5 g) in 100 ml of spectroquality acetone. After filtration the acetone was reduced in volume on a rotary evaporator to 40 ml and 60 ml of ethanol (100%) added. Further removal of solvent until solid began to appear gave 1.77 g (82%) of the desired chalcone as bright yellow needles. m.p. 94-95°C, (Found: C, 79.43; H, 5.85%; m+, 450.1808. C2gH2604 requires C, 79.42; H, 5.98%; m+, 450.1831); Amax (methylene dichloride) 236 (3.26) and 340 nm (3.48); vmax (CHC13) 3001, 1601, 1250, 1163, 1129, 1020br, 830 cm"1; 6 (100 MHz, CDC13) 3.78 (3H, s, -OCI^ ), 5.01 and 5.03 (4H, 2xs, OCH2*), 6.50-6.64 (2H, m, 3', 5'), 6.91 (2H, d, J 8 Hz, 3, 5), 7.18-7.76 (15H, m, a, ,g, 2, 6, 6\ 2x-<j>); MS 450(14), 359(12), 197(12), 92(15), 91(100). 4,4'-Pi hydroxy-2'-methoxychal cone  Method A 4'-Hydroxy-2'-methoxyacetophenone (489 mg, 2.95 mmoles) and p_-hydroxybenzaldehyde (396 mg, 3.25 mmoles) were dissolved in 8 ml of 95% ethanol. 10 ml of 60% (w/wjaqueous potassium hydroxide was added. The reaction flask was stoppered and set aside for 4 weeks. The impure product was precipitated from the reaction mixture by acidification with 25% aqueous HC1 and collected. The solid was crystallized from aqueous ethanol as slightly impure (TLC) red microcrystals which resisted subsequent purifica-tion procedures. Yield: 165 mg (21%). 119 Method B 4,4'-Bis(benzyloxy-2'-methoxychalcone (230 mg, .510 mmoles) was debenzylated in cone. HCl/glacial acetic acid [2/3 (v/v), 25 ml] by stirring the reaction mixture for 2 hours in a water bath at 48°C. The product was extracted into ether after the addition of water (100 ml) to the reaction mixture. The ether was successively extracted with water, 5% aqueous sodium bicarbonate and water. After drying over anhydrous sodium sulfate and filtration the solvent was removed on a rotary evaporator. The residue was chromatographed on silica gel (15 g, 20x1 cm column) with methylene dichloride/acetone (4/1) and the resultant solid crystallized from aqueous ethanol to give 70 mg of the product (51%) as orange microcrystals. m.p. 210-212°C, (Found: C, 70.90; H, 5.11%. C]6H1404 requires C, 71.10: H, 5.22%); xmax (methanol) 234 (4.00) and 349 nm (4.25); Amax (NaOMe added) 260, 419 nm; vmax (nujol) 1560br, 1330, 1315, 1250br, 1218br, 1170, 1120, 825 cnfl; <5 (100 MHz, (CD3)2C0) 3.93 (3H, s, -OCHg), 6.53 (IH, dxd, J 2, 8 Hz, 5'), 6.58 (IH, d, J 2 Hz, 3'), 6.91 (2H, d, J 8 Hz, 3, 5), 7.28 (IH, d, J 18 Hz, a), 7.50-7.66 (5H, m, 6, 2, 6, 6'); MS 271(20), 270(100), 269(19), 255(26), 242(23), 164(39), 163(17), 151(71), 147(29), 119(19), 107(22). 120 4-Hydrdxy-(2',4'-dihydroxybenzyl)styrene £-Hydroxycinnamyl alcohol (.750 g, 5 mmoles) was dissolved with resorcinol (1.10 g, 10 mmoles) in 50 ml of 75% (v/v) acetic acid/water and stirred for 15 minutes at 70°C. The product was extracted into ether after the addition of 100 ml of water. The ether layer was extracted successively with water, 5% aqueous sodium bicarbonate and water followed by drying over anhydrous sodium sulfate, filtration and removal of the solvent on a rotary evaporator. Column chromatographic purification of the residue on silica gel (100 g, 45x4 cm column) with petroleum ether (30-50°C)/ether (1/3; TLC = .65) and subsequent removal of the solvent gave .449 g of the TLC pure product (37%) as a glass which resisted all attempts at crystallization. The compound proved to be too unstable for adequate microanalysis but unambiguous reaction to the trimethoxy- derivative, which gave a satisfactory analysis, along with the usual NMR and MS data proved its structure. Found: m+, 242.0925. C^H^Og requires m+, 242.0943; Amax (methanol) 262 nm (ca. 4.20); Amax (NaOMe added) 240sh, 283 nm; 6 (270 MHz, (CD3)2C0) 3.37 (2H, d, J 6 Hz, -benzyl), 6.16 (TH, txd, J 6, 16 Hz, a ) , 6.23-6.33 (2H, m, 3, 5'), 6.37 (IH, d, J 2 Hz, 3'), 6.72 (2H, d, J 9 Hz, 3, 5), 6.87 (IH, d, J 8 Hz, 6'), 7.17 (2H, d, J 9 Hz, 2, 6); MS 243(12), 242(100), 241(10), 225(17), 149(10), 147(40), 135(10), 133(22), 132(13), 131(28), 123(42), 119(29), 107(69). 