Plant Cell Reports (1981) 1 : 36-39

Plant Cell Reports © Springer-Verlag 1981

Indole Alkaloids from Cell Suspension Cultures of Rauwolfia serpentinaBenth. Joachim St6ckigt ~, Artur Pfitzner ~, and Joachim Firl ~,2 Lehrsmhl Pharmazeutische Biologie, Ludwig-Maximilians-UniversitSt, D-8000 Miinchen, Federal Republic of Germany 20rganisch-Chemisches Institut der TU Mfinchen, D-8046 Garching, Federal Republic of Germany Received July 22, 1981

ABSTRACT Twelve i n d o l e a l k a l o i d s b e l o n g i n g to the Ajmaline-, S a r p a g i n e - , Y o h i m b i n e - , and H e t e r o y o h i m b i n e - t y p e have been i s o l a t e d and i d e n t i f i ed from c e l l s u s p e n s i o n c u l t u r e s of Rauwolfia e e r p e n t i n a . Ten o f the a l k a l o i d s were f o u n d f o r the f i r s t time i n c u l t u r e d R. s e r p e n t i n a cells. The y i e l d o f the main a l k a l o i d v o m i l e n i n e was 51 t i m e s more t h a n t h a t o f d i f f e r e n t i a t e d p l a n t s . Crude enzymes i s o l a t e d from this cell suspension culture c o m p l e t e l y meta b o l i z e the b i o g e n e t i c p r e c u r s o r s t r i c t o s i d i ne u n d e r f o r m a t i o n o f s e v e r a l a l k a l o i d a l compounds. INTRODUCTION The b i o s y n t h e t i c pathway l e a d i n g to i n d o l e a l k a l o i d s o f the h e t e r o y o h i m b i n e - t y p e has been successfully investigated a t the c e l l - f r e e level u s i n g c e l l s u s p e n s i o n c u l t u r e s as an e n z yme s o u r c e (Zenk 1 9 8 0 ) . Our p r e s e n t knowledge on the b i o g e n e t i c f o r m a t i o n o f a l k a l o i d s of the y o h i m b i n e - and a j m a l i n e - t y p e s , however,is rather small and o n l y based on a l i m i t e d number o f i n vimo f e e d i n g e x p e r i m e n t s (see Cotd e l l 1 9 7 4 ) . One o f the b a s i c p r e r e q u i s i t e s for elucidating t h o s e b i o g e n e t i c pathways at a cell-free l e v e l w o u l d be the s y n t h e s i s o f the corresponding indole alkaloids i n c u l t u r e d cel l s . We, t h e r e f o r e , d e t e r m i n e d the g e n e r a l a l k a l o i d p a t t e r n i n two s t r a i n s o f c e l l s u s p e n sion cultures, initiated from seeds o f Rauwo-

lfia

serpentina.

EXPERIMENTAL AND RESULTS Spectrometric Determinations: UV s p e c t r a were o b t a i n e d on a P e r k i n Elmer S p e c t r o p h o t o m e t e r 551S, i n case o f b e i n g n e c c e s s a r y UV maxima and minima were d e t e r m i n e d by the method o f the second d e v i a t i o n . Mass s p e c t r a (MS) w e r e measured on a F i n n i g a n MAT 44S a t 70 eV, NMR a n a l y s e s were p e r f o r m e d on a B r u c k e r WP 200 instrument. Plant Material, Extraction, S e p a r a t i o n and Identification of Alkaloids: Cell suspension c u l t u r e s o f Rauwolfia serpentina ( s t r a i n C and F) were grown f o r 13 days i n a s t a n d a r d p r o d u c t i o n medium, d e v e l o p e d by Zenk et a l . (1977). After filtration by s u c t i o n , c e l l s were f r o z e n w i t h l i q u i d n i t r o g e n and the a l k a l o i d a l prod-

0721-085X/81/0001/0036/$

01.00

d u c t s e x t r a c t e d as d e s c r i b e d p r e v i o u s l y by St~Sckigt and S o l l ( 1 9 8 0 ) , e x c e p t t h a t 1 M p h o s p h a t e b u f f e r (pH 8 . 5 ) was added i n s t e a d o f ammonia. 2 kg o f c e l l s ( c o r r e s p o n d i n g to 127 9 d r y w e i g h t ) were s t i r r e d w i t h 3 I ethyl a c e t a t e and 200 ml b u f f e r f o r 24 h The org a n i c l a y e r was s e p a r a t e d ( f r a c t i o n i ) and the r e m a i n i n g c e l l m a t e r i a l e x t r a c t e d w i t h methanol ( f r a c t i o n ii). F r a c t i o n I was w o r k ed up as p u b l i s h e d by S t b c k i g t and S o l l (1980) and the r e s i d u e c r y s t a l l i z e d 3 t i m e s from methanol yielding 160 mg c o l o u r l e s s c r y s t a l l s (m.p. 1 9 6 - 2 0 4 ° C ) , UV (MeOH) x max 218, 257; h min 237 nm. MS m/e ( t e l . i n t . % ) 1 6 8 ( 1 9 ) ;

169 i00

170 18 ; 1831=) 50 71 IR (KBr

1030. Th e -OH), 1740 cm-Z 29 ) ,• 123 UV s p e c t r u m and the bathochrome s h i f t in aci d i c s o l u t i o n were t y p i c a l f o r an i n d o l e n i n e structure. MS and IR data were i n agreement with those published for vomilenine (Taylor e t a l . 1 9 6 2 ) . The g e n e r a l s t r u c t u r e o f the i s o l a t e d compound was f u r t h e r d e t e r m i n e d by a c e t y l a t i o n ( a c e t y l v o m i l e n i n e , MS m/e ( r e l . i n t . % ) 169(100); 183(15); 291(21); 333(42); 349(6); 392(21) and by the chemical conversion to perakine (Taylor et al. 1962). Even a f t e r repeated r e c r y s t a l l i s a t i o n of vomilenine the reported m.p. of 207°C was not reached. The IH-NMR-spectrum c l e a r l y i s i n agreement w i t h the e s t a b l i s h e d s t r u c t u r e of vomilenine [IH-NMR(CDCI3): 7.64 ( d , C12H), 7.46 (d,C9H), 7.42 ( t r , C 1 1 H ) , 7.24 ( t r ~ CIOH), 5.74 ( q , C19H), 5.03 (s b r o a d , C~±H), 4.98 ( s , D17H), 4.33 (m, C3H), 3.95 ( t r b r o a d , CZH), 3.28 j m , C15H), 2.77 (dd, C6H#), 2.42 ( t r b r o a d , CIOH), 2.17 ( s , OCOCH3), 1.89 (m, C14H2), 1.72 ( d , C6Ha), 1.67 (d, C 1 8 H 3 ) ] . However, the s p e c t rum a l s o d e m o n s t r a t e d the presence o f a s e c ond compound ( a b o u t 15 % o f the t o t a l ) which most p r o b a b l y i s an i s o m e r o f v o m i l e n i n e . T h i s m i x t u r e o f isomers c o u l d n o t be s e p a r a t e d by crystallisation, TLC, HPLC ( s t r a i g h t or r e v erse phase) or c a p i l l a r y GC-MS o f the c o r r e sponding acetylated mixture (condition for GC-MS a n a l y s i s : 20 m c a p i l l a r y column SE 54, f l o w 2.5 ml, program 240-260°C w i t h 30°C/min). The m o t h e r l i q u o r s of the vomilenine crystalls contained different alkaloids separated as f o l l o w s : A f t e r e v a p o r a t i o n o f the s o l v e n t the r e s i d u e was s t i r r e d w i t h 1.7 % p h o s p h o r i c a c i d , the s o l u t i o n a d j u s t e d to pH 3 and e x t r a c t e d w i t h c h l o r o f o r m . The aquous l a y e r was

3? i n a y i e l d o f 0.2 % o r 57 mg/l medium (Tabl e i ) . I t is w o r t h m e n t i o n i n g t h a t t h i s a l k ~ l o i d has n o t been found in d i f f e r e n t i a t e d p l a n t s o f R. s e r p e n t i n a but o n l y as a m i n o r a l k a l o i d i n R. v o m i t o r i a (Hofmann and Frey L957). M o r e o v e r , the c o n t e n t o f v o m i l e n i n e in c u i t u r e d c e l l s exceeded t h a t found in R. v o m i t o r i a p l a n t s by a f a c t o r o f 51. As a l r e a d y d e s c r i b e d f o r t a b e r s o n i n e and v i n e r v i n e in C a t h a r a n t h u s roseus c u l t u r e s by S t ~ c k i g t and S o l l (1980)~ t h i s i s the t h i r d example i n the a l k a l o i d f i e l d showing t h a t trace alkaloids of differentiated p l a n t s can be formed as main p r o d u c t s i n c e l l s u s p e n s i on c u l t u r e s . 1H-and 13C-NMR a n a l y s i s o f the isolated vomilenine revealed that it represe n t e d an a b o u t 85 : 15 i s o m e r m i x t u r e o f vom i l e n i n e and an i s o m e r o f i t . But the s e p a r a t i o n of the isomers has n o t been a c h i e v e d by the methods d e s c r i b e d in the e x p e r i m e n t a l part till now. The o t h e r a l k a l o i d s o f t h i s group found were a j m a l i n e ( 0 . 0 0 7 %), 1 7 - 0 acetylajmaline, 1 7 - O - a c e t y l n o r a j m a l i n e and the i n d o l e n i n e v i n o r i n e . P r i o r to t h i s i n v e stigation, 17-O-acetylnorajmaline has n o t been d e t e c t e d as a n a t u r a l l y occurring alkal o i d . Since the i n d o l e n i n e a l k a l o i d v i n o r i n e has been o b s e r v e d to be p r e s e n t o n l y in V i n ca minor p l a n t s ( M e i s e l and Dbpke 1 9 7 1 ) , the f o r m a t i o n in the s p e c i e s o f R a u w o l f i a i s r e p o r t e d here f o r the f i r s t time. C o n c e r n i n g the y o h i m b i n e - t y p e , two a l k a l o i d s were s y n t h e z i s e d by the c u l t u r e s , y o h i m b i n e o n l y as a m i n o r p r o d u c t (