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UBC Theses and Dissertations

Studies on the biosynthesis of coumarins Verma, Ashok Kumar 1972

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STUDIES ON THE BIOSYNTHESIS OF COUMARINS BY ASHOK K. VERMA M.Sc. Honors, Panjab U n i v e r s i t y , I n d i a , 1968 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department of CHEMISTRY We accept t h i s t h e s i s as conforming to the re q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA August, 1972 In 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 the r e q u i r e m e n t s f o r an advanced degree a t the U n i v e r s i t y of 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 agree 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 the Head o f my Department 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 not 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 . Department The U n i v e r s i t y o f B r i t i s h C o l umbia Vancouver 8, Canada - i i -ABSTRACT This thesis describes the investigations on the biosynthesis of coumarins from Thamnosma montana Torr. and Frem plants and tissue cultures. Part I of this thesis discusses the degradative sequences developed for the furanocoumarins, isoimperatorin (13), allimperatorin methyl ether (7) and isopimpinellin (2) and for the coumarin, umbelliprenin (9). These degradative sequences were developed to gain information as to the distribution of radioactivity in the radioactive compounds made available from the subsequent biosynthetic studies. In Part II of this thesis, the role of mevalonate (85) in the biosynthesis of the alkyl side chains and the furan ring of furano-coumarins of Thamnosma montana tissue cultures is described. In 14 preliminary studies, i t was shown that D,L-phenylalanine-[3- C] was being efficiently incorporated into three furanocoumarins, isoimperatorin (13), alloimperatorin methyl ether (7) and isopimpinellin (2). Incorporation experiments with various tritium labelled forms of 3 mevalonic acid showed that mevalonic acid-[5- H] was being incorporated into the alkyl side chain and the 6-position of the furan ring of the 3 furanocoumarins. Similarly, experiments with mevalonic acid-[4- H] indicated i t to be a precursor of the alkyl side chains and the 7— position of the furan ring of the furanocoumarins. Mevalonic acid-3 [2- H] was incorporated into the alkyl side chains but was not incorporated into the furan ring. Incorporation studies with mevalonic 14 acid-[5- C] supported the data already obtained with mevalonic acid-- i i i -3 [5- H] and revealed that any activity in the methoxy groups of furano-3 3 coumarins obtained from the [5- H]- and [2- H]-mevalonic acid feeding experiments was either due to a tritium exchange between the tritiated mevalonic acid and the C^-pool in the tissue culture system or by some other unknown mechanism. Part III of this thesis describes the role of glycine (124) in the biosynthesis of the coumarins, umbelliprenin (9), alloimperatorin methyl ether (7) and isopimpinellin (2), in Thamnosma montana plants. 14 By specific degradations, i t was shown that glycine-[2- C] was acting as an efficient precursor of the methoxyl groups of alloimperatorin methyl ether (7) and isopimpinellin (2). Glycine-[2-^^C] was also shown to incorporate almost exclusively into the farnesyl-ether side chain of umbelliprenin (9) and to a small extent into the C^-alkyl side chain of allimperatorin methyl ether (7). However, very l i t t l e activity could be found in the furan ring and the coumarin portion of alloimperatorin methyl ether (7) and isopimpinellin (2). These 14 results suggest that glycine-[2- C] is acting as an efficient precursor of the source in Thamnosma montana plants and can also be utilized by the plant system for the biosynthesis of the C^-alkyl side chain and, in turn, the C^^-alkyl-ether side chain of alloimperatorin methyl ether (7) and umbelliprenin (9), respectively. - iv -TABLE OF CONTENTS Page TITLE PAGE i ABSTRACT i i TABLE OF CONTENTS iv LIST OF FIGURES v LIST OF TABLES v i i ACKNOWLEDGEMENT ix INTRODUCTION 1 DISCUSSION (PART I) 36 EXPERIMENTAL (PART I) 59 DISCUSSION (PART II) 66 EXPERIMENTAL (PART II) 87 DISCUSSION (PART III) 112 EXPERIMENTAL (PART III) 128 BIBLIOGRAPHY 143 - v -LIST OF FIGURES Fig u r e Page 1 Iso m e r i z a t i o n s i n v o l v i n g coumarin (19) 8 2 The biogenesis of aromatic compounds v i a s h i k i m i c a c i d (34) 10,11 3 Proposed b i o s y n t h e t i c r e a c t i o n s l e a d i n g to 7-hydroxycoumarin (49) 13 4 B i o s y n t h e s i s of coumarin (19) 15 5 The b i o s y n t h e s i s of u m b e l l i f e r o n e (49) 18 6 Proposed b i o s y n t h e s i s of h e r n i a r i n (53) i n Lavendula 21 7 Degradation of p i m p i n e l l i n (76) 26 8 Proposed scheme f o r furanocoumarin b i o s y n t h e s i s i n P i m p i n e l l a magna 27 9 A l t e r n a t i v e pathway of furanocoumarin b i o s y n t h e s i s i n P i m p i n e l l a magna 28 10 The b i o s y n t h e t i c route to marmesin (74) 30 11 The acetate b i o g e n e s i s of mevalonic a c i d (85) 33 12 Proposed mechanism f o r a l k y l a t i o n of phenols 34,35 13 Degradative scheme of i s o p i m p i n e l l i n (2) 42 14 Nmr spectrum of 6-acetoxymethyl-7-acetoxy-5,8-dimethoxycoumarin (103b) „ 44 15 Degradative scheme of a l l o i m p e r a t o r i n methyl ether (7) 51 16 Nmr spectrum of 6-formyl-7-hydroxy-5-methoxycoumarin (114) 57 3 17 I n c o r p o r a t i o n of D,L-mevalonic a c i d - [ 5 - H] i n t o coumarins of Thamnosma montana t i s s u e c u l t u r e s versus time 72 - vi -Figure Page 18 Degradation of radioactive isopimpinellin (2) 73 19 Degradations of radioactive alloimperatorin methyl ether (7) 76 20a The photosynthetic fixation of C02 113 20b The glycolytic pathway to pyruvate 114 21 The Shah and Rogers chloroplastidic acetyl-CoA biogenesis 116 22 Degradation of radioactive umbelliprenin (9) 121 23 Degradation of radioactive isopimpinellin (2) 123 24 Degradation of radioactive alloimperatorin methyl ether (7) 125 - v i i -LIST OF TABLES Table Page 1 Co n s t i t u e n t s of Thamnosma montana 6 14 2 I n c o r p o r a t i o n of D,L-phenylalanine-[3- C] i n t o coumarins of Thamnosma montana t i s s u e c u l t u r e s 69 3 3 I n c o r p o r a t i o n of D,L-mevalonic a c i d - [ 5 - H] i n t o t i s s u e c u l t u r e s of Thamnosma montana 71 4 D i s t r i b u t i o n of r a d i o a c t i v i t y i n i s o p i m p i n e l l i n (2) 3 from D,L-mevalonic a c i d - [ 5 - H] i n c o r p o r a t i o n experiments 74 5 D i s t r i b u t i o n of r a d i o a c t i v i t y i n a l l o i m p e r a t o r i n 3 methyl ether (7) f o r D,L-mevalonic a c i d - [ 5 - H] i n c o r p o r a t i o n experiments 76 3 6 I n c o r p o r a t i o n of D,L-mevalonic a c i d - [ 4 - H] i n t o the t i s s u e c u l t u r e s of Thamnosma montana 79 3 7 I n c o r p o r a t i o n of D,L-mevalonic a c i d - [ 2 - H] i n t o the t i s s u e c u l t u r e s of Thamnosma montana 82 14 8 I n c o r p o r a t i o n of D,L-mevalonic a c i d - [ 5 - C] i n t o t i s s u e c u l t u r e s of Thamnosma montana 84 14 9 I n c o r p o r a t i o n of D,L-phenylalanine-[3- C] (sodium s a l t ) i n t o t i s s u e c u l t u r e s of Thamnosma montana .... 92 3 10a,b I n c o r p o r a t i o n of D,L-mevalonic a c i d - [ 5 - H] (DBED s a l t ) i n t i s s u e c u l t u r e s 94 4 11a,b I n c o r p o r a t i o n of D,L-mevalonic a c i d - [ 4 - H] (sodium s a l t ) i n t o t i s s u e c u l t u r e s 103 - v i i i -Table Page 3 12a,b Incorporation of D,L-mevalonic acid-[2- H] (sodium salt) into tissue cultures D6 14 13 Incorporation of D,L-mevalonic acid-[5- C] (sodium salt) .into tissue cultures 110 14 14 Incorporation of D,L-phenylalanine-[3- C] into Thamnosma montana plants 119 14 15 Incorporation of glycine-[2- C] into Thamnosma montana plants 120 16 Degradation of radioactive isopimpinellin (2) (Experiments 2, 3, and 4) 124 17 Degradations of radioactive alloimperatorin methyl ether (7) 126 14 18 Incorporation of D,L-phenylalanine-[3- C] into young Thamnosma montana plants 130 14 19a,b Incorporation of glycine-[2- C] into Thamnosma montana plants 131 - i x -ACKNOWLEDGEMENTS I wish to express my thanks to Dr. J.P. Kutney f o r h i s e x c e l l e n t guidance throughout the course of t h i s research. The support provided by h i s unbounded optimism and u n f a i l i n g encouragement g r e a t l y f a c i l i t a t e d the s u c c e s s f u l completion of t h i s work. I would l i k e to thank Dr. P h i l i p J . S a l i s b u r y f o r h i s e x p e r t i s e i n c u l t i v a t i n g the p l a n t s and t i s s u e c u l t u r e s and a l s o f o r many h e l p f u l suggestions. S p e c i a l thanks are due to Miss Diane Johnson f o r her e x c e l l e n t t y p i n g of the manuscript. F i n a n c i a l support from the U n i v e r s i t y of B r i t i s h Columbia i s g r a t e f u l l y acknowledged. - 1 -INTRODUCTION The world of nature abounds i n organic compounds of n e a r l y every conceivable s t r u c t u r a l c l a s s and the study of these c o n s t i t u e n t s represent a f a s c i n a t i n g and f r u i t f u l area of s c i e n t i f i c i n v e s t i g a t i o n . The c e l l s of l i v i n g organisms - p l a n t s , f u n g i , i n s e c t s , and higher animals - are the s i t e s of i n t r i c a t e and complex s y n t h e t i c a c t i v i t i e s t hat r e s u l t i n the formation of a remarkable a r r a y of organic compounds, many of them of great p r a c t i c a l importance to mankind. Of i n c r e a s i n g i n t e r e s t to organic chemists and b i o l o g i s t s a l i k e , i s the i n v e s t i g a t i o n of b i o s y n t h e t i c pathways and precursors u t i l i z e d by p l a n t s to s y n t h e s i z e these n a t u r a l products. The members of the Rutaceae f a m i l y (of which the C i t r u s genus i s perhaps the best known) are mostly t r e e s and shrubs, w i d e l y d i s t r i b u t e d i n t r o p i c a l and s u b t r o p i c a l h a b i t a t s , and p a r t i c u l a r l y abundant i n A u s t r a l i a and South A f r i c a . This f a m i l y i s w e l l known to c o n t a i n a l a r g e number of benzenoid compounds, coumarins, flavones and some q u i n o l i n e a l k a l o i d s . Thamnosma montana Torr. and Frem. (Rutaceae), more commonly known as t e r p e n t i n e broom and found i n desert mesas and slopes of South Western United S t a t e s , has over the years a t t r a c t e d the i n t e r e s t s of s e v e r a l groups of research workers. I t represents a source of coumarins - 2 -which exhibit plant-growth-inhibitor properties, ' and its use in 3 1 folk medicine is also reported. Bennett and Bonner, while studying the toxicity of aqueous extracts of the leaves of desert plants found Thamnosma montana to be the most toxic as judged by the response to young tomato plants. The crude material when exposed to young plants over a period of about seven days caused death of these plants at concentrations of about 1 mg/ml of the administered solution. These workers isolated three crystalline compounds from Thamnosma  montana and identified two of them as byakangelicin (1) and isopimpinellin The structure of the third and most toxic compound was elucidated by Dreyer^ and found to be 5-(3*-methyl-2',3'-dihydroxy :butany 1)-8-methoxy psoralen (3), hereafter known as alloimperatorin methyl ether diol. • Dreyer developed a better extraction scheme.and by chromatography of Hie acetone extract of Thamnosma montana on alumina was able to isolate not only the three coumarins obtained previously by Bennett and Bonner, but six other compounds as well. These compound:.; were identified as throe known alkaloid:;; N-mcthyl acrid one (M , ';k i mmiairi no. (2). OH OH 1 2 3 - 3 -(5a) and y~fagarine (5b); $-sitosterol (6); a known furano coumarin, alloimperatorin methyl ether (7) and an unknown compound, thamnosin. This was the first report of N-methyl acridone (4), the parent member of the acridine alkaloids, occurring as a natural product. Dreyer proposed a partial structure for thamnosin based on preliminary evidence, which proved to be incorrect. Inaba and Kutney^'^ working in our laboratories were able to determine the structure of thamnosin as a novel dimeric system (8), a heretofore unknown coumarin. Dreyer could find no trace of the epoxide of alloimperatorin methyl ether, which he had occasion to prepare during the synthesis of alloimperatorin methyl ether diol (3). The co-occurrence of several types of coumarins in Thamnosma  montana, including the interesting dimeric system thamnosin (8), appeared to offer a genuine opportunity to study the biosynthesis of - 4 -coumarins. During the course of preliminary investigations for the purpose of biosynthetic studies, i t became apparent that Thamnosma montana contained a large number of isolable constituents in addition 4 to those identified by Dreyer. Thus to gain more complete appreciation of the plant system and to determine i f these unknown compounds might offer further opportunities for biosynthetic experi-ments, an exhaustive isolation and structure elucidation study was carried out. Thamnosma montana shoots and roots, obtained from the region in the Mojave Desert of California, were extracted with acetone and the acetone extract was chromatographed on alumina. By successive chromatography of various fractions, eight new compounds were isolated 4 in addition to the nine already isolated by Dreyer. Six of these compounds were identified to be already known coumarins; umbelliprenin (9); psoralen (10a); bergapten (10b); xanthotoxin (11); phellopterin (12); and isoimperatorin (13). The remaining two compounds were characterized as two novel epoxy coumarins; alloimperatorin methyl ether epoxide (14) and thamnosmin (15). R - 5 -13 14 The detailed study on the isolation and structure elucidation of these new compounds was done in collaboration with Dr. R.N. Young and as this work has already been reported in detail in Dr. Young's Ph.D. thesis,^ I do not wish to discuss this aspect in any detail. Thus the known chemical constituents of Thamnosma montana can be summarized as in Table 1. The co-occurrence of thamnosmin (15) and thamnosin (8) within the same plant raises the question of whether they are biogenetically related; since thamnosmin (15) is closely related to the diene (16) which would be produced by a retro Diels-Alder reaction of the co-occurring dimer thamnosin (8). Such a process is actually observed in the mass spectrum of thamnosin (8)."*'^ - 6 -Table 1. Constituents of Thamnosma montana Name Formula m.p. Reference g-sitosterol (6) C29 H50° 137-9° 4 alloimperatorin methyl ether (7) C17H16°4 108-10° 4 isopimpinellin (2) C11H10°5 148-9° 1,4,; thamnosin (8) C30H28°6 244-6° 4,5,i N-methyl acridone (4) C14 H11° N 202-3° 4 skimmianine (5a) C14 H13°3 N 173-5° 4 y-fagarine (5b) C 1 3H 1 10 3N 140-2° 4 byakangelicin (1) C17H18°7 105-7° 4 alloimperatorin methyl ether diol (3) C17H18°6 174-6° 4 umbelliprenin (9) C24H30°3 61-2° 7,8 psoralene (10a) C11H6°3 163.5-164. 5° 7,8 bergapten (10b) C12 H8°4 186.5-188. 5° 7,8 xanthotoxin (11) C12 H8°4 146-7° 7,8 phellopterin (12) C17H16°5 92-4° 7,8 isoimperatorin (13) C16H14°4 97-8° 7,8 alloimperatorin methyl ether epoxide (14) C17H16°5 101-3° 7,8 thamnosmin (15) C14H13°3 101-4° 7,8 - 7 -9 10 Recently Guise et al. and Reyes have reported the isolation of the novel dimeric coumarin thamnosin (8) from two other plant species of Rutoideae subfamily of Rutaceae,'''''" namely Zanthoxylum  dominianum Merr. and Perry and Ruta pinnata L . f i l . Zanthoxylum  dominianum also contains, two monor.ieric coumarins which possibly would serve as biogenetic precursors of thamnosin (8); suberosin (17), a known coumarin, and suberenol (18), a new coumarin. Biosynthesis of Coumarins Despite the large number of investigations in recent years into the formation of phenolic compounds in plants, the problem of the origin of the coumarins has attracted relatively l i t t l e attention. This is perhaps surprising in view of the wide spread distribution 12 and variety of structures associated with this class of compounds. It is notable that with few exceptions, such as coumarin (19) itsel f , a l l naturally occurring coumarins have an oxygen atom at the 7-position, i.e. they can be regarded as derivatives of umbelliferone (49), which is one cf the most widely distributed compounds of this class. Before discussing the question of biosynthesis, one important feature of coumarin chemistry which has proved to be relevant to biosynthetic studies should be mentioned. 8 20a, R = H 20b, R = Na 19 C00H 21 Figure 1. Isomerizations involving coumarin (19) Coumarin (1.9) is a lactone of a cis- o-hydroxy cinnamic acid (20a) or coumarinic acid. The lactone ring is easily opened by heating with alkali to form the sodium salt of this coumarinic acid (20b). Upon acidification, the free acid lactonizes spontaneously, but i f the salt is treated with mercuric oxide, a cis-trans isoinerization takes place to yield stable _o-coumaric acid (21). Ultraviolet radiations can 13 catalyze the reverse isomerization to form the coumarin (19). Before the application of radioactive tracers became possible, attention was focussed on structural regularities within this family of compounds and in this way attempts were made to predict plausible biogenetic pathways. Robinson's"^ theories of biogenesis postulated - 9 -that the coumarin system arose by union of and units whilst Pavolino, in 1932, suggested pentose sugars as the basis of coumarin biosynthesis, and felt that this would account for the great variety of positions occupied by hydroxy1 groups. The C^ -C-j unit is present in phenylalanine (22) and tyrosine (23) , but in earlier speculations these were not considered as direct precursors but rather as related compounds. However in subsequent isotopic labelling studies, both these amino acids (22,23) were in fact found to be the key intermediates. COOH Before discussing the role of these intermediates in the biosynthesis of coumarins, i t is instructive to consider their biogenesis. The Biosvnthesis of the C,-C„ Unit 5 — 3 Several metabolic pathways for the biosynthesis of aromatic compounds are now known but the phenyl propane unit in many natural products is considered to be biosynthesized from cyclohexane derivatives arising from cyclization of carbohydrates. Davis and coworkers"*""* ^ established the biogenetic route to phenylalanine (22) (later modified to include chorismic acid) in their work with nutritionally deficient microbial mutants. However, even earlier, when the structures of quinic acid (33a) and shikimic acid (34) were established, their - 10 -possible function as intermediates in the biosynthesis of aromatic natural 18 products were suggested by Fischer and Dangschat. The formation of shikimic acid (34) proceeds from the three- and four-carbon atom precursors, phosphoenolpyruvate (24) and erythrose-4-phosphate (25) , through a series of steps as shown in Figure (2). 24 CH20P 25 C00H f c=o I CH. I 1 HOCH I HCOH I HCOH I CH20P 26 C00H I C=0 I CH-I 1 HOCH I C=0 I HCOH i CH20P 27 HO C00H OH e OH 31 COOH f C=0 I CH-I 2 HOCH I HCOH i C=0 I CH„ COOH f C=6 ! CH0 I 2 HOCH I C=0 I C-OH II CH2 30 29 31 COOH OH OH 32 11 - HO ,. . COOH RO* OH / / / I COOH OH 33a, R 33b, R = H = phosphate HO'° OH OH 34 COOH PO' \ ^ ' OH OH 35 ii P O ^ ^ COOH 24 COOH H PO A »o x COOH OH 36 COOH . OH 37 ' 40 COOH COOH 22 COOH COOH COOH 39 COOH COOH HOOC, 38 OH HOOC COOH 41 F i g u r e 2. The b i o g e n e s i s of aromatic compounds v i a s h i k i m i c a c i d (34). - 12 -Further r e a c t i o n of s h i k i m i c a c i d (34) w i t h phosphoenolpyruvate (24), f o l l o w e d by a s t e r e o s p e c i f i c t r a n s - 1 , 4 - e l i m i n a t i o n of phosphate 19 y i e l d s the key intermediate chorismic a c i d (37) which then rearranges i n a r e a c t i o n reminiscent of the C l a i s e n rearrangement to give prephenic a c i d (38). Decarboxylation w i t h accompanying e x p u l s i o n of a h y d r o x y l group y i e l d s phenylpyruvic a c i d (39). Transamination of phenyl p y r u v i c a c i d (39) then leads to phenylalanine (22). The deamination of 22 by phenylalanine ammonia-lysase y i e l d s cinnamic 20 21 a c i d (40). ' S i m i l a r l y transamination of p_-hydroxyphenyl p y r u v i c a c i d (42) y i e l d s t y r o s i n e (23) and subsequent deamination leads to p_-hydroxy cinnamic a c i d (43). Tyrosine (23) can a l s o be formed by h y d r o x y l a t i o n of phenylalanine (22) and p_-hydroxy cinnamic a c i d (43) can s i m i l a r l y be formed from cinnamic a c i d (40) , but these are minor r e a c t i o n s . Most of the work i n the s h i k i m i c a c i d route to aromatic compounds was done w i t h microorganism (E. c o l i mutants). However, s t u d i e s done w i t h p l a n t s suggest the pathway i s s i m i l a r i f not i d e n t i c a l to that 20 e l u c i d a t e d i n microorganism . One p o s s i b l e d i f f e r e n c e may be that p l a n t s p r e f e r q u i n i c a c i d (33a) to s h i k i m i c a c i d (34) as a key i n t e r m e d i a t e . I t i s p o s s i b l e that q u i n i c a c i d (33a) can be dehydrated d i r e c t l y to s h i k i m i c a c i d (34) or that 33a i s converted to 5-phospho-s h i k i m i c a c i d (35) v i a 5-phosphoquinic a c i d (33b). B i o s y n t h e s i s of Coumarin (19) The f i r s t p r o p o s a l f o r coumarin b i o s y n t h e s i s from cinnamic aci d s 22 was o f f e r e d by Haworth, who p o s t u l a t e d para o x i d a t i o n of a - 13 -p_-hydroxy cinnamic acid (4 3) to yield (45) which could then cyclize by a Michael addition of carboxyl group and finally dehydrate to yield 7-hydroxy coumarin (49) (Figure 3). Figure 3. Proposed biosynthetic reactions leading to 7-hydroxy coumarin (49). - 14 -23 Grisebach and O l l i s suggested the d i r e c t o x i d a t i v e c o u p l i n g of the p a r a - p o s i t i o n w i t h the cinnamoyl c a r b o x y l , as i n (46), whereas 24 Kenner et a l . have favored i n i t i a l t wo-electron o x i d a t i o n of the c a r b o x y l , as i n (44), so as not to have the process dependent on the p_-hydroxy f u n c t i o n . However, the f i r s t t r a c e r experiment on the b i o s y n t h e s i s of 25 coumarin (19) was done by Brown e t a l . By feeding v a r i o u s l a b e l l e d compounds i n t o the p e r e n n i a l grass, Hierochloe odorata, they found that o-coumaric a c i d (21) and cinnamic a c i d (40) were u t i l i z e d most e f f i c i e n t l y , w h i l e phenylalanine (22) and s h i k i m i c a c i d (34) were l e s s e f f i c i e n t l y u t i l i z e d , w i t h acetate and s a l i c y l i c a c i d being i n c o r p o r a t e d at very low l e v e l s . These r e s u l t s i n d i c a t e d that cinnamic a c i d (40) i s being converted to o-coumaric a c i d (21) by a mechanism found i n 26 M e l i l o t u s species (sweet c l o v e r ) to be c o n t r o l l e d by a s p e c i f i c gene. The a b i l i t y to hydroxylate cinnamic a c i d i n the ortho p o s i t i o n seems to be a key f a c t o r i n a p l a n t ' s a b i l i t y to s y n t h e s i z e coumarin (19). _o-Coumaric a c i d (21) i s next converted to o-coumaryl g l u c o s i d e (50) and 50 has been shown to be a very good p r e c u r s o r of coumarin (19), 13 having been found i n a l l species c o n t a i n i n g 19 thus f a r examined. The g l u c o s i d e (50) i s isomerized to the c i s - i s o m e r , coumarinyl g l u c o s i d e (51) which may c y c l i z e to form coumarin (19). 