121 4-Methoxy-(2',4'-dimethoxybenzyl)styrene 4-Hydroxy-2',4'-dihydroxybenzyl)styrene (108 mg, .446 mmoles) was refluxed in 25 ml of spectroquality acetone with excess methyl iodide (1.3 ml) and potassium carbonate (1.1 g) for 8 hours. After filtration the solvent was removed on a rotary evaporator and the residue purified by thick layer chromatography on silica gel (2000u layer, 40x40 cm plate) with petroleum ether (30-50°C)/ether (4/1, Rf = .75) solvent. After extraction from the plate with ether, filtration and removal of solvent the residue was crystallized from aqueous methanol to give 72 mg (57%) of the product as white plates. m.p. 66-67°C, (Found: C, 75.60; H, 6.82%; m+, 284.1432. C18H2003 requires C, 76.03; H, 7.09%; m+, 284.1412); Max (methylene dichloride) 262 nm (4.35); vmax (CHC13) 1611, 1506, 1465br, 1296, 1252br, 1184, 1165, 1127, 1045, 975, 845 cm"1; 6 (270 MHz, (CD3)2C0) 3.43 (2H, d, J 6 Hz, - benzyl), 3.75, 3.76, 3.80 (9H, 3xs, -0CH3), 6.19 (IH, txd, J 6, 16 Hz, a), 6.33 (IH, d, J 16 Hz, 3 ) , 6.41 (IH, dxd, J 2, 8 Hz, 5'), 6.44 (IH, d, J 2 Hz, 3'), 6.80 (2H, d, J 9 hz, 3, 5), 7.06 (IH, d, J 8 Hz, 6'), 7.26 (2H, d, J 9 Hz, 2, 6); MS 284(20), 283(100), 282(13), 269(11), 253(58), 151(18), 146(20), 145(42), 121(39). 122 4-Hydroxy-(4'-hydroxy-2'-methoxybenzyl)styrene (Sativastyrene) and  4-Hydroxy-(2'-hydroxy-4'-methoxybenzyl)styrene (IsosatiVastyrene) Method A p_-Hydroxycinnamyl alcohol (.750 g, 5 mmoles) was dissolved with m-methoxyphenol (1.24 g, 10 mmoles) in 20 ml of 75% (v/v) acetic acid/water and stirred for 15 minutes at 60°C. The products were isolated as for the trihydroxybenzylstyrene with column chromatography using petroleum ether (30-50°C)/ether (1/1; TLC upper isomer Rf = .65, lower isomer Rf = .57) to give .444 g of the mixed isomers (30%). Separation of the isomers by column chromatography on silica gel as above using petroleum ether (30-50°C)/ ether (2/1) and methylene dichloride/ethanol (19/1; TLC upper isomer Rf = .40, lower isomer Rf = .35) gave 205 mg of TLC pure isomer L (lower R^.) and 190 mg of TLC pure isomer U (upper Rf) as glasses. Both isomers resisted all attempts at crystallization and proved to be too unstable for satisfactory microanalysis. However, both isomers, when reacted with methyl iodide in the usual manner, gave the expected trimethoxybenzylstyrene. Analysis of the two isomers gave: Isomer U Found: m+, 256.1079. C16H1603 requires m+, 256.1099; Amax (methanol) 262 nm (ca. 4.0); Amax (NaOMe added) 282 nm; 6 (270 MHz, (CD3)2C0) 3.43 . (2H, d, J 4 Hz, - benzyl), 3.70 (3H, s, -0CH3), 620.(IH, txd, J 6, 16 Hz, a), 6.30-6.39 (2H, m, 3, 5'), 6.44 (HI, d, J 2 Hz, 3'), 6.76 (2H, d, J 9 Hz, 3, 5), 7.01 (IH, d, J 8 Hz, 6'), 7.21 (2H, d, J 9 Hz, 2, 6); MS 257(18), 256(100), 255(18), 225(10), 161(11), 150(27), 149(14), 137(65), 133(19), 131(17), 121(18), 120(31), 119(48), 107(25). 123 Isomer L Found: m+, 256.1105. C^H^Og requires m+, 256.1099; Amax (methanol) 261 nm (ca. 4.0); Amax (NaOMe added) 281 nm; 6 (270 MHz, (CD3)2C0) 3.36 (2H, d, J 6 Hz, - benzyl), 3.82 (3H, s, -OCHg), 6.16 (IH, txd, J 6, 16 Hz, a), 6.33 (IH, d, J 16 Hz, g), 6.38 (IH, dxd, J 2, 8 Hz, 5'), 6.45 (IH, d, J 2 hz, 3'), 6.76 (2H, d, J 9 Hz, 3, 5), 6.96 (IH, d, J 8 Hz, 6'), 7.22 (2H, d, J 9Hz, 2, 6); MS 257(19), 256(100), 255(20), 241(14), 239(18), 225(20), 161(10), 150(36), 149(15), 148(10), 147(12), 137(49), 133(14), 132(12), 131(26), 122(38), 121(37), 107(92). From these data a definite assignment of isomer structure could not be made. Method B For the assignment of structure to each of the isolated isomers the corresponding chalcones (4,4'-dihydroxy-2'-methoxy- and 2',4-dihydroxy-4'-methoxy-) were reduced using A1H3>61 The chalcone (10 mg) was dissolved in ether (50 ml) and a solution of A l i n ether (prepared by adding 207 mg of AlCI3 to a solution of 120 mg of LiAlH^ in 30 ml of ether) added until precipitation was complete. On extraction of the reaction mixture with 5% aqueous HC1 followed by chromatography a small amount (TLC: ca_. 