Science, Faculty of
Chemistry, Department of
DSpace
UBCV
Kuri-Brena, Francisco
Doctor of Philosophy - PhD
University of British Columbia
This thesis investigates the use of plant cell cultures in combination with synthetic chemistry to provide new routes to biologically active compounds. The first chapter consists of approaches to the lignan podophyllotoxin (4), by means of enzyme catalyzed ring closure reactions of dibenzylbutyrolactone intermediates 56 and 57 mediated by Podophyllum peltatum cells. The substrates 56 and 57 studied were synthesized from readily available aromatic aldehydes employing a "one pot", 1,4addition- enolate alkylation sequence, as the key step. Semi-continuous biotransformation experiments performed with 56 illustrate the potential of the technique yielding the ring-closed products 75 and 76. A possible mechanism for the ring closure in vivo is provided, as well as suggestions to alter the stereochemical outcome of the process. The second chapter of this thesis concerns the attempts to oxidize dehydroisoabietanolide (132) with crude enzyme preparations (CFEs) from Tripterygium wilfordii, as part of an ongoing study regarding the biogenesis of the diterpcne triepoxides tripdiolide (5) and triptolide (91). The 12-hydroxy analogue of 132, isotriptophenolide (190), is also studied. Both compounds were prepared in gram quantities from dehydroabietic acid (122). The results from the biotransformation experiments, suggest that triptophenolide (163), an isomer of 190 and bearing a phenolic group at C-14, is a likely candidate for future biotransformation experiments. The present studies also suggest that an "activated" diterpene, that is, a substrate bearing a hydroxyl group in the aromatic ring (190 or an isomer), is a biogenetic precursor for the triepoxide system present in 5 and 91.
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S T U D I E S O N P L A N T C E L L C U L T U R E S O F P O D O P H Y L L U M P E L T A T U M A N D T R I P T E R Y G I U M W I L F O R D I I F O R B I O S Y N T H E S I S O F B I O L O G I C A L L Y A C T I V E C O M P O U N D S . by F R A N C I S C O K U R I - B R E N A B.Sc. (Chemistry), Universidad Nacional Autonoma de Mexico, 1985 M.Sc. (Chemistry), Universidad Nacional Autonoma de Mexico, 1987 A THESIS S U B M I T T E D I N P A R T I A L F U L F I L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E OF D O C T O R O F P H I L O S O P H Y in T H E F A C U L T Y O F G R A D U A T E STUDIES C H E M I S T R Y We accept this thesis as conforming to the required standard. T H E 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 26 August 1992 ® Francisco Kur i -Brena , 1992. In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Chemis t ry The University of British Columbia Vancouver, Canada Date Oc t . 1992 DE-6 (2/88) A B S T R A C T This thesis investigates the use of plant cell cultures in combination with synthetic chemistry to provide new routes to biologically active compounds. The first chapter consists of approaches to the lignan podophyllotoxin (4), by means of enzyme catalyzed ring closure reactions of dibenzylbutyrolactone intermediates 56 and 57 mediated by Podophyllum peltatum cells. The substrates 56 and 57 studied were synthesized from readily available aromatic aldehydes employing a "one pot", 1,4-addition-enolate alkylat ion sequence, as the key step. Semi-continuous biotransformation experiments performed with 56 illustrate the potential of the technique yielding the ring-closed products 75 and 76. A possible mechanism for the ring closure in vivo is provided, as well as suggestions to alter the stereochemical outcome of the process. The second chapter of this thesis concerns the attempts to oxidize dehydroisoabietanolide (132) with crude enzyme preparations (CFEs) from Tripterygium wilfordii, as part of an ongoing study regarding the biogenesis of the diterpcne triepoxides tripdiolide (5) and triptolide (91). The 12-hydroxy analogue of 132, isotriptophenolide (190), is also studied. Both compounds were prepared in gram quantities from dehydroabietic acid (122). The results from the biotransformation experiments, suggest that triptophenolide (163), an isomer of 190 and bearing a phenolic group at C-14, is a l ikely candidate for future biotransformation experiments. The present studies also suggest that an "activated" diterpene, that is, a substrate bearing a hydroxyl group in the aromatic ring (190 or an isomer), is a biogenetic precursor for the triepoxide system present in 5 and 91. OH OH OCH3 Podophyllotoxin (4) 56: Ri= H, Rg» i-Pr 57: Ri,R2= methylene Dehydroisoabietanoiide (132) Isotriptophenolide (190) R= H ; Triptolide (91 ) Dehydroabietic acid (122) R= OH; Tripdiolide (5) TABLE OF CONTENTS A b s t r a c t i i Table o f Co: c e n t s i v L i s t o f F i g u r e s v i i i L i s t o f Schemes i x L i s t o f Tables x i i L i s t o f A b b r e v i a t i o n s x i i i Acknowledgements xv Foreword 1 Chapter 1. The P o d o p h y l l o t o x i n F a m i l y . 8 I n t r o d u c t i o n . 8 T o t a l Syntheses . 11 1. K i n e t i c p r o t o n a t i o n o f p i c r o p o d o p h y l l o t o x i n 11 2. D i e l s - A l d e r approach 13 3. Tandem M i c h a e l a d d i t i o n - a l d o l condensa t ion approach 16 The B i o s y n t h e s i s o f P o d o p h y l l o t o x i n s . 22 Techniques o f C e l l Suspension C u l t u r e . Premise o f t h i s Work. 27 P r o d u c t i o n o f Podophy l lo tox ins by P l a n t T i s sue C u l t u r e s . 30 O b j e c t i v e s o f t h i s I n v e s t i g a t i o n . 33 R e s u l t s and D i s c u s s i o n . 34 Syn thes i s o f r r a i3 s -2 - (4 -hyd roxy -3 ,5 -d ime thoxybenzy l ) -3 -(3 -hyd roxy -4 - i sop ropoxy - Q -hyd roxybenzy l )bu t ano l i de (56) . 34 The B i o t r a n s f o r m a t i o n o f r r a i7S-2 - (4 -hydroxy-3 ,5 -d ime thoxybenzy l ) -3 - ( 3 - h y d r o x y - 4 - i s o p r o p o x y - Q - h y d r o x y b e n z y l ) b u t a n o l i d e (56) w i t h P . peltatum C e l l Suspension C u l t u r e s . 43 I s o l a t i o n and S t r u c t u r e E l u c i d a t i o n o f the Major B i o t r a n s f o r m a t i o n Products o f 56 w i t h P . peltatum C e l l Suspension C u l t u r e s . 44 The Semi-cont inuous B i o t r a n s f o r m a t i o n o f Subs t ra te 56 w i t h P. peltatum C e l l Suspension C u l t u r e s . 54 B i o t r a n s f o r m a t i o n R e p r o d u c i b i l i t y . Summary o f the Semi-Continuous B i o t r a n s f o r m a t i o n Exper iments . 