27 Haskins and Gorz have shown t h a t , at l e a s t i n M e l i l o t u s , coumarin (19) as such i s an a r t i f a c t , and that what e x i s t s i n the i n t a c t p l a n t 28 c e l l i s a c t u a l l y the coumarinyl g l u c o s i d e (51). Kosuge and Conn have i d e n t i f i e d and c h a r a c t e r i z e d a 8-glucosidase i n M e l i l o t u s which s p e c i f i c a l l y hydrolyses the c i s - g l u c o s i d e , r e l e a s i n g coumarin (19), when - 15 -52 19 51 Figure 4. Biosynthesis of coumarin (19). the cells are disrupted. Free coumarin (19), which probably does exist in some plants, may be formed by a route involving ortho-hydroxylation of cis-cinnamic acid (52); the evidence for this has 29 been presented by Stoker and Bellis. The isomerization of o_-coumaryl glucoside (50) to coumarinyl glucoside (51), in sweet clover, has been shown to be photochemically . , » 30,31 induced. Biosynthesis of 7-0xygenated Coumarins The formation of 7-oxygenated coumarins has been the subject of increasing study in recent years. A few of the simple members are - 16 -herniarin (53), esculetin (54a) and scopolctin (55). The corresponding cinnamic acid, p-methoxy cinnamic acid (56), caffeic acid (57) and ferulic acid (58) are also naturally occurring, thereby suggesting the existence of a biogenetic relationship between these compounds. A body 13 32 of evidence supports this hypothesis. ' 54a, R^R^OR^R^H 54b, RX=H, R2=R3=OH COOH HO COOH H3CO 00H 57 58 I t i s noteworthy t h a t the co-occurrence of coumarin (19) and a 33 7-oxygenated coumarin i s a r a r e phenomenon. However, lavender (Lavandula o f f i c i n a l i s ) i s one of the few sp e c i e s which e l a b o r a t e both coumarin (19) and a 7-oxygenated coumarin, i n t h i s case, 7-methoxy 34 coumarin (53). On t h i s . b a s i s , t h i s p l a n t was chosen as a convenient s p e c i e s i n which to compare the b i o s y n t h e s i s of coumarin (19) and h e r n i a r i n (53). 34 Using lavender p l a n t s , Brown s t u d i e d the b i o s y n t h e s i s of these 14 two coumarins w i t h a number of C - l a b e l l e d compounds. He found that - 17 -such precursors as glucose and phenylalanine (22) were incorporated equally well into both, as was cinnamic acid, while oxygenated cinnamic acids showed striking differences. For example, o_-coumaric acid (21) and its glucoside (50) were selectively used for the synthesis of coumarin (19) while p_-coumaric acid (A3) and trans-2-glucosyloxy-A-methoxy cinnamic acid (59) were utilized selectively for herniarin (53) biosynthesis. COOH 59 The results of these investigations strongly suggest that the diversion in the pathway occurs at the trans-cinnamic acid stage. ortho-Hydroxylation apparently leads to coumarin (19), and para-hydroxylation to herniarin (53). As with coumarin (19), herniarin (53) is also found in lavender in the bound state (as a glucoside) and, as with 19, intermediary glucosides appear to be important. Subsequent study has shown that biosynthesis of umbelliferone (A9) can proceed by analogous pathways. The formation of umbelliferone (A9) 35 36 has been studied by Brown,. Towers and Chen and by Austin and Meyers using Hydrangea macrophylla and the feeding and trapping experiments with 1A C-labelled compounds allowed the proposal for umbelliferone biosynthesis as represented in Figure (5). - 18 -Figure 5. The biosynthesis of umbel]iferone (49). 36 Austin and Meyers have reported that umbelliferone (49) exists as the free coumarin only to a very small extent, i f at a l l , and have identified two bound forms in Hydrangea. The two forms are skimmin (63) and. cis-2,4-di-g-D-glucosylo>:ycinnarnic acid (62), the former being predominant. The conversion 63—*49 may only occur when plant cells are disrupted. Reactions 61—• 62 and 62—*• 63 are assumed to be fast as these intermediates cannot bo isolated, although the conversion of p_-hydroxycinnamic acid (4 3) to umbellic acid (60) has 35 been detected by trapping experiments. - 19 -The final step in the sequence, the trans-cis isomerization, has not been completely clarified. Current evidence suggests that It 30 may be catalyzed in part by ultraviolet radiation and in part by 37 31 a specific isomerase. However, recently, Edward and Stoker have shown that in lavender, the trarts-cis isomerization reaction in herniarin (53) biosynthesis is entirely photochemical with no isomerase involved. They consider that this situation is applicable to 38 a l l plant coumarin biosynthesis. However, Ourisson and coworkers feel the trans-Cis isomerization in scopoletin (55) formation in tobacco tissue cultures is not purely photochemical. Thus there seems to be some doubt about the general validity of Edward and Stoker's statement. Two other aspects of the format'.!on of coumarins has been studied; one is the origin of the lactone ring, and the other is the stage at which methylation occurs in such coui'u<rins as herniarin (53) and scopoletin (55). After the studies on coumarin (19) , i t was assumed that the sequence of events was ortho-hydroxylation, glucosylation, trans-cis inversion, hydrolysis and lactonization; the last two being minor 24 39 reactions (see Figure 4). However Kenner and coworkers ' have shown that the lactone ring in the coumarin residue of the antibiotic novobiocin (64), formed by Streptomyces niveus, originates in a different way. By the use of ^C and ^0 experiments, they showed that the carboxy oxygen atoms of tyrosine (23) served as a source of the heterocyclic coumarin oxygen atom. They postulated an oxidative 23 AO cyclization of the amino acid to explain their results. Others ' have raised the question whether a similar mechanism may not also operate in higher plants, and Scott and Meyers have suggested the involvement of a spiro lactone (46) (see Figure 3) but subsequent'work by Austin 36 1A and Meyers with a C-labelled spirolactone did not bear out the theory. The fact that the coumarins in question actually exist in the cell as glucosides of coumarinic acids argues strongly for an o^-hydroxylation mechanism, as opposed to oxidative cyclization, in 41 * plants. The stage at which o_-methylation occurs remains uncertain. 34 Brown found p_-methoxy cinnamic acid (56) a much better precursor of herniarin (53) than was umbellic acid (60), which suggests that methylation occurs prior to ortho-hydroxylation, but trapping 42 32 experiments failed to detect 56 in Lavandula. Brown feels that this plant can utilize 56 but the main pathway is by way of umbellic acid (60). He proposed that the poor incorporation of 60 could be explained i f p_-hydroxycinnamic acid (43) proceeded to trans-2-glucosyloxy-4-hydroxycinnamic acid (65) via enzyme substrate complex (X) with ortho-hydroxylation and glucosylation both occurring without - 21 -leaving the enzyme surface. The product, tran.s-2-glucosyloxy-4-hydroxycinnamic acid (65), could then undergo _0-methylation to trans-2-glucosyloxy-4-methoxycinnamic acid (59), a known intermediate, followed by isomerization of the double bond to give the bound form of herniarin (53) (see Figure 6). 65 59 53 Figure 6. Proposed biosynthesis of herniarin (53) in Lavendula. - 22 -Ourisson however suggests that methylat ion i n s c o p o l e t i n (55) b i o s y n t h e s i s , i n tobacco t i s s u e c u l t u r e s , occurs p r i o r to o r t h o -h y d r o x y l a t i o n , as supported by i n c o r p o r a t i o n of f e r u l i c a c i d (58) i n t o 55. However, he f a i l e d to f i n d evidence of the o_-hydroxylated f e r u l i c a c i d intermediate and suggests that c y c l i z a t i o n may occur v i a r a d i c a l c o u p l i n g without intermediary o r t h o - h y d r o x y l a t i o n . Recent ly , a p u b l i c a t i o n has appeared on the b i o s y n t h e s i s of the 43 dihydroxy coumarin daphnin (54b) and i t s corresponding 8 -g lucos ide . The authors suggest these compounds are produced mainly v i a p_-coumaric 32 a c i d (43) and not v i a c a f f e i c a c i d (57) as has been suggested e a r l i e r . Thus the o v e r a l l p i c t u r e of the cor rec t sequence of events i s s t i l l somewhat confused. B i o s y n t h e s i s of Furanocoumarins 44 The furanocoumarins are f i s h poisons and i n s e c t i c i d e s . P l a n t s of the Rutaceae and U m b e l l i f e r a e f a m i l i e s are the p r i n c i p l e source of the many n a t u r a l l y o c c u r r i n g members of t h i s group. S e v e r a l l i n e a r and n o n l i n e a r s t r u c t u r a l l y i someric furanocoumarins are t h e o r e t i c a l l y p o s s i b l e , but w i t h few except ions , d e r i v a t i v e s of only two of these isomers have been obtained from n a t u r a l sources . The two ca tegor ies are i ) the l i n e a r system portrayed by p s o r a l e n (10a) (6,7-furanocoumarin) and i i ) a n g e l i c i n (66) (7,8-furanocoumarin) represent ing the n o n l i n e a r s k e l e t o n . - 23 -The biogenesis of the two extrn carbon atoms of the furan ring (C-6 and C-7) of the furanocoumarins has been a source of controversy 45 over the years. Haworth suggested that theoretically the unsubstituted furan rings of these natural coumarins could be derived by elimination of propane from a hypothetical a-isopropyldihydrofuran (67). Geissman 46 and Hinreiner proposed a two carbon phosphorylated keto alcohol moiety (68), which could cyclize to furan-3-one (69) and subsequently yield a furan ring by reduction and dehydration. 1 1/ 70 69 68 A review of natural products within the coumarin and related families reveals a remarkable feature that, frequently, definite isopentane units and unsubstituted furan rings arc found incorporated - 24 -into a structure of a given natural product. Based on this close 47 association, Seshadri and coworkers suggested structure 71 to be the precursor of the simple furan ring. The transformation of 71 into the glycol (72) and oxidative cleavage of the latter, would result in the loss of three carbons, leaving a residue of two carbon atoms as shown in 73 which on cyclization and dehydration would form unsubstituted furans. H3C H3C HO CH-HC I OH HO "H^ C" CH*H„ H3° OH OH HO OHC-H2C 71 72 73 OCH H0-10a 10b 74 The first tracer study on the biosynthesis of furanocoumarins 48 was done by Caporale et al. They reported the incorporation of radioactivity from acetate-[2- H], tyrosine-[2- C] (23), [U- H]-14 3 tyrosine, mevalonic acid-[2- C] (85), and succinic acid-[2,3- H] into bergapten (10b) and psoralene (10a) utilizing leaves of Ficus carica. However, no degradations were performed to determine the position of the 14 labels. The incorporation of mevalonic acid-[2- C] into these furano-coumarins is difficult to reconcile with the proposed formation of the furan ring via a marmesin (74) type intermediate, as C-2 of mevalonic acid should be lost with the degradation of the three extra carbon atoms i f Seshedri's hypothesis is followed. The f i r s t direct evidence as to the origin of furanocoumarins in 49 Pimpinella magna was provided by Floss and Mothes. By incorporating radioactively labelled precursors into the plant, they demonstrated that the coumarin nucleus, C^ -C^  portion, of sphondin (75) is derived from cinnamic acid (40). Umbelliferone (49), a natural constituent of P.magna roots, proved to be a more efficient precursor than coumarin (19) indicating that para-hydroxylation occurs prior to 75 14 oftho-hydroxylation. By incorporating mevalonic acid-[4- Cj (85) into Pimpinella, they were able to isolate radioactive pimpinellin (76) and its subsequent degradation provided good evidence of specific incorporation of C-4 of mevalonic acid into the 7-position of . 49 pimpinellin (76) (Figure 7). 49 Floss and Mothes also presented evidence as to the general mechanism of furanocoumarin biosynthesis, especially the sequence of the individual steps. On the basis of comparison of the specific activities of the various furanocoum;irins after feeding cinnamic acid-[ l - l 4 C j and mevalonic acid-[b^^C] , they suggested a biogenetic scheme - 26 -Figure 7. Degradation of piminellin (76). which involved isoprenylation occurring after the final oxygenation pattern of the coumarin portion had been established. Since the 7-hydroxy group of umbelliferone (49) moiety must not be methylated, they also suggested that umbelliferone-7-glucoside (63) might be an intermediate and that further hydroxylation and methylations might occur at the glucoside stage. However in latter work, Floss and Paikert"^ tested this proposed scheme and found evidence which did not support prenylation as a last step in the biosynthetic pathway. They found that umbelliferonc-7-glucoside (63) was not a better precursor of the 3 furanocoumarins than umbelliferone (49) itself and that [CH^- H]-scopoletin (55) was not incorporated preferentially into any one furanocoumarin as would be expected i f isoprenylation were a late step in the biogenetic pathway. It was observed that the labelled methyl group of 55 was being rapidly equilibrated, by demethylation and reniethylation, with the pool of the plant. They proposed an alternative scheme for furanocoumarin biosynthesis, involving - 28 -isoprenylation of umbelliferone (49) followed by further modification of the resulting 6- and 8-dimethylallyl-umbelliferone (82,83). They feel that such a pathway (Figure 9) would also explain the almost exclusive occurrence, in nature, of 6,7- and 7,8-furanocoumarins. This opinion is supported by Steck and coworkers. Figure 9. Alternative pathway of furanocoumarin biosynthesis in .. . „ 50 Pimpmella magna. - 29 -Recently Steck, El-Dakhnkhny and Brown"'"'' have demonstrated the intermediacy of mermesin (74) (in Ruta graveolens) in the biosynthesis of psoralen (10a), bergapten (10b) and xanthotoxin (11) and the intermediacy of columbianetin (84) (in Heracleum lanatum) in the biosynthesis of angelicin (66), isobcrgapten (81) and pimpinellin (76). By feeding umbelliferone-[2-"^C] (49) to Ruta graveolens and 14 skimmin-[2- C] (63) to Heracleum lanatum, they were able to 14 demonstrate the intermediacy of umbelliferone-[2- C] (49) in mermesin (74) and columbianetin (84). Furthermore, by direct feeding of tritiated marmesin (74) and columbianetin (84), they established their conversion to the appropriate furanocoumarins. R 81 76 - 30 -Little is known as to the actual mechanism of furan ring formation but Brown and Stock"*"*" envisage the formation of marmesin (74) as depicted in Figure 10. Figure 10. The biosynthetic route to marmesin (74). 52 ' Brown has recently attempted to test some of the proposed pathways to furanocoumarins, utilizing parsnips (Pastinaca sativa L.) which contains the furanocoumarins bergapten (10b), imperatorin (86), isopimpinellin (2), and xanthotoxin (11). In his incorporation 14 studies, with 5-methoxy-7-hydroxycoumarin-[2- C] (87), Brown could only conclude that there is no evidence to indicate that 87 is a precursor of bergapten (10b). From incorporation of mevalonic acid-[2- UC] and -[5- 1 4C] (85), sodium acetate- [1- UC] and -[2- 1 4C] and 14 umbelliferone-[2- C] (49), Brown showed that mevalonatc (85) and acetate were very poorly utilized relative tc umbellifcrone (49), and - 31 that in general acetate was a better precursor than mevalonate. He 14 reports that mevalonate-[2- C] was incorporated into a l l the furano-14 coumarins to approximately the same extent as the -[5- C] compound. These results seem to raise doubts as to the role of mevalonate in the furanocoumarin formation, at least in parsnips. Brown suggests that i t may be necessary to consider other possible origins of the 3-methylbutanoid moiety in marines in (74) and thus also the furan ring carbons in furanocoumarins. Alkylated Coumarins Little information is available as to the origin of alkyl side chains often found in natural coumarins. Floss et a l . " ^ have presented evidence which shows that formate or the S-methyl group of methionine may act as a carbon source for the methoxyl group in xanthotoxin (11). 86 87 OCH 3 11 Isoprenoid groups are present in many coumarins; they may appear as 0- or C-alkyl substituents or involved in ring formation with an adjacent hydroxyl group (see section on furanocoumarins). Multiples of the unit, such as the geranyl ( C ^ Q ) or farnesyl ( C ^ ^ ) side chains are also found. Little definite information is presently known about 46 the origin of these isoprenoid groups. Geissman and Hinreiner have adopted the earlier ideas that the C , . unit is the result of condensation o f a n d C ^ . Robinson"*"4 has suggested senecioic acid (8 ,8-dimethyl-acrylic acid) (88) as the terpene precursor, and the carbonyl group of 88 as the spearhead of the attack on the aromatic nuclei. However, more recent work has emphasized the importance of mevalonic acid (85) as theprecursor to these side chains. Mevalonic acid (85) itself is the product resulting from the combination of three 53 units of acetyl coenzyme-A and as shown in Figure (11), its biogenesis is accomplished via Claisen and aldol-like condensations and the reduction of the resultant ^-hydroxy-3-methyl glutaryl CoA (89). C O O H 88 2CH3'CO-SCoA 0 II CH -C-CH -CO-SCoA Cll3-C0-SCoA OH I CHQ-C-CH_-CO-SCoA 3 , 2 CH -COOH 8 9 Figure 11. The acetate biogenesis of mevalonic acid (85). Although no direct evidence is available to substantiate this hypothesis in coumarin biosynthesis, experiments with radioactively labelled mevalonic acid (85) have shown i t to be incorporated into 54 the prenyl side chains of some natural phenols and quinones. Recently Hamada and Chabachi"'"' have reported that mevalonic acid is utilized in the formation of the isoprene side chain of rotenone (90) OCH, OCH, However, Kunesch and Polonsky"^ have shown isoleucine to be the presursor of the tigloyl side chain of the 4-phenyl coumarin calophyllolide (91). - 34 -91 C- and O-alkylations of these phenolic compounds are assumed to occur via attack of the phenolic oxygen or of phenol activated ortho or para attack on Y,Y-dimethylallylpyrophosphate. This would give rise to any of the four possible products by the mechanisms indicated 54 in Figure 12. (a) ( b ) - 35 -OPP Figure 12. Proposed mechanism for alkylation of phenols. The two alkylation reactions (a) and (c) leading to C-yjy-dimethylallyl derivatives and y, y-^imethyl a l l y l ethers are more likely to occur for obvious steric reasons. However, compounds of a l l four types have been isolated from natural sources and as expected, the products of the first two processes greatly outnumber the rather rare a,a-dimethylallyl derivatives. The seemingly general confusion as to the nature of prenylation in coumarin biosynthesis, and the presence in Thamnosma montana of a number of interesting prenylated coumarins and furanocoumarins, led us to consider further experiments which might serve to help clarify some of the many questions that remain unanswered. Such experiments will be discussed in Parts II and III of this thesis. - 36 -DISCUSSION (PART I) Degradations of Coumarins from Thamnosma montana Before the advent of modern spectroscopy,many compounds were i s o l a t e d and t h e i r s t r u c t u r e s determined by the use of exhaustive degradative techniques. Coumarins are c e r t a i n l y no exception i n t h i s regard. Thus there are a wide v a r i e t y of methods a v a i l a b l e i n the l i t e r a t u r e f o r the degradation of the coumarin system. However, f o r the purpose of b i o s y n t h e t i c i n v e s t i g a t i o n s , i t i s e s s e n t i a l t h a t the r e a c t i o n s u t i l i z e d be a p p l i c a b l e to the s m a l l q u a n t i t i e s of m a t e r i a l that would be a v a i l a b l e from the r a d i o a c t i v e i n c o r p o r a t i o n experiments. Keeping t h i s i n mind, a d e t a i l e d study on the degradations of coumarins a v a i l a b l e from Thamnosma montana was undertaken. The coumarins u m b e l l i p r e n i n (9), i s o p i m p i n e l l i n (2) and a l l o -i m p e r a t o r i n methyl ether (7) were s e l e c t e d f o r t h i s purpose as they are present i n reasonable q u a n t i t i e s i n Thamnosma montana, are e a s i l y i s o l a t e d and p u r i f i e d and are r e p r e s e n t a t i v e examples of three types of coumarins. However, the l a t e r s t u d i e s on the t i s s u e c u l t u r e s of Thamnosma montana revealed the absence of u m b e l l i p r e n i n (9), whereas i s o i m p e r a t o r i n (13) was present i n i s o l a b l e q u a n t i t i e s i n the t i s s u e c u l t u r e e x t r a c t along w i t h i s o p i m p i n e l l i n (2) and a l l o i m p e r a t o r i n methyl ether (7). Therefore i t was decided to study the b i o s y n t h e s i s of - 37 -a l l four coumarins from Thamnosma montana plants and tissue cultures and the degradations utilized in these investigations are discussed below. To study precursors other than mevalonic acid which could be utilized by plants in the biosynthesis of the isoprenoid side chains in coumarins, umbelliprenin (9), a simple coumarin with a farnesol ether side chain, was considered to be the ideal compound. Since mevalonic acid has been shown to be the precursor of these isoprenoid units in many other compounds (see Introduction), umbelliprenin (9) could also act as an internal standard in any subsequent mevalonic acid feeding experiment. On the other hand, isopimpinellin (2) is a relatively simple furanocoumarin whose chemistry can be correlated with that of other such furanocoumarins as bergapten (10b) and xanthotoxin (11). Thus, although l i t t l e has been reported on the degradative chemistry of isopimpinellin (2) itself, related compounds, such as bergapten (10b), have been studied in detail. Alloimperatorin methyl ether (7) is also a furanocoumarin but with the added complication of a alkyl side chain whereas in isoimperatorin (13), a C,. alkyl-ether side chain is present. Thus isopimpinellin (2), alloimperatorin methyl ether (7) and isoimperatorin (13) offer the possibility of studying the mode of furan ring formation and the origin of isoprenoid side chains in these compounds. With these various objectives in mind, a detailed degradative scheme was prepared for these compounds. It is necessary to mention at this point that the degradations on umbelliprenin, isopimpinellin and alloimperatorin methyl ether were performed in collaboration with Dr. R.N. Young. A detailed account of - 38 -these degradations along w i t h complete c h a r a c t e r i z a t i o n of the degradative products can be found i n the d o c t o r a l t h e s i s of R.N. Young,^ and consequently f o r the sake of c l a r i t y f o r f u r t h e r d i s c u s s i o n s only a b r i e f d e s c r i p t i o n of these degradations w i l l . b e presented here together w i t h f u l l d e t a i l s of any d e v i a t i o n from t h i s sequence. A complete degradation of i s o i m p e r a t o r i n (13) was developed more r e c e n t l y and i s presented i n d e t a i l . Degradations of U m b e l l i p r e n i n (9) As mentioned p r e v i o u s l y , i t was our i n t e n t i o n to study the b i o s y n t h e s i s of the f a r n e s o l ether s i d e chain present i n u m b e l l i p r e n i n (9) and t h e r e f o r e i t was d e s i r a b l e to determine the amount of r a d i o a c t i v i t y a s s o c i a t e d w i t h t h i s s i d e chain i n any r a d i o a c t i v e i n c o r p o r a t i o n experiment. Kariyone and Matsumo"^ found that the coumarin auraptene (the g e r a n y l ether of u m b e l l i f e r o n e (49)) would undergo e f f i c i e n t h y d r o l y s i s i n hot g l a c i a l a c e t i c a c i d to give u m b e l l i f e r o n e (69) and g e r a n y l acetate. However i f the h y d r o l y s i s was c a r r i e d out i n g l a c i a l a c e t i c a c i d i n the presence of a s m a l l amount of s u l f u r i c a c i d , no g e r a n y l acetate could be i s o l a t e d due to i t s p o l y m e r i z a t i o n . When the h y d r o l y s i s of u m b e l l i p r e n i n (9) was c a r r i e d out i n hot g l a c i a l a c e t i c a c i d , umbelliferone (49) was obtained i n good y i e l d (75%) but no f a r n e s y l acetate could be i s o l a t e d . I t was observed that under these r e a c t i o n c o n d i t i o n s , the f a r n e s y l acetate being produced was undergoing a s e r i e s of f u r t h e r r e a c t i o n s . However, s i n c e the r a d i o a c t i v i t y present i n umbelliferone (49) derived from the r e a c t i o n would a l l o w c a l c u l a t i o n of the r a d i o a c t i v i t y i n the s i d e chain by d i f f e r e n c e , i t was decided to abandon further attempts to isolate the entire side chain. In order to determine the amount of radioactivity present (if any) in the 2, 3 and 4-positions of umbelliprenin (9), umbelliferone (49) was treated with potassium hydroxide at high temperature. This 5 8 reaction was previously reported to give resorcinol. However very l i t t l e resorcinol could be obtained and the major component isolated proved to be 2,4-dihydroxy benzoic acid (48% yield). Thus this degradation allows the determination of radioactivity at the 2- and 3-positions of umbelliprenin (9). - 40 -As i t was also of interest to determine the distribution of radioactivity in the farnesol ether side chain of umbelliprenin (9), 59 a suitable degradation scheme was devised. Caldwell and Jones have reported the isolation of both acetone and levulinaldehyde (94a) as their 2,4-dinitrophenylhydrazone derivatives, from 7-methoxy-5-geranyl coumarin (92) by ozonolysis and steam distillation of the reaction mixture into a solution of 2,4-dinitrophenylhydrazine (2,4-DNP). 