10% yield) of a single product was obtained from each chalcone which on HPLC (gradient chromatography, solvent systems A and C) and TLC (PE/E; as described above) comparison with the isolated isomers U and L identified the isomers as: U - 4-Hydroxy-(2'-hydroxy-4'-methoxybenzyl)styrene (Isosativastyrene) L - 4-Hydroxy-(4'-hydroxy-2'-methoxybenzyl)styrene (Sativastyrene) 124 4' -Benzyloxy-2'methoxychalcone 4'-Benzyloxy-21-hydroxychalcone (614 mg, 1.86 mmoles) was refluxed for 1 hour with an excess of methyl iodide (1.5 ml) and potassium carbonate (3 g) in 50 ml of spectroquality acetone. After filtration 50 ml of methanol was added and the solution reduced in volume on a rotary evaporator to 20 m l . Addition of water (10 ml) and cooling gave 606 mg (95%) of the desired chalcone as pale yellow needles. m.p. 88-89°C, (Found: C, 80.46; H, 5.66. C23H2003 requires C, 80.21; H, 5.85); Amax (methylene dichloride) 306 nm (4.30); vmax (CHC13) 1603, 1333, 1167, 1131, 1025br, 840br cm'1; 6 (100 MHz, CDC13) 3.84 (3H, s, -0CH3), 5.08 (2H, s, -OCH^ ), 6.50-6.66 (2H, m, 3', 5'), 7.18-7.78 (13H, m, 6', a , 3 , -4>); MS 344(50), 253(13), 91 (100). 4'-Hydroxy-2'-methoxychalcone 4'-Benzyloxy-2'-methoxychalcone (204 mg, .593 mmoles) was debenzylated in cone. HCl/ethanol (3/10, v/v) by refluxing the solution for 36 hours. After the addition of water the reaction solution was extracted with ether. The ether layer was subsequently extracted with 1% K0H which was in turn acidified with 5% HC1 and extracted with ether. The ether solution was dried over sodium sulfate, filtered and the solvent removed on a rotary evaporator. The residue was chromatographed on silica gel (15 g, 20x1 cm column) with methylene dichloride/acetone (19/1) and the resultant solid crystallized from aqueous methanol to give 81 mg of the product (54%) as yellow needles. m.p. 143-144°C. The literature m.p. (crystallized from ethanol) was 152-153°C. (Found: C, 76.0,0; H, 5.41. C16H]403 requires C, 75.57; H, 5.55); 125 Xmax (methanol) 305.(4.37) and 334sh nm (4.26); xmax (NaOMe added) 298 and 386 nm; vmax (CH2C12) 1610, 1305br, 1210, 1120br, 960 cm"1; 6(100 MHz, (CD3)2C0) 3.88 (3H, s, - O C H 3 ) , 6.42-6.58 (2H, m, 3', 5'), 7.28-7.44 (2H, m, a, 6'), .7.54-7.74 (6H, m, @, MS 255(20), 254(100), 253(36), 239(18), 226(33), 151(62), 136(30). 41-Hydroxy-2'-methoxybenzylstyrene (Obtustyrene) The synthesis of this compound, via the chalcone and the condensa-50 tion of m-methoxyphenol and cinnamyl alcohol, has been described. Conse-quently, only the analytical data, which is more complete than that given in the literature, is presented here. Found: m+, 240.1152. CifiH-|602 requires m+, 240.1151 ; xmax (methanol) 253 (4.22) and 282sh nm (3.84); Amax (NaOme added) 246, 287 and 292sn nm; vmax ( C H C I 3 ) 3 6 0 1• 1 5 9 8» 1 4 9 3» 1 4 7 0» 1290br, 1150, lllObr, 1041, 962br, 840 cm"1 ; 6 (270 MHz, C D C I 3 ) 3.47 (2H, d, J 6 Hz, -benzyl), 3.83 ( 3 H , s, - O C H 3 ) , 6.31-6.46 (4H, m, a, e, 3', 5'), 7.03 (IH, d, J 8 Hz, 6'), 7.19-7.39 ( 5 H , m, -*); MS 241(19), 240(100), 239(22), 225(19), 223^ 35), 209(26), 163(12), 149(12), 137(15), 117(12), 116(13), 115(36), 91(23). 2'-Hydroxy-41-methoxybenzylstyrene This compound, which is isomeric with obtustyrene, was obtained as a second product from the obtustyrene m-methoxyphenol and cinnamyl 42 alcohol condensation (.01 mole scale, 14% yield). It was isolated as a TLC pure oil (PE/E, 50/1; = .62 : obtustyrene = .53) which could not oe distilled without decomposition. Found: m+, 240.1143. C^ gH^ O,, requires m+, 240.1151 ; Amax (methanol) 254 (ca_.4.2) and 281 sh nm (ca.3.8); xmax (NaOMe added) 249, 286 and 292sh 125 Xmax (methanol) 305 (4,37) and 334sh nm (4.26); xmax (NaOMe added) 298 and 386 nm; vmax (CH2C12) 1610, 1305br, 1210, 1120br, 960 cm"1; 6(100 MHz, (CD3)2C0) 3.88 (3H, s, - O C H 3 ) , 6.42-6.58 (2H, m, 3', 5'), 7.28-7.44 (2H, m, a, 6'), 7.54-7.74 (6H, m, 3, -$); MS 255(20), 254(100), 253(36), 239(18), 226(33), 151(62), 136(30). 4'-Hydroxy-21-methoxybenzylstyrene (Obtustyrene) The synthesis of this compound, via the chalcone and the condensa-50 tion of m-methoxyphenol and cinnamyl alcohol, has been described. Conse-quently, only the analytical data, which is more complete than that given in the literature, is presented here. Found: m+, 240.1152. C^ H^ O,, requires m+, 240.1151 ; Amax (methanol) 253 (4.22) and 282sh nm (3.84); Amax (NaOme added) 246, 287 and 292sn nm; vmax (CHC13) 3601, 1598, 1493, 1470, 1290br, 1150, lllObr, 1041, 962br, 840 cm"1 ; $ (270 MHz, CDClg) 3.47 (2H, d, J 6 Hz, -benzyl), 3.83 ( 3 H , s, - O C H 3 ) , 6.31-6.46 (4H, m, o, 3, 3', 5'), 7.03 (IH, d, J 8 Hz, 6'), 7.19-7.39 (5H, m, -<(.); MS 241 (19), 240(100), 239(22), 225(19),'223(35), 209(26), 163(12), 149(12), 137(15), 117(12), 116(13), 115(36), 91(23). 