63 The B i o t r a n s f o r m a t i o n o f 1 - (3 ,5 -d ime thoxy-4-hydroxypheny l ) -6 ,4 -d i h y d r o x y - 3 - h y d r o x y m e t h y l - 7 - i s o p r o p o x y - 1 , 2 , 3 , 4 - t e t r a h y d r o - 2 -naphthoic a c i d 7 - l a c tone (75) w i t h P. peltatum C e l l Suspension C u l t u r e s . 64 Syn thes i s o f r r a n 5 - 2 - ( 4 - h y d r o x y - 3 , 5 - d i m e t h o x y b e n z y l ) - 3 - ( 3 , 4 -me thy l ened ioxy ) -7 -hyd roxybenzy l )bu t ano l ide (57). 67 B i o t r a n s f o r m a t i o n o f r r a n s - 2 - ( 4 - h y d r o x y - 3 , 5 - d i m e t h o x y b e n z y l ) -3 - ( 3 , 4 - m e t h y l e n e d i o x y ) - 7 - h y d r o x y b e n z y l ) b u t a n o l i d e (57) w i t h P . peltatum C e l l Suspension C u l t u r e s . 70 Pha rmaco log ica l Screen ing o f the C e l l - p r o d u c e d M e t a b o l i t e s , and S y n t h e t i c Subs t r a t e s . 73 Summary. 74 Future Work. 74 Chapter 2 . The T r i p d i o l i d e F a m i l y . 78 I n t r o d u c t i o n . 78 Chemical Impairment o f Spermatogenesis . 80 Other C l i n i c a l and Pharmaceut ica l Uses o f the P l a n t . 81 T o t a l Syntheses . 82 Abie tane and abeo-abietane Bios3rn thes i s . 93 P r o d u c t i o n o f T r i p d i o l i d e (5) and T r i p t o l i d e (91) by P l a n t C e l l T i s s u e C u l t u r e s . 101 O b j e c t i v e s o f T h i s I n v e s t i g a t i o n . 103 R e s u l t s and D i s c u s s i o n . 104 Syn thes i s o f D e h y d r o i s o a b i e t a n o l i d e (132) . 104 D e h y d r o i s o a b i e t a n o l i d e (132) B i o t r a n s f o r m a t i o n w i t h C e l l Free E x t r a c t s (CFE) d e r i v e d from the TRP4a C e l l L i n e . 108 D e h y d r o i s o a b i e t a n o l i d e (132) B i o t r a n s f o r m a t i o n w i t h TRP4a C e l l s . 112 D e h y d r o i s o a b i e t a n o l i d e (132) O x i d a t i o n w i t h Hortierella isabellina. 113 The Synthes i s o f a Ring C - a c t i v a t e d Subs t r a t e . 114 I s o t r i p t o p h e n o l i d e (190) B i o t r a n s f o r m a t i o n w i t h TRP4a C e l l s . 118 C o n c l u s i o n s . 120 Exper imen ta l S e c t i o n . 122 G e n e r a l . 123 3-Hydroxy-4- isopropoxybenzaldehyde (63) 125 3-Benzyloxy-4- i sopropoxybenza ldehyde (64) 127 3- B e n z y l o x y - 1 - b i s ( p h e n y l t h i o ) m e t h y l - 4 - i s o p r o p o x y b e n z e n e (65) 128 4- Benzyloxy-3 ,5-d imethoxybenzaldehyde (67a) 129 4 -Benzy loxy -3 ,5 -d ime thoxybenzy l a l c o h o l (67b) 130 4 -Benzy loxy -3 ,5 -d ime thoxybenzy l bromide (67) 131 r r a n s - 2 - ( 4 - b e n z y l o x y - 3 , 5 - d i m e t h o x y b e n z y l ) - 3 - ( 3 - b e n z y l o x y - 4 -i s o p r o p o x y - a , a - b i s ( p h e n y l t h i o ) b e n z y l ) b u t a n o l i d e (68) 132 Trans-2-(4-benzyloxy-3,5-dimethoxybenzyl)-3-(3-benzyloxy-4-i s o p r o p o x y - Q - o x o - b e n z y l ) b u t a n o l i d e (69) 133 Trans-2-(4-benzyloxy-3,5-dimethoxybenzyl)-3-(3-benzyloxy-4-i sopropoxy-7 ' /9 -hydroxybenzy l )bu tano l ide (70) 135 r r a n s - 2 - ( 4 - h y d r o x y - 3 , 5 - d i m e t h o x y b e n z y l ) - 3 - ( 3 - h y d r o x y - 4 -i sopropoxy-7 'y9-hydroxybenzy l )bu tano l ide (56) 136 r r a n s - 2 - ( 4 - H y d r o x y - 3 , 5 - d i m e t h o x y b e n z y l ) - 3 - ( 3 - b e n z y l o x y - 4 -i sopropoxy-7 'y9-hydroxybenzy l )bu tano l ide (71) 137 r r a n s - 2 - ( 4 - h y d r o x y - 3 , 5 - d i m e t h o x y b e n z y l ) - 3 - ( 3 - h y d r o x y - 4 -i sop ropoxybenzy l )bu t ano l i de (54) 138 l -B i s (pheny l t h io )me thy l -3 ,4 -me thy l ened ioxybenzene (82) 138 r r a n s - 2 - ( 4 - b e n z y l o x y - 3 , 5 - d i m e t h o x y b e n z y l ) - 3 - [ 3 , 4 - m e t h y l e n e d i o x y -û , a - b i s ( p h e n y l t h i o ) b e n z y l ] b u t a n o l i d e (83) 139 r r a n s - 2 - ( 4 - b e n z y l o x y - 3 , 5 - d i m e t h o x y b e n z y l ) - 3 - ( 3 , 4 - m e t h y l e n e d i o x y -û - o x o - b e n z y l ) b u t a n o l i d e (84) 140 r r a n s - 2 - ( 4 - b e n z y l o x y - 3 , 5 - d i m e t h o x y b e n z y l ) - 3 - ( 3 , 4 - m e t h y l e n e d i o x y -7 ' /3 -hydroxybenzyl )bu tano l ide (85) 142 Trans-2-(4-hydroxy-3,5-dimethoxybenzyl)-3-(3,4-methylenedioxy-7 ' ;9 -hydroxybenzyl )bu tano l ide (57) 143 Genera l Procedure f o r Subcul tu re of P l a n t Suspension C u l t u r e s . 144 Media P r e p a r a t i o n s . 145 B i o t r a n s f o r m a t i o n o f Subs t ra te 56 w i t h P . peltatum C e l l Suspension C u l t u r e . 148 A r y l T e t r a l i n 75. S t a b i l i t y S t u d i e s . 153 B i o t r a n s f o r m a t i o n o f Subs t ra te 57 w i t h P . peltatum C e l l Suspension C u l t u r e . 154 Dehydroab ie t i c A c i d (122) P u r i f i c a t i o n . 157 Dehydroab ie t i c A c i d A n a l y s i s by Gas Chromatography. 157 18- N o r a b i e t a - 4 ( 1 9 ) 8 , 1 1 . 1 3 - t e t r a e n e (150) 158 1 8 , 1 9 - D i n o r a b i e t a - 8 , l l , 1 3 - t r i e n - 4 - o n e (151) 160 3 - D i m e t h y l t h i o m e t h y l e n e - 1 8 , 1 9 - d i n o r a b i e t a - 8 , l l , 1 3 - t r i e n - 4 - o n e (152) 161 19- Hydroxy-18(4 -• 3 ) a b e o a b i e t a - 3 , 8 , 1 1 , 1 2 - t e t r a e n - 1 8 - o i c a c i d l a c tone (132) 162 12 -Ace ty l -19 -hyd roxy -18 (4 3 ) a b e o a b i e t a - 3 , 8 , 1 1 , 1 3 - t e t r a e n -1 8 - o i c a c i d l a c tone (192) 164 12-Acetoxy-19-hydroxy-18(4 3 ) a b e o a b i e t a - 3 , 8 , 1 1 , 1 3 - t e t r a e n -1 8 - o i c a c i d l ac tone (193) 165 12 ,19-Dihydroxy-18(4 3 ) a b e o a b i e t a - 3 , 8 , 1 1 , 1 3 - t e t r a e n - 1 8 -o i c a c i d l ac tone (190) 166 D e h y d r o i s o a b i e t a n o l i d e B i o t r a n s f o r m a t i o n : TRP4a C e l l Free E x t r a c t Exper iments , and C u l t u r e C h a r a c t e r i z a t i o n . Attempted B i o t r a n s f o r m a t i o n o f D e h y d r o i s o a b i e t a n o l i d e (132) . 167 Measurement o f P r o t e i n C o n c e n t r a t i o n : B i o - R a d P r o t e i n Assay . 168 Measurement o f Peroxidase A c t i v i t y : P y r o g a l l o l - P u r p u r o g a l l i n Assay . 168 D e h y d r o i s o a b i e t a n o l i d e B i o t r a n s f o r m a t i o n : Assessment o f Membrane-bound Enzymes and C o f a c t o r s . 169 D e h y d r o i s o a b i e t a n o l i d e B i o t r a n s f o r m a t i o n : TRP4a Whole C e l l Exper iments . 170 D e h y d r o i s o a b i e t a n o l i d e B i o t r a n s f o r m a t i o n : Hortierella isabellina Exper iments . 171 I s o t r i p t o p h e n o l i d e B i o t r a n s f o r m a t i o n w i t h T. wilfordii Whole C e l l s . 172 B i b l i o g r a p h y 173 LIST OF FIGURES F i g u r e 1. C o u p l i n g o f ca tha ran th ine (1) and v i n d o l i n e ( 2 ) . 