93, R=2,4-DNP 94a, R=0 94b, R=2,4-DNP This procedure when applied in the case of umbelliprenin (9) turned out to be unsatisfactory as the yield of levulinaldehyde-2,4-DNP (94b) was very poor (< 1%). However when the ozonide of 9 was worked up 5 8 under reductive conditions utilizing catalytic hydrogenation and the resulting reaction mixture treated with a solution of 2,4-DNP reagent in methanolic hydrogen chloride, orange coloured crystals of 94b precipitated (26% yield). In order to isolate the acetone-2,4-DNP produced in the reaction, acetone-free methanol (methanol distilled from iodine and aqueous base) proved to be unsuitable as i t was found that the 2,4-DNP reagent would not dissolved satisfactorily in the acetone-free methanolic hydrogen chloride. - 41 -0 R H R 9 94b, R=2,4-DNP As mentioned p r e v i o u s l y , i t was d e s i r a b l e to i s o l a t e the t e r m i n a l three carbon system of the f a r n e s o l ether s i d e chain of 9 as acetone i n an o z o n o l y s i s r e a c t i o n , but the l a c k of m a t e r i a l prevented f u r t h e r e v a l u a t i o n of t h i s r e a c t i o n . Degradation of I s o p i m p i n e l l i n (2) I s o p i m p i n e l l i n ( 2 ) , as an abundant component of Thamnosma montana shoots, was s e l e c t e d f o r study of the b i o s y n t h e s i s of the furan r i n g 49 i n furanocoumarins. As mentioned p r e v i o u s l y , F l o s s and Mothes have shown s p e c i f i c i n c o r p o r a t i o n of C-4 of mevalonic a c i d (85) i n t o C -7 of p i m p i n e l l i n (76). However, the f i n d i n g s of Rodighiero and coworkers 4^ and of Brown"*2 that the C-2 of mevalonic a c i d i n c o r p o r a t e s i n t o simple furanocoumarins as e f f i c i e n t l y as C~4 l a b e l l e d m a t e r i a l 49 c a l l s i n t o question the r e s u l t s of F l o s s and Mothes. Brown has a l s o found acetate to be a much b e t t e r precursor of furanocoumarins than was mevalonic a c i d . Therefore, to c l a r i f y these u n c e r t a i n t i e s , i t was decided that an extensive degradation procedure to determine the d i s t r i b u t i o n of r a d i o a c t i v i t y i n i s o p i m p i n e l l i n (2) was needed. A summary of these degradations i s given i n Figure 13. - 43 -In order to determine the amount of r a d i o a c t i v i t y present at the 7 - p o s i t i o n , i s o p i m p i n e l l i n (2) was ozonized under c o n t r o l l e d c o n d i t i o n s . Previous workers^"*" have found that by o z o n o l y s i s , furano-coumarins could be converted to the p h e n o l i c aldehydes where the furan r i n g had undergone degradation i n preference to the pyrone r i n g . To accomplish such a s e l e c t i v e o z o n o l y s i s , a c e t i c a c i d was s a t u r a t e d w i t h ozone and the ozone c o n c e n t r a t i o n determined by t i t r a t i o n of the i o d i n e produced when the a l i q u o t of t h i s - s o l u t i o n was reacted w i t h aqueous potassium i o d i d e . Thus the o z o n o l y s i s of i s o p i m p i n e l l i n (2) w i t h a 60-70% molar excess of ozone fo l l o w e d by r e d u c t i o n of the ozonide w i t h z i n c dust y i e l d e d a product which when chromatographed on a s i l i c a g e l column f o l l o w e d by f r a c t i o n a l c r y s t a l l i z a t i o n gave pure 6-formyl-7-hydroxy-5,8-dimethoxy coumarin (95), m.p. 214-216° (45% y i e l d ) . I t was c h a r a c t e r i z e d on the b a s i s of i r , uv, nmr, mass spectrometry and elemental a n a l y s i s . However, i t was observed that when i s o p i m p i n e l l i n (2) was ozonized i n a s i m i l a r manner as above but the r e d u c t i o n of the ozonide was c a r r i e d out w i t h a l a r g e amount of z i n c and over a longer p e r i o d of time, no aldehyde (95) could be obtained. The nmr spectrum of the r e a c t i o n mixture revealed that the d e s i r e d aldehyde was being reduced to the corresponding a l c o h o l (103a). However, the a l c o h o l (103a) was not i s o l a t e d but was a c e t y l a t e d w i t h a c e t i c anhydride and p y r i d i n e and a f t e r p r e p a r a t i v e l a y e r chromatography of the r e a c t i o n mixture pure 6-acetoxy methyl-7-acetoxy-5,8-dimethoxy coumarin (103b), m.p. 139-14L° was obtained i n 66% o v e r a l l y i e l d . Thus the nmr spectrum (Figure 14) was very s i m i l a r to that of i s o p i m p i n e l l i n (2) but the s i g n a l s due to the furan protons i n the s t a r t i n g m a t e r i a l were now absent and i n s t e a d a - 45 -sharp two proton singlet at T 4.81 was assigned to the benzylic methylene group containing the acetyl function. A sharp three proton singlet at T 7.65 was assigned to the aromatic acetyl group and another at x 7.98 was assigned to the aliphatic acetyl group. Elemental analysis and high resolution mass spectrometry supported the assigned structure. Next i t was of interest to determine the proportion of radio-activity in isopimpinellin (2) which resided in the 6-position. The phenolic aldehyde (95) was felt to be the obvious starting material and i t was apparent that some kind of oxidative procedure would be necessary for such a degradation. As phenols are generally unstable to oxidative conditions, 95 was methylated with methyl iodide and anhydrous potassium carbonate in acetone to give 6-formyl-5,7,8-tri-methoxy coumarin (96), mp 152.5-154° (75% yield). This compound was fully characterized on the basis of analytical and spectral data. The removal of the formyl group from 96 was achieved by utilizing 6 ? a modified Dakin reacti on. Thus when 96 in glacial acetic acid was treated with a mixture of hydrogen peroxide and sulfuric acid and the residue after work up of the reaction mixture chromatographed on preparative layer chromatography, the major component, after crystalliza-tion from 95% ethanol, yielded pure 6-hydroxy-5,7,8-trimethoxycoumarin (97), mp 198.5-199.5° (75% yield). Next of interest was the determination of the radioactivity in the pyrone portion of isopimpinellin (2). Treatment of furanocoumarins with a large excess of ozone is known to cause degradation of both the 6 3 furan and the pyrone ring. Therefore, isopimpinellin, in a mixture of - 46 -a c e t i c a c i d and e t h y l acetate was ozonized w i t h a l a r g e excess of ozone and the r e s u l t a n t ozonide was reduced w i t h z i n c dust. The work up of the reaction mixture gave, a f t e r c r y s t a l l i z a t i o n , pure 1,3-diformyl-4,6-dihydroxy-2,5-dimethoxybenzene (98), mp 162-164° i n 42% y i e l d . Thus t h i s r e a c t i o n allowed the determination of r a d i o a c t i v i t y a s s o c i a t e d w i t h the 2- and 3 - p o s i t i o n s of i s o p i m p i n e l l i n . In order to o b t a i n a degradative procedure which would a l l o w the determination of r a d i o a c t i v i t y a s s o c i a t e d w i t h the 4 - p o s i t i o n of i s o p i m p i n e l l i n ( 2 ) , the dialdehyde (98) was choosen as the s t a r t i n g m a t e r i a l . I t was considered that a Dakin-type of r e a c t i o n could be u t i l i z e d f o r such a purpose on the methylated product of 98. Therefore, 98 was methylated under c o n d i t i o n s p r e v i o u s l y d escribed and the product l,3-diformyl-2,4,5,6-tetramethoxybenzene (99) mp 49-50°, was obtained i n 86% y i e l d . This compound had s p e c t r a l and a n a l y t i c a l data completely c o n s i s t e n t w i t h the assigned s t r u c t u r e . The treatment of the methylated dialdehyde (99) i n a c e t i c a c i d w i t h a mixture of hydrogen peroxide and s u l f u r i c a c i d f o r 16 hours i n 6 2 c o l d ( i . e . under the c o n d i t i o n s described by Schonberg et a l . ) y i e l d e d only a complex mixture of h i g h l y coloured products. Thus i t was evident that the diphenol (100 a) apparently being formed i n the r e a c t i o n was decomposing under these c o n d i t i o n s . In order to minimize the suspected decomposition, the r e a c t i o n time was reduced to only 15 minutes, l e s s hydrogen peroxide and a n i t r o g e n atmosphere was used and die r e a c t i o n was then q u i c k l y worked up i n the c o l d . The nmr spectrum of the product mixture revealed no dialdehyde protons but the presence of s i g n a l s due to a formate e s t e r (T 1.62) and a phenol (T 4.70). I t - 47 -was apparent that the reaction had proceeded but the hydrolysis of the intermediate formate esters was incomplete. Due to the apparent instability of the diphenol (100a), i t was decided to trap i t as the diacetate derivative (100b). However, when the formate ester mixture was treated with aqueous base and acetic anhydride (to trap the resulting diphenolate anion), only a complex mixture of products could be obtained. Considering the instability of 100a to hydrolysis conditions, a more rigorously controlled method of formate ester hydrolysis was devised. It was expected that a strong nucleophile such as methyl-lithium could be utilized to effect rapid and complete transformation of the formate ester to the dilithio salt of diphenol (100a). Under strictly anhydrous conditions, the salt would be expected to precipitate from the organic solvent and thus as a solid, perhaps would be less prone to decomposition. Quenching such a reaction mixture with acetic anhydride would then afford the diacetate (100b). Thus the dialdehyde (99) in acetic acid was treated with a hydrogen peroxide and sulfuric acid mixture at 0°C under a nitrogen atmosphere for 20 minutes and the reaction was worked up quickly in the cold as before. The product mixture was dissolved in anhydrous ether and treated with excess methyllithium. As expected, a precipitate formed and after treatment with acetic anhydride (with some pyridine added to ensure complete acetylation), the work up of this complex mixture yielded a near quantitative yield of 1,3-diacetoxy-2,4,5,6-tetramethoxy-benzene (100b) (97% yield from preparative layer chromatography) as a colourless o i l which could be induced to crystallize, mp 57-58°. It - 48 -was c h a r a c t e r i z e d completely on the b a s i s of a n a l y t i c a l and s p e c t r a l data. Thus by comparison of the molar a c t i v i t y of 100b w i t h that of the dialdehyde (98), the a c t i v i t y a s s o c i a t e d w i t h the 4- and 6 - p o s i t i o n s of i s o p i m p i n e l l i n (2) could be obtained. Since the r a d i o a c t i v i t y of the 6 - p o s i t i o n could be determined from previous degradations, the percentage of r a d i o a c t i v i t y r e s i d i n g at the 4 - p o s i t i o n of i s o p i m p i n e l l i n (2) i s thus determinable. I t was f i n a l l y of i n t e r e s t to determine the percentage of r a d i o a c t i v i t y of i s o p i m p i n e l l i n (2) which might be a s s o c i a t e d w i t h the two methoxyl groups. To accomplish t h i s , i s o p i m p i n e l l i n (2) was 64 demethylated by r e f l u x i n g w i t h h y d r i o d i c a c i d and the r e s u l t i n g methyl i o d i d e was swept from the r e a c t i o n mixture w i t h a stream of n i t r o g e n and trapped as tetramethylammonium i o d i d e (101). A f t e r s c i n t i l l a t i o n counting of 101, i t was converted to i t s p i c r a t e d e r i v a t i v e (102), mp 323-325°C. Elemental a n a l y s i s was c o n s i s t e n t w i t h the molecular formula C, _H, .0..N,. 10 14 7 4 Thus by a s e r i e s of degradations, i s o p i m p i n e l l i n (2) could be degraded to determine the r a d i o a c t i v i t y a s s o c i a t e d w i t h a l l the carbon atoms attached to the benzene p o r t i o n of the molecule. Degradations of A l l o i m p e r a t o r i n Methyl Ether (7) A l l o i m p e r a t o r i n methyl ether (7) contains a f u r a n r i n g and a d i m e t h y l a l l y l s i d e chain. Although no d i r e c t evidence i s a v a i l a b l e as to the o r i g i n of these side chains i n furanocoumarins, experiments w i t h s i m i l a r coumarins have shown them to be mevalonic a c i d d e r i v e d (see I n t r o d u c t i o n ) . Thus i n order to gain some i n f o r m a t i o n as to the - 49 -specificity of incorporation of such precursors into 7, a series of degradations were devised which would allow the determination of the distribution of radioactivity in alloimperatorin methyl ether (7) in the course of biosynthetic experiments. Thus to determine the distribution of radioactivity in the prenyl side chain of 7, a cleavage reaction was indicated. It was felt that although 7 has three double bonds which would be reactive to ozone, the partial aromatic character of both the furan and the pyrone rings might allow selective ozonization of the side chain double bond. Thus alloimperatorin methyl ether (7) was treated with 1.5 molar equivalents of ozone in acetic acid and the resultant ozonide was reductively cleaved with zinc dust. The resulting mixture was steam distilled and the 2,4-dinitrophenylhydrazone derivative of acetone (93) was isolated in 43% yield. The non-volatile portion of the reaction mixture was extracted and subsequent preparative layer chromatography allowed the isolation of unreacted 7 (37% yield) and the expected aldehyde (104) in 49% yield. This compound appeared to be unstable to air and therefore i t was decided to reduce i t to the corresponding alcohol (105). Thus the aldehyde (104) was treated with sodium borohydride and alcohol (105) was isolated in 85% yield, mp 167-169°. The spectra data was consistent with the assigned structure. 0 R OCH 3 93, R=2,4-DNP 7 - 50 -This degradation, w h i l e g i v i n g the d e s i r e d products, was found to have some s e r i o u s drawbacks when performed on r a d i o a c t i v e 7. The most s i g n i f i c a n t problem was that 7 proved to be very d i f f i c u l t to o b t a i n r a d i o c h e m i c a l l y pure. A l s o , the h i g h l y coloured nature of acetone-2,4-DNP (93) made s c i n t i l l a t i o n counting i n a c c u r a t e when low l e v e l s of r a d i o a c t i v i t y were present. Thus an a l t e r n a t i v e scheme f o r the degradation of a l l o i m p e r a t o r i n methyl ether (7) was considered. The scheme must i n c o r p o r a t e the main o b j e c t i v e of determining the d i s t r i b u t i o n of r a d i o a c t i v i t y i n the f u r a n and the pyrone r i n g s of 7. This scheme i s summarized i n Figure 15. As has been shown p r e v i o u s l y , ozone a t t a c k s p r e f e r e n t i a l l y the s i d e chain double bond and thus i t was apparent that i f an o z o n o l y s i s procedure was to be used to cleave the f u r a n and the pyrone r i n g s , i t would be necessary to f i r s t modify the s i d e chain double bond to make i t r e s i s t a n t to o z o n o l y s i s . A l l o i m p e r a t o r i n methyl ether d i o l (3) was considered to be the i d e a l i ntermediate as t h i s could be used f o r the cleavage of the s i d e chain as w e l l as f o r the degradation of the furan 4 and the pyrone r i n g . Dreyer had p r e v i o u s l y shown that a l l o i m p e r a t o r i n methyl ether (7) could be converted to d i o l (3) v i a the epoxide (14) i n good o v e r a l l y i e l d . Thus a l l o i m p e r a t o r i n methyl ether (7) was t r e a t e d w i t h m-chloroperbenzoic a c i d and the epoxide (14) was i s o l a t e d i n 80% y i e l d . Treatment of the epoxide (14) w i t h 5% o x a l i c a c i d gave the d e s i r e d d i o l (3) i n 70% y i e l d . This compound was i d e n t i c a l w i t h a u t h e n t i c a l l o i m p e r a t o r i n methyl ether d i o l (3) k i n d l y s u p p l i e d by Dreyer. To g a i n i n f o r m a t i o n as to the d i s t r i b u t i o n of r a d i o a c t i v i t y i n the s i d e chain of 3, d i o l (3) was t r e a t e d w i t h p e r i o d i c a c i d and the acetone Figure 15. Degradative schemes of alloimperatorin methyl ether (7). - 52 -removed from the r e a c t i o n mixture i n the form of the c o l o u r l e s s p_-bromobenzenesulfonylhydrazone d e r i v a t i v e (106). The n o n - v o l a t i l e p o r t i o n gave aldehyde (104) which upon r e d u c t i o n w i t h sodium borohydride gave a l c o h o l (105) i n 58% y i e l d . Next i t was of i n t e r e s t to determine the d i s t r i b u t i o n of r a d i o a c t i v i t y i n the fu r a n p o r t i o n of a l l o i m p e r a t o r i n methyl ether (7). For t h i s purpose, d i o l (3) was a c e t y l a t e d w i t h a c e t i c anhydride i n p y r i d i n e to 4 form a monoacetate d e r i v a t i v e (107) i n 90% y i e l d . The monoacetate (107) was then t r e a t e d w i t h a s l i g h t excess of ozone and a f t e r the r e d u c t i o n of the ozonide w i t h z i n c dust, work up of the mixture y i e l d e d the d e s i r e d p h e n o l i c aldehyde (108) which could be separated from the s t a r t i n g m a t e r i a l (107) by p a r t i t i o n i n g the mixture between chloroform and aqueous base. The base s o l u b l e m a t e r i a l , a f t e r a c i d i f i c a t i o n and e x t r a c t i o n , y i e l d e d the pure p h e n o l i c aldehyde (108) i n 35% y i e l d , mp 162-164°. The s p e c t r a l data was c o n s i s t e n t w i t h the assigned s t r u c t u r e . Thus t h i s r e a c t i o n allows the determination of r a d i o a c t i v i t y i n the 7 - p o s i t i o n of a l l o i m p e r a t o r i n methyl ether ( 7 ) . In order to determine the amount of r a d i o a c t i v i t y which might r e s i d e i n the 6 - p o s i t i o n of 7, the removal of the aldehyde group of 108 i n the manner p r e v i o u s l y found s u c c e s s f u l i n the degradations of i s o p i m p i n e l l i n ( 2 ) , was considered. For t h i s purpose, the p h e n o l i c aldehyde (108) was methylated w i t h methyl i o d i d e and potassium carbonate i n acetone and the methylated coumarin (109), mp 116-118°, was i s o l a t e d i n 75% y i e l d . This m a t e r i a l (109) was t r e a t e d w i t h a mixture of hydrogen peroxide and s u l f u r i c a c i d i n the c o l d and p r e p a r a t i v e l a y e r chromatography of the mixture provided a major band i n 90% y i e l d . The nmr spectrum of t h i s - 5 3 -m a t e r i a l revealed that the d e s i r e d phenol (110a) had been obtained. However, t h i s compound could not be induced to c r y s t a l l i z e and t h e r e f o r e i t was a c e t y l a t e d w i t h a c e t i c anhydride and p y r i d i n e to give 110b i n 45% o v e r a l l y i e l d . This m a t e r i a l was h i g h l y c r y s t a l l i n e , mp 143-144°, and had a n a l y t i c a l and s p e c t r a l p r o p e r t i e s completely c o n s i s t e n t w i t h the expected product. To allow determination of r a d i o a c t i v i t y a s s o c i a t e d w i t h the pyrone r i n g of 7, the d i o l acetate (107) was ozonized i n the manner des c r i b e d 6 3 by Hegarty and Lahey. The r e s i d u e , a f t e r work up, was c r y s t a l l i z e d to provide i n 48% y i e l d a compound which was f u l l y c h a r a c t e r i z e d as the expected product 111, mp 188-190°. This r e a c t i o n allows the determination of r a d i o a c t i v i t y a s s o c i a t e d w i t h the 2- and 3 - p o s i t i o n s of a l l o i m p e r a t o r i n methyl ether (7). To determine the amount of r a d i o a c t i v i t y a s s o c i a t e d w i t h the 4 - p o s i t i o n of 7, a sequence of r e a c t i o n s s i m i l a r to those performed s u c c e s s f u l l y on i s o p i m p i n e l l i n (2) was attempted. The dialdehyde (111) was methylated by standard procedures and the product (112), mp 79.5-80.5°, gave s p e c t r a l and a n a l y t i c a l data c o n s i s t e n t w i t h the assigned s t r u c t u r e . However, t h i s m a t e r i a l when t r e a t e d under the c o n d i t i o n s developed i n the degradation of i s o p i m p i n e l l i n ( 2 ) , gave only a complex mixture of coloured products. Thus i t was evident that even under h i g h l y c o n t r o l l e d c o n d i t i o n s of the r e a c t i o n , the r e s u l t a n t diphenol (or perhaps the i n t e r m e d i a t e diformate e s t e r ) was decomposing as f a s t as i t was being formed. Thus attempts to e f f e c t t h i s conversion were abandoned. F i n a l l y , i n order to determine the amount of r a d i o a c t i v i t y r e s i d i n g i n the methoxyl group of a l l o i m p e r a t o r i n methyl ether ( 7 ) , a l c o h o l (105), - 54 -der i v e d from the cleavage of d i o l ( 3 ) , was demethylated w i t h h y d r i o d i c a c i d and the r e s u l t a n t methyl i o d i d e was trapped as tetramethylammonium i o d i d e (101). Conversion to the p i c r a t e d e r i v a t i v e was c a r r i e d out as b e f o r e . Thus these s e r i e s o f r e a c t i o n s a l l o w the determination of r a d i o -a c t i v i t y a s s o c i a t e d w i t h most of the carbon atoms of 7. I t should a l s o be noted that these degradations are e q u a l l y a p p l i c a b l e to the degradations of a l l o i m p e r a t o r i n methyl ether epoxide (14). Degradations of I s o i m p e r a t o r i n (13) I s o i m p e r a t o r i n (13) contains an a l k y l - e t h e r s i d e chain and a furan r i n g . Thus to study i t s b i o s y n t h e s i s , i t was considered e s s e n t i a l to determine the amount of r a d i o a c t i v i t y a s s o c i a t e d w i t h the e n t i r e s i d e chain and the furan r i n g . I t was f e l t that an a c i d h y d r o l y s i s of 13 i n a manner s i m i l a r to