2'-Hydroxy-4'-methoxybenzylstyrene This compound, which is isomeric with obtustyrene, was obtained as a second product from the obtustyrene m-methoxyphenol and cinnamyl 50 alcohol condensation (.01 mole scale, 14% yield). It was isolated as a TLC pure oil (PE/E, 50/1; Rf = ..62 : obtustyrene Rf = .53) which could not be distilled without decomposition. Found: m+, 240.1143. C16Hlg02 requires m+, 240.1151; A.max (methanol) 254 (ca_.4.2) and 281sh nm (ca.3.8); Amax (NaOMe added) 249, 286 and 292sh 126 nm; 6 (270 MHz, CDC13) 3.48 (2H, d, J 6 Hz, -benzyl), 3.77 (3H, s, -0CH3), 6.28-6.47 ( 4 i i , m, a, 3 , 3',5'), 7.06 (IH, d, J 8 Hz, 6 ' ) , 7.17-7.37 (5H, m, -<{,); MS 241 (19), 240(100), 239(20), 225(11 ), 223(16), 209(11 ), 149J2), 137(18), 117(12), 115(21), 91(16). 2',4'-Dimethoxybenzylstyrene This compound has been previously synthesized by the reduction of 42 the appropriate chalcone and the methylation of obtustyrene. This procedure includes the analytical data on this known compound because it is more complete than that found in the literature. 2',4'-Dihydroxybenzylstyrene (500 mg, 2.21 mmoles) was refluxed in 25 ml of acetone with excess methyl iodide (2.0 ml) and potassium carbonate (3.0 g) for 8 hours. After filtration and removal of excess solvent on a rotary evaporator the residue was purified by distillation (68°C at .01 mm) to give 360 mg (64%) of the product. Found: C, 80.53; H, 7.26. C17H1802 requires C, 80.28; H, 7.13; xmax (methanol) 253 (4.38), 282sh nm (3.86); vmax (CHCU) 1610, 1588, ISOObr, 1460br, 1290, 1262, 1160, 1124, 1039, 969, 839 cm"1; 6 (270 MHz, CDC13) 3.45 ( 2 l i , d, J 6 Hz, -benzyl), 3.79 and 3.81 (6H, 2xs, -0CH_3), 6.28-6.48 (2H, m, a, 3 ) , 6.40-6.48 (2H, m, 3', 5'), 7.06 (IH, d, J 8 Hz, 6'), 7.22-7.36 (5H, m, -<j>); MS 255(24), 254(100), 253(25), 239(20), 223(34), 151(28), 115(29), 91(20). 4'-Methoxybenzylstyrene This compound has been previously synthesized by the sequential reduction of a chalcone intermediate.100'101 In this study the desired compound was prepared by methylation of 4'-hydroxybenzylstyrene in the 127 usual manner with methyl iodide/potassium carbonate. 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DAVID, Chem. Ber., 70B, 183 (1937). 93. G. SIIPOS, I. DOBO and B. CZUKOU, Acta Phys. Chem., 8, 160 (1962). 94. W.D. OLLIS, CA. RHODES and 1.0. SUTHERLAND, Tet., 23, 4741 (1967). 95. A.C. JAIN, P.D. SARPAL and T.R. SESHADRI, Indian J. Chem., 3_, 369 (1965). 96. S.K. GROVER, A.C. JAIN and T.R. SESHADRI, Indian J. Chem., 1 , 517 (1963). 97. M. UCHIYAMA and M. MATSUI, Agr. Biol. Chem., 31_, 1490 (1967). 135 98. M. NAIM, B. GESTETNER, S. ZIIKAH, Y. BIRK and A. BONDI, J. Agr. Food Chem., 22, 806 (1974). 99. H. WYNBERG, Chem. Rev., 60, 169 (1960). 100. CS. RONDESTEVEDT, J. Am. Chem. Soc, 73, 4509 (1951). 101. S.S. HIXSON, J. Am. Chem. Soc, 94, 2505 (1972). 136 APPENDIX METABOLITE ISOLATION AND IDENTIFICATION DATA 137 The purpose of this appendix is to provide a convenient source of all of the data used to identify the compounds observed in G_. max, IP. vulgaris and P_. sativum. To facilitate the inspection of this data, it has been presented in a uniform layout format. All of the metabolites were initially isolated by HPLC on solvent system A. Consequently, the HPLC retention values given for each isolate refer to this system. The isolates are given in the order; Isolate Number Identification * 1 4',7-Dihydroxyisoflavone (Daidzein) [27] 2 4',5,7-Trihydroxyisoflavone (Genistein) [27] 3 5-Hydroxymethyl-2-furaldehyde [27] 4 Phased 1 in [32] 5 3,9-Dihydroxy-!O-isopentenylpterocarpan (Phased!idin) [32] 6 Phaseollinisoflavan [32] 7 4',7-Dihydroxycoumestan (Coumestrol) [40] 8 2' ,4',5,7-Tetrahydroxy-8-isopentenylisoflavone (Kievitone) [32] 9 6a-Hydroxy-3-methoxy-8,9-methylenedioxypterocarpan (Pisatin) [32] 10 2,3,9-Trimethoxy-, 3-Hydroxy-2,9-dimethoxy- and 4-Hydroxy-2,3,9-trimethoxypterocarpan [32] 11 4,4'-Dihydroxy-2'-methoxychalcone (Sativone) [53] 12 4',7-Dihydroxyflavanone (Liquiritigenin) [53] 13 2',4,4'-Trihydroxychalcone (Isoliquiritigenin) [53] 14 4-Hydroxy-(4'-hydroxy-2'-methoxybenzyl)styrene (Sativastyrene) [53] * - Page of the first reference to this compound in the text. 138 7-Hydroxy-4'-methoxyisoflavone (Formononetin) [53] 