6 F i g u r e 2 . B r i e f h i s t o r y o f e topos ide development - from f o l k medic ine to c l i n i c a l d rug . 9 F i g u r e 3. The s t r u c t u r e o f 4 ' - d e m e t h y l e p i p o d o p h y l l o t o x i n ( 6 ) , e topos ide ( 7 ) , and t e n i p o s i d e ( 8 ) . 10 F igu re 4 . The e q u i l i b r i u m between p o d o p h y l l o t o x i n (4) and p i c r o p o d o p h y l l o t o x i n ( 9 ) . 11 F igu re 5. The s y n t h e t i c p recu r so r s used i n p rev ious b i o t r a n s -fo rmat ion experiments i n P r o f . K u t n e y ' s group, and i n v o l v i n g c e l l f ree e x t r a c t s o f C. roseus. 26 F igu re 6. Model curve r e l a t i n g c e l l number per u n i t volume o f c u l t u r e to time i n a ba tch grown p l a n t c e l l suspens ion c u l t u r e . Phases o f the growth c y c l e are l a b e l l e d . 28 F igu re 7. Some o f the l i g n a n s o f P . peltatum. 31 F igu re 8. E x t r a c t i o n procedure f o r the P . peltatum c e l l s . 45 F igu re 9. E x t r a c t i o n procedure f o r P . peltatum spent medium. 46 F igure 10. Chromatographic s epa ra t i on o f the b r o t h e x t r a c t . 47 F igure 11. B i o t r a n s f o r m a t i o n products from subs t ra t e 56 w i t h P. peltatum c e l l s . 49 F igu re 12. Schematic r e p r e s e n t a t i o n o f the semi-cont inuous b i o t r a n s f o r m a t i o n p rocess . 56 F igu re 13. Time course s tudy to check s t a b i l i t y o f a r y l t e t r a l i n 75 under the b i o t r a n s f o r m a t i o n c o n d i t i o n s . 65 F igu re 14 The s t r u c t u r e o f t r i p d i o l i d e (5) and t r i p t o l i d e (91) . 78 F igure 15. A l k y l a t i o n o f t r i p t o l i d e (91) w i t h propane t h i o l . 81 F igure 16. Di te rpenes i s o l a t e d from Tripterygium p l a n t s . 94 F igure 16a Di te rpenes i s o l a t e d from T, wilfordii t i s s u e c u l t u r e s . 96 F igure 17. S y n t h e t i c p r e cu r so r u t i l i z e d i n p rev ious b i o t r a n s f o r m a t i o n s tud i e s i n P r o f . Ku tney ' s group. 98 F igure 18. The i somer i c o l e f i n s from the o x i d a t i v e decarboxy-l a t i o n o f dehydroab ie t i c a c i d . 105 LIST OF SCHEMES Scheme 1. I d e a l i n t e r a c t i o n s between subs t ra t e and c e l l . 3 Scheme 2 . B a s i c problems and s o l u t i o n s f o r s u b s t r a t e - c e l l i n t e r a c t i o n s . 4 Scheme 3. R e t r o s y n t h e t i c a n a l y s i s o f p o d o p h y l l o t o x i n (4) by G e n s l e r . 12 Scheme 4 . T o t a l s jmthes i s o f p i c r o p o d o p h y l l o t o x i n (9) by G e n s l e r . 14 Scheme 5. Syn thes i s o f p o d o p h y l l o t o x i n (4) from the t e t r a h y d r o p y r a n y l e ther o f p i c r o p o d o p h y l l o t o x i n (24) by G e n s l e r . 16 Scheme 6. Syn thes i s o f p o d o p h y l l o t o x i n (4) from 25 by Rodr igo e t a l . 17 Scheme 7. Z i e g l e r ' s approach to a r y l t e t r a l i n s y n t h e s i s . 19 Scheme 8. The syn thes i s o f e topos ide (7) from p o d o p h y l l o t o x i n ( 4 ) . 20 Scheme 9. B i o s y n t h e s i s o f f e r u l i c a c i d (43) from s h i k i m i c a c i d (39) . 22 Scheme 10. Proposed b i o s y n t h e s i s o f y a t e i n (49) from c o n i f e r y l a l c o h o l (44) . 23 Scheme 11. The i n c o r p o r a t i o n o f y a t e i n (49) i n t o p o d o p h y l l o t o x i n ( 4 ) . 25 Scheme 12. The syn thes i s o f subs t r a t e 56 from 3 ,4 -d ihydroxybenz-aldehyde (62) . 35 Scheme 13. The hydrogeno lys i s o f compound 70. 40 Scheme 14. The proposed b i o t r a n s f o r m a t i o n o f subs t r a t e 56. 42 Scheme 15. H y p o t h e t i c a l mechanism f o r the r i n g c l o s u r e o f subs t r a t e 56 to a r y l t e t r a l i n 60. 43 Scheme 16. The in te rmediacy o f a h y p o t h e t i c a l quinone methide i n the format ion o f products 74 and 75. 62 Scheme 17. The proposed b i o t r a n s f o r m a t i o n o f subs t r a t e 57. 66 Scheme 18. Proposed C ( l ) e p i m e r i z a t i o n sequence from compound 79 to e p i p o d o p h y l l o t o x i n ( 6 ) . 66 Scheme 19. The syn thes i s o f subs t r a t e 57 from p i p e r o n a l (81 ) . 67 Scheme 20. Proposed mechan i s t i c pathway f o r the fo rmat ion o f products 74 and 75 i n the b i o t r a n s f o r m a t i o n o f subs t ra te 56 by P. peltatum c e l l suspens ion c u l t u r e s . 72 Scheme 21 . The i n c o r p o r a t i o n o f m a t a l r e s i n o l (47) i n t o p o d o p h y l l o t o x i n (4) by P. hexandrum p l a n t s . 75 Scheme 22. Proposed b i o t r a n s f o r m a t i o n f o r subs t r a t e 87. 76 Scheme 23. Proposed s y n t h e s i s o f 3 - i sop ropoxy -4 -benzy loxy -benzaldehyde (90) from 3,4-dihydroxybenzaldehyde (62) . 76 Scheme 24. Syn thes i s o f t r i p t o l i d e (91) by B e r c h t o l d e t a l . 83 Scheme 25. Syn thes i s o f 97 by Garver and van Tamelen. 85 Scheme 26. T r i e p o x i d e c o n s t r u c t i o n from l aevop imar i c a c i d (112) by Tokoroyama et a l . 86 Scheme 27. Syn thes i s o f the b u t e n o l i d e r i n g o f t r i p d i o l i d e and t r i p t o l i d e from d e h y d r o a b i e t i c a c i d (122) by Tokoroyama et a l . 87 Scheme 28. B i o g e n e t i c - t y p e s y n t h e s i s o f 97 by van Tamelen and L e i d e n . 90 Scheme 29. C h i r a l syn thes i s o f 97 by van Tamelen e t a l . 91 Scheme 30. C h i r a l syn thes i s o f d e h y d r o i s o a b i e t a n o i i d e (132) by Robe r t s . 92 Scheme 31. B i o s y n t h e t i c gene ra t ion o f the abie tane s k e l e t o n . 93 Scheme 32. Proposed b i o s y n t h e t i c pathway to t r i p d i o l i d e (5) v i a d e h y d r o a b i e t i c a c i d (122) . 97 Scheme 33. B i o t r a n s f o r m a t i o n o f d e h y d r o i s o a b i e t a n o i i d e (132) w i t h Tripterygium c e l l s . 98 Scheme 34. B i o s y n t h e t i c pathway to the d i t e rpene t r i e p o x i d e s proposed by Robe r t s . 99 Scheme 35. O x i d a t i o n o f benzene by p r o k a r y o t i c and e u k a r y o t i c organisms. 100 Scheme 36. P r o d u c t i o n o f the e x o - o l e f i n 150 from d e h y d r o a b i e t i c a c i d (122) . 105 Scheme 37. The e p i m e r i z a t i o n of C5 i n ketone 151. 107 Scheme 38. The s y n t h e s i s o f d e h y d r o i s o a b i e t a n o i i d e (132) from ketone 151. 108 Scheme 39. P r e p a r a t i o n o f the c e l l homogenate, resuspended p e l l e t and CFE, and b i o t r a n s f o r m a t i o n . I l l Scheme 40. The b i o t r a n s f o r m a t i o n o f d e h y d r o i s o a b i e t a n o l i d e (132) i n t o 175 by T. wilfordii c e l l suspens ion c u l t u r e . 