t h a t of u m b e l l i p r e n i n (9) would a l l o w the determination of r a d i o a c t i v i t y a s s o c i a t e d w i t h the C ^ - a l k y l s i d e chain of i s o i m p e r a t o r i n (13). Thus when 13 was r e f l u x e d w i t h g l a c i a l a c e t i c a c i d and the r e s i d u e , a f t e r work up, was chromatographed on a p r e p a r a t i v e l a y e r chromatoplate, b e r g a p t o l (113), mp 275°, could be i s o l a t e d i n 85% OH 13 113 - 55 -y i e l d . The nmr spectrum was c h a r a c t e r i s t i c of a simple furanocoumarin. The s i g n a l s at x 1.72 (doublet of doublets, J = 9.50 Hz and 0.5 Hz) and at x 3.80 (doublet, J = 9.50 Hz) could be assigned to H(4) and H(3) r e s p e c t i v e l y . S i g n a l s a t t r i b u t e d to the furan r i n g were observed at x 2.49 (doublet, J = 2.50 Hz, H(7)) and at x 3.01 ( m u l t i p l e t , H(6)). A m u l t i p l e t at x 2.82 was assigned to H(8) and a broad s i g n a l at x 7.40 (disappearing on a d d i t i o n of D^O) was assigned to the p h e n o l i c proton. However, recovery during c r y s t a l l i z a t i o n of 113 was poor and t h e r e f o r e i t was methylated w i t h methyl i o d i d e and potassium carbonate i n acetone to give bergapten (10b), mp 186-188° ( i n 90% y i e l d ) . I t s s t r u c t u r e was e s t a b l i s h e d by a n a l y t i c a l and s p e c t r a l data and comparison w i t h an a u t h e n t i c sample. In order to determine the amount of r a d i o a c t i v i t y a s s o c i a t e d w i t h the furan r i n g of 13, bergapten (10b) was f e l t to be the i d e a l s t a r t i n g m a t e r i a l . I t was expected that by u t i l i z i n g a c o n t r o l l e d o z o n o l y s i s procedure bergapten (10b) could be degraded to the corresponding p h e n o l i c aldehyde, a r e a c t i o n p r e v i o u s l y employed i n the case of i s o p i m p i n e l l i n (2). Thus bergapten (10b) was t r e a t e d w i t h a s l i g h t excess of ozone i n g l a c i a l a c e t i c a c i d and a f t e r r e d u c t i o n of the OH 113 10b - 56 -ozonide w i t h z i n c and work up of the mixture y i e l d e d a residue which when analyzed by t i c was observed to be a mixture of the s t a r t i n g m a t e r i a l and a more p o l a r y e l l o w compound. This r e a c t i o n mixture was chromatographed on a s i l i c a g e l column and e l u t i o n w i t h benzene and benzene-chloroform mixtures a f f o r d e d the y e l l o w compound. Fur t h e r p u r i f i c a t i o n by means of c r y s t a l l i z a t i o n gave pure 6-formyl-7-hydroxy-5-methoxy coumarin (114), mp 220-221° (30% y i e l d ) . This compound was c h a r a c t e r i z e d on the b a s i s of the f o l l o w i n g data. The molecular formula, G^l^gO,., was e s t a b l i s h e d by elemental a n a l y s i s and h i g h Me OH r e s o l u t i o n mass spectrometry. The uv spectrum of 114 (X 266 and max 312 nm) revealed the p h e n o l i c nature of the compound, as, on a d d i t i o n Me OH of a l k a l i , the spectrum showed a marked bathochromic s h i f t (X (+Na0H) r max 238, 262, 347, and 394 nm). A c i d i f i c a t i o n of the uv sample reversed Me OH the spectrum to i t s o r i g i n a l form (X (+ HCI) 267 and 314 nm). The max i r spectrum of 114 i n d i c a t e d that the phenol was s t r o n g l y hydrogen bonded to the adjacent aldehyde carbonyl group. Thus no hydroxy1 ab s o r p t i o n was i n evidence. The carbonyl r e g i o n of the spectrum had an a b s o r p t i o n at 1742 cm ^ and another at 1647 cm ^ (aldehyde C=0). An a b s o r p t i o n at 1592 cm was assigned to the a-pyrone system. The nmr Figure 16. Nmr spectrum of 6-formyl-7-hydroxy-5-methoxycoumarin (114). - 58 -spectrum of 114 (Figure 16) fully confirmed the structure of this compound. Thus the spectrum was very similar to that of bergapten (10b) but the signals due to the furan protons in the starting material were now absent and instead low field singlets at T -0.23 and atT -1.96 (disappearing on addition of D^O) were readily assigned to the aldehyde and phenol protons respecitvely. Thus this reaction allowed the determination of radioactivity in the C-7 position of 13. It was also of interest to determine the amount of radioactivity which resided in the 6-position and in the pyrone ring of isoimperatorin (13). However, the lack of material prevented any further development of these degradations. - 59 -EXPERIMENTAL (PART I) Melting points were determined on a Kofler block and are uncorrected. The ultraviolet (uv) spectra were recorded in methanol solution utilizing a Cary 11 or a Unicam, model SP800 spectrophotometer. The infrared (ir) spectra were recorded on Perkin-Elmer model 21 or 457 spectrometers,utilizing a potassium bromide disc. The position of the absorption maxima are quoted in wave numbers (cm ^). Nuclear magnetic resonance (nmr) spectra were recorded in deuterochloroform solution (unless otherwise indicated) at 100 MHz on a Varian HA-100 or a Varian XL-100 instrument and at 60 MHz on a Varian T-60 spectrometer. Chemical shifts are given in the Tiers T scale with reference to tetramethylsilane as the internal standard. Mass spectra were recorded on an Atlas CR-4 mass spectrometer and high resolution mass spectra were carried out on an AEI-MS 902 instrument. Woelm neutral alumina and si l i c a gel G (acc. to Stahl) containing 1% by weight electronic phosphor were used for analytical and preparative layer chromatography (tic), unless otherwise noted. Woelm neutral alumina (activity IV - unless otherwise indicated) was used for column chromatography. The tic plates were activated in an oven at 90° for one hour. For qualitative chromatography, layers of 0.3 mm thickness were used and spots were visualized by viewing under ultraviolet (uv) light. For preparative tic, large (20 x 20 cm) plates with a thicker - 60 -l a y e r (0.5 mm) were used. Developing s o l v e n t s used were; A anhydrous ether-hexane (1:1) or B, e t h y l acetate-chloroform (1:1), unless otherwise noted. M i c r o a n a l y s i s were performed by Mr. P. Borda, M i c r o a n a l y t i c a l Laboratory, U n i v e r s i t y of B r i t i s h Columbia. Methanol was made acetone-free by treatment w i t h i o d i n e and 65a aqueous sodium hydroxide. Chloroform was made acetone-free by f l u s h i n g through a column of C e l i t e impregnated w i t h 2 , 4 - d i n i t r o p h e n y l hydrazine and the eluent was d i s t i l l e d . For d e t a i l e d experimental procedures on the v a r i o u s degradations discussed above, the reader i s r e f e r r e d to the Ph.D. t h e s i s of R.N. Young. Only those procedures developed more r e c e n t l y and not discussed i n that t h e s i s are presented below. P r e p a r a t i o n of a Standard S o l u t i o n of Ozone i n G l a c i a l A c e t i c A c i d G l a c i a l a c e t i c a c i d was placed i n a f l a s k equipped w i t h a bubbler and ozone enriched oxygen was allowed to bubble through the s o l u t i o n f o r 30 minutes at room temperature, at which time the s o l u t i o n had a d e f i n i t e blue t i n g e . The bubbler was then removed and the f l a s k was t i g h t l y stoppered. A l i q u o t s (20 ml) of t h i s s o l u t i o n were added to a s o l u t i o n of potassium i o d i d e ( 1 g) i n water ( 20 ml) and the i o d i n e which was l i b e r a t e d was t i t r a t e d w i t h a standard s o l u t i o n of sodium t h i o s u l f a t e using s t a r c h as an i n d i c a t o r . The sodium t h i o s u l f a t e s o l u t i o n was standardized against a standard potassium dichromate s o l u t i o n . In a t y p i c a l experiment, g l a c i a l a c e t i c a c i d was sa t u r a t e d w i t h ozone as described above and two a l i q u o t s (20 ml) were removed - 61 -and added individually to aqueous solutions of potassium iodide (1 g per flask in two flasks). The iodine liberated was titrated with 0.0125 N sodium thiosulfate solution requiring respectively 17.1 and 16.8 ml to reach the end point. Thus the average of these two values (16.95 ml) required that the ozone concentration at room temperature be 0.106 mmole per 20 ml gla c i a l acetic acid. 6-Formyl-7-hydroxy^-5,8-dimethoxy coumarin (95) Isopimpinellin (2) (45 mg; 0.183 mmole) was treated with ozone saturated gl a c i a l acetic acid (60 ml; 0.30 mmole) and the mixture stirred for one hour at room temperature. Zinc dust (100 mg) was then added and s t i r r i n g continued for further 10 minutes. The mixture was then f i l t e r e d and solvent was then evaporated in Vacuo. The residue (^  70 mg) was dissolved in chloroform-methanol mixture and was chromatographed on s i l i c a gel (6 g). The fractions eluted with benzene and benzene-chloroform contained isopimpinellin (2) and a more polar compound (yellow spot; uv and v i s i b l e ) . These fractions were combined (33 mg) and crystallized from acetone to yield 6-formyl-7-hydroxy-5,8-dimethoxy coumarin (95) (23 mg; 50% yield), mp 214-216°; i r (KBr) 1758, 1730, MPOH 1625, 1592 (a-pyrone), 1640 (aldehyde C=0); uv X (e) 275 (27,100); LT13X uv x M e 0 H (e) (+Na0H) 238 (19,200), 269 (16,600), 299 (12,900), 360 n i c i x (14,200); uv X (e) (+HC1) 208 (29,000), 226 (sh) (15,600), 263 IH3X (12,800), 320 (15,600); nmr (100 MHz) in CDC± 3, TMS lock, -2.03 (1H, singlet,disappears on addition of D^ O, phenolic OH), -0.23 (1H, singlet, aromatic CH0), 2.17 (1H, doublet, J = 10 Hz, H(4) of coumarin), 3.73 (1H, doublet, J = 10 Hz, H(3) of coumarin), 6.00, 6.02 (6H, two singlets, two aromatic 0CH_3) ; mass spectrum m/e 250 (M) , 235 (M-15) , 221 (M-29), - 62 -207 and 179. A n a l . Calcd. f o r C,_H i n0,: C, 57.61; H, 4.03. Found: C, 57.38; 1Z 1(J o H, 4.07. High r e s o l u t i o n molecular weight determination. Calcd. f o r C 1 oH..0, : 250.048. Found: 250.046 1 Z 1U O 6-Acetoxy methyl-7-acetoxy-5,8-dimethoxy coumarin (103b) I s o p i m p i n e l l i n (2) (23.5 mg; 0.095 mmole) was t r e a t e d w i t h ozone sa t u r a t e d g l a c i a l a c e t i c a c i d (32 ml; 0.160 mmole) and the mixture s t i r r e d f o r 3 hours at room temperature. Zinc dust (400 mg) was added and s t i r r i n g continued f o r f u r t h e r one hour. S o l u t i o n was f i l t e r e d and s o l v e n t was evaporated i n vacuo. The residue (30 mg) showed no aldehyde on t i c p l a t e . The nmr of the r e s i d u e revealed i t to be the corresponding a l c o h o l (103a). This (103a) was t r e a t e d w i t h a c e t i c anhydride and p y r i d i n e and s o l u t i o n was s t i r r e d f o r 10 hours. The s o l v e n t was evaporated i n vacuo and the residue gave almost a s i n g l e spot on t i c . I t was separated on p r e p a r a t i v e t i c and c r y s t a l l i z e d from e t h y l acetate to y i e l d 6-acetoxy methyl-7-acetoxy-5,8-dimethoxy coumarin (103b) (21 mg; 66% y i e l d ) , mp 139-141°; i r (KBr) 1780 (aromatic i MeOH acetate C=0), 1735 ( a l i p h a t i c acetate C=0), 1598 (a-pyrone); uv A max ( E ) (23,000), 225 (sh) (15,000), 251 (6, 940), 293 (11,670); nmr (100 MHz) i n CDC1 3, TMS lock,2.06 (IH, doublet, J = 10 Hz, H(4) of coumarin), 3.59 (IH, doublet, J = 10 Hz, H(3) of coumarin), 4.81 (2H, s i n g l e t , CH_2OCOCH3) , 6.00, 6.08 (6H, two s i n g l e t s , two aromatic OCE^), 7.65 (3H, s i n g l e t , aromatic 0C0CH_3) , 7.98 (3H, s i n g l e t , a l i p h a t i c CH2OCOCH_3); mass spectrum m/e 336 (M) , 294 (M-42) , 251 (M-85), 234 (base peak), 219 and 205. - 63 -Axial. Calcd. f o r C,,H1,C' : C, 57.14; H, 4.76. Found: C, 56.99; lb ID O H, 4.88. High r e s o l u t i o n molecular weight determination. C a l c d . f o r C ^ H 1 c 0 o : 336.084. Found: 336.086. Io l o o A c i d Catalyzed H y d r o l y s i s of I s o i m p e r a t o r i n (13) Is o i m p e r a t o r i n (13) (21 mg; 0.078 mmole) was d i s s o l v e d i n g l a c i a l a c e t i c a c i d (5 ml) and the s o l u t i o n was r e f l u x e d f o r 10 hours. The r e a c t i o n mixture was allowed to c o o l to room temperature. Water was added and the s o l u t i o n was e x t r a c t e d w i t h chloroform (5 x 20 m l ) . Chloroform e x t r a c t was washed w i t h water (20 m l ) , d r i e d over anhydrous sodium s u l f a t e and s o l v e n t was removed under reduced pressure. Residue (23 mg) was c r y s t a l l i z e d from e t h a n o l (95%) to give b e r g a p t o l (113) (11.8 mg, 85% y i e l d ) , mp 275° ( l i t . 6 6 mp 278°); nmr (100 MHz) i n CDC1 3, TMS l o c k , 1.72 (1H, doublet of doubl e t s , J = 9.50 Hz and 0.5 Hz, H(4) of furanocoumarin), 2.49 (1H, doublet, J = 2.5 Hz, H(7) of furanocoumarin), 2.82 (1H, m u l t i p l e t , H(8) of furanocoumarin), 3.01 (1H, m u l t i p l e t , H(6) of furanocoumarin), 3.80 (1H, doublet, J = 9.50 Hz, H(3) of furanocoumarin), 7.40 (1H, broad s i n g l e t , d i s a p p e a r i n g on a d d i t i o n of D^O, p h e n o l i c h y d r o x y l group). M e t h y l a t i o n of Bergaptol (113) Bergaptol (113) (11.8 mg; 0.054 mmole) from previous r e a c t i o n was d i s s o l v e d i n acetone (1 ml) and anhydrous potassium carbonate (500 ng) and methyl i o d i d e (3 ml) were added. The mixture was r e f l u x e d f o r 15 minutes, cooled to room temperature and s t i r r i n g continued f o r f u r t h e r 10 minutes. Water (10 ml) was added, s o l u t i o n was a c i d i f i e d - 64 -w i t h concentrated h y d r o c h l o r i c a c i d and was e x t r a c t e d w i t h chloroform (4 x 20 ml). The chloroform e x t r a c t was washed w i t h water (20 m l ) , d r i e d over anhydrous sodium s u l f a t e and the s o l v e n t was removed under reduced pressure to y i e l d a re s i d u e (18 mg) which was observed to be e s s e n t i a l l y one component on t i c p l a t e . P r e p a r a t i v e t i c from c h l o r o f o r m - e t h y l acetate mixture (1:1) gave pure bergapten (10b) (12 mg, 90% y i e l d ) which was c r y s t a l l i z e d from e t h y l acetate as c o l o u r l e s s 6 7 p l a t e s , mp 186-188° ( l i t . mp 191°), mixed mp w i t h a u t h e n t i c bergapten (10b) (obtained from Dr. D.L. Dreyer) 186.5-187.5°; i r (KBr) 1726, 1623, 1580 (a-pyrone): uv X (e) 221 (21,400), 248 (17,800), 257.5 (16,200), 267 (17,500), 309 (14,700); nmr (100 MHz) i n CDC1 3, TMS l o c k , 1.91 (IH, doublet of doub l e t s , J = 9.75 Hz and 0.6 Hz, H(4) of furano-coumarin), 2.47 (IH, doublet, J = 2.5 Hz, H(7) of furanocoumarin), 2.92 (IH, m u l t i p l e t , H(8) of furanocoumarin), 3.05 (IH, doublet of doub l e t s , J = 2.5 Hz and 1.0 Hz, H(6) of furanocoumarin), 3.80 (IH, doublet, J = 9.75 Hz, H(3) of furanocoumarin), 5.80 (3H, s i n g l e t , aromatic OCH^); mass spectrum m/e 216 (M), 201 (M-15), 188 (M-28), 173 and 145. Anal . Calcd. f o r C.„H o0.: C, 66.67; H, 3.73. Found: C, 66.57; LZ o 4 H, 3.80. 6-Formyl-7-hydroxy-5-methoxy coumarin (114) Bergapten (10b) (24 mg; 0.111 mmole) was d i s s o l v e d i n g l a c i a l a c e t i c a c i d (4 ml) and 30 ml of ozone s a t u r a t e d g l a c i a l a c e t i c a c i d was added and the mixture was s t i r r e d f o r one hour. Zinc dust (50 mg) was added and s t i r r i n g continued f o r another 10 minutes. S o l u t i o n was f i l t e r e d aid the so l v e n t was evaporated in_ vacuo. The res i d u e (50 mg) - 65 -was d i s s o l v e d i n chloroform-methanol mixture and was chromatographed on s i l i c a g e l (6 g ) . E l u t i o n w i t h benzene and benzene-chloroform mixture gave the d e s i r e d p h e n o l i c aldehyde (114) (14 mg) which was c r y s t a l l i z e d from acetone to give pure 6-formyl-7-hydroxy-5-methoxy coumarin (114) (8 mg; 33% y i e l d ) , mp 220-221° ( l i t . mp 222-223°); i r (KBr) 1742, 1592 (a-pyrone), 1697 (aldehyde C=0); uv X M e ° H (e) 205.5 (5,870), 225 (sh) (2,690), 266 (19,900), 312 TT13X Me OH (3,180), 340 (sh) (1,345); uv X (e) (+Na0H) 206 (18,850), 238 max (13,700), 262 (8,940), 285 (sh) (4,160), 347 (8,800), 394 (9,800); Me OH uv A (e) (+HC1) 207 (11,370), 222 (sh) (5,740), 267 (15,400), UlclX 314 (5,630); nmr (100 MHz) i n CDC1 3, TMS l o c k , -1.96 (1H, s i n g l e t , disappears on a d d i t i o n of D^O, p h e n o l i c OE), -0.23 (1H, s i n g l e t , aromatic CHO), 2.14 (1H, doublet of doublets, J = 9.75 Hz and 0.6 Hz, H(4) of coumarin), 3.35 (1H, m u l t i p l e t , H(8) of coumarin), 3.72 (1H, doublet, J = 9.75 Hz, H(3) of coumarin), 5.95 (3H, s i n g l e t , aromatic 0CH_3); mass spectrum m/e 220(M), 202(M-18), 191 (M-29), 174, 146 (base peak). Anal. Calcd. f o r C^HgO^ C, 60.00; H, 3.64. Found: C, 60.06; H, 3.71. High r e s o l u t i o n molecular weight determination. Calcd. f o r C11 H8°5 : 2 2 0 ' 0 3 7 * F° u nd: 220.036. - 66 -DISCUSSION (PART II) Biosynthetic Studies on Coumarins from Thamnosma montana Tissue Cultures The Introduction l i s t e d many questions on the biosynthesis of coumarins that remain to be answered. Of p a r t i c u l a r i n t e r e s t to us i s the r o l e of mevalonic acid i n the formation of the furan r i n g of furanocoumarins and the o r i g i n of the C^-units found i n many coumarins. 49 Floss and Mothes have shown that C-4 of mevalonic a c i d i s incorporated s p e c i f i c a l l y i n t o the 7-position of p i m p i n e l l i n (76). However reports 48 52 14 of Caporale et a l . and of Brown that mevalonic a c i d - [ 2 - C] 14 incorporates as e f f i c i e n t l y as [5- C]-mevalonic a c i d i n t o simple furanocoumarins reopens the question as to the true r o l e of mevalonic acid i n the biosynthesis of these furanocoumarins. Recent work i n our l a b o r a t o r y 7 on young Thamnosma montana plants supports 49 the r e s u l t s of Floss and Mothes but the very low incorporation of mevalonic acid makes the s i g n i f i c a n c e of these observations marginal. Thus the main object of t h i s work was to more c l o s e l y define, through s p e c i f i c degradations, the r o l e of mevalonic acid i n the biosynthesi.; of furanocoumarins of Thamnosma montana tis s u e cultures. Plant tissue culture systems have often been used to study many fundamental problems of plant cytology and physiology. Since the e a r l y p u b l i c a t i o n s by Tuleck^^ and N i c k e l l ? ^ there has been considerable i n t e r e s t - 67 -in the possibility of using plant tissue cultures for secondary product biosynthesis. It has long been known that tobacco root cultures can 71 72 biosynthesize the alkaloids nicotine and anabasine. Routien and 73 Nickell have reported plant suspension cultures to produce coumarin 74 and melilotic acid from sweet clover. Similarly, Steck and his group have isolated various coumarins and alkaloids from the cell cultures of Ruta Graveolens and Ourisson et al. 7~* have studied the biosynthesis of the coumarin, scopoletin (55) from tobacco tissue cultures. Frequently higher levels of incorporation of precursors of certain metabolites can be achieved in plant tissue cultures than in normally grown plants or in stem-and-leaf or in root cuttings from plants. Since tissue cultures can be manipulated to minimize woody or conductive tissue which may be of low metabolic activity, they often contain a much higher percentage of actively metabolizing cells than normal plants do. Since aggregation of cells in tissue cultures can be divided before they get too large, the distance from the exterior to the inner-most cell is so much less than in the normally grown plant that the problem of transporting precursors to a l l the cells in the tissue cultures is much easier than for the normal plants. The i n i t i a l requirement for preparing tissue cultures from green plants is to eliminate microorganisms from the system which is being set up for the purpose. Microorganisms may be aggressive competitors of green-plant tissue on the nutrient media used and in any event, their biochemical activities might interfere with, or be confused with, those of seed plants being studied. To obtain plant cultures free of microorganisms, the procedure generally followed was to surface-sterilize seeds, germinate them on sterile water-agar in - 68 -P e t r i p l a t e s and then use a s e p t i c techniques to t r a n s f e r s e e d l i n g s that appear to be f r e e of f u n g i and b a c t e r i a to the appropriate s t e r i l e media. N u t r i e n t requirements i n c u l t u r e media vary w i t h the k i n d of p l a n t and the purpose f o r which the c u l t u r e i s prepared. Ourisson and h i s group^ 6 have found c a l l u s t i s s u e to produce coumarins normal to the plant being c u l t u r e d and t h i s has been our experience w i t h the c a l l u s t i s s u e of Thamnosma montana. Under the circumstances, a gene r a l purpose l i q u i d medium (a modified White's n u t r i e n t s o l u t i o n ) has produced t i s s u e c u l t u r e s adequate f o r our purpose. For s o l i d media, 1% agar i s added. Before adopting an experimental design w i t h t i s s u e c u l t u r e s r o r b i o s y n t h e t i c s t u d i e s , a p r e l i m i n a r y f e a s i b i l i t y study was conducted. This was done by determining the major coumarins of the Thamnosma montana t i s s u e c u l t u r e s , confirming r e g u l a r and measurable b i o s y n t h e s i s of these c o n s t i t u e n t s , and f i n a l l y , p o s t u l a t i n g 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 between the coumarins of Thamnosma montana t i s s u e c u l t u r e s . P r e l i m i n a r y s t u d i e s on the c o n s t i t u e n t s of t i s s u e c u l t u r e s of Thamnosma montana revealed the presence of i s o i m p e r a t o r i n (13), a l l o -i m p e r a t o r i n methyl ether (7) and i s o p i m p i n e l l i n (2) i n i s o l a b l e q u a n t i t i e s whereas no u m b e l l i p r e n i n (9) could be detected. Presence of s m a l l amounts of thamnosmin (15) was a l s o i n d i c a t e d but no attempt was made to i s o l a t e t h i s or to i d e n t i f y other c o n s t i t u e n t s of the t i s s u e c u l t u r e e x t r a c t . I t was considered that the i s o l a t i o n of i s o i m p e r a t o r i n (13), a l l o i m p e r a t o r i n methyl ether (7) and i s o p i m p i n e l l i n (2) would o f f e r an opportunity to study the b i o s y n t h e s i s of these three d i f f e r e n t types of furanocoumarins. - 69 -In order to determine i f the b i o s y n t h e s i s of these furanocoumarins 14 was o c c u r r i n g on a r e g u l a r b a s i s , D,L-phenylalanine-[3- C] was fed to the 5 week o l d t i s s u e c u l t u r e s . Three experiments were s e t up f o r d i f f e r e n t time i n t e r v a l s andin two of these experiment, an Erlenmeyer f l a s k was used c o n t a i n i n g l i q u i d growth medium, c u l t u r e s and r a d i o a c t i v e p r e c u r s o r and the f l a s k was put on a r o t a r y shaker f o r i n c u b a t i o n . In the t h i r d experiment, the c u l t u r e s were t r a n s f e r r e d onto P e t r i p l a t e s c o n t a i n i n g normal growth medium w i t h 1% agar added and the r a d i o a c t i v e precursor was a p p l i e d on the surface of the c u l t u r e s . A f t e r the p r e s e l e c t e d time p e r i o d , t i s s u e c u l t u r e s were f r e e z e - d r i e d and the major c o n s t i t u e n t , i s o p i m p i n e l l i n ( 2 ) , was i s o l a t e d i n each case and was c r y s t a l l i z e d to constant r a d i o a c t i v i t y . The r e s u l t s are given i n Table 2. 