4',6-Dimethoxy-7-hydroxyisoflavone (Afromosin) [53] Unidentified isoflavone [53] 4'-Hydroxy-2'-methoxybenzylstyrene (Obtustyrene) [53] 6a,7a-Dehydro-3-methoxy-8,9-methylenedioxypterocarpan (Anhydropisatin, Flemichapparin B) [53] Unidentified flavanone or dihydroflavonol [46] 3,6a-Dihydroxy-8,9-methylenedioxypterocarpan [85] 139 Isolate 1 4'7-Dihydroxyisoflavbne (Daidzein) The chromatographic isolate (28.5 minutes) was further purified by TLC (chloroform/methanol; 9/1: R^  = .60). Analysis of the resultant sample gave: UV: xmax(nm): Solvent MeOH Added NaOMe NaOAc NaOAc/H3B03 AlClg A1C13/HC1 249, 259sh, 303sh 259, 289sh, 328 253sh, 272sh, 310 261sh, 303 249, 260sh, 300sh 249, 262sh, 302sh Proof of the structure presented was obtained by comparison of 94 these results with the data from a synthetic sample 140 Isolate 2 4',6,7-Trihydroxyisoflavone (Genistein) The chromatographic isolate [25.0 minutes) was further purified by TLC (chloroform/methanol; 9/1: Rf = .66). Analysis of the resultant sample gave: UV: Amax(nm): Solvent MeOH : 261, 328sh Added NaOMe 276, 327sh NaOAc 271, 325 NaOAc/H3B03 262, 336sh A1C13 272, 307sh A1C13/HC1 : 273, 309sh Proof of the structure presented was obtained by comparison of these results with the data from a sample isolated by a literature98 procedure. 141 Isolate 3 5-HydfOxymethyl-2-furaldehyde OHC o CHoOH The chromatographic isolate 09 minutes) was further purified by TLC (PE/E; 1/2: = .46). Analysis of the resultant sample gave: UV: xmax(nm): Solvent MeOH : 280 nm Added NaOMe : No change NMR: Solvent (CD^CO (100 MHz, FT) Coupling 6 Constant Integration Assignment (ppm) (Hz) 9.62 s - 1 -CH0 7.38 d 4 1 3 6.60 d 4 1 4 4.66 s - 2 -CH20H 142 MS: Low Resolution m/e Found 326 (74) 126.Q318 125 (24) 109 (39) 109.Q286 98 (10) 97 (100) 97.0270 Higli Resolution formula Expected C6H603 126.0318 C6H502 109.0290 C5K502 97.0290 Proof of the structure presented was obtained by comparison of these results with the data from a commercial (Aldrich Chemical Co.) 143 Isolate 4 Phaseollin The chromatographic isolate 00.2 minutes) was further purified by TLC (PE/E; 2/1: Rf = .47). Analysis of the resultant sample gave: UV: xmax(nm): Solvent EtOH : 280, 286sh, 315 Added NaOMe : 253, 281, 290 Proof of the structure presented was obtained by comparison of these results with the data from a sample which had been received as a gift. 144 Isolate 5 3,9-Dihydroxy-!O-isoperitehylpterocarpan (Phaseoll idin) A sample of this compound was not available. However, analysis of all of the major peaks in a typical chromatogram on the chloroform/ 93 methanol TLC system which had been previously used for phaseollidin did not exhibit an observable quantity of compound at the appropriate R^ . This TLC system gave an R^- for phaseollidin betv.een phaseollin and phaseollinisoflavan. Consequently, the similarity of the HPLC and TLC systems in combination with the absence of any major peaks in this HPLC region was another indication that this compound had not accumulated in copper(II) chloride stressed beans. 145 Isolate 6 Phaseoll iiiisoflavan The chromatographic isolate 05.2 minutes) was further purified by TLC (PE/E; 1/1: Rf = .54). Analysis of the resultant sample gave: UV: xmax (nm): Solvent EtOH : 280, 310sh Added NaOMe : 293, 330sh Proof of the structure presented was obtained by comparison of these results with the data from a sample which had been received as a gift. 146 Isolate 7 4',7-DihydrOxycoumestan (COumestrOl) The chromatographic isolate (20.4 minutes) gave a bright blue fluorescent spot on TLC (PE/E; 1/1: Rf = .24). Analysis of the TLC sample gave: UV; Xmax (nm): Solvent EtOH : 244, 304, 343 Added NaOMe : 260, 281, 320, 387 NaOAc : 244, 264, 311, 362 47 Tne UV data is in good agreement with the literature values for this compound which has been previously observed in stressed bean. However, in the absence of confirmatory evidence from a known sample the identification is still tentative. 