113 Scheme 41 . Syn thes i s o f 14 -ace toxydehydroab ie t i c a c i d (189) by Demers. 115 Scheme 42 . The proposed b i o t r a n s f o r m a t i o n o f subs t r a t e 190. 116 Scheme 43 . Syn thes i s o f subs t r a t e 190 from d e h y d r o i s o a b i e t a -n o l i d e (132) . 117 Scheme 44. The b i o t r a n s f o r m a t i o n o f i s o t r i p t o p h e n o l i d e (190) by T. wilfordii whole c e l l s . 119 Scheme 45. The proposed b i o g e n e s i s o f the t r i e p o x i d e system. 121 LIST OF TABLES Table 1. Semi-cont inuous b i o t r a n s f o r m a t i o n o f sub t ra t e 56 w i t h 7-day o l d P . peltatum c e l l s . I ncuba t i on time was 22 h , pH - 6 . 5 . 57 Table 2 . Semi-cont inuous b i o t r a n s f o r m a t i o n o f subs t r a t e 56 w i t h 17-day o l d P . peltatum c e l l s . E n t r i e s 1-5 w i t h pH c o n t r o l l e d to 6 . 5 . 60 Table 3. Semi-cont inuous b i o t r a n s f o r m a t i o n o f subs t r a t e 56 w i t h 7-day o l d P . peltatum c e l l s . pH was not c o n t r o l l e d . 61 Table A . Average y i e l d s f o r the semi-cont inuous b io t r ans fo rma-t i o n o f subs t r a t e 56 w i t h P . peltatum c e l l s . 63 List of Abbreviations A c A C - 3 aq. B-5 br Bn C F E d dd D M F D M S O D N A D C C D M E D Y E Et G C [H] H P L C H R M S hexanes H M P A hv /-Pr IR J K B L-1210 L D A m M C P B A Me M E S MSNA0.5K0.5 N a 2 E D T A NBS N O E n-Bu nmr N i R a 10] P-388 F C C P D A Ph PP acetyl a cell line of tissue culture developed from Catharanthus roseus aqueous standard tissue culture medium developed by Gamborg and Eveleigh broad benzyl cell free extract doublet doublet of doublets dimethylformamide dimethylsulphoxide deoxyribonucleic acid dicyclohexyl carbodiimide dimethoxy ethane dextro yeast extract ethyl gas chromatography reduction high pressure l iqu id chromatography high resolution mass spectrum generally a mixture of several isomers of hexane (C6H]4) predominantly w-hexane, and methylcyclopentane (C6H]2) hexamethylphosphoramide light radiation /50-propyl infra-red coupling constant a tissue culture cell line derived from human carcinoma of the nasopharynx a tissue culture eel! line derived from mouse leukemia l i th ium diisopropyl amide multiplet mé'/fl-chloroperoxybenzoic acid methyl 2-(4-morpholino)-ethane sulfonic acid MS medium of Murashige and Skoog supplemented with naphthalene acetic acid ( N A , 0.5 mg/L) and kinetin ( K , 0.5 mg/L) ethylene diamine tetraacetic acid disodium salt w-bromosuccinimide Nuclear Overhauser Effect «ormaZ-butyl nuclear magnetic resonance Nickel-Raney oxidation a tissue culture cell line derived from mouse leukemia pyr id in ium chlorochromate potato dextrose agar phenyl pyrophosphate ppm PRI2C0100 PRD2C0100 P R L - 4 pyr R R I R.T. s sp. t T F A T H F T L C TMS TRP-4a T H P U V A parts per mi l l ion P R L - 4 medium of Gamborg and Eveleigh supplemented with indole-3-acetic acid (I, 2 mg/L) and coconut milk (100 m l / L ) P R L - 4 medium of Gamborg and Eveleigh supplemented with 2,4-dichlorophenoxy acetic acid (D, 2 mg/L) and coconut milk (Co, 100 m L / L ) standard tissue culture medium developed by Gamborg and Eveleigh. pyridine a lky l residue refractive index room temperature singlet species triplet trifluoroacetic acid tetrahydrofuran thin layer chromatography tetramethylsilane a cell line of tissue culture developed from Tripterygium wilfordii tetrahydropyrane ultraviolet heat wave number (cm" ) A C K N O W L E D G E M E N T S I would l ike to thank my supervisor. Professor J. P. Kutney, for his guidance and Support throughout this work. I would l ike to thank also the following people for their contribution: Dr. Malcolm Roberts, Mi jo Samija, Carles Cirera , and K a n g Han for their help and invaluable suggestions during the preparation of this thesis; Gary Hewitt , David Chen, Fay Hutton, and Elizabeth Bugante for their hard work in producing the cultures and for having the patience to teach me how to work with them; Radka Milanova and Nikolay Stoynov who were never too busy to lend a hand; Johanne Renaud, Jacques Roberge and Krys tyna Piotrowska for their advice throughout my synthetic studies; Andres Gonzalez, Martha K l i n e and Antonio K u r i -Brena who helped keep my sanity; the Staff and Faculty of this Chemistry Department. Financia l support from Syntex, S.A. is gratefully acknowledged. Thank you, family and friends. FOREWORD Biotechnology has been widely used since the early days of mankind; microbial and yeast-mediated transformations in particular, were applied to the production of bread, dairy products, and alcoholic beverages. A l l of these early applications used mixed cultures of microorganisms, and pertained to agriculture and human nutrit ion. In 1862 Pasteur* la id the scientific foundation of one of these early applications, namely, the oxidation of alcohol to acetic acid by using a pure culture of Bacterium xylinum^. Investigations of the oxidation of glucose to gluconic acid"' by Acetobacter aceti and of sorbitol to sorbose by Acetobacter sp. followed these earlier studies'*. The reducing action of fermenting yeast, Saccharomyces cerevisiae, was first observed by Dumas in 1874^. He reported that, on addition of finely powdered sulfur to a suspension of fresh yeast in a sugar solution, hydrogen sulfide was liberated. The reduction of furfural to fur furyl alcohol under the anaerobic conditions of fermentation by means of l iv ing yeast^'^ was the first "phytochemical reduction"^ of an organic molecule described in the literature. Numerous further enzymatic or microbial biotransformations, b iodégradat ions and fermentations followed, and in the in i t ia l excess enthusiasm that invariably accompanies the birth of a new field, biotransformations were hailed as a panacea that would ultimately displace traditional organic chemistry. However, the role of such biotransformations is one of support rather than supplantation; biotransformations should be employed when a given reaction step is Q not easily accomplished by "ordinary" chemical methods . Contrary to the very early applications, biotransformations are carried out today by pure cultures of microorganisms or animal and plant cells or with purif ied enzymes, and they should always be considered as a way of performing selective modifications of defined pure compounds into defined final products*^. The main differences between biotransformation and fermentation have been clearly listed by Y a m a d a ^ ^ The general goal of biotransformations may be considered to be