14 a TABLE 2. I n c o r p o r a t i o n of D,L-phenylalanine-[3- C] i n t o coumarins of Thamnosma montana t i s s u e c u l t u r e s . E x p e r i -ment no. Feeding time (hours) A c t i v i t y fed Dry weight (dpm) of t i s s u e c u l t u r e s (g) 8 10.93xl0 6 1.20 0.029 48 11.30x10 6 1.38 0.71 72 11.00x10 1.30 0.48 a b c Precu r s o r administered i n water as sodium s a l t . P recursor fed i n l i q u i d growth medium. Precursor a p p l i e d on the surface of the c u l t u r e s i n s o l i d medium. - 70 -These r e s u l t s i n d i c a t e that i s o p i m p i n e l l i n (2) i s being b i o s y n t h e s i z e d by 5 week o l d t i s s u e c u l t u r e s and that the optimum time p e r i o d f o r the b i o s y n t h e s i s of i s o p i m p i n e l l i n i s 2 days. To gain some i n f o r m a t i o n as to the b i o s y n t h e t i c i n t e r r e l a t i o n s h i p between these various furanocoumarins of Thamnosma montana t i s s u e c u l t u r e s and to study the r o l e of mevalonate i n t h e i r b i o s y n t h e s i s , s i x f eeding experiments were s e t up f o r d i f f e r e n t time i n t e r v a l s 3 usin g D,L-mevalonic a c i d - [ 5 - H] as the r a d i o a c t i v e p r e c u r s o r . A f t e r the d e s i r e d feeding time, each experiment was worked up and is o i m p e r a -t o r i n (13), a l l o i m p e r a t o r i n methyl ether (7) and i s o p i m p i n e l l i n (2) were i s o l a t e d by the d i l u t i o n technique ( i . e . the t i s s u e c u l t u r e e x t r a c t was d i l u t e d w i t h n o n - r a d i o a c t i v e coumarins before column chromatography). I s o i m p e r a t o r i n (13) and i s o p i m p i n e l l i n (2) were c r y s t a l l i z e d to constant a c t i v i t y and t h e i r r a d i o a c t i v i t y determined by the s c i n t i l l a t i o n counting method. A l l o i m p e r a t o r i n methyl ether (7) was converted to i t s d i o l (3) before counting. The r e s u l t s are given i n Table 3 and are represented g r a p h i c a l l y i n Figure 17. I t should be noted t h a t i n each of these experiments, 5-week o l d t i s s u e c u l t u r e s were used and the r a d i o a c t i v e precursor was mixed w i t h the t i s s u e -c u l t u r e s i n s t e r i l e d i s t i l l e d water. Only i n experiment no. 4, normal l i q u i d growth medium was used i n s t e a d of d i s t i l l e d water. I t i s apparent from these r e s u l t s that the furanocoumarins i s o l a t e d 3 i n c o r p o r a t e D,L-mevalonic a c i d - [ 5 - H] at d i f f e r e n t time i n t e r v a l s . Thus i s o p i m p i n e l l i n (2) reaches a maximum i n c o r p o r a t i o n a f t e r 2 days and then f a l l s o f f r a p i d l y reaching a minimum i n 7 days. The r i s e between 7 and 10 days i s questionable. A l l o i m p e r a t o r i n methyl ether (7) TABLE 3. Incorporation of D,L-mevaIonic acid-[5- H] into tissue cultures of Thamnosma montana. Experi-ment no. Feeding time (days) Activity fed (dpm) Dry weight of tissue cultures (g) % Incorporation isoimperatorin (13) a. alloimperatorin methyl ether (7) isopimpinellin (2) 4b 2 l.llxlO 9 2.54 — 0.000040 0.0010 5 C 2 l.llxlO 9 3.00 — 0.000045 0.00164 6 C 4 l.llxlO 9 1.45 0.00182 0.00165 0.00015 7C 7 8.0 xlO 8 2.50 0.0025 0.00055 0.000114 8C 7 l.llxlO 9 3.50 0.0021 0.00011 0.00017 9° 10 8.0 xlO 8 3.00 0.00113 0.00036 0.00076 All compounds were isolated by dilution technique, Alloimperatorin methyl ether (7) was converted to its corresponding diol (3) before counting. Precursor mixed with tissue cultures in liquid'growth medium. Precursor mixedvith tissue cultures in distilled water. 0 2 4 6 8 10 days 2 Figure 17. Incorporation of D,L-mevalonic acid-[5- H] into coumarins of Thamnosma nontana tissue cultures versus time. - 73 -reaches maximum incorporation after 4 days whereas isoimperatorin (13) has a maximum value after 7 days. These results indicate that 3 mevalonic acid-[5- H] is indeed being incorporated into a l l three furanocoumarins studied and also the level of incorporation is about five to ten times higher than that achieved in our laboratory'' with young Thamnosma montana plants. To determine the location of radioactivity, isopimpinellin (2) from these various feeding experiments was degraded according to the scheme previously described and as illustrated in Figure 18. The distribution of label determined as a result of these degradations is listed in Table 4. OCH 101 97 Figure 18. Degradation of radioactive isopimpinellin ( 2). - 74 -TABLE 4. D i s t r i b u t i o n of r a d i o a c t i v i t y i n i s o p i m p i n e l l i n (2) from P , L - m e v a l o n i c a c i d - [ 5 - H] i n c o r p o r a t i o n e x p e r i m e n t s . E x p e r i - S p e c i f i c i c t i v i t y of the compounds i s o l a t e d (dpm/mmole) ment no. . . . ,,. a ._ i s o p i m p i n e l l i n 95 97 101 (2) 4 2.34xl0 4 2.25xl0 4 1.025xl0 4 (100%) (96.5%) (44%) 5 2.42xl0 4 2 . 5 1 x l 0 4 1.067xl0 4 1 . 1 7 x l 0 4 dpm/3 mmole) (100%) (103%) (44%) (48%) 9 1.722xl0 4 3 4.18x10 dpm/2 mmole (100%) (24.3%) The t o t a l a c t i v i t y i n i s o p i m p i n e l l i n (2) i s set at 100%. From these r e s u l t s , i t i s evident that there i s no l o s s of a c t i v i t y i n the 7 - p o s i t i o n of i s o p i m p i n e l l i n (2) and that the 6 - p o s i t i o n contains approximately 56% of the t o t a l r a d i o a c t i v i t y of i s o p i m p i n e l l i n ( 2 ). 3 This data i n d i c a t e s that mevalonic-[5- H] i s being i n c o r p o r a t e d s p e c i f i c a l l y i n t o the 6 - p o s i t i o n of i s o p i m p i n e l l i n (2) as would be expected i f Seshadri's h y p o t h e s i s 4 ^ f o r the furanocoumarin b i o s y n t h e s i s ( i . e . the C-4 and C-5 p o s i t i o n s of mevalonic a c i d (85) serve as the pre c u r s o r of C-7 and C-6 of furanocoumarin) i s f o l l o w e d . Somewhat s u r p r i s i n g l y , the remaining r a d i o a c t i v i t y i s found 7 e s s e n t i a l l y i n the methoxyl groups (between 24-44%) of i s o p i m p i n e l l i n ( 2 ). I t i s d i f f i c u l t to e x p l a i n these observed r e s u l t s on the b a s i s of invoked t h e o r i e s about the metabolism of mevalonic a c i d (85). Mevalonic a c i d i s not considered to be an important source of the "C^-pool" i n p l a n t systems. One p o s s i b l e e x p l a n a t i o n could be that there i s a t r i t i u m - 75 -exchange between the tritiated mevalonic acid and the biological medium in the system being studied. To determine the distribution of radioactivity in alloimperatorin methyl ether (7), i t was converted to its diol (3). The diol (3) was then acetylated to the monoacetate (107) which was degraded according to the scheme already dscribed and as illustrated in Figure 19. The results are given in Table .5. - 76 -110a; R=H 110b; R=COCH Figure 19. Degradation of r a d i o a c t i v e a l l o i m p e r a t o r i n methyl ether (7). TABLE 5. D i s t r i b u t i o n of r a d i o a c t i v i t y i n al l o i m p e r a t o r i n methyl ether 3 from D,L-mevalonic acid-[5- H] incorporation experiments. Experi- S p e c i f i c a c t i v i t y of the compounds i s o l a t e d (dpm/mmole) ment no. 107 a 109 110b 101 6 6.63xl0 3 (100%) 6.5xl0 3 (100%) 4.28xl0 3 (68%) 6 7.12xl0 3 (100%) 6.8xl0 3 (95.5%) 5.0xl0 3 (70%) 7 1.944xl0 4 (100%) — — 6.06xl0 2 (3%) The t o t a l a c t i v i t y of monoacetate (107) i s set at 100%. - 77 -These results again indicate that there is no loss of activity in the 7-position of alloimperatorin methyl ether (7) and that the 6-position contains between 30-32% of the total activity. The loss of activity in the 6-position is in accord with the proposed hypothesis of furan ring formation in these furanocoumarins. Since alloimperatorin methyl ether also contains a dimethylallyl side chain and a methoxyl group, the remaining activity (68-70%) would be expected in the methoxyl group and/or in the alkyl side chain. However, the demethylation of 107 gave very l i t t l e activity in the methoxyl group (<v 3%). Since the coumarin portion of these furanocoumarins has been shown to be 49 cinnamic acid derived, the remaining activity (^  65%) in 7 must be present in the alkyl side chain. Thus i t is evident that mevalonic 3 acid-[5- H] is acting as a specific precursor of the 6-position and the alkyl side chains of alloimperatorin methyl ether (7). To determine the distribution of label in isoimperatorin (13), i t was converted to bergapten (10b) as described previously. Thus isoimperatorin (13) (1.728x10"* dpm/mmole) from experiment 6 was degraded and bergapten (10b) (1.77x10^ dpm/mmole) was found to contain 10.3% of the total activity of 13. In a similar experiment, isoimpera-torin (13) (1.458x10"* dpm/mmole) from experiment 8 was converted to bergapten (10b) and i t was found to have a specific activity of 2.268x10 dpm/mmole or 15.5% of the total activity of 13. Therefore, i t is evident that 85-90% of the total activity of 13 resides in the alkyl-ether side chain and that only 10-15% of the radioactivity is in the rest of the furanocoumarin molecule. Since isoimperatorin (13) contains 3 a furan ring and as i t has already been shown that mevalonic acid-[5- H] - 78 -incorporates into the 6-position of the furanocoumarins, the residual (10-15%) of activity in bergapten (10b) will be expected to reside in the 6-position of isoimperatorin (13). However, due to lack of cold material, this degradation could not be pursued further. 3 These results show that mevalonic acid-[5- H] is indeed a specific precursor of the furan ring and the alkyl and alkyl-ether side chains in furanocoumarins. 3 In order to establish the role of mevalonic acid-[4- H] in the biosynthesis of furanocoumarins, two feeding experiments were set up for a period of 2 and 4 days and the results are listed in Table 6. 3 Thus mevalonic acid-[4- H] is also incorporated into a l l three furanocoumarins and the level of incorporation is again 5 to 10 times higher than that achieved in young Thamnosma montana plants.^ Again to determine the location of radioactivity in various furano-coumarins isolated, isopimpinellin (2) (1.96x10^ dpm/mmole) from experiment 10 was degraded to 6-acetoxymethyl-7-acetoxy-5,8-dimethoxy-coumarin (103b) according to the scheme previously described and 103b was found to be completely inactive. In a similar experiment, 3 isopimpinellin (2) (8.4x10 dpm/mmole) from experiment 11 was selectively ozonized to 6-formyl-7-hydroxy-5,8-dimethoxycoumarin (95) which was shown to lack any measurable amount of radioactivity. Thus i t is 3 evident that mevalonic acid-[4- H] is being specifically incorporated into the 7-position of isopimpinellin (2). It is necessary to note that no radioactivity could be found in the methoxyl groups of 3 isopimpinellin (2) as was the case in the mevalonic acid-[5- H] experiments. TABLE 6. Incorporation of D,L-mevalonic acid-[4- H] into the tissue cultures of Thamnosma montana. Experi- Feeding Activity Dry weight % Incorporation ment no. time (days) fed (dpm) of tissue cultures (g) isoimperatorin (13) alloimperatorin methyl ether (7) isopimpinellin (2) 10 2 5.50xl08 2.1 0.0057 0.0015 0.001 11 4 5.50xl08 1.7 0.0124 0.00107 0.00117 - 80 -Similarly, alloimperatorin methyl ether diol (3) from experiment 10 was converted to its monoacetate (107). The monoacetate (107) 4 (1.60x10 dpm/mmole) was then selectively ozonized to the corresponding phenolic aldehyde, 5-(2'-acetoxy-31-hydroxy-3'-methylbuty1)-6-formyl-7-hydroxy-8-methoxycoumarin (108). The phenolic aldehyde (108) 3 (7.11x10 dpm/mmole) was found to contain 42% of the radioactivity 4 of 107. In a similar experiment, the monoacetate (107) (1.44x10 dpm/mmole) from experiment 11 was converted to the corresponding phenolic 3 aldehyde (108) (5.76x10 dpm/mmole) which was shown to have 40% of the original activity of 107. It is clear from these results that about 58% of the activity of 3 7 resides in the 7-position as would be expected i f mevalonic acid-[4- H] was acting as a specific precursor of the furan ring. Since there is no activity found in the methoxyl groups of isopimpinellin (2) from experiments 10 and 11 and since the coumarin portion of furanocoumarins 49 has been shown to be cinnamic acid derived, the remaining activity (40-42%) in 7 must reside in the alkyl side chain which would be expected to be mevalonic acid derived. Location of radioactivity in isoimperatorin (13) (3.46x10"* dpm/mmole) from experiment 10 was determined by converting 13 to bergapten (10b) 4 and bergapten (8.64x10 dpm/mmole) was found to contain 25% of the total activity of 13. Similarly isoimperatorin (13) (5.65x10"* dpm/mmole) from experiment 11 was degraded to bergapten (10b) and the latter 4 (3.54x10 dpm/mmole) was found to contain 6.1% of the activity of isoimperatorin. Thus i t is apparent that between 75-94% of the activity in isoimperatorin is located in the C,.-alkyl-ether side chain and only - 81 -6-25% of the a c t i v i t y i s present i n the furanocoumarin molecule. In order to determine the l o c a t i o n of r a d i o a c t i v i t y i n the furanocoumarin 4 p o r t i o n of 13, bergapten (10b) (1.296x10 dpm/mmole) from experiments 10 and 11 was combined and ozonized s e l e c t i v e l y to 6-formyl-7-hydroxy-5-methoxycoumarin (114) and 114 was shown to be completely i n a c t i v e . Thus a l l the remaining a c t i v i t y (6-25%) i n i s o i m p e r a t o r i n (13) r e s i d e s i n the 7 - p o s i t i o n of 13. Thus the r e s u l t s of experiments 10 and 11 c l e a r l y i n d i c a t e that 3 mevalonic a c i d - [ 4 - H] i s a c t i n g as a s p e c i f i c p r e c u r s o r of the fur a n r i n g and the a l k y l s i d e chains of furanocoumarins. These r e s u l t s are 49 i n complete agreement w i t h the r e s u l t s of F l o s s and Mothes that 3 mevalonic a c i d - [ 4 - H] i s a s p e c i f i c p r e c u r s o r of the 7 - p o s i t i o n of the furan r i n g of furanocoumarins. F i n a l l y to determine the r o l e of mevalonic a c i d - [ 2 - H] i n the b i o s y n t h e s i s of furanocoumarins, two feeding experiments f o r a time p e r i o d of 2 and 4 days were set up and three furanocoumarins were i s o l a t e d . The r e s u l t s are given i n Table 7. 3 Thus mevalonic a c i d - [ 2 - H] i s i n c o r p o r a t e d i n t o a l l three furano-coumarins. However, the l e v e l of i n c o r p o r a t i o n i s much lower than i n 3 3 the cases of [4- H]- or [5- H]-mevalonic a c i d . A l s o , the i n c o r p o r a t i o n TABLE 7. Incorporation of D,L-mevalonic acid-[2- H] into the tissue cultures of Thamnosma montana. Experi- Feeding Activity Dry weight % Incorporation  ment no. time fed of tissue . j ^ , n j ^ j .» j - I U / I N . . . / , \ -, isoimperatorin (13) alloimperatorin isopimpinellin (2) (days) (dpm) cultures r , n> ^ v i ' methyl ether (7) (g) 12 2 l.llxlO 9 1.70 0.00083 0.00012 0.0002 13 4 l.llxlO 9 1.69 0.0054 0.00038 0.00036 - 83 -3 of mevalonic a c i d - [ 2 - H] i n t o i s o p i m p i n e l l i n ( 2 ) , a simple furano-coumarin, i s hard to r e c o n c i l e w i t h the previous r e s u l t s . Therefore to determine the l o c a t i o n of r a d i o a c t i v i t y , i s o p i m p i n e l l i n (2) 3 (9.0x10 dpm/mmole) from experiment 12 was demethylated and t e t r a m e t h y l -3 ammonium i o d i d e (101) (9.05x10 dpm/2 mmole) was found to c o n t a i n a l l the r a d i o a c t i v i t y of 2. In a s i m i l a r experiment, i s o p i m p i n e l l i n (2) (4.182 x l 0 4 dpm/mmole) from experiment 13 was demethylated and t e t r a -4 methylammonium i o d i d e (101) (4.0x10 dpm/2 mmole) was found to c o n t a i n a l l the r a d i o a c t i v i t y of i s o p i m p i n e l l i n ( 2 ) . Thus i t i s evident that a l l the r a d i o a c t i v i t y i n i s o p i m p i n e l l i n (2) r e s i d e s i n the methoxyl groups and no a c t i v i t y i s present i n the r e s t of the furanocoumarin molecule. To determine the l o c a t i o n of r a d i o a c t i v i t y i n a l l o i m p e r a t o r i n 3 methyl ether ( 7 ) , a l l o i m p e r a t o r i n methyl ether d i o l (3) (6.36x10 dpm/mmole) from experiment 12 was cleaved w i t h p e r i o d i c a c i d and the 3 a l c o h o l (105) (1.30x10 dpm/mmole) was found to co n t a i n 20% of the 3 o r i g i n a l a c t i v i t y of 3. In a s i m i l a r experiment, d i o l (3) (7.63x10 dpm/mmole) from experiment 13 was converted to a l c o h o l (105) which was shown to l a c k any measurable amount of a c t i v i t y . Thus i t i s evident that between 80-100% of the r a d i o a c t i v i t y i n a l l o i m p e r a t o r i n methyl ether (7) r e s i d e s i n the t e r m i n a l three carbon atoms of the d i m e t h y l a l l y l 3 s i d e c h a i n , i n d i c a t i n g that mevalonic a c i d - [ 2 - H] i s a c t i n g as a s p e c i f i c precursor of t h i s s i d e chain. Since the furanocoumarin p o r t i o n of a l l o i m p e r a t o r i n methyl ether (7) and of i s o p i m p i n e l l i n (2) w i l l be expected to be b i o s y n t h e s i z e d i n a s i m i l a r manner, any r e s i d u a l amount (0-20%) of r a d i o a c t i v i t y i n 7 w i l l be expected to r e s i d e i n the - 84 -methoxyl group of a l l o i m p e r a t o r i n methyl ether (7) as has been shown to be the case i n i s o p i m p i n e l l i n ( 2 ) . Since i s o i m p e r a t o r i n (13) i s a simple furanocoumarin w i t h an a l k y l - e t h e r s i d e chain and no methoxyl group, a l l the a c t i v i t y i n 13 would be expected to r e s i d e i n t h i s C,. s i d e chain. Therefore, i s o i m p e r a t o r i n (13) (1.08x10^ dpm/mmole) from experiment 12 was 3 hydrolyzed and bergapten (10b) (5.85x10 dpm/mmole) was shown to have about 5% of the t o t a l a c t i v i t y of 13. In a s i m i l a r experiment, i s o i m p e r a t o r i n (8.1x10^ dpm/mmole) from experiment 13 was degraded to bergapten (10b) (1.728x10 dpm/mmole) and t h i s was shown to have about 2.7% of the t o t a l r a d i o a c t i v i t y of 13. Thus i t i s apparent from these r e s u l t s t h a t between 95-97% of the r a d i o a c t i v i t y of isoimpera-t o r i n (13) r e s i d e s i n the C ^ - a l k y l - e t h e r s i d e chain i n d i c a t i n g that nevalonic 3 a c i d - [ 2 - Hj i s a c t i n g as a s p e c i f i c precursor of t h i s s i d e chain. F i n a l l y to determine i f mevalonic a c i d i s being degraded to the C^-pool and i s thus being i n c o r p o r a t e d i n t o the methoxyl groups of furanocoumarins or i f there i s some s o r t of t r i t i u m exchange between the t r i t i a t e d mevalonic a c i d and the C^-pool i n the t i s s u e c u l t u r e 14 system, mevalonic a c i d - [ 5 - C] was fed to the 5-week o l d t i s s u e c u l t u r e s over a 2 day p e r i o d and i s o p i m p i n e l l i n (2) was i s o l a t e d . The r e s u l t s are given i n Table 8. 14 TABLE 8. I n c o r p o r a t i o n of D,L-mevalonic a c i d - [ 5 - C] i n t o t i s s u e c u l t u r e s of Thamnosma montana E x p e r i -ment no. Feeding time (days) A c t i v i t y fed (dpm) Dry weight of t i s s u e c u l t u r e s (g) % I n c o r p o r a t i o n i s o p i m p i n e l l i n (2) 14 2 1.11x10 9 3.33 0.002 - 85 -To determine the d i s t r i b u t i o n of r a d i o a c t i v i t y , i s o p i m p i n e l l i n 4 (2) (5.00x10 dpm/mmole) from experiment 14 was degraded according to 3 the scheme i n Figure 18 and phenol (97) (2.75x10 dpm/mmole) was shown to c o n t a i n only 5.5% of the r a d i o a c t i v i t y of 2. This c l e a r l y i n d i c a t e s t h a t about 95% of the r a d i o a c t i v i t y i n i s o p i m p i n e l l i n ( 2 ) r e s i d e s i n the 6 - p o s i t i o n . This i s i n c o n t r a s t to only 56% of the a c t i v i t y 3 present i n the 6 - p o s i t i o n i n the mevalonic a c i d - [ 5 - H] fe e d i n g experiments. Thus i f mevalonic a c i d was being degraded to the C^-pool and thus a c t i n g as a precursor of the methoxyl groups of i s o p i m p i n e l l i n ( 2 ) , an e q u i v a l e n t amount of a c t i v i t y would be expected i n the methoxyl 3 14 groups of i s o p i m p i n e l l i n i n both [5- H] and [5- C]-mevalonic a c i d 14 feedings. Since t h i s i s not the case and i n the mevalonic a c i d - [ 5 - C] experiment, about 95% of the r a d i o a c t i v i t y i s present i n the 6 - p o s i t i o n of i s o p i m p i n e l l i n ( 2 ) , i t i s c l e a r l y evident that mevalonic a c i d i s not being degraded to the C^-pool and thus i s not a pr e c u r s o r of the methoxyl groups. Any a c t i v i t y found i n the methoxyl groups of furano-3 3 coumarins i n the [5- H]- and [2- H]-mevalonic a c i d feedings must come from e i t h e r a t r i t i u m exchange between the t r i t i a t e d mevalonic a c i d and the C^-pool i n the system or by some other unknown mechanism. The r e s u l t s of experiments 4-14 are i n complete agreement w i t h . 49 47 the r e s u l t s of F l o s s and Mothes and thus support Seshadri's proposal f o r furanocoumarin b i o s y n t h e s i s . However, these r e s u l t s are not i n agreement w i t h the r e s u l t s of Brown^ 2 and of Caparole et a l . 4 8 These workers have i n d i c a t e d that the i n c o r p o r a t i o n of mevalonic a c i d i n t o furanocoumarins was n o n s p e c i f i c . However, our r e s u l t s c l e a r l y i n d i c a t e - 86 -that mevalonic acid is acting as a specific precursor of the furan ring and the alkyl groups of furanocoumarins in the tissue cultures of 52 Thamnosma montana. Since in the investigations of Brown and 48 Caparole et al. no degradations to determine the distribution of radio-activity were performed, the significance of their results is questionable. - 87 -EXPERIMENTAL (PART I I ) For general experimental i n f o r m a t i o n see page 59. R a d i o a c t i v i t y was measured w i t h a Nuclear Chicago Mark I or Mark I I L i q u i d S c i n t i l l a t i o n counter i n counts per minute (cpm). The r a d i o a c t i v i t y of the sample i n d i s i n t e g r a t i o n per minute (dpm) was subsequently c a l c u l a t e d using the counting e f f i c i e n c y which was determined f o r each sample by the e x t e r n a l standard t e c h n i q u e 7 7 u t i l i z i n g the b u i l t - i n barium-133 gamma source. The organic s c i n t i l l a t o r s o l u t i o n used w i t h the counter was made up of the f o l l o w i n g components: toluene (1 2,) » 2,5-diphenyloxazole (4 g) and 1 , 4 - b i s [ 2 - ( 5 - p h e n y l o x a z o l y l ) ] -benzene (0.05 g). In p r a c t i c e , a sample was d i s s o l v e d i n benzene (lml) or i n methanol (1 ml) i f the compound was not s u f f i c i e n t l y s o l u b l e i n benzene, i n a counting v i a l . The volume was then made up to 15 ml w i t h the above s c i n t i l l a t o r s o l u t i o n . In case of water s o l u b l e counting samples, an aqueous s c i n t i l l a t o r s o l u t i o n was u t i l i z e d made up of the f o l l o w i n g components: toluene (385 m l ) , dioxane (385 m l ) , methanol (230 m l ) , naphthalene (80 g ) , 2,5-diphenyloxazole (5 g) and 1,4- b i s [ 2 - ( 5 - p h e n y l o x a z o l y l ) ] benzene (0.0625 g) . In p r a c t i c e , a sample was d i s s o l v e d i n water (as required) and methanol (1 ml) i n the counting v i a l . The s o l u t i o n was made up to 15 ml w i t h the aqueous s c i n t i l l a t o r s o l u t i o n . For each sample counted, the background was - 88 -determined f o r the counting v i a l to be used by f i l l i n g the v i a l w i t h the a p p r o p r i a t e s o l v e n t and s c i n t i l l a t o r s o l u t i o n and counting (3 x 40 min; 3 x 100 min; 2 x 100 min). The d i f f e r e n c e i n the cpm between the background count and the sample count was used f o r subsequent c a l c u l a t i o n s . Unless otherwise noted, r a d i o a c t i v i t y was determined by s c i n t i l l a t i o n counting w i t h organic s c i n t i l l a t o r s o l u t i o n . D e v i a t i o n from these normal counting procedures w i l l be discussed i n the s p e c i f i c i n s tances i n which they a r i s e . C u l t i v a t i o n of Tissue C u l t u r e s of Thamnosma montana A l l c u l t u r i n g was done at room temperature (24°C). Seeds were r i n s e d w i t h 70% e t h a n o l , soaked f o r approximately 2 minutes i n 0.1% HgC^, r i n s e d w i t h s t e r i l e water and were pl a n t e d on water-agar. Microorganism-free s e e d l i n g s were cut i n t o 3 or 4 pieces and t r a n s f e r r e d to the su r f a c e of the modif i e d White's agar medium i n Erlenmeyer f l a s k s . The s e e d l i n g pieces were incubated u n t i l c a l l u s t i s s u e was observed at the cut ends. Then the fragments of c a l l u s t i s s u e were t r a n s f e r r e d to l i q u i d medium, 100 ml per 250 ml Erlenmeyer f l a s k , which was placed on a r o t a r y shaker (about 120 rpm and 30 mm r a d i u s ) . Whenever t i s s u e was t r a n s f e r r e d to f r e s h media, the pi e c e s were cut up i n t o s m a l l e r p i e c e s . For n u t r i e n t purposes, the f o l l o w i n g general-purpose medium (a mod i f i e d White's n u t r i e n t s o l u t i o n ) was used. For s o l i d medium, 1% agar was added. - 89 -Component mg/£ of distilled water sucrose 20,000 NH.NO- 400 4 3 KC1 65 KN03 80 KH.PO. 12.5 2 4 Ca(N03)2-4H20 144 MgS04.7H20 72 NaFeEDTA 25 H3 B 03 1 , 6 MnSO.-4Ho0 6.5 4 2 ZnS0.-7Ho0 2.7 4 2 KI 0.75 3IAA 2.0 kinetin 0.2 thiamine HCI 10 nicotinic acid 1.0 pyridoxine HCI 1.0 glycine 2.0 myoinositol 100 casein hydrolysate 1,000 Usually the culturing was done in 250 ml flasks. The amount of tissue cultures in a flask varied according to the size and number of pieces originally transferred to the flask. In a typical experiment, five flasks contained 35 g of wet tissue which when air-dried gave 4.7 g. - 90 -When cultures were used for biosynthetic experiments, most of the pieces of wet tissue were 5 mm or less in diameter. The tissue cultures were cultivated by Dr. P. Salisbury of this department. Constituents of tissue cultures of Thamnosma montana Five flasks containing tissue cultures (5 week old) were decanted and the solid residue was put on an aspirator for 30 minutes. The residue was washed with water (2 times) and weighed to give 35 g of wet tissue. This was air-dried at room temperature and the dry residue (4.7 g) was extracted with acetone in a Soxhlet extractor. The acetone extract was evaporated to dryness, the residue dissolved in chloroform, filtered and dried over sodium sulfate. The solvent was removed under reduced pressure to give 100 mg of the residue which was pre-adsorbed on 1 g of alumina (activity IV) and was chromatographed on alumina (10 g, neutral activity III). Preparative layer chromatography gave isopimpinellin (2) (1.5 mg), alloimperatorin methyl ether (7) (0.8 mg) and isoimperatorin (13) (1.6 mg). No umbelliprenin (9) was indicated by tic. Some thamnosmin (15) was present but no attempt was made to isolate this or to identify other components of the tissue culture extract. In subsequent radioactive experiments, tissue cultures were freeze-dried (unless otherwise noted) and the chloroform soluble extract of the tissue cultures was diluted with cold furanocoumarins before column chromatography. In each experiment, 5 week old tissue cultures were utilized for biosynthetic investigation. - 91 -Feeding Experiments 1 and 2 In each of these experiments, two f l a s k s containing t i s s u e cultures were decanted and the s o l i d was transferred to 50 ml of fresh growth 14 medium. D,L-Fhenylalanine-[3- C] (1.2 mg, obtained from New England Nuclear Corp., Boston, Mass.) was dissolved i n d i l u t e sodium bicarbonate s o l u t i o n (2 mg i n 10 ml of d i s t i l l e d water) and an a l i q u o t was removed, weighed and the r a d i o a c t i v i t y per unit weight of s o l u t i o n was determined (11.0 x 10 dpm/g). Two aliquots were then removed from t h i s s o l u t i o n (a. 1 g each) and mixed with the t i s s u e cultures i n the f l a s k s with fresh growth media, and these f l a s k s were placed on a rotary shaker. When the preselected time period was over, the ti s s u e c u l t u r e s , i n c l u d i n g the solutions, were freeze-dried. The dry material was worked up as usual. The chloroform soluble extract was d i l u t e d with cold i s o p i m p i n e l l i n (2) and chromatographed on n e u t r a l alumina ( a c t i v i t y IV). Is o p i m p i n e l l i n was i s o l a t e d by preparative layer chromatography, c r y s t a l l i z e d and counted to constant r a d i o a c t i v i t y . The r e s u l t s and experimental d e t a i l s are presented i n Table 9. Feeding Experiment 3 In t h i s experiment, two f l a s k s containing t i s s u e cultures on a 14 s o l i d growth medium were used and D,L-phenylalanine-[3- C] s o l u t i o n (^  1 g) was applied to the surface of the tissue cultures with the help of a p i p e t t e . The f l a s k s were l e f t under fluorescent i l l u m i n a t i o n for 72 hours a f t e r which the tissue cultures were removed from the s o l i d medium with spatula, freeze-dried and worked up i n a usual manner. Isopimpinellin (2) was i s o l a t e d and the r e s u l t s are given i n Table 9. 14 TABLE 9. Incorporation of D,L-phenylalanine-[3- C] (sodium salt) into tissue cultures of Thamnosma montana Experi- Activity Specific Weight Dry weight Feeding Weight Specific % Incorporation ment fed activity fed of tissue time isolated 3 activity isolated Isopimpinellin no. (dpm) fed (mg) cultures (hr) isopimpinellin isopimpinellin (2) (2) (dpm/mmole) (g) (2) (dpm/mmole) (mg) 1 1.093xl07 1.55xl010 0.12 1.20 8 6.5 l.LSxlO5 0.029 2 1.13xl07 1.55xl010 0.12 1.38 48 7.5 2.62xl06 0.71 3 l.lOxlO 7 1.55xl010 0.12 1.30 72 6.5 1.98xl06 0.48 Isopimpinellin (2) was diluted with inactive material before isolation. - 93 -Feeding Experiments 4, 5, 6, 7, 8, and 9 In each of these experiments, three to four f l a s k s c o n t a i n i n g t i s s u e c u l t u r e s were used and the t i s s u e c u l t u r e s t r a n s f e r r e d i n t o 25 ml of d i s t i l l e d water. Only i n experiment 4, 25 ml of n u t r i e n t 3 s o l u t i o n was used i n s t e a d of d i s t i l l e d water. D,L-Mevalonic a c i d - [ 5 - H] as d i b e n z o y l ethylene diamine (DBED) s a l t (obtained from New England Nuclear Corp.) i n methanol was used as precursor and the f l a s k s were put on r o t a r y shaker f o r a p r e s e l e c t e d time p e r i o d . Tissue c u l t u r e s were f r e e z e - d r i e d and worked up i n the u s u a l manner. In each case, chloroform s o l u b l e e x t r a c t was d i l u t e d w i t h i n a c t i v e i s o p i m p i n e l l i n ( 2 ) , a l l o i m p e r a t o r i n methyl ether (7) and i s o i m p e r a t o r i n (13) before chromatography. The experimental d e t a i l s and the r e s u l t s are given i n Table 10a and 10b. In each case, a l l o i m p e r a t o r i n methyl ether (7) was converted to i t s d i o l (3) before counting. The s p e c i f i c a c t i v i t y and i n c o r p o r a t i o n values quoted f o r a l l o i m p e r a t o r i n methyl ether (7) are c a l c u l a t e d from the ap p r o p r i a t e i n f o r m a t i o n obtained f o r the d i o l ( 3 ) , c o r r e c t i n g f o r the y i e l d i n con v e r t i n g 7 to 3. The coumarins were d i l u t e d w i t h i n a c t i v e m a t e r i a l as necessary to ob t a i n q u a n t i t i e s which could be c r y s t a l l i z e d to constant r a d i o a c t i v i t y and to perform degradations Degradations of I s o p i m p i n e l l i n (2) from Experiment 4 Rad i o a c t i v e 6-formyl-7-hydroxy-5,8-dimethoxycoumarin (95) I s o p i m p i n e l l i n (2) (45 mg, 2.34 x 1 0 4 dpm/mmole) from Experiment 4 7 w a s ^ s e l e c t i v e l y ozonized as described p r e v i o u s l y and 6-formyl-7-hydroxy-5,8-dimethoxycoumarin (95)( (19.5 mg) was i s o l a t e d and shown to have a 4 s p e c i f i c a c t i v i t y of 2.25 x 10 dpm/mmole or 96.5% of the o r i g i n a l a c t i v i t y of i s o p i m p i n e l l i n ( 2). TABLE 10a. Incorporation of D,L-mevalonic acid-[5- H] (DBED salt) in tissue cultures. Experi- Activity Specific Weight Dry weight Feeding Weight of compound isolated (mg) ment no. sfed activity fed fed of tissue time . . -, •, . . j . . , , S / J # i \ / \ -i / J \ isoimpera- alloimperatorin isopimpii (dpm) (dpm/mmole) (mg) cultures (days) t o r l n ( 1 3 ) m e t h y l £ t h £ r ( ? ) ( g ) 4 l.llxlO 9 13.86xl012 0.0214 2.54 2 — 5.0 4.5 5 l.llxlO 9 13.86xl012 0.0214 3.00 2 -- 6.2 9 6 l.llxlO 9 13.86xl012 0.0214 1.45 4 4.9 2.5 7.1 7 8.0xl08 13.86xl012 0.0172 2.50 7 0.8a 5.6 6.4 8 l.ll x l O 9 13.86xl012 0.0214 3.50 7 4.0 3.5 7.5 9 8.0xl08 13.86xl012 0.0172 3.00 10 2.9 5.0 6.8 Isoimperatorin (13) from experiment 7 was not diluted with inactive material before chromatography. TABLE 10b. Experi- Specific activity isolated (dpm/mmole) % Incorporation ment no. isoimperatorin (13) alloimperatorin methyl ether (7) isopimpinellin (2) isoimperatorin (13) alloimperatorin methyl ether (7) isopimpinellin (2) 4 — 2.52xl04 6.08xl05 — 0.000040 0.0010 5 — 2.28xl04 4.98xl05 — 0.000045 0.00164 6 l.ll x l O 6 2.34xl04 5.78xl04 0.00182 0.00165 0.00015 7 9.35xl06 3.49xl05 4.87xl04 0.0025 0.00055 0.000114 8 1.57xl06 9.90xl04 6.20xl04 0.0021 0.00011 0.00017 9 1.17xl06 2.27xl05 3.22xl05 0.00113 0.00036 0.00076 - 96 -Radioactive 6-formyl-5,7,8-trimethoxycoumarin (96) Pure 6-formyl-7-hydroxy-5,8-dimethoxycoumarin (95) and i t s mother liquor from crystallization were methylated separately and the preparative layer chromatography gave combined 6-formyl-5,7,8-trimethoxy-coumarin (6) (23 mg). Radioactive 6-hydroxy-5,7,8-trimethoxycoumarin (97) 4 6-Formy1-5,7,8-trimethoxycoumarin (97) (23 mg, 2.25x10 dpm/mmole) from the previous reaction was degraded to 6-hydroxy-5,7,8-trimethoxy-coumarin (97) (14.5 mg) and 97 was shown to have a specific activity 4 of 1.025x10 dpm/mmole or 44% of the original activity of isopimpinellin (2). Degradations of Isopimpinellin (2) from Experiment 5 Radioactive 6-formyl-7-hydroxy-5,8-dimethoxycoumarin (95) 4 Isopimpinellin (2) (50 mg, 2.42x10 dpm/mmole) from Experiment 5 was converted to 6-formyl-7-hydroxy-5,8-dimethoxycoumarin (95) (24 mg) 4 and this (95) was shown to have a specific activity of 2.51x10 dpm/mmole or a l l the original activity of isopimpinellin (2). Radioactive 6-formyl-5,7, 8-trimethoxycoumarin (96) 6-Formyl-7-hydroxy-5,8-dimethoxycoumarin (95) from previous reaction was methylated as described previously^to give 6-formyl-5,7,8-trimethoxycoumarin (96) (25 mg) by preparative layer chromatography. It was not counted but used as such in the next reaction. - 97 -6-Hydroxy-5,7,8-trimethoxycoumarin (97) Radioactive 6-formyl-5,7,8-trimethoxycoumarin (96) (25 mg) from the above reaction was treated with hydrogen peroxide and sulfuric acid to give 6-hydroxy-5,7,8-trimethoxycoumarin (97) (16.7 mg). This was 4 shown to have a specific activity of 1.067x10 dpm/mmole or 44% of the original activity of isopimpinellin (2). Radioactive tetramethylammonium iodide (101) Radioactive 6-hydroxy-5,7,8-trimethoxycoumarin (97) (10 mg, 4 1.067x10 dpm/mmole) from a previous reaction was demethylated as described previously^and tetramethylammonium iodide (101) (14 mg) was isolated. This was counted by the following method. The salt (^  1.5 mg) was dissolved in aqueous sodium thiosulfate solution (0.1 N, 10 drops) and methanol (1 ml) and the solution was made up to 15 ml with aqueous s c i n t i l l a t o r solution. A blank sample of the same constitution (except that non-radioactive 101 was used) was counted in the same v i a l to determine background. By this method, 101 gave a specific activity 4 of 1.17x10 dpm/mmole or 48% of the original activity of isopimpinellin (2). Radioactive tetramethylammonium iodide from Experiment 9 4 Radioactive isopimpinellin (2) (14.5 mg, 1.722x10 dpm/mmole)1'- from Experiment 9 was demethylated as described previously and tetramethyl-ammonium iodide (101) (10 mg) was isolated. It was counted by dissolving the salt (^  1.5 mg) in sodium thiosulfate solution (0.1 N, 10 drops) and methanol (1 ml) and making the solution up to 15 ml with aqueous - 98 -s c i n t i l l a t o r s o l u t i o n . By t h i s method, 101 gave a s p e c i f i c a c t i v i t y 3 of 4.18x10 dpm/2mmole or 24.3% of the o r i g i n a l a c t i v i t y of i s o p i m p i n e l l i n (2 ) . Tetramethylammonium i o d i d e (101) (7 mg) from the above r e a c t i o n was converted to i t s p i c r a t e ( 1 0 2 ) (6 mg) which was counted as f o l l o w s : p i c r a t e (a. 2 mg) was d i s s o l v e d i n a c e t i c anhydride (5 drops) and a c e t i c a c i d (5 dr o p s ) , z i n c dust (10 mg) was added to d e c o l o r i z e the s o l u t i o n . Sodium m e t a b i s u l f i t e (100 mg) was added and the mixture was then f i l t e r e d d i r e c t l y i n t o the counting v i a l . The o r i g i n a l c o n t a i n e r was washed w i t h methanol (1 ml) and t h i s wash was a l s o f i l t e r e d i n t o the counting v i a l . The s o l u t i o n was made up to 15 ml w i t h organic s c i n t i l l a t o r s o l u t i o n and then counted a f t e r standing at l e a s t 1 hour i n the c o l d and i n the dark. As b e f o r e , an i n a c t i v e sample of 102 was counted i n the same manner to determine the background p r i o r to counting the r a d i o a c t i v e sample. By t h i s method, tetramethylammonium p i c r a t e (102) 3 i n d i c a t e d a s p e c i f i c a c t i v i t y of 4.107x10 dpm/2mmole or 24% of the o r i g i n a l a c t i v i t y of i s o p i m p i n e l l i n ( 2). Degradations of A l l o i m p e r a t o r i n Methyl Ether (7) from Experiment 6 Ra d i o a c t i v e 5-(2'-acetoxy-3 1-hydroxy-3'-methyl butyl)-8-methoxy-i p s o r a l e n (107) Rad i o a c t i v e a l l o i m p e r a t o r i n methyl ether (7) from Experiment 6 was converted to i t s d i o l ( 3). The d i o l (3) was d i l u t e d w i t h n o n - r a d i o a c t i v e m a t e r i a l and was a c e t y l a t e d to 5-(2'-acetoxy-3'-hydroxy-3'-methyl b u t y l ) -8-methoxypsoralen (107). - 99 -T r i a l a Ra d i o a c t i v e 5-(2 '-acetoxy-3'-hydroxy-3'-methyl b u t y l ) - 6 - f o r m y l - 7 - hydroxy-8-methoxy coumarin (108) Rad i o a c t i v e 5-(2'-acetoxy-3'-hydroxy-3'-methyl butyl)-8-methoxy-3 pso r a l e n (107) (42 mg, 6.3x10 dpm/mmole) from Experiment 6 was s e l e c t i v e l y ozonized as p r e v i o u s l y described 7and a f t e r c r y s t a l l i z a t i o n , 5-(2'-acetoxy-3'-hydroxy-3'-methyl butyl)-6-formyl-7-hydroxy-8-methoxy coumarin (108) (30 mg) was i s o l a t e d . This m a t e r i a l (108) was not counted but was used d i r e c t l y i n the next r e a c t i o n . R a d i o a c t i v e 5-(2'-acetoxy-3'-hydroxy-3 1-methyl b u t y l ) - 6 - f o r m y l - 7 , 8 - dimethoxy coumarin (109). 5-(2'-Acetoxy-3'-hydroxy-3'-methylbutyl)-6-formyl-7-hydroxy-8-methoxy coumarin (108) (30 mg) from previous r e a c t i o n was methylated an 109 (25 mg) was i s o l a t e d and shown to have a s p e c i f i c a c t i v i t y of 3 6.5x10 dpm/mmole or a l l the o r i g i n a l a c t i v i t y of 107. Rad i o a c t i v e 5-(2'-acetoxy-3'-hydroxy-3'-methyl b u t y l ) - 6 - a c e t o x y -7,8-dimethoxy coumarin (110b) \ 3 Rad i o a c t i v e 109 (24.5 mg, 6.5x10. dpm/mmole) from the previous r e a c t i o n was converted to 5-(2'-acetoxy-3'-hydroxy-3'-methyl b u t y l ) - 6 -acetoxy-7,8-dimethoxy coumarin (110b) (16 mg) as p r e v i o u s l y d e s c r i b e d 7 3 and 110b was shown to have a s p e c i f i c a c t i v i t y of 4.28x10 dpm/mmole or 68% ofthe t o t a l a c t i v i t y of 107. - 100 -T r i a l b Radioactive 5-(2'-acecoxy-3'-hydroxy-3'-methyl butyl)-6-formy1-7- hydroxy-8-methoxy coumarin (108) Radioactive 5-(2'-acetoxy-3'-hydroxy-3'-methyl butyl)-8-methoxy 3 coumarin (107) (38 mg, 7.12x10 dpm/mmole) was converted to 5-(2'-acetoxy-3'-hydroxy-3'-methyl butyl)-6-formyl-7-hydroxy-8-methoxy coumarin (108) (25 mg). The product 108 was not counted but used directly for the next reaction. Radioactive 5-(2'-acetoxy-31-hydroxy-3'-methyl butyl)-6-formy1-7,8- dimethoxy coumarin (109) Radioactive 5-(21-acetoxy-3'-hydroxy-3'-methyl butyl)-6-formy1-7-hydroxy-8-methoxy coumarin (108) (25 mg) from the previous reaction was methylated to give 5-(2'-acetoxy-3'-hydroxy-3'-methyl butyl)-6-formy1-7,8-dimethoxycoumarin (109) (23 mg). The product 109 was shown to have 3 a specific activity of 6.8x10 dpm/mmole or 95.5% of the original activity of 107. Radioactive 5-(2'-acetoxy-3'-hydroxy-3'-methyl butyl)-6-acetoxy- 7,8-dimethoxy coumarin (110b) 3 Radioactive 109 (22 mg, 6.8x10 dpm/mmole) from the previous reaction was converted to 5-(2'-acetoxy-3'-hydroxy-3'-methyl bu t y l ) - 6 -acetoxy-7,8-dimethoxy coumarin (110b) (12 mg) and i t was shown to have 3 a specific activity of 5.0x10 dpm/mmole or 70% of the original activity of 107. - 101 -Radioactive tetramethylammonium iodide (101) from Experiment 7 Radioactive 5-(2'-acetoxy-3'-hydroxy-3'-methyl butyl)-8-methoxy 4 psoralen (107) (19.5 mg, 1.944x10 dpm/mmole) was demethylated to give tetramethylammonium iodide (101) (7.3 mg). It was counted as before 2 and was shown to have a specific activity of 6.06x10 dpm/mmole or about 3% of the original activity of 107. Degradations of Isoimperatorin (13) from Experiments and 8  Radioactive bergapten (10b) Isoimperatorin (13) (24.5 mg, 1.728x10"* dpm/mmole) from Experiment 6 was converted to bergapten (10b) (10 mg) as previously described and was shown to have a specific activity of 1.771xl04 dpm/mmole or 10.3% of the original activity of isoimperatorin (13). Radioactive bergapten (10b) Radioactive isoimperatorin (13) (21 mg, 1.458x10"* dpm/mmole) from Experiment 8 was converted to bergapten (10b) (9 mg) and was shown to 4 have a specific activity of 2.268x10 dpm/mmole or 15.5% of the original activity of isoimperatorin (13). Feeding Experiments 10 and 11 In each of these experiments, two flasks containing tissue cultures were used and the tissue cultures were transferred into 25 ml of 3 d i s t i l l e d water. D,L-Mevalonic acid-[4- H] lactone (obtained from Amersham/Searle Corp.) was converted to i t s sodium salt by evaporating the benzene solution of the lactone and dissolving the residue in dilute sodium carbonate solution (5 mg in 1 ml of water). This solution was - 102 -then added to the t i s s u e c u l t u r e s i n d i s t i l l e d water and the f l a s k s were put on r o t a r y shaker f o r a p r e s e l e c t e d time p e r i o d . Tissue c u l t u r e s were f r e e z e - d r i e d and worked up as us u a l . A l l o i m p e r a t o r i n methyl ether (7) was converted to i t s d i o l (3) before counting. The ' experimental d e t a i l s and the r e s u l t s are given i n Table 11a and l i b . Degradation of I s o p i m p i n e l l i n (2) from Experiments 10 and 11 Rad i o a c t i v e 6-acetoxymethyl-7-acetoxy-5,8-dimethoxycoumarin (103b) I s o p i m p i n e l l i n (2) (23.5 mg, 1.96xl0 4 dpm/mmole) from Experiment 10 was degraded to 6-acetoxymethyl-7-acetoxy-5,6-dimethoxycoumarin (103b) (21 mg) and was shown to be completely i n a c t i v e . R a d i o a c t i v e 6-formyl-7-hydroxy-5,8-dimethoxycoumarin (95) 3 I s o p i m p i n e l l i n (2) (19 mg, 8.4x10 dpm/mmole) from Experiment 11 was s e l e c t i v e l y ozonized as described p r e v i o u s l y and 6-formyl-7-hydroxy-5,8-dimethoxycoumarin (95) (9 mg) was i s o l a t e d . This was shown to be completely i n a c t i v e . Degradations of A l l o i m p e r a t o r i n Methyl Ether (7) from Experiments 10 and 11  Rad i o a c t i v e 5-(2'-Acetoxy-3'-hydroxy-3'-methylbutyl)-8-methoxy- p s o r a l e n (107) Rad i o a c t i v e a l l o i m p e r a t o r i n methyl ether d i o l (3) from Experiments 10 and 11 was d i l u t e d w i t h n o n - r a d i o a c t i v e 3 and was converted to 107 se p a r a t e l y . 3 TABLE 11a. Incorporation of D,L-mevalonic acid-[4- H] (sodium salt) into tissue cultures. Experi- Activity Specific Weight Dry weight Feeding Weight of compounds isolated (mg)  ment no. fed activity fed fed of tissue time . , ,. . . /, v / J / -I \ / \ i / J \ isoimpera-. alloimperatorin isopimpinellin (dpm) (dpm/mmole) (mg) cultures (days) t o r i nl ( 1 3 ) m e t h y ]/ e t h e r ( 7 ) (2J 10 5.55xl08 5.55X1011 0.132 2.1 2 6.9 6.3 9.6 11 5.55xl08 5.55X1011 0.132 1.7 4 6.5 6.6 7.6 TABLE lib. o Experi- Specific activity isolated (dpm/mmole) % Incorporation  ment no. i s o-L mp e r a t- o r :£ n alloimperatorin isopimpinellin isoimperatorin alloimperatorin isopimpinellin (13) methyl ether (7) (2) (13) methyl ether (7) (2) 10 1.24xl06 3.76xl05 1.45xl05 0.0057 0.0015 0.001 11 3.38xl06 2.56xl05 2.1xl05 0.0124 0.00107 0.00117 - 104 -Radioactive 5-(2'-acetoxy-3'-hydroxy-3'-methylbutyl)-6-formy1-7- hydroxy-8-methoxycoumarin (108) Radioactive 5-(2'-acetoxy-3'-hydroxy-3'-methylbutyl)-8-methoxy-4 psoralen (107) (28 mg, 1.69x10 dpm/mmole) from Experiment 10 was ozonized selectively to 5-(2'-acetoxy-3'-hydroxy-3'-methylbutyl)-6 -formyl-7-hydroxy-8-methoxycoumarin (108) (6 mg). This substance (108) 3 was shown to have a specific activity of 7.11x10 dpm/mmole or 42% of the original activity of 107. Radioactive 5-(2'-acetoxy-3'-hydroxy-3'-methylbutyl)-6-formyl-7- hydroxy-8-methoxycoumarin (108) Radioactive 5-(2'-acetoxy-3'-hydroxy-3'-methylbutyl)-8-methoxypsoralen 4 (107) (28 mg, 1.44x10 dpm/mmole) from Experiment 11 was selectively ozonized as described previously and 5-(2"-acetoxy-3'-hydroxy-3'-methylbutyl)-formyl-7-hydroxy-8-methoxycoumarin (108) (9 mg) was 3 isolated. This was shown to have a specific activity of 5.76x10 dpm/mmole or 40% of the original activity of 107. Degradations of Isoimperatorin (13) from Experiments 10 and 11  Radioactive bergapten (10b) Isoimperatorin (13) (13 mg, 3.46x10"* dpm/mmole) from Experiment 10 was converted to bergapten (10b) (9 mg) as described previously 4 and bergapten (10b) was shown to have a specific activity of 8.64x10 dpm/mmole or 25% of the original activity of isoimperatorin (13). - 105 -Rad i o a c t i v e bergapten (10b) Rad i o a c t i v e i s o i m p e r a t o r i n (13) (11.3 mg, 5.65x10"* dpm/mmole) from Experiment 11 was converted to bergapten (10b) (7.4 mg) and t h i s 4 compound (10b) was shown to have a s p e c i f i c a c t i v i t y of 3.45x10 dpm/mmole or 6.1% of the o r i g i n a l a c t i v i t y of i s o i m p e r a t o r i n (13). R a d i o a c t i v e 6-formyl-7-hydroxy-5-methoxycoumarin (114) 4 R a d i o a c t i v e bergapten (10b) (24 mg, 1.296x10 dpm/mmole) from Experiments 10 and 11 was combined and was s e l e c t i v e l y ozonized to give 6-formyl-7-hydroxy-5-methoxycoumarin (114) (8 mg). This (114) was shown to be completely i n a c t i v e . Feeding Experiments 12 and 13 In each experiment, two f l a s k s c o n t a i n i n g t i s s u e c u l t u r e s were used and t i s s u e c u l t u r e s were t r a n s f e r r e d i n t o 25 ml of d i s t i l l e d water. D,L-Mevalonic a c i d - [ 2 - H] lactone (obtained from Amersham/ Se a r l e Corp.) was converted to i t s sodium s a l t and mixed w i t h the f r e s h t i s s u e c u l t u r e s . The f l a s k s were put on a r o t a r y shaker f o r p r e s e l e c t e d time p e r i o d and t i s s u e c u l t u r e s were worked up as u s u a l . The a l l o i m p e r a t o r i n methyl ether (7) was converted to i t s d i o l (3) before counting. The experimental d e t a i l s and the r e s u l t s are given i n Tables 12a and 12b. TABLE 12a. Incorporation of D,L-mevalonic acid-[2- H] (sodium salt) into tissue cultures. Experi- Activity ment no. fed (dpm) Specific Weight Dry weight Feeding .activity fed fed of tissue time (dpm/mmole) (mg) cultures (days) (g) Weight of compounds isolated (mg)  isoimpera- alloimperatorin isopimpinellin torin (13) methyl ether (7) (2) 12 13 1.11x10' 1.11x10" 1.82X1011 0.794 ai 1.92x10 0.794 1.70 1.69 8.6 4.3 6.6 7.1 3.9 3.8 TABLE 12b. Experi- Specific activity isolated (dpm/mmole) % Incorporation  isoimperatorin alloimperatorin isopimpinellin isoimperatorin alloimperatorin isopimpinellin (13) methyl ether (7) (2) (13) methyl ether (7) (2) 12 2.89xl05 5.75xl04 1.4xl05 0.00083 0.00012 0.0002 13 3.76xl06 1.69xl05 2.46xl05 0.0054 0.00038 0.00036 - 107 -Degradation of I s o p i m p i n e l l i n from Experiments 12 and 13  Rad i o a c t i v e tetramethylammonium i o d i d e (101) 3 Ra d i o a c t i v e i s o p i m p i n e l l i n (2) (13.5 mg, 9.0x10 dpm/mmole) from Experiment 12 was demethylated and tetramethylammonium i o d i d e (101) (12.5 mg) was i s o l a t e d . This was counted by d i s s o l v i n g 101 Ov 2 mg) i n 10 drops of sodium t h i o s u l f a t e (0.1 N) s o l u t i o n , 1 ml of methanol was added and the s o l u t i o n was made up to 15 ml w i t h aqueous s c i n t i l l a t o r s o l u t i o n . By t h i s method, 101 gave a s p e c i f i c a c t i v i t y 3 of 9.05x10 dpm/mmole or a l l the o r i g i n a l a c t i v i t y of i s o p i m p i n e l l i n ( 2). R a d i o a c t i v e tetramethylammonium i o d i d e (101) 4 I s o p i m p i n e l l i n (16 mg, 4.182x10 dpm/mmole) from Experiment 13 was demethylated and tetramethylammonium i o d i d e (101) (18 mg) was i s o l a t e d . This was converted to tetramethylammonium p i c r a t e (102) (10 mg). I t was counted by d i s s o l v i n g the p i c r a t e (y 2 mg) i n a c e t i c a c i d (10 drops) and a c e t i c anhydride (10 drops). Enough z i n c dust was added to d e c o l o r i z e the s o l u t i o n . Sodium m e t a b i s u l f i t e (100 mg) was added and the s o l u t i o n was f i l t e r e d i n t o the counting v i a l . The o r i g i n a l c o n t a iner was washed w i t h methanol (1 ml) and the wash was a l s o f i l t e r e d i n t o the counting v i a l . The s o l u t i o n was made up to 15 ml w i t h organic s c i n t i l l a t o r s o l u t i o n and then counted a f t e r standing at l e a s t 1 hour i n the c o l d and i n the dark. By t h i s method, tetramethylammonium p i c r a t e (102) was shown to have a s p e c i f i c a c t i v i t y 3 of 4.0x10 dpm/2 mmole or a l l the o r i g i n a l a c t i v i t y of i s o p i m p i n e l l i n ( 2 ) . - 108 -Degradations of A l l o i m p e r a t o r i n Methyl Ether (7) from Experiments 12 and 13  P e r i o d i c a c i d cleavage of r a d i o a c t i v e 5-(2',3'-dihydroxy-3'-methyl- b u t y l ) -8-methoxypsoralen (3) ( a l l o i m p e r a t o r i n methyl ether d i o l ) 3 A l l i m p e r a t o r i n methyl ether d i o l (3) (18.5 mg, 6.36x10 dpm/mmole) from Experiment 12 was cleaved w i t h p e r i o d i c a c i d as d e s c r i b e d p r e v i o u s l y and 5-(2'-hydroxy ethyl)-8-methoxypsoralen (105) (5 mg) 3 was i s o l a t e d and shown to have a s p e c i f i c a c t i v i t y of 1.3x10 dpm/mmole or 20.4% of the o r i g i n a l a c t i v i t y of 3. P e r i o d i c a c i d cleavage of r a d i o a c t i v e 5-(2',3'-dihydroxy-3'-methyl- b u t y l ) -8-methoxypsoralen (3) 3 A l l i m p e r a t o r i n methyl ether d i o l (3) (36.5 mg, 7.36x10 dpm/mmole) from Experiment 13 was cleaved w i t h p e r i o d i c a c i d and 5-(2'-hydroxy-ethyl)-8-methoxypsoralen (105) (9.3 mg) was i s o l a t e d and was shown to be completely i n a c t i v e . Degradations of I s o i m p e r a t o r i n (13) from Experiments 12 and 13  Rad i o a c t i v e bergapten (10b) Is o i m p e r a t o r i n (13) (12.7 mg, 1.08x10"* dpm/mmole) from Experiment 12 was converted to bergapten (10b) (7.5 mg) and t h i s was shown to 3 have a s p e c i f i c a c t i v i t y of 5.85x10 dpm/mmole or about 5.5% of the o r i g i n a l a c t i v i t y of i s o i m p e r a t o r i n (13). Radi o a c t i v e bergapten (10b) Is o i m p e r a t o r i n (13) (14 mg, 8.1x10"* dpm/mmole) from Experiment 13 was degraded to bergapten (10b) (5.6 mg) and t h i s was shown to have a 4 s p e c i f i c a c t i v i t y of 1.728x10 dpm/mmole or about 2.7% of the o r i g i n a l - 109 -a c t i v i t y of isoimperatorin (13). Feeding Experiment 14 In t h i s experiment, three f l a s k s containing t i s s u e cultures were 14 transferred to 25 ml of d i s t i l l e d water and D,L-mevalonic acid-[5- c] (obtained from Schwarz/Mann Corp.) was fed as a sodium s a l t i n water. The f l a s k was put on a rotary shaker f o r 2 days, worked up i n the usual manner and only i s o p i m p i n e l l i n (2) was i s o l a t e d and c r y s t a l l i z e d to constant a c t i v i t y . The experimental d e t a i l s and the r e s u l t s are given i n Table 13. Degradations of Is o p i m p i n e l l i n (2) from Experiment 14 6-Formyl-7-hydroxy-5,8-dimethoxycoumarin (95) 4 Isopimpinellin (2) (45 mg, 4.92x10 dpm/mmole) from Experiment 14 was ozonized s e l e c t i v e l y and 6-formyl-7-hydroxy-5,8-dimethoxycoumarin (95) (22 mg) was i s o l a t e d . This was shown to have a s p e c i f i c a c t i v i t y 4 of 4.90x10 xpm/mmole or a l l the o r i g i n a l a c t i v i t y of i s o p i m p i n e l l i n (2). Radioactive 6-formy1-5,7,8-trimethoxycoumarin (96) Radioactive 6-formyl-7-hydroxy-5,8-dimethoxycoumarin (95) (22 mg) from the previous reaction was methylated as described previously and 6-formy1-5,7,8-trimethoxycoumarin (96) (20 mg) was i s o l a t e d . This was not counted but used as such f o r the next r e a c t i o n . TABLE 13. Incorporation of D,L-mevalonic acid-[5- C] (sodium salt) into tissue cultures. Experi- Activity Specific Dry weight Feeding Weight of Specific activity of % Incorporation ment no. fed activity of tissue time isopimpinellin (2) isopimpinellin (2) isopimpinellin (dpm) fed cultures (days) (mg) (dpm/mmole) (2) (dpm/mmole) (g) 14 l.llxlO 9 2.62xl010 3.33 2 12 4.55xl05 0.002 - I l l -6-Hydroxy-5,7,8-trime thoxycoumarin (97) Radioactive 6-formy1-5,7,8-trimethoxycoumarin (96) (20 mg) from the previous reaction was converted to 6-hydroxy-5,7,8-trimethoxy-coumarin (97) (12 mg) as described previously. This was shown to have 3 a specific activity of 2.75x10 dpm/mmole or about 5.5% of the original activity of isopimpinellin (2). - 112 -DISCUSSION (PART III) Biosynthetic Studies on Coumarins from Young Thamnosma montana Plants As documented in the Introduction, l i t t l e information is available as to the origin of alkyl side chains often found in many natural coumarins. Although no direct evidence is available, these alkyl groups are often considered to be mevalonate derived. As shown in Figure 11, mevalonic acid (85) itself is the product resulting from 53 the combination of three units of acetyl coenzyme-A. The principal cellular source of acetyl-CoA is the intramitochondrial oxidation of 78 ¥X^ pyruvate (126), which is itself the major product of glucose catabolisrs —^^  78 in most cells (Figure 20b). Thus the origin of the C,. unit of mevalonate can be directly retraced to the carbohydrate products of photosynthesis. This process of carbon dioxide fixation is outlined in Figure 20a. The starred atom illustrates the path of the carbon dioxide carbon atom from its i n i t i a l fixation by ribulose-l,5-diphosphate 79 (115) through to glucose-6-phosphate (124). In Figure 20b, the glycolytic pathway from glucose-6-phosphate (124) to pyruvate (126) is presented. If the phospho-3-glycerate (118) is directly derived from the carbon dioxide fixation process, then the shortest route to pyruvate (126), and thereby acetyl-CoA is: carbon dioxide ->• 2-carboxy-3-ketopentitol (117) -»- phospho-3-glycerate (118) •> phospho-2-glycerate - 113 -CHo0P I l C=0 I H-C-OH I H-C-OH I CH OP CH.OP I 2 C-OH II C-OH I H-C-OH I CH OP CO, CH.OP * | 2 OOC-C-OH I C=0 I H-C-OH I CH OP H20 115 116 117 CHo0P I 2 HO-C-H * l _ COO coo" I H-C-OH I CH2OP 118 ATP,  : ADP CO-OP I H-C-OH I CH OP 119 TPNH TPN CHO I H-C-OH I CH2OP 120 CHOH I C=0 I CH OP 121 Jt 0 ! PH-P=0 !_ o ' CHO I H-C-OH * l HO- C-H * l H- C-OH I H-C-OH 1 CH OP 124 CH„OH I 2 C=0 * l HO- C-H * l H- C-OH I H-C-OH CH„OP I 2 C=0 * l HO- C-H * l H- C-OH I H-C-OH CH2OP CH2OP 123 122 Figure 20a. The photosynthetic fixation of CO - 114 -CHO I H-C-OH I HO-C-H I H-C-OH I H-C-OH I CH2OP 124 CHo0H I 2 C=0 I HO-C-H I H-C-OH I H-C-OH I CH2OP 123 CHOP I 2 C=0 I HO-C-H I H-C-OH I H-C-OH I CH2OP 122 CO-OP I H-C-OH I CH2OP 119 TPN TPNH CHO I H-C-OH I CH2OP 120 V CH„OH I 2 C=0 I CH2OP 121 ATP ADP COO I H-C-OH I CH2OP 118 COO I H-C-OP I CH2OH 125 COO I C-OP II CH2 24 ADP COO C02 + CH3C0S-CoA CoA-SH C=0 I CH3 126 Figure 20b. The glycolytic pathway to pyruvate. - 115 -(125) -> phosphoenolpyruvate (24) -* pyruvate (126) -> acetyl-CoA. The purpose of this precursor delineation is to show that a unit becomes involved with the biosynthesis of the C,. unit of mevalonate only at the acetate stage. It was, therefore, of some interest when 80 Shah and Rogers postulated the intermediacy of glycollate (127) via glycine (129) and serine (130) in the biosynthesis of acetyl-CoA. This proposal was based on the work with greening etiolated maize seedlings and involved intra- and extra-chloroplastidic terpenoid synthesis. By means of a wide range of radioisotopic-incorporation studies, radioisotopic dilution studies and experiments with inhibitors 80 of the proposed pathway, Shah and Rogers were able to demonstrate the existence of the pathway, carbon dioxide ->- glycollate (127) -> glyoxylate (128) -> glycine (129) -> serine (130) -> pyruvate (126) acetyl-CoA, within the chloroplasts (Figure 21). Extrachloroplastidic sterols displayed incorporation patterns consistent with the established sequence, carbon dioxide -> glucose (124) -*• pyruvate (126) -> acetyl-CoA -> mevalonate (85). The authors felt that their work offered strong evidence for a number of proposals, specifically the relatively direct synthesis of amino acids from carbon dioxide by-passing the carbohydrate intermediates and the involvement of these amino acids in terpenoid biosynthesis. Both 81 82 of these proposals are supported in the literature. ' In an attempt to determine a specifically incorporated (i.e., non-randomized) precursor of mevalonate and the unit of caphaeline 83 (135), Gear and Garg administered labelled glycollic acid and glycine 14 to three-year-old cephaelis ipecacuanha plants. The [1- C]-labelled - 116 -* o CH OH ^ * Z01 Z CHO ^ CH-NH-CO • u *• I y I 2 1 COOH o C 0 0 H o C 0 0 H C l 127 128 129 CH.OH tl 2 CH NH, 'COOH 130 * CH„ * l 3 C=0 °COOH 126 -CO, CH. * l 3 C=0 I S-CoA CH^ OH *l C=0 'COOH CH„OH I 2 *CHOH I "COOH CH-OH t l 2 CH-OP I 'COOH CH-kll 2 C-OP I 'COOH 1 3 1 132 133 134 Figure 21. The Shah and Rogers chloroplastidic acetyl-CoA biogenesis. - 117 -forms of both these compounds gave either inactive or only slightly 80 active cephaeline (135). This was not unexpected as Shah and Rogers 14 had likewise reported negative incorporation for glyoxylic acid-[l- C] (128) into g-carotene. The rationale for this finding can be seen in the biogenesis of acetyl-CoA as postulated by these authors (Figure 14 14 21). When, however, glycine-[2- C] (129) and glycollic acid-[2- C] (127) were fed to the aforementioned plant system, active cephaeline 14 (135) was isolated. It was found that glycollic acid-[2- C] (127) 14 was being randomized whereas glycine-[2- C] (129) was acting as a specific precursor of the Cg unit of cephaeline (135). H3CO OCH, 136 135 14 14 In subsequent publications, glycine-[2- C] and acetate-[2- C] were fed to C_. acumentata84 and R. serpentina8"* plants. In the former, cephaeline (135) and the phytosterol, 8-sitosterol (136), were isolated and examined. It was found that, whereas acetate was specifically incorporated into the sterol, its activity was randomized 14 whereas glycine-[2- C] was again reported to be specifically incorporated into the C^  unit of cephaeline (135) but not at a l l into - 118 -the phytosterol. This result was taken to indicate that glycine and acetate "act as specific and exclusive precursor of different monoterpene moieties, in different compounds, in the same plant." Since Thamnosma montana contained a large number of alkylated coumarins and the incorporation of mevalonic acid into these coumarins was very poor,'' i t was decided to study precursors other than mevalonic acid which could be utilized by plants in the biosynthesis of these 14 alkyl groups. It was felt that i f glycine-[2- C] can act as a specific precursor of the Cg unit of cephaeline (135), which has been known to be mevalonate derived, i t might also act as a specific precursor of the alkyl side chains found in many natural coumarins. Therefore, i t was decided to study the role of glycine in the biosynthesis of these alkyl groups. Thamnosma montana contains a large array of coumarins (see Introduction) but for the purpose of these biosynthetic investigations, three coumarins, umbelliprenin (9), alloimperatorin methyl ether (7) and ispimpinellin (2) were selected. Since umbelliprenin (9) contains a farnesyl-ether side chain and alloimperatorin methyl ether (7), a C,--alkyl side chain, i t was felt that these two coumarins offer an opportunity to study the role of glycine in the biogenesis of these alkyl groups. Isopimpinellin (2), a simple furanocoumarin with two methoxyl groups, was chosen to evaluate the role of glycine in the biosynthesis of the furan ring and the origin of these methoxyl groups in furanocoumarins. However, before discussing the studies performed in this regard i t is pertinent to discuss some preliminary work done to determine - 119 -i f the b i o s y n t h e s i s of these coumarins was o c c u r r i n g at a r e g u l a r and measurable b a s i s . For t h i s purpose young Thamnosma montana p l a n t s (about 2 to 3 years o l d ) , which had been grown from seeds, were 14 s e l e c t e d and the p r e c u r s o r , D,L-phenylalanine-[3- C] was fed by the hydroponic method to the roo t s of these p l a n t s . A f t e r the p r e s e l e c t e d feeding time, the p l a n t s were mechanically ground to a coarse powder and e x t r a c t e d i n a Soxhlet apparatus w i t h acetone. The e x t r a c t was concentrated and the residue was t r e a t e d w i t h hot chloroform. The chloroform s o l u b l e p o r t i o n was chromatographed on an alumina column and the compounds were i s o l a t e d by p r e p a r a t i v e l a y e r chromatography, c r y s t a l l i z e d to constant a c t i v i t y and the r a d i o a c t i v i t y determined by the s c i n t i l l a t i o n counting method. The r e s u l t s are given i n Table 14. 14 TABLE 14. I n c o r p o r a t i o n of D,L-phenylalanine-[3- C] i n t o Thamnosma  montana. E x p e r i - Feeding A c t i v i t y Weight of % I n c o r p o r a t i o n  ment no. time fed the p l a n t , ,.. . .,., . s , , f u m b e l l i - allxmpera- xsopim-(days) (dpm) (g) . . , . -,, . J p r e n i n t o r i n methyl p x n e l l z n (9) ether (7) (2) 1 2 1 3 . 8 x l 0 6 46.5 -- 0.08 0.2 Activity corrected for radioactivity isolated outside the plant. Thus i t i s evident that young Thamnosma montana p l a n t s were bio-s y n t h e s i z i n g v a rious coumarins i s o l a t e d and that D,L-phenylalanine-14 [3- C] was being u t i l i z e d w i t h c o n s i d e r a b l e e f f i c i e n c y . S i m i l a r experiments done i n our l a b o r a t o r y ^ w i t h young Thamnosma montana - 120 -p l a n t s i n d i c a t e d that the optimum time p e r i o d f o r the b i o s y n t h e s i s of these coumarins was between 7 and 10 days. Therefore, i n the next s e r i e s of experiments, a t t e n t i o n was focussed on the r o l e of g l y c i n e i n the b i o s y n t h e s i s of these coumarins 14 i n Thamnosma montana. In these experiments, g l y c i n e - [ 2 - C] was administered to young Thamnosma montana p l a n t s by the hydroponic method and the p l a n t s were allowed to grow f o r a p e r i o d of 7 days. The p l a n t s were worked up i n the normal manner and a l l o i m p e r a t o r i n methyl ether (7) was converted to the d i o l (3) f o r counting purposes. The r e s u l t s are presented i n Table 15. 14 TABLE 15. I n c o r p o r a t i o n of g l y c i n e - [ 2 - C] i n t o Thamnosma montana. E x p e r i - A c t i v i t y Weight of ment no. fed p l a n t (dpm) (g) 2 8.8x10 15 3 2 . 5 3 c l 0 8 20 4 5 . 5 5 x l 0 8 20 % I n c o r p o r a t i o n u m b e l l i p r e n i n a l l o i m p e r a - i s o p i m p i n -(9) t o r i n methyl e l l i n (2) ether (7) 0.031 0.073 0.082 0.019 0.029 0.15 0.001 0.01 0.022 Corrected f o r a c t i v i t y recovered outside the p l a n t , k Counted as a l l o i m p e r a t o r i n methyl ether d i o l ( 3 ) . 14 Thus the r e s u l t s i n Table 15 i n d i c a t e that g l y c i n e - [ 2 - C] i s being i n c o r p o r a t e d i n t o a l l three coumarins and the i n c o r p o r a t i o n l e v e l i s reasonable. - 121 -To determine the d i s t r i b u t i o n of r a d i o a c t i v i t y i n u m b e l l i p r e n i n ( 9 ) , i t was degraded according to the scheme p r e v i o u s l y d e s c r i b e d and as shown i n F i g u r e 22. \ H 94a; R=0 94b; R=2,4-DNP Figure 22. Degradation of r a d i o a c t i v e u m b e l l i p r e n i n ( 9 ) . Thus u m b e l l i p r e n i n (9) (2.23x10"* dpm/mmole) from experiment 2 was subjected to a c i d h y d r o l y s i s as p r e v i o u s l y described and umb e l l i f e r o n e (49) was i s o l a t e d and shown to have a s p e c i f i c a c t i v i t y of 2.43x10^ dpm/mmole or 10.9% of the o r i g i n a l r a d i o a c t i v i t y of 9. In a s i m i l a r experiment, u m b e l l i p r e n i n (9) (2.16x10"* dpm/mmole) from experiment 3 was 4 degraded and umbelliferone (49) (3.24x10 dpm/mmole) was shown to have 15% of the o r i g i n a l a c t i v i t y of u m b e l l i p r e n i n (9). Thus i t i s c l e a r that between 85-90% of the r a d i o a c t i v i t y of 9 r e s i d e s i n the f a r n e s y l s i d e chain and only 10-15% i s present i n the r e s t of the coumarin molecule. - 122 -I t was also of i n t e r e s t to gain i n f o r m a t i o n as to the d i s t r i b u t i o n of r a d i o a c t i v i t y present i n the f a r n e s y l s i d e chain of u m b e l l i p r e n i n 4 ( 9 ) . To t h i s end, u m b e l l i p r e n i n (9) (2.23x10 dpm/mmole) from e x p e r i -ment 3 was ozonized as p r e v i o u s l y described and l e v u l i n a l d e h y d e b i s - 2 , 4 -dinitrophenylhydrazone (94b) was i s o l a t e d . This compound was.shown 3 to have a s p e c i f i c a c t i v i t y of 4.55x10 dpm/mmole or 20.4% of the o r i g i n a l a c t i v i t y of 9. As two molar e q u i v a l e n t s of l e v u l i n a l d e h y d e (94a) should be produced i n t h i s r e a c t i o n , t h i s r e s u l t i n d i c a t e s that only 41% of the r a d i o a c t i v i t y i n the s i d e chain r e s i d e s i n the i n t e r n a l ten carbon p o r t i o n . Whether t h i s represents an unequal l a b e l i n g of the f a r n e s o l or r e f l e c t s some e r r o r i n the counting method i s d i f f i c u l t to determine at t h i s time as the l a c k of nonactive u m b e l l i -p r e n i n (9) precludes f u r t h e r experimentation. To determine the d i s t r i b u t i o n of r a d i o a c t i v i t y , i s o p i m p i n e l l i n (2) from experiment 2, 3 and 4 was degraded according to the scheme p r e v i o u s l y described and as depicted i n Figure 23. The r e s u l t s are presented i n Table 16. From these r e s u l t s i t i s evident that more than 90% of the a c t i v i t y i n i s o p i m p i n e l l i n (2) r e s i d e s i n the two methoxyl groups and very l i t t l e ( l e s s than 10%) a c t i v i t y i s present i n the r e s t of the molecule. The i n c o r p o r a t i o n of g l y c i n e i n t o the methoxyl groups of i s o p i m p i n e l l i n (2) i s e x p l i c a b l e i n terms of the degradation of g l y c i n e 86 to a C^ - u n i t , a process observed p r e v i o u s l y . Byerrum and coworkers showed that i n p l a n t systems, the C-2 of g l y c i n e could f u n c t i o n as a C^-unit as e f f i c i e n t l y as the methyl groups of methionine and c h o l i n e , and ten times as e f f i c i e n t l y as formate whereas the c a r b o x y l carbon of - 123 -OCH_ OCH„ OCH 100b; R=COCH3 Figure 23. Degradation of radioactive isopimpinellin (2). TABLE 16. Degradation of radioactive isopimpinellin (2) (Experiments 2, 3 and 4). Compound Specific activity (% total activity in isopimpinellin) Experiment 2 Experiment 3 Experiment 4 isopimpinellin (2) 3.208xl05 (100%) dpm/mmole 2.80xl04 (100%) dpm/mmole l.OOxlO5 (100%) dpm/mmole 6-formyl-7-hydroxy-5,8-dimethoxycoumarin (95) 3.12xl05 (97.5%) 2.57xl04 (91.8%) 8.2xl04 (82%) 6-formy1-5,7,8-trimethoxy-coumarin (96) — — — 6-hydroxy-5,7,8-trimethoxy-coumarin (97) 2.91xl05 (90.5%) — — i ro 1,3-diformy1-4,6-dihydroxy-2,5-dimethoxybenzene (98) 3.03xl05 (94.5%) 2.57xl04 (91.8%) 9.27xl04 (92.7%) 1,3-diformy1-2,4,5,6-tetra-methoxybenzene (99) 3.06xl05 (95.5%) — — 1,3-diacetoxy-2,4,5,6-tetra-methoxybenzene (100b) 3.08xl05 (96%) — 9.17xl04 (91.7%) tetramethylammonium iodide (101) 2xl.44xl05 (89.8%) 2xl.44xl05 (100%) 2x4.75xl04 (95%) tetramethylammonium picrate (102) 2xl.38xl05 (86%) 2xl.26xl05 (90.5%) — The total activity in isopimpinellin is set at 100%. - 125 -g l y c i n e showed no such a c t i v i t y . Our s t u d i e s are c o n s i s t e n t w i t h these f i n d i n g s . To determine the d i s t r i b u t i o n of r a d i o a c t i v i t y i n a l l o i m p e r a t o r i n methyl ether (7) from experiment 2, 3, and 4, i t was degraded according to the scheme already described and as depicted i n F i g u r e 24. The r e s u l t s are given i n Table 17. Figure 24. Degradations of r a d i o a c t i v e a l l o i m p e r a t o r i n methyl ether ( 7 ) . TABLE 17. Degradations of radioactive alloimperatorin methyl ether (7). Compound Specific activity (% of alloimperatorin methyl ether) Experiment 2 Experiment 3 Experiment 4 Trial a Trial b alloimperatorin methyl ether diol (3) 5-(21-hydroxyethyl)-8-methoxy-psoralen (105) acetone p_-bromobenzenesulfonyl-hydrazone (106) tetramethylammonium iodide (101) tetramethylammonium picrate (102) 5-(2'-ace toxy-3'-hydroxy-3'-methylbutyl)-8-methoxypsoralen (107) 5-(2'-acetoxy-3'-hydroxy-3'-methylbutyl)-6-formy1-7-hydroxy-8-methoxycoumarin (108) 5-(2'-acetoxy-3'-hydroxy-3'-methylbutyl)-6-acetoxy-7,8-dimethoxycoumarin (110b) 1-(21-acetoxy-3'-hydroxy-3'-me thylbutyl)-2,6-diformy1-3,5-dihydroxy-4-methoxybenzene (111) 3.29x10* (100%) dpm/mmole 2.19x10 (6.7%) 3.29x10 (100%) 2.91x10 (88.5%) 1.10x10 (100%) dpm/mmole l.OOxlO4 (91.5%) 5.72x10 (5.2%) 7.7x10 (70%) 1.10x10 (100%) 1.22x10 (100%) dpm/mmole l.l l x l O 4 (92%) 1.12x10* (92%) £ 1.18xl04 (97.5%) ' 1.22xl04 (100%) 4.85xl04(100%) l.lOxlO 4 (100%) 1.18xl04 (96.6%) 4.75xl04(97.5%) 1.06x10 (96.5%) 1.04x10 (94.5%) - 127 -14 These results indicate that glycine-[2- C] is being incorporated into the dimethylallyl side chain of alloimperatorin methyl ether (7). Also i t is clear from these results that most of the activity in 7 is present in the methoxyl groups. It is significant to note that very l i t t l e activity could be found in the furanocoumarin portion 14 of the molecule indicating that glycine-[2- C] is acting as a specific precursor of the C^-pool and the C^-alkyl side chain. This result 14 is in contrast to the experiments with acetate-[2- C] done in our laboratory^ with young Thamnosma montana plants. In these experiments, 14 it was found that acetate-[2- C] was being incorporated into the furanocoumarin portion of the molecule as ,well as in the methoxyl groups and the alkyl side chain indicating a randomization of the activity. - 128 -EXPERIMENTAL (PART III) For general experimental information, see page 59 and 87. Thamnosma montana plants used in this study were collected in the summer as seeds and mature plants from the north-facing slopes of small h i l l s in the vicinity of Joshua Tree National Monument, in the Mojave Desert area of Southern California. Some seeds could be propagated by Dr. P. Salisbury of our Department. Small Thamnosma montana plants (2-3 years old) were obtained from Molecular Biochemical Corporation, Tempe, Arizona. Isolation of the Constituents of Young Thamnosma montana Plants Whole Thamnosma montana plants (wet weight 15 g) which had been grown from seeds (^  16 months old) were ground in a Waring blender to a coarse powder and extracted with acetone (300 ml) in a Soxhlet extractor for 3 hours. The acetone extract was reduced to dryness under reduced pressure to yield a residue (0.6 g) which was treated with hot chloroform (100 ml). The chloroform soluble portion was filtered and the solvent was removed under reduced pressure to yield a residue (0.28 g) which was preadsorbed on alumina (neutral, 3 g, activity IV). The preadsorbed material was placed on top of a column of alumina (neutral, 27 g, activity IV) which had been made up in petroleum ether and elution was begun immediately. The fractions - 129 -e l u t e d w i t h petroleum ether and petroleum ether-benzene contained waxy m a t e r i a l and were not examined f u r t h e r . A l s o e l u t e d w i t h petroleum ether-benzene was a f r a c t i o n (20 mg) noted as a purple (uv) band on the column. P r e p a r a t i v e l a y e r chromatography on t h i s f r a c t i o n allowed the i s o l a t i o n of u m b e l l i p r e n i n (9) (10 mg), mp 62-63°. A l s o noted i n t h i s f r a c t i o n was a y e l l o w (uv) band corresponding to i s o i m p e r a t o r i n (13), but t h i s m a t e r i a l was not i s o l a t e d . L a t e r f r a c t i o n s e l u t e d w i t h petroleum ether-benzene and benzene were noted (by t i c ) to c o n t a i n a l l o i m p e r a t o r i n methyl ether ( 7 ) , thamnosmin (15) and p h e l l o p t e r i n (12). P r e p a r a t i v e l a y e r chromatography on these combined (25 mg) allowed i s o l a t i o n of a l l o i m p e r a t o r i n methyl ether (7) (8 mg), mp 108-110°. The f r a c t i o n s e l u t e d w i t h benzene gave i s o p i m p i n e l l i n (2) (9.4 mg) by p r e p a r a t i v e l a y e r chromatography, mp 150-151°. Examination of other f r a c t i o n s by t i c suggested the presence of a l l o i m p e r a t o r i n methyl ether epoxide (14), bergapten (10b), xanthotoxin (11) and p o s s i b l y p s o r a l e n (10a) but the q u a n t i t y of these m a t e r i a l s was s m a l l . Feeding Experiment 1 14 In t h i s experiment, D,L-phenylalanine-[3- C] (obtained from New England Nuclear Corp., Boston, Mass.) was administered to whole young Thamnosma montana p l a n t s (^  16 months old) by hydroponic methods. 9 The precursor (2.22 xlO dpm, 37.4 mg) was d i s s o l v e d i n d i s t i l l e d water (25 ml) and an a l i q u o t was removed, weighed and the r a d i o a c t i v i t y per u n i t weight of s o l u t i o n determined (9.11 x 10^ dpm/g). Young p l a n t s were c a r e f u l l y uprooted so as to cause minimum damage to the f i n e r o o t l e t s and the roots were placed i n the t e s t tube c o n t a i n i n g precursor - 130 -i n s o l u t i o n . Several p l a n t s (5 to 10) were used and the precursor s o l u t i o n was d i s t r i b u t e d e q u a l l y among the t e s t tubes to be used. A f t e r the p l a n t s had absorbed the precursor s o l u t i o n , the o r i g i n a l c o n t a i n e r of the precursor s o l u t i o n was washed w i t h d i s t i l l e d water and the p l a n t s were allowed to absorb the washings. The p l a n t s were l e f t to grow under continuous fluorescent i l l u m i n a t i o n . A f t e r the p r e s e l e c t e d time, the p l a n t s were worked up as p r e v i o u s l y d e s c r i b e d . The experimental d e t a i l s are given i n Table 18. As necessary, the compounds i s o l a t e d were d i l u t e d w i t h the corresponding i n a c t i v e compound to al l o w the necessary c r y s t a l l i z a t i o n s to be performed. 14 TABLE 18. I n c o r p o r a t i o n of D,L-phenylalanine-[3- C] i n t o young Thamnosma montana p l a n t s E x p e r i - Feeding A c t i v i t y P l a n t S p e c i f i c a c t i v i t y [% i n c o r p o r a t i o n ]  -- time fed weight • ^ • • • • i-i • x , , N , ? u m b e l l i - a l l o i m p e r a t o r i n i s o p i m i n e l l i n (days) (dpm) (g) p r e n i n ( g ) m e t h y l e t h e r ( y ) ( 2 ) ment no. 13.8x 46.5 — 4.629xl0 5[0.08] 1.279xl0 5[0.2] ^Q6 dpm/mmole dpm/mmole Corrected f o r a c t i v i t y i s o l a t e d o u t s i d e the p l a n t . Feeding Experiment 2, 3, and 4 14 In these experiments, g l y c i n e - [ 2 - C] (obtained from New England, Nuclear Corp., Boston, Mass.) was administered to young whole p l a n t s (18-24 months old) by the hydroponic technique (feeding d i r e c t l y i n t o the r o o t s ) . The p r e c u r s o r , obtained as 0.1 N HCI s o l u t i o n , was used TABLE 19a. Incorporation of glycine-[2- C] into Thamnosma montana Experi- Activity Specific Weight Wet weight Weight of compounds isolated (mg) ment No. fed (dpm) activity fed (dpm/mmole) fed (mg) of plant (g) umb elliprenin (9) alloimperatorin methyl ether (7) isopimpinellin (2) 2 8.8xl0? 5.9xl010 0.116 15 10 16 9.4 3 2.53xl08 5.9xl010 0.350 20 23.5 13.8 17.3 4 5.55xl08 5.9xl010 0.705 20 7.5 12.0 20 TABLE 19b. Experi- Specific activity isolated (dpm/mmole) % Incorporation  ment no. u mbelliprenin alloimperatorin isopiminellin umbelliprenin alloimperatorin isopimpinellin (9) methyl ether (7) (2) (9) methyl ether (7) (2) 2 9.99xl05 11.4xl05 18.9xl05 0.031 0.073 0.082 3 4.13xl05 15.1xl05 5.4xl06 0.019 0.029 0.15 4 2.72xl05 13.1xl05 15.0xl05 0.001 0.01 0.022 - 132 -as such and the p l a n t s were allowed to grow f o r 7 days. A f t e r the s o l u t i o n was absorbed, the o r i g i n a l v i a l c o n t a i n i n g the precursor was r i n s e d w i t h d i s t i l l e d water ( 2 ml) and these washes were allowed to be absorbed by the p l a n t s . The procedure was continued throughout the course of the feedings. In each case, the p l a n t s were worked up as before and u m b e l l i p r e n i n ( 9 ) , a l l o i m p e r a t o r i n methyl ether (7) and i s o p i m p i n e l l i n (2) were i s o l a t e d . In each case, a l l o i m p e r a t o r i n methyl ether (7) was converted to d i o l ( 3 ) . The values quoted f o r 7 are based on the appropriate values obtained from 3, c o r r e c t e d f o r the y i e l d of con v e r t i n g 7 to 3. Where necessary, d i l u t i o n s were performed to provide s u f f i c i e n t samples f o r p u r i f i c a t i o n and degradations. The experimental d e t a i l s are presented i n Table 19a and 19b. Degradations of U m b e l l i p r e n i n (9) from Experiments 2 and 3  A c i d - c a t a l y z e d h y d r o l y s i s of u m b e l l i p r e n i n (9) Um b e l l i p r e n i n (9) (40 mg, 2.23x10"* dpm/mmole) from Experiment 2 was hydrolyzed w i t h a c e t i c a c i d as des c r i b e d p r e v i o u s l y 7 and u m b e l l i -ferone (49) (12 mg) was i s o l a t e d and shown to have a s p e c i f i c a c t i v i t y 4 of 2.43x10 dpm/mmole or 10.9% of the o r i g i n a l a c t i v i t y of u m b e l l i p r e n i n (9). A c i d - c a t a l y z e d h y d r o l y s i s of u m b e l l i p r e n i n (9) Rad i o a c t i v e u m b e l l i p r e n i n (9) (50 mg, 2.16x10"* dpm/mmole) from Experiment 3 was degraded as described p r e v i o u s l y 7 and um b e l l i f e r o n e (49) 4 (14 mg) was i s o l a t e d and shown to have a s p e c i f i c a c t i v i t y of 3.24x10 dpm/mmole or 15% of the t o t a l a c t i v i t y of 9. - 133 -Ozonolysis of r a d i o a c t i v e u m b e l l i p r e n i n (9) 4 U m b e l l i p r e n i n (9) (25 mg, 2.23x10 dpm/mmole) from Experiment 3 was ozonized under optimum c o n d i t i o n s as described p r e v i o u s l y ^ and le v u l i n a l d e h y d e bis-2,4-dinitrophenylhydrazone (94b) (20 mg) was i s o l a t e d a f t e r c r y s t a l l i z a t i o n . This m a t e r i a l (94b) was counted i n the f o l l o w i n g manner. The d e r i v a t i v e (94b) (^  2 mg) was d i s s o l v e d i n the counting v i a l i n a mixture of g l a c i a l a c e t i c a c i d (10 drops), a c e t i c anhydride (5 drops) and dimethylformamide (20 drops). The mixture was then heated to complete d i s s o l u t i o n and z i n c dust (^  50 mg) was added to d e c o l o u r i z e the s o l u t i o n . Sodium m e t a b i s b u l f i t e (100 mg) was added, then benzene (^0.5 ml) and the s o l u t i o n was made up to 15 ml w i t h organic s c i n t i l l a t o r s o l u t i o n . A f t e r standing i n the c o l d and dark f o r 1 hour, the sample was counted. Due to the unorthodox counting s o l u t i o n employed, counting e f f i c i e n c y was determined by 14 adding an a c c u r a t e l y weighed sample of C-hexadecane standard to the alre a d y counting sample and i t was counted again. The r a t i o of the expected dpm to found cpm f o r hexadecane determined the counting e f f i c i e n c y (^  64%). In each case a blank sample c o n t a i n i n g an equal amount of i n a c t i v e l e v u l i n a l d e h y d e bis-2,4-DNP (94b) was counted f i r s t to determine the accurate background. In t h i s manner, the r a d i o a c t i v e l e v u l i n a l d e h y d e bis-2,4-DNP (94b) was shown to have a 3 s p e c i f i c a c t i v i t y of 4.55x10 dpm/mmole or 20.4% of the t o t a l a c t i v i t y of u m b e l l i p r e n i n (9). - 134 -Degradations of I s o p i m p i n e l l i n (2) from Experiment 2 Ra d i o a c t i v e 6-formyl-7-hydroxy-5,8-dimethoxycoumarin (95) Radio a c t i v e i s o p i m p i n e l l i n (2) (50 mg, 3.208x10"* dpm/mmole) was s e l e c t i v e l y ozonized as p r e v i o u s l y d e s c r i b e d 7 and 6-formyl-7-hydroxy-5,8-dimethoxycoumarin (95) (20 mg) was i s o l a t e d . I t was shown to have a s p e c i f i c a c t i v i t y of 3.12x10"* dpm/mmole or 97.5% of the t o t a l a c t i v i t y of i s o p i m p i n e l l i n ( 2). Radioactive 6-formy1-5,7,8-trimethoxycoumarin (96) 6-Formyl-7-hydroxy-5,8-dimethoxycoumarin (95) (20 mg, 3.12x10"* dpm/mmole) from the above r e a c t i o n was methylated and 6-formy1-5,7,8-trimethoxycoumarin (96) (18 mg) was i s o l a t e d by p r e p a r a t i v e l a y e r chromatography. I t was not counted but was used as such i n the next r e a c t i o n . R a d i o a c t i v e 6-hydroxy-5,7,8-trimethoxycoumarin (97) Radioactive 6-formy1-5,7,8-trimethoxycoumarin (96) (18 mg) from the previous r e a c t i o n was t r e a t e d w i t h hydrogen peroxide and s u l f u r i c a c i d and 6-hydroxy-5,7,8-trimethoxycoumarin (97) (10 mg) was i s o l a t e d and shown t o have a s p e c i f i c a c t i v i t y of 2.99x10"* dpm/mmole or 90.5% of the t o t a l a c t i v i t y of i s o p i m p i n e l l i n (2). Radi o a c t i v e 1,3-diformy1-4,6-dihydroxy-2,5-dimethoxybenzene (98) I s o p i m p i n e l l i n (2) (60 mg, 3.208x10"* dpm/mmole) was ozonized as described p r e v i o u s l y 7 and c r y s t a l l i n e 1,3-diformy1-4,6-dihydroxy-2,5-diraethoxybenzene (98) (23 mg) was i s o l a t e d and shown to have a s p e c i f i c - 135 -activity of 3.03x10 dpm/mmole or 94.5% of the total activity of isopimpinellin (2). Radioactive l>3-diformyl-2>4,5,6-tetramethoxybenzene (99) Radioactive l,3-diformyl-4,6-dihydroxy-2,5-dimethoxybenzene (98) (23 mg, 3.03x10"* dpm/mmole) from the above reaction was methylated and l,3-diformyl-2,4,5,6-tetramethoxybenzene (99) (19 mg) was isolated. This was shown to have a specific activity of 3.06x10"* dpm/mmole or 95.5% of the total activity of isopimpinellin (2). Radioactive 1,3-diacetoxy-2,4,5,6-tetramethoxybenzene (100b) l,3-Diformyl-2,4,5,6-tetramethoxybenzene (99) (19 mg, 3.06xl05 dpm/mmole) from the previous reaction was degraded as described previously'' and l,3-diacetoxy-2,4,5,6-tetramethoxybenz.ene (100b) (12 mg) was isolated and shown to have a specific activity of 3.08x10"* dpm/mmole or 96% of the total activity of isopimpinellin. Radioactive tetramethylammonium iodide (101) Radioactive isopimpinellin (2) (40 mg, 3.208x10"* dpm/mmole) was demethylated with hydriodic acid and tetramethylammonium iodide (101) (48 mg) was isolated. It was counted as previously described (page 97) and was shown to have a specific activity of 2x1.44x10^ dpm/mmole or 89.8% of the total activity of isopimpinellin. Tetramethylammonium iodide (101) was converted to tetramethylammonium picrate (102) and this was counted as previously described (page 107 ) and was shown to have a specific activity of 2x1.38x10"* dpm/mmole or 86% of the total activity of isopimpinellin (2). - 136 -Degradations of Isopiminellin (2) from Experiment 3 Radioactive 6-formyl-7-hydroxy-5,8-dimethoxycoumarin (95) 4 Isopimpinellin (2) (50 mg, 2.80x10 dpm/mmole) from Experiment 3 was ozonized and 6-formyl-7-hydroxy-5,8-dimethoxycoumarin (95) (20 mg) was isolated and shown to have a specific activity of 2.57x10* dpm/mmole or 91.8% of the total activity of isopimpinellin (2). Radioactive 1,3-diformyl-4,6-dihydroxy-2,5-dimethoxybenzene (98) 4 Isopimpinellin (2) (43 mg, 2.80x10 dpm/mmole) was ozonized as described previously'' and 1,3-diformyl-4,6-dihydroxy-2 ,5-dimethoxybenzene (98) (8.5 mg) was isolated and shown to have a specific activity of 4 2.57x10 dpm/mmole or 91.8% of the total activity of isopimpinellin (2). Radioactive tetramethylammonium. iodide (101) 4 Isopimpinellin (2) (23 mg, 2.80x10 dpm/mmole) was demethylated and tetramethylammonium iodide (101) (25 mg) was isolated and shown to have a specific activity of 2x1.44x10* dpm/mmole or a l l the original activity of isopimpinellin (2). Tetramethylammonium iodide (101) was converted to tetramethylammonium picrate (102) and this was shown to 4 have a specific activity of 2x1.26x10 dpm/mmole or 90% of the activity of isopimpinellin. Degradations of Isopimpinellin (2) from Experiment 4  6-Formyl-7-hydroxy-5,8-dimethoxycoumarin (95) Radioactive isopimpinellin (35 mg, 1.00x10"* dpm/mmole) was selectively ozonized and 6-formyl-7-hydroxy-5,8-dimethoxycoumarin (95) - 137 -(15 mg) was isolated and was shown to have a specific activity of 4 8.2x10 dpm/mmole or 82% of the total activity of isopimpinellin (2). Radioactive 1,3-diformy1-4, 6-dihydroxy-2,5-dimethoxybenzene (98) Isopimpinellin (55 mg, 1.00x10"* dpm/mg) was ozonized as described previously'' and 1,3-dif ormy 1-4,6-dihydroxy-2,5-dimethoxybenzene (98) (19.5 mg) was isolated and shown to have a specific activity of 4 9.27x10 dpm/mmole or 92.7% of the total activity of isopimpinellin (2). Radioactive 1,3-diformy1-2,4,5,6-tetramethoxybenzene (99) Radioactive 1,3-diformyl-4,6-dihydroxy-2,5-dimethoxybenzene (98) 4 (19.5 mg, 9.27x10 dpm/mmole) from the previous reaction was methylated and l,3-diformyl-2,4,5,6-tetramethoxybenzene (99) (16 mg) was isolated. It was not counted but was used as such in the next reaction. Radioactive 1,3-diacetoxy-2,4,5,6-tetramethoxybenzene (100b) l,3-Diformyl-2,4,5,6-tetramethoxybenzene (99) (16 mg) from the previous reaction was degraded to 1,3-diacetoxy-2,4,5,6-tetramethoxy-benzene (100b) (10 mg). This was shown to have a specific activity of 9.17x10* dpm/mmole or 91.7% of the total activity of 2. Radioactive tetramethylammonium iodide (101) Isopimpinellin (2) (25 mg, 1.00x10"* dpm/mmole) from Experiment 4 was demethylated and tetramethylammonium iodide (101) (28 mg) was 4 isolated and shown to have a specific activity of 2x4.75x10 dpm/mmole or 95% of the total activity of isopimpinellin. - 138 -Degradations of Alloimperatorin Methyl Ether (7) from Experiment 2 Periodic acid cleavage of 5-(2',3'-dihvdroxy-3'-methylbutyl)-8- methoxypsoralen (3) (alloimperatorin methyl ether diol) Radioactive alloimperatorin methyl ether diol (3) (67.5 mg, 3.29x10 dpm/mmole) from Experiment 2 was cleaved with periodic acid and acetone p_-bromobenzenesulfonylhydrazone (106) (41 mg) was isolated and shown 3 to have a specific activity of 2.19x10 dpm/mmole or 6.7% of the total activity of 3. Radioactive 5-(2'-acetoxy-3'-hydroxy-3'-methylbutyl)-8-methoxy- psoralen (107) Alloimperatorin methyl ether diol (3) from Experiment 2 was acetylated to 5-(2'-acetoxy-3'-hydroxy-3'-methylbutyl)-8-methoxypsoralen (107). Radioactive 5-(2'-acetoxy-3'-hydroxy-3'-methylbutyl)-6-formy1-7- hydroxy-8-methoxycoumarin (108) Radioactive 5-(2'-acetoxy-3'-hydroxy-3'-methylbutyl)-8-methyl-4 psoralen (107) (56 mg, 3.29x10 dpm/mmole) was selectively ozonized to 5-(2'-acetoxy-3'-hydroxy-3'-methylbutyl)-6-formyl-7-hydroxy-8-methyoxy-coumarin (108) (36 mg). This was not counted but was used as such in the next reaction. Radioactive 5-(2'-acetoxy-3'-hydroxy-3'-methylbutyl)-6-formy1-7,8- dimethoxycoumarin (109) 5-(2'-Acetoxy-3'-hydroxy-3'-methylbutyl)-6-formyl-7-hydroxy-8-methoxycoumarin (108) (36 mg) from the previous reaction was methylated - 139 -to give 5-(2'-acetoxy-3'-hydroxy-3'-methylbutyl)-6-formyl-7,8-dimethoxycoumarin (109) (15 mg). This was shown to have a s p e c i f i c 4 a c t i v i t y of 2.91x10 dpm/mmole or 88.5% of the t o t a l a c t i v i t y of 107. Degradations of A l l o i m p e r a t o r i n Methyl Ether (7) from Experiment 3 P e r i o d i c a c i d cleavage of a l l o i m p e r a t o r i n methyl ether d i o l (3) Rad i o a c t i v e a l l o i m p e r a t o r i n methyl ether d i o l (3) (55 mg, 1.10x10 dpm/mmole was cleaved w i t h p e r i o d i c a c i d as p r e v i o u s l y d e s c r i b e d 7 and 5-(2'-hydroxyethyl)-8-methoxypsoralen (105) (25.8 mg) was i s o l a t e d and was shown to have a s p e c i f i c a c t i v i t y of 1.00x10^ dpm/mmole or 91.5% of the t o t a l a c t i v i t y of 3. A l s o i s o l a t e d was acetone p_-bromo-benzenesulfonylhydrazone (106) (11 mg). This was shown to have a 2 s p e c i f i c a c t i v i t y of 5.72x10 dpm/mmole or 5.2% of the t o t a l a c t i v i t y of 3. Demethylation of r a d i o a c t i v e 5-(2'-hydroxyethyl)-8-methoxy  psoralen (105) Radio a c t i v e 5-(2'-hydroxyethyl)-8-methoxypsoralen (105) (23 mg, 4 1.00x10 dpm/mmole) from the previous r e a c t i o n was demethylated and tetramethylammonium i o d i d e (101) (10 mg) was i s o l a t e d and was shown 3 to have a s p e c i f i c a c t i v i t y of 7.70x10 dpm/mmole or 70% of the t o t a l a c t i v i t y of 3. Radioactive 5-(2'-acetoxy-3'-hydroxy-3'-methylbutyl)-8-methoxy- pso r a l e n (107) Radioactive a l l o i m p e r a t o r i n methyl ether d i o l (3) from Experiment 3 was a c e t y l a t e d to 5-(2'-acetoxy-3'-hydroxy-3'-methylbutyl)-8-- 140 -methoxypsoralen (107). Ra d i o a c t i v e 5-(2'-acetoxy-3'-hydroxy-3'-methylbutyl)-6-formyl-7- hydroxy-8-methoxycoumarin (108) 4 R a d i o a c t i v e 107 (67 mg, 1.10x10 dpm/mmole) was s e l e c t i v e l y ozonized and 5-(2'-acetoxy-3'-hydroxy-3'-methylbutyl)-6-formy1-7-hydroxy-8-methoxycoumarin (108) (32 mg) was i s o l a t e d . This was shown 4 to have a s p e c i f i c a c t i v i t y of 1.10x10 dpm/mmole or a l l the a c t i v i t y of 107. Rad i o a c t i v e 5-(2'-acetoxy-3'-hydroxy-3'-methylbutyl)-6-formy1-7,8- dimethoxycoumarin (109) Rad i o a c t i v e 5-(2'-acetoxy-3'-hydroxy-3'-methylbutyl)-6-formy1-7-4 hydroxy-8-methoxycoumarin (108) (32 mg, 1.10x10 dpm/mmole) from the previous r e a c t i o n was methylated and 5-(2'-acetoxy-3'-hydroxy-3 1-methylbutyl)-6-formyl-7,8-dimethoxycoumarin (109) (18.5 mg) was i s o l a t e d . This was not counted but was used as such f o r the next r e a c t i o n . R a d i o a c t i v e 5-(2'-acetoxy-3'-hydroxy-3'-methylbutyl)-6-acetoxy- 7,8-dimethoxycoumarin (110b) Radi o a c t i v e 109 (18.5 mg) from the above r e a c t i o n was degraded and 110b (8.3 mg) was i s o l a t e d and shown to have a s p e c i f i c a c t i v i t y 4 of 1.06x10 dpm/mmole or 96.4% of the t o t a l a c t i v i t y of 107. - 141 -R a d i o a c t i v e 1-(2'-acetoxy-3'-hydroxy-3'-methylbutyl)-2,6-dlformyl- 3,5-dihydroxy-4-methoxybenzene (111) Rad i o a c t i v e 107 (50 mg, 1.10x10* dpm/mmole) from Experiment 3 was ozonized as described p r e v i o u s l y ^ and 111 (15 mg) was i s o l a t e d . This 4 was shown to have a s p e c i f i c a c t i v i t y of 1.04x10 dpm/mmole or 99.5% ofthe t o t a l a c t i v i t y of 107. Degradations of A l l o i m p e r a t o r i n Methyl Ether (7) from Experiment 4 P e r i o d i c a c i d cleavage of a l l o i m p e r a t o r i n methyl ether d i o l (3) 4 R a d i o a c t i v e a l l o i m p e r a t o r i n methyl ether d i o l (3) (50 mg, 1.22x10 dpm/mmole)'from Experiment 4 was cleaved w i t h p e r i o d i c a c i d and 5-(2'-hydroxyethyl)-8-methoxypsoralen (105) (32 mg) was i s o l a t e d and shown 4 to have a s p e c i f i c a c t i v i t y of 1.11x10 dpm/mmole or 92% of the t o t a l a c t i v i t y of 3. Demethylation of r a d i o a c t i v e 5-(2'-hydroxyethyl)-8-methoxy- ps o r a l e n (105) Radio a c t i v e 5-(2'-hydroxyethyl)-8-methoxypsoralen (105) (20 mg, 4 1.11x10 dpm/mmole) from the previous r e a c t i o n was demethylated and tetramethylammonium i o d i d e (101) (12 mg) was i s o l a t e d and was shown 4 to have a s p e c i f i c a c t i v i t y of 1.12x10 dpm/mmole or 92% of the o r i g i n a l a c t i v i t y of 3. Tetramethylammonium i o d i d e (101) was converted to tetramethylammonium p i c r a t e (102) which was shown to have a s p e c i f i c 4 a c t i v i t y of 1.18x10 dpm/mmole or 96.6% of the t o t a l a c t i v i t y of 3. - 142 -Radio a c t i v e 5-(2'-acetoxy-3'-hydroxy-3'-methylbutyl)-6-formy1-7- hydroxy-8-methoxycoumarin (108). T r i a l a Radio a c t i v e 5-(2'-acetoxy-3'-hydroxy-3'-methylbutyl)-8-methoxy-4 p s o r a l e n (107) (60 mg, 1.22x10 dpm/mmole) was s e l e c t i v e l y ozonized and 5-(2'-acetoxy-3'-hydroxy-3'-methylbutyl)-6-formyl-7-hydroxy-8-methoxycoumarin (108) (25 mg) was i s o l a t e d and was shown to have a 4 s p e c i f i c a c t i v i t y of 1.18x10 dpm/mmole or 96.6% of the t o t a l a c t i v i t y of 107. Radioactive 5-(2'-acetoxy-3'-hydroxy-3'-methylbutyl)-6-formyl-7-hydroxy-8-methoxycoumarin (108). T r i a l b 4 R a d i o a c t i v e 107 (60 mg, 4.85x10 dpm/mmole) from Experiment 4 was s e l e c t i v e l y ozonized and 5-(2'-acetoxy-3'-hydroxy-3'-methylbutyl)-6-formyl-7-hydroxy-8-methoxycoumarin (108) (15 mg) was i s o l a t e d and 4 shown to have a s p e c i f i c a c t i v i t y of 4.73x10 dpm/mmole or 97.5% of the t o t a l a c t i v i t y of 107. - 143 -BIBLIOGRAPHY 1. E.L. Bennett and J . Bonner, Amer. J . Botany, 40, 29 (1953). 2. W.H. M u l l e r and C H . M u l l e r , Amer. J . Botany, 43, 354 (1956). 3. T.H. Kearney and R.H. Peebles, A r i z o n a F l o r a , U n i v e r s i t y of C a l i f o r n i a P r e s s , Berkeley and Los Angeles, 1960, p. 494. 4. D.L. Dreyer, Tetrahedron, 22_, 2923 (1966). 5. J.P. Kutney, T. Inaba and D.L. Dreyer, J . Amer. Chem. Soc., 90, 813 (1968). 6. J.P. Kutney, T. Inaba and D.L. Dreyer, Tetrahedron, 26_, 3171 (1970). 7. R.N. 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