147 Isolate 8 2' ,4',5,7 - Tetrahydroxy-8-isopehtenylisoflavone (Kievitone) The chromatographic isolate (23 minutes, tailed) was further purified by TLC (PE/E; 2/1: Rf = .28). Analysis of the resultant sample gave: UV: xmax (nm): Solvent MeOH : 294, 340sh Added NaOMe : 33Q Proof of the structure presented was obtained by comparison of these results with the data from a sample which was obtained as a gift. 148 Isolate 9 6a-Hydr0xy-3-methoxy-8,9-methylenedioxypterocarpan (.Pisatin) The chromatographic isolate (12.2 minutes) was further purified by TLC (PE/E; 1/1: Rf = .69). Analysis of the resultant sample gave: UV: Xmax (nm): Solvent MeOH : 280, 286, 309 Added NaOMe : No change m/e 315 (19) 314 (100) 296 (16) 295 (12) Proof of the structure presented was obtained by comparison of these results with the data from a sample isolated from copper(II) 43 chloride stressed F\ sativum by literature procedures. M S : Low Resolution 149 Isolate 10 2,3,9-Trimethoxy- Ca)> 3-Hydroxy-2,9-dimethox,y- (h), and 4-Hydroxy-2,3,9-trimethbxypterocarpan (c) A comparison of the chromatograms of copper(II) chloride and Fusarium stressed samples with standards for the above three compounds gave the results illustrated (Figure). Subsequent analysis for resolution of pi satin from 3-hydroxy-2,9-dimethoxypterocarpan on solvent system 2 indicated that this compound was not present at an observable concentration. 150 GRADIENT CHROMATOGRAM OF THE PTEROCARPAN REGION OF A FUSARIUM STRESSED SAMPLE. Solvent system A. 50 pi of a .1 ml/g dilution injected. 7-12 minute segment of the chromatogram illustrated. 151 Isolate 11 4,4'-Pihydroxy:2'-methoxychalcone (Sativone) The chromatographic isolate (28.3 minutes) was further purified by TLC (PE/E; 1/2: Rf = .38). Analysis of the resultant sample gave: UV: xmax (nm] : Solvent MeOH : 253, 349 Added NaOMe : 257, 419 NaOAc : 255sh, 348sh, 393 NaOAc/H3B03 : 351 A1C13 : 235, 350 A1C13/HC1 : 235, 350 NMR: Solvent (CD3)2C0 (100 MHz, FT) Coupling Constant (Hz) Integration Assignme 7.50 -7.64 m 4 e , 2, 6, 7.28 d 18 1 a 6.91 d 8 2 3, 5 6.59 d 2 1 3' 6.52 dxd 8, 2 1 5' 3.92 s 3 -OCH0 152 MS: Low Resolution High Resolution m/e Found Formula Expected 271 (21) 270 (100) 270.0885 C 1 6 H 1 4 ° 4 2 7 0 - 0 8 9 2 269 (23) 255 (38) 2 5 5 . 0 6 6 3 C 1 5 H 1 1 ° 4 2 5 5 . 0 6 5 7 242 (31 ) 242.0919 C l 5 H n ° 3 2 4 2 . 0 9 4 3 164 (38) 164.0483 c g H o 0 3 164.0473 163 (18) 151 (80) 151.0383 C 8 H 7 ° 3 151.0395 147 (33) 147.0426 CgH^ 147.0446 119 (25) 119.0481 C8H70 119.0497 107 (89) 107.0469 C7H70 107.0497 MS Diagnostic Fragments:96 Proof of the structure presented was obtained by comparison of these results with the data from a sample synthesized during the course of this investigation. 153 Isolate 12 4',7-Dihydroxyflavanoiie (Liquiritigenin) chromatographic isolate (25.3 minutes) was further purified by TLC (PE/E; 1/2: Rf = .46). Analysis of the resultant sample gave: UV: xmax (nm): Solvent MeOH : 276, 312 Added NaOMe : 250, 298sh, 327sh, 336 MS: Low Resolution 256 (96) 255 (53) 137 (100) 120 (84) 107 (67) I I O m/e 154 MS Diagnostic Fragments: Proof of the structure presented was obtained by comparison of these results with the data from a synthetic57 sample. 155 Isolate 13 . 2',4,4'-Tfihydr6xychalcone (Isoliquiritigenin) The chromatographic isolate (25.5 minutes, tailed) was collected by detection at 360 nm. The sample was further purified by TLC (PE/E; 1/2: Rf = .61). Analysis of the resultant sample gave: UV; Xmax (nm); Solvent MeOH Added NaOMe NaOAc NaOAc/H3B03 A1CU A1C13/HC1 260sh, 295sh, 367 251sh, 280sh, 320sh, 345sh, 429 275, 338, 402 285, 375 440sh 317, 385sh, 422 305, 380sh, 418 Proof of the structure presented was obtained hy comparison of these results with the data from a synthetic 89 sample 156 Isolate 14 4-Hydroxy-(4'-hydroxy-2'-methoxybenzyl)styrene (Sativastyrerie) 6' £ 6 The chromatographic isolate (.20.6 minutes) was further purified by TLC (PE/E; 1/1: Rf = .58). Analysis of the resultant sample gave: UV: Amax (nm): Solvent MeOH : 261.5 Added NaOMe : 281 NaOAc : No change NaOAc/H3B03 No change A1C13 No change A1C13/HC1 : No change NMR: Solvent (CD3)2C0 (100 MHz, FT) Integration .6 (ppm) Coupling Constant (Hz) 7.22 6.97 6.76 d d d 9 8 9 6.38 - 6.50 m 6.16 - 6.32 m 3.80 s 3.36 d 2 1 2 2 2 3 2 Assignment 2,6 6' 3,5 3',5' a,6 -0CH_3 -CH^ -cj) 157 High Resolution Found Formula Expected 256.1104 C16H16°3 256.1100 MS: Low Resolution m/e 256 (100) 255 (19) 241 (17) 241.0869 239 (17) 239.1088 225 (21) 225.0931 161 (11 ) 161.0606 150 (15) 149 (23) 149.0591 137 (63) 137.0599 133 (26) 132 (23) 131 (41) 131.0496 122 (36) 122.0375 121 (61 ) 121.0648 121.0291 107 (56) 107.0509 MS Diagnostic Fragments C 1 5 H 1 3 ° 3 241.0865 C 1 6 H 1 5 ° 2 239.1072 C 1 5 H 1 3 ° 2 225.0916 C 1 0 H 9 ° 2 161.0603 C9Hg°2 149.0602 C 8 H 9 ° 2 137.0602 C9H70 131.0497 C 7 H 6 ° 2 122.0368 w 121.0654 C 7 H 5 ° 2 121.0290 C7H70 107.0497 158 Additional structural information was provided by reaction of the isolate with methyl or ethyl iodide by the usual potassium carbonate procedure. TLC isolation of the products (methyl iodide: PE/E; 2/1: = .87 (corresponds to a synthetic sample of 4-methoxy-(2',4'-dimethoxy-benzyl ) styrene); ethyl iodide: PE/E; 8/1: R^  = .76) and MS analysis gave the expected results (methyl iodide: m = 284; ethyl iodide: m = 312) for 2 hydroxyls and no carbonyl oxygens in the isolate. Proof of the structure presented was obtained by comparison of these results with the data from a sample which was synthesized during the course of this investigation. 159 Isolate 15 7-Hydroxy-4'-methoxyisoflaVone (Formononetin) The chromatographic isolate 08.9 minutes) was analyzed on an HPLC system for the separation of formononetin and pseudobaptigenin (see Section II). This procedure showed that the isolate was predominantly (>90%) what was presumed to be formononetin with the remainder identified as pseudobaptigenin by comparison of its MS (m/e; 282, 146) and HPLC characteristics with a synthetic96 sample. Analysis of the major isolate gave: TLC: PE/E; 1/1 UV: xmax (nm): : Rf = .37 Solvent MeOH Added NaOMe NaOAc NaOAc/H3B03 A1C13 A1CU/HC1 248, 261sh, 305 255, 270sh, 332 255, 310sh, 330 Not done 248, 260sh, 299 248, 260sh, 299 160 MS: Low Resolution m/e 268 (100) 267 (74) 253 (22) 132 (78) MS Diagnostic Fragments: Proof of the structure presented was obtained by comparison of these results with the data from a synthetic sample. 161 Isolate 16 4' ,6-Dimethbxy-7-hydrbxyisoflavbne (Afromosin) The chromatographic isolate (16.2 minutes) was further separated on a less polar HPLC gradient system (Figure). This showed that the isolate FIGURE. GRADIENT CHROMATOGRAM OF ISOLATED 'AFROMOSIN'. Solvent system D. 162 was a mixture of 2 components. The apparent ratio of the 2 compounds was found to be quite variable from sample to sample. Analysis of the later eluting component gave: TLC: Benzene/ethyl acetate; 5/1 UV: xmax (nm): Solvent MeOH Added NaOMe Rf = .45 256, 320 255, 347 MS: Low Resolution m/e 298 (100) 297 (85) 283 (27) 166 (55) 151 (32) 149 (.22) 132 (45) High Resolution Found Formula 298.0823 283.0580 166.0267 C17H14°5 C16H11°5 C8H6°4 132.0584 CgHg0 Expected 29.8,0841 283.0606 166.0266 132.0575 MS Diagnostic Fragments: H 3 C O + • C = C H + • Proof of the structure presented was obtained by comparison of 95 these results with the data from a synthetic sample. 163 The other component of the isolate mixture, which was not conclusively identified, gave essentially the same TLC and UV data as afromosin. However, the most significant peaks in its mass spectrum were: Low Resolution High Resolution m/e Found Formula Expected 314 (51) 314.0807 C17H14°6 314.0790 148 (100) 148.0509 cgH8°2 148.0524 A compound which is consistent with this data, the accepted pathway of pterocarpan biogenesis, and the 2,3,9-trisubstituted pterocarpans 44 isolated by VanEtten from stressed P_. sativum is 2',7-dihydroxy-4',6-dimethoxyi sof1avone: Although this isolate represents a variable mixture of 2 components it has been referred to in the text as 'afromosin'. 164 Isolate 17 Unidentified Isoflavone The chromatographic isolate [10.6 minutes) was further purified by TLC (PE/E; 1/1: Rf = .39). Analysis of the resultant sample gave: UV: Xmax (nm): Solvent MeOH : 247sh, 260, 292sh Added NaOMe ; 289 MS: Low Resolution High Resolution m/e Found Formula Expected 298 (100) 298.0847 C17H14°5 298.0841 297 (21) 283 (7) 283.0573 C1CH,,0C 283.0606 l b I I o 149 (17) 148 (8) 148.0505 cgH8°2 148.0524 133 (16) 133.0269 C8H5°2 133.0289 105 (8) 105.0373 C7H50 105.0340 This sample, based on its UV and m+ data, is probably an isoflavone. The MS fragment at m/e 148 suggests that the general structure of the isolate would be: 165 However, the UV band II absorption at 260 nm suggests that ring A , not ring B, should be di-oxygenated. Since this isolate was a minor component of the sample and the data indicate that it may be a mixture of two components further analysis of its structure was not performed. 166 Isolate 18 41-Hydroxy-2'-methoxybenzylstyrene (Obtustyrene) The chromatographic isolate (11.6 minutes) was further purified by TLC (PE/E; 1/1: Rf = .84). Analysis of the resultant sample gave: UV: Xmax (nm): Solvent MeOH : 253, 282sh Added NaOMe : 246, 287, 292sh MS: Low Resolution m/e 241 (14) 240 (100) 239 (32) 225 (21) 209 (35) 137 (27) 117 (11) 116 (19) 115 (62) 91 (38) Found 240.1136 225.0904 209.0957 137.0609 115.0554 91.0550 Resolution Formula C16H16°2 C15H13°2 C15H13° C8H9°2 C7H7 Expected 240.1151 225.0916 209.0966 137.0602 115.0548 91.0548 167 MS Diagnostic Fragments: Additional structural information was provided by reaction of the isolate with methyl iodide by the usual potassium carbonate procedure. TLC isolation of the product (PE/E; 40/1: Rf = .50 (corresponds to a synthetic sample of 2',4'-dimethoxybenzylstyrene)) and MS analysis gave the expected result (m+ = 254) for 1 hydroxyl and no carbonyl oxygens in the isolate. Proof of the structure presented was obtained by comparison of 42 50 these results with the data from a synthetic ' sample. 168 Isolate 19 6aJa-Dehydrb-S-methoxy-S^ -methylenedioxypterocarpan (Arihydropisatin; Flemichapparin B) H 3 C O The chromatographic isolate [3.2 minutes), which was conveniently separated from the solvent front by a slight variation in solvent polarity (Figure), was further purified by TLC (PE/MDC; 1/1: Rf = .48). Analysis of the resultant sample gave: m/e 297 (19) 296 (100) 295 (61) Proof of the structure presented was obtained by comparison of these results with the data from a sample synthesized by acidic dehydration 51 of an authentic sample of isolated pisatm. UV: xmax (nm): Solvent Ether: 233sh, 244sh, 250sh. 291, 339, 358 MS: Low Resolution 169 FIGURE. ISOCRATIC CHROMATOGRAM OF ANHYDROPISATIN. Solvent system 5. 170 Isolate 20 Unidentified Flavanoiie (a) or Dihydroflavonol (b) The chromatographic isolate (27,5 minutes, Figure) which was observed as a minor novel metabolite in the aqueous extraction samples of copper(II) chloride stressed P_. sativum, was further purified by TLC (PE/E; 1/2: Rf = .33). Analysis of the resultant sample gave: UV: Xmax (nm): Solvent MeOH Added NaOMe NaOAc NaOAc/H3R03 A1CU A1C13HC1 275, 305 241, 333 282, 336 280, 314sh 311, 360sh 282, 305, 355sh published data The tentative identification of this isolate was on the basis of 84 *See p. 46. 171 SATIVONE 3 0 2 5 T I M E, m in FIGURE. GRADIENT CHROMATOGRAM OF THE FLAVANONE OR DIHYDROFLAVONOL. Solvent system A. 0-25 minutes omitted for clarity. 172 Isolate 21 3,6a-Dihydroxy-8,9-methyleriedioxypterocarpan The chromatographic isolate (.21.6 minutes) was further purified by TLC (PE/E; 1/5: = .30). Analysis of the resultant sample gave: UV: Xmax (nm): Solvent MeOH : 281, 287, 309 UV after acidic dehydration: Xmax (nm): Solvent MeOH : 340, 357 MS: Low Resolution m/e 300 (100) 282 (69) 281 (65) This data is comparable to the literature74 values for thi compound. PUBLICATIONS 1. "Facial Incorporation of Chlorine into Aromatic Systems During Aqueous Chlorination Processes", Environmental Science and Technology, 9_, 674 (1975) with R.M. Carlson, H.L. Kopperman and R. Caple. 2. "Determination of Partition Coefficients by Liquid Chroma-tography", Journal of Chromatography, 107, 219 (1975) with R.M. Carlson and H.L. Kopperman. 3. "HPLC Techniques for the Separation of Complex Mixtures of Naturally Occuring Porphyrins" in M. Doss, Ed., Porphyrins  in Human Diseases, S. Karger, New York (1976) p. 465 with D. Dolphin. 4. "Application of HPLC to the Analysis of Clinically Important Porphyrins" in P.F. Dixon, CH. Gray, C.K. Lim and M.S. Stoll, Eds., HPLC in Clinical Chemistry, Academic Press, San Francisco (1976) p. 87 with D. Dolphin. 5. "Copper Coproporphyria Excretion in Familial Coproporphyria", Clinical Chemistry, 24, 2009 (1978) with D. Dolphin and M. Bernstein. 6. "The Chromatographic Separation of Uroporphyrin I and III Octamethyl Esters", Analytical Biochemistry, 95, 444 (1979) with J.C. Bommer, B.F. Burnham and D. Dolphin. 7. "An HPLC Method for the Analysis of Isoflavones", submitted for publication with D. Dolphin. 

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