as follows: resolution of racemates, selective conversion of functional groups among several groups of similar reactivities, introduction of a chiral center, and functionalization of a certain nonactivated carbon^^. There are two different biotransformation systems: cells or isolated enzymes. Ava i l ab i l i t y of a certain organism can be a deciding factor for an organic chemist turning to biotransformations in synthesis. For example. Baker's yeast (Saccharomyces cerevisiae) is readily available and easily used, but access to other microorganisms may require help by a microbiologist and access to fermentation facilities. Disadvantages of the use of whole cells for laboratory or industrial-scale operations are that aseptic growth of the cells is usually required, and the work-up is time consuming and messy due to separation of the product from the huge amount of biomass. The advantages of isolated enzymes are specificity for selected reactions and that their use may require small equipment and simple work-up '^ . But enzymes are more expensive, and addition of enzyme cofactors or enzyme cofactor recycling might be necessary^"*'^^. The present work consists of general biotransformation methodology applied to plant cell cultures and derived crude enzyme preparations (cell free extracts). Ideal interaction between substrate and cell (Scheme 1)^^ is rarely found in practice. Some advice on how to deal with basic problems often encountered with such biotransformations is provided in Scheme 2^^. Plants are the most important source of land-based foods, oils, and fibers, and represent an immense repository of biochemicals, including flavors, essences, pigments, fine chemicals, pharmaceuticals, and novel biologically active s u b s t a n c e s ^ I n most instances, plant-derived biochemicals are secondary metabolites with wide structural variety, although specific taxonomic plant groups yield specific homologues. Gives high turn-over rates. Gives high regio-and stereospecificity. \ IDEAL SUBSTRATIE Is able to pass the cell membrane. Is soluble in the culturing medium. \ The catalytic activity of the cell is not inacti-vated by the substrate or by the product Scheme 1. Ideal interactions between substrate and cell. The plants sought for their secondary products arc sometimes native to remote or poli t ical ly unstable countries. Biotechnology provides a potential means of securing stable supplies of these compounds^^ Secondary metabolites are derived biosynthetically from the products of primary metabolism. The main categories include compounds derived from 1) acetate, 2) mevalonate, 3) shikimic acid, 4) non-aromatic amino acids, or 5) are of mixed biosynthetic origin. The structural diversity and complexity of plant secondary products account for the unique properties and biological activities of these compounds. Dissolve in a non-toxic water miscible solvent prior to addition The reaction is carried out with a cell homogenate, a cell free enzyme preparation or a purified enzyme. Add an emulsi-fying agent Is insoluble in the reaction medium. Cannot pass the cell membrane Chemical modification of the substrate SUBSTRATE Use a ceU fiee extract Affects the viability and reproduction of cells. Shows slow turn-over rate and/or low regio-and Stereoselectivity The substrate is dispersed after completion of growth at a defmed rate in small portions (semi-continuosly) or continuosly (at low rate) such that the cells can convert the substrate before it be-comes toxic. Change the strain Change cell density, aeiadon, pH shift or medium composition. Scheme 2. Basic problems and solutions for substrate-cell interactions. Chemical synthesis is the obvious approach for obtaining supplies of these compounds; however, even in cases where synthetic routes can be established, the total synthesis of the desired product cannot always be achieved economically. In vitro plant culture systems provide an alternate route to plant biochemicals. Plants cultured in vitro represent an excellent experimental system for biochemical investigations and potentially a vast commercial source of valuable chemicals. For example, plantlets, specific plant organs or cells growing on solid medium may produce the substances of interest. However, these cultures grow relatively slowly and are labour intensive because they require extensive subculture to generate the large quantities of biomass needed for commercial product isolation. However, differentiated plant tissue may not be required. If one puts an excised plant tissue on semi-solid nutrient media, an undifferentiated callus tissue may form. This can be dissociated into a fine cell suspension by putting the callus into l iquid media and al lowing further growth in rotary shakers. The advantages of cell suspensions as potential sources of secondary metabolites include a defined culture environment, the ease of manipulation of culture conditions, and the rapid generation of large volumes of relatively uniform tissue. But, despite the tremendous versatility of the system, the technique has as yet resulted in few commercial successes. One of the major reasons is that in contrast to microbial fermentations, where many years of research have been available, the technique of producing secondary metabolites in plant cell cultures is a relatively new area of research, so much further development must occur. Another way to exploit a specific enzyme of a cultured plant cell for the purpose of biotransformation of a relatively inexpensive substrate to a more valuable product is found in a cell free system. This involves the isolation and use of enzyme(s), thus maintenance of the biomass is not required. Chemical production based on isolated enzymes, either in solution or as an immobilized system, is an established technology; the production of plant derived chemicals by using this technique, however, is not yet common, again for the reasons noted above. The potential of this approach to commercial phytochcmical production has been demonstrated in our laboratories. For example, the discovery of a catharanthine-vindoline coupling enzyme which yields 3 ' ,4 ' -anhydrovinblast ine (3)^^'^^, an analogue of the anticancer drugs vinblastine and vincristine, has resulted in an important process (Figure 1). CO2CH3 Catharanthine (1) CHaQ OCOCH3 'OH NCH3 C02CH3 Vindoline (2) Coupling Enzyme CH3O' NCH3 CO2CH3 3',4'-anhydrovinblastine (3) Figure 1. Coupling of catharanthine (1) and vindoline (2). This example couples the use of "precursors" believed to be the biosynthetic bui lding units with an elegant biotransformation step. As chemists, the clue to the use of cultured cells for biochemical synthesis is the understanding of secondary metabolic processes, so that we may logically and consistently control and manipulate the relevant pararteters. In the present study, two different pharmacologically interesting and synthetically challenging products were chosen for studies of plant-cell mediated biotransformation: (i) the lignan podophyllotoxin (4), the commercial starting material in the synthesis of the anti-cancer drugs etoposide and teniposide, and (ii) t r ipdiol ide (5), a diterpene with significant male anti-fert i l i ty act ivi ty . One chapter of this thesis is devoted to each of these studies. Podophyllotoxin (4) Tripdiolide (5) C H A P T E R 1. T H E P O D O P H Y L L O T O X I N F A M I L Y I N T R O D U C T I O N Podophyllotoxin (4)^^'^^ is an interesting member of the naturally-occurring family of compounds known as lignans which are derived biosynthetically from the shikimate pathway^^"^^. Podophyllotoxin has been the subject of much study (Figure 2) as it presents a challenging synthetic target and, more importantly, has proven anti-cancer properties. Natural sources of podophyllotoxin are relatively few''^'"' , but species of Podophyllum produce quite large amounts of this lignan in their roots^*"^^. Podophyllotoxin is isolated commercially from the American Mandrake {Podophyllum peltatum Linnaeus) and the related Indian species P. emodi Wallich (P. hexandrum Royle). The dried roots and rhizomes, known as podophyllum, are extracted with alcohol to produce a resin (podophyllin) containing podophyllotoxin. For example, P. peltatum yields 4-6% podophyllin^'* which affords 9-10% podophyllotoxin upon chromatographic separation^^. Although the medicinal properties of P. peltatum had long been known by North American natives, it was not unti l 1942 that Kap lan^^ demonstrated the efficacy of podophyllin in the treatment of venereal warts. This f inding sparked much interest in the biological and chemical properties of podophyll in and its constituents, and this continues today as can be seen from the present publications on this topic. It has been found that certain glycosides of 4'-demethylepipodophyllotoxin (6) (Figure 3) are also effective anti-cancer drugs but lack the unacceptable side effects of podophyllotoxin"^ . The most important compound of this class is etoposide (1) , which is used c l in ica l ly in the treatment of myelocytic leukemia, neuroblastoma, bladder cancer, testicular cancer, and small-cell lung cancer^^. Un l ike podophyllotoxin, these compounds do not inhibit microtubule assembly; they are believed to derive their anticancer properties from interference with D N A topoisomerase II, an enzyme which makes reversible double-stranded breaks in D N A 1700's: Folklore medicine. Uses of podophyllin, a resin obtained from alc»hol extraction of Podophyllum peltatum. North American Indians used preparations as a purgative and emetic and also as a suicide agent. It was also used for diseases of the liver, kidneys, syphilis gonorrhea, urinary obstmction, and as a cathartic. 1820: Included in the first US pharmacopoeia as a cathartic. 1850: Commercial production of podophyllin begins. 1880: Isolation of components from podophyllin starts. Active compounds believed to be podophyllotoxin and peltatin. 1942: Pharmacological studies are continued and reports about possible use of podophyllotoxin in cancer treatment emerge. 1950: Clinical studies initiated at Sandoz Laboratories. Basel, Switzerland with a variety of synthetic anatogues of podo-phyllotoxin are discontinued due to toxicity. 1970: Clinical studies with synthetic analogues of podophyllotoxin are reassumed. 1978: Etoposide is selected as clinical drug, licensed by Sandoz to Bristol-Myers, USA. 1983: FDA approves dmg etoposide (VP-16-213), commercial name: vepesid. Figure 2. Br ie f history of etoposide development - from folk medicine to cl inical drug. 6: R«H, 4-demGthylepipoclophyllotoxin 7:Ra ° " ' ^ 2 r \ ^ p etoposide teniposide Figure 3. The structures of 4 '-demethylepipodophyllotoxin (6), etoposide (7), and teniposide (8). in order to prevent the tangling resulting from D N A replication'*^. Recent work has suggested that the role of etoposide is to promote both D N A cleavage and inhibi t its subsequent ligation**' '^^, the presence of a hydroxyl group at C(4')'*^ and a /9-orientation of the C(4) substituent have been shown to be cr i t ical for such anti-tumor activity. The actual nature of the C(4) substituent is much less important"*^. The drugs are produced from podophyllotoxin by a demethylation and epimerization sequence^^. Podophyllotoxin, rather than its demethylated analogue, 4 ' -demethylpodophyllotoxin, which is also present in Podophyllum species, was chosen as starting material because of its greater natural abundance. The structure of podophyllotoxin is only deceptively simple as the l,2-c/5-2,3-trans stereorelationship, which is of crucial importance for the biological activity^'*, constitutes a synthetic d i f f i cu l ty , due to facile epimerization to the less strained, but inactive c/5-lactone picropodophyllotoxin (9)^^* (Figure 4). It should be noted that the stereochemistry of C(4) at this point is of less concern as podophyllotoxin and epipodophyllotoxin are easily interconvertible'*^''*^. OH Base 0CH3 Podophyllotoxin (4) OH CHaO' 0CH3 Picropodophyllotoxin (9) Figure 4. Tlie equi l ibr ium between podophyllotoxin (4) and picropodophyllotoxin (9). Furthermore, for the synthesis of etoposide and teniposide the C-4 hydroxyl group can either be a- or ;3-oriented because in the glycosidation reaction only the C-4 0-glycoside is obtained'*^. T O T A L S Y N T H E S E S The first total synthesis of podophyllotoxin was achieved in 1966 by Gensler^^. Now, a quarter of a century later, synthetic chemistry continues to be enchanted with the tactical complexity of establishing the four contiguous stereocenters and trans-fused 7-lactone ring of this natural product^^*^^ Three different strategies have been used; and I have arbi trar i ly selected three syntheses from the literature to illustrate these approaches. 1. Kinet ic protonation of picropodophyllotoxin. Several approaches^'*'^^'^^, including the first enantioselective synthesis reported by Meyers et a l^^ , are directed towards picropodophyllotoxin and rely on Gensler's observation of kinetic protonation of the enolate anion of A Q picropodophyllotoxin yielding podo- and picropodophyllotoxin in a 45:55 ratio. More recently, equilibration at an earlier stage of the sequence was found to be more efficient^. Gensler's retrosynthetic analysis is shown in Scheme 3. OH Scheme 3. Retrosynthetic analysis of podophyllotoxin (4) by Gensler. Disconnection on ring C and dehydration leads to DL-a-apopodophyllinic acid (10) which can be thought of as being derived from intermediate 11 via formylation followed by induction and dehydration. The ketone functionality in 11 is derivable from the intermediate product 12, the latter arising from condensation between a succinic ester and the ketone 13. This d iary l ketone could, in turn, be prepared via Friedel-Crafts acylation of an appropriately substituted benzene with 3,4,5-trimethoxy benzoyl chloride (15). R ing B of podophyllotoxin was constructed by a condensation reaction between the ketone 13 and diethyl succinate (Scheme 4) to produce the unsaturated acid 12. This acid was further elaborated to produce the anhydride 17, which was cyclized using tin (IV) chloride to afford intermediate 18. R i n g C was constructed by the sequence, 19 -» 20 -» 21 22 (Scheme 4). Reaction of intermediate 19 with sodium hydride and ethyl formate provided enol 20, the latter was then further elaborated to the diol 21 which was then cyclized under acidic conditions to produce the lactone 22. The key intermediate 22 was then hydrolyzed to the corresponding acid 23, resolved with quinine and relactonized to give the (-)-enantiomer of 22. The synthesis was completed when the tetrahydropyranyl ether of picropodophyllotoxin (24) (Scheme 5) was treated with triphenylmethyl sodium to produce the enolate. Irreversible protonation of the enolate with glacial acetic acid followed by removal of the protective group with dilute aqueous acid gave a 45:55 mixture of podophyllotoxin (4) and picropodophyllotoxin (9) which could be chromatographically separated. 2. Diels-AIder approach. The intermediacy of picropodophyllotoxin was avoided first by Rodrigo et al who constructed ring B via a Diels-Alder reaction involving an isobenzofuran. a) diethyl succinate b) NaOH c) separation (continued) 17 18 Scheme 4. Total synthesis of picropodophyllotoxin (9) by Gensler. CH,0 *0CH3 OCH3 19 CH3O' J OCH3 OCH3 (±)22 NaOH aq. CHjOH CO2H CH3O' J OCH3 23 Scheme 4 - Continued. a) NaH, HCOaEt b) H2. RO2 HzSO^ a) Quinine b) Resolution 0) H2SO4 0 8 3 0 - ^ ^ ^ ° ^ " » OCH3 20 a) NaBH4 b) H3O* c) KOH CH3O' J OCH3 OCH3 21 CH,0' (.)22 'OCH, OCH3 a) HCI b) CaCOa OH CH3O OCH, OCH, < OTHP OH C H , 0 ^ ^ ^OCHa OCH3 24 a) PhaCNa b) HOAc c) H2SO4 d) Chromatography 0CH3 Podophyllotoxin (4) Scheme 5. Synthesis of podophyllotoxin (4) from the tetrahydropyranyl ether of picropodophyllotoxin (24) by Gensler. The i n i t i a l step in this synthesis (Scheme 6) involves a cycloaddition of compound 25 (prepared by l i thiat ion of the dimethylacetal of 6-bromo piperonal and reaction with 3,4,5-trimethoxybenzaldehyde) with dimethylacetylene dicarboxylate to give the isobenzofuran 26. This is further elaborated to produce the diol 29. The acid 31 produced from the diol 29 by protection as an acetonide and saponification, proved to be the key intermediate in the synthesis. When this compound was treated with dilute acid for 24 h, the acetonide was cleaved and subsequent treatment with dicyclohexylcarbodiimide (DCC) yielded epipodophyllotoxin (33). However, i f compound 31 was treated with aqueous acid for 48 h, then neopodophyllotoxin (32) was produced. On treatment of 32 with sodium hydroxide followed by treatment with D C C , podophyllotoxin was produced. Other groups have also drawn on this strategy^*'^^'^'*'^^. 3. Tandem Michael addition-aldoi condensation approach. A n interesting approach has been explored by Ziegler et a l^^ . In this approach (Scheme 7) the carbon framework is assembled during a one-pot tandem Michael 9CH3 CO2CH3 AcOH, trace C H j O ^ ' ^ Y ^ O C H j OCH325 CH30^ OCH3 OCH3 28 NiRa C H 3 0 ^ ^ V ^ O C H 3 AcH3 26 a) H2. Pd b) MeONa UEtgBH pHaOH acetone CO2CH3 CHaO^ OCH3 OCH3 29 CH30^ ^ ^OCH, OCH3 30 Scheme 6. Synthesis of podophyllotoxin (4) from 25 by Rodrigo et al. dilute H* 'CO2H 48 h. OCH3 31 a) dilute H*. 24 h b) DCC CH3O I OCH; OCH, CH3O a) NaOH b) DCC OH C H g O ^ ' j l ^ O C H a OCH, epipodophyllotoxin (33) podophyllotoxin (4) Scheme 6 - Continued. Scheme 7. Ziegler's approach to aryl tetralin synthesis. addition-aldol condensation resulting in the desired 2,3-trans relationship. However, electrophilic r ing closure (i.e. formation of the l-8a bond) of 34 leads to the al l trans product 35, belonging to the pharmacologically uninteresting isopodophyllotoxin series. Also a "biomimetic" ring closure with thal l ium (III) trifluoroacetate has provided only the l,2-rrfl«5-substitution pa t te rn" . a) HBr b) BaCOs C H a O ^ J ^ O C H , 0CH3 4 a OH; pcxiophyllotoxin (4) 4 p OH; epipodophyllotoxin (33) CH3O' J 0CH3 OH AoO AcO AcO AcO OAc OH BFg.EtzO CH,0 Y o 36 OCH3 O ^ ^Ph a) H2, Pd/G b) CH3CHO CH3O J OCH3 O C H 3 0 ' ' ] ' ' ' ' ^ O C H 3 OH 38 etoposide (7) Scheme 8. The synthesis of etoposide (7) from podophyllotoxin (4). Deoxypodophyllotoxin-' , and epipodophyllotoxin*''* have also been synthesized. Other routes into the lignan system have also been investigated and are discussed in a review by Whi t ing^ ' . The etoposide precursor, 4 '-dcmethylepipodophyllotoxin (6) was synthesized by 49 Kende in 1981 with an overall yield of 2.4% in a 13 step sequence. The stereochemical problem of the lactone (ring C) was solved by Gensler's methodology. The conversion of podophyllotoxin (4), epipodophyllotoxin (33) or 4 '-dcmethylepi-podophyllotoxin (6) to etoposide is outlined in Scheme i^^'^^, in which 4'-dcmethyl-epipodophyllotoxin (6) is treated with benzyl chloroformate to produce the protected derivative 36. Treatment of 36 with 2,3,4,6-tetra-0-acetyl-;3-D-glucopyranose and boron trif luoride etherate followed by zinc acetate afforded the glycosylated intermediate 38. Deprotection via catalytic hydrogenolysis of the carbonate and condensation with acetaldehyde produced the drug etoposide (7). Because Ziegler's strategy is inherently straightforward and easily adaptable to different ring substitution patterns, the reinvestigation of a stereoselective l-8a bond formation would be highly rewarding. A n approach which may solve this problem could involve biotransformations by plant cell cultures in which suitable precursors are subjected to such enzymatic processes. Such processes may be more amenable to scale up than the corresponding synthetic counterparts. A n y investigation of the use of biotransformations in the production of the podophyllotoxins must first consider their biosynthesis. It is this information which w i l l direct research towards the most potentially rewarding precursors. B I O S Y N T H E S I S O F T H E P O D O P H Y L L O T O X I N S The biosynthesis of lignans is a rather neglected experimental area. Studies that have been reported to date have concentrated on the demonstration of the incorporation of C6-C3 units into lignans and on the nature of the preferred units. Li t t le is known about the nature or the transformations of the immediate products formed from the coupling of such C6-C3 units. The skeleton of the Podophyllum lignans is known to be derived from two C6-C3 molecules, and a phenolic oxidative coupling process is generally believed to operate^^. The biosynthesis of C6-C3 compounds is clearly germane to the origin of lignans. Some of the intermediates believed to be involved in this pathway are shown in Scheme 9^°. Scheme 9. Biosynthesis of ferulic acid (43) from shikimic acid (39). The aromatic rings are formed from shilcimic acid (39), which is enzymatically transformed into (L)-phenyl alanine (40) and (L)-tyrosine (41). These intermediates are then converted by further enzymatic processes into 4-hydroxycinnamic acid (42) and then into ferulic acid (43). Two of these nine carbon units arc then envisaged to couple together in a head-to-head fashion to produce the lignan intermediate (46) (Scheme 10). 49 R= CH3: yatein Scheme 10. Proposed biosynthesis of yatein (49) from coniferyl alcohol (44). From the results of the feeding experiments with Podophyllum hexandrum l iv ing plants, this coupling has been shown to involve two phenylpropane "precursors" with the same 4-hydroxy-3-methoxy substitution pattern^' , and coniferyl alcohol (44) is almost certainly the intermediate concerned^^. Stereospecific enzyme-catalyzed coupling of free radical mesomers 45 derived from coniferyl alcohol would lead to a diquinomethide (46). The dibenzobutyrolactones are thought to be produced from 46 via reduction, lactone ring formation and appropriate modification of the aromatic substitution pattern. Matairesinol (47) has been confirmed to be a common intermediate in the pathway of Podophyllum lignans^^, such as yatcin (49). Other experiments involving labelled compounds have shown that matairesinol and yatein are incorporated into podophyllotoxin. The incorporation of yatein (49) into podophyllotoxin (4) involves a stereoselective cyclization in which the newly created chiral centers (CI and C2) place the protons attached to these centers in a cis relationship. These results are summarized in Scheme 11. In considering which substrates are suitable for a potential biotransformation, a number of factors must be taken into account. Firs t ly , the substrate must be easily synthesized on a large scale; secondly, the substrate must possess structural features similar to presumed late stage biosynthetic intermediates, for example, 49, in order that its transformation to the desired product may "mimic" the reactions thought to occur in the biosynthetic process. Only in cases where the substrate is very inexpensive can low incorporation into the product be tolerated. For these reasons, compounds structurally related to yatein were chosen, and it was hoped that a stereoselective ring closure to produce compounds with the podophyllotoxin structure could be achieved. Terry Jarvis^'* and Jan Palaty^^ in our group have addressed some of these problems in investigating the biotransformation of compounds 52 to 55 (Figure 5) with Catharanthus roseus cell free extracts (crude enzymes). Scheme 11. The incorporation of yatein (49) into podophyllotoxin (4). Prel iminary evaluation of compound 56 by these workers showed some encouraging results. However, even in the cases where they achieved ring closure, the stereochemistry at C j was incorrect. It was therefore concluded that enzymes from the C. roseus cell line, the latter producing indole alkaloids as secondary metabolites, are not suitable for the stereoselective ring closure, for example, 49 51. In these studies, high yields (approximately 90%) of ring closure 4 id occur to achieve the "wrong" cycl ic isomer in which the aromatic unit at C j in 51, for example, was attached to the ^-orientation. While the above studies wi th C. roseus were underway, a cell line of P. peltatum (see next section), the plant from which the podophyllotoxin family of compounds was isolated, was developed. It was shown that this cell line produces the "podophyllotoxins" and it was presumed that enzymes for the correct ring closure (49 51, for example) were now available. It was therefore of interest to turn our attention to studies of biotransformations of appropriate precursors wi th this cell line. C H p ' OCH3 OH OCH, 52; R=H 53; R=CH3 54; R= /-Pr 55 CHP - ^^Y^OCH, OH 56 Figure 5. The synthetic precursors used in previous biotransformation experiments in Prof. Kutney's group, and involving cell free extracts of Catharanthus roseus. T E C H N I Q U E S O F C E L L S U S P E N S I O N C U L T U R E S . P R E M I S E O F T H I S W O R K . A n appreciation of the basic techniques of cell suspension culture is essential to any assessment of their potential application. Suspension cultures are normally init iated by transfer of undifferentiated callus pieces to a l iqu id medium which is agitated during growth. Successful establishment of a fine suspension culture depends on the in i t i a l callus being friable, a condition that may depend upon appropriate phytohormone supplements. Dur ing the in i t ia l growth phase in the l iqu id medium, some cells are released from the callus and multiply. For subculture, a wide-mouth pipette is usually used to exclude any persistent large cell aggregates derived by only partial break-up of the initiate callus fragment. The suspension transferred on subculture should consist of free cells and some cell aggregates. The culture medium used for suspension culture is usually based upon that which maintains good growth of callus; it may, however, be necessary to modify this medium (particularly in its phytohormone content) to achieve a high growth rate and good cell separation in l iqu id medium. Batch cultures (or cultures in a f ixed volume of medium) increase in biomass by cell division and cell growth until a factor in the culture environment (nutrient or oxygen avai labi l i ty) becomes l imit ing. The cells then enter a stationary phase and final ly a declining phase in which cell dry weight decreases: the stability of the cells in stationary phase depends upon the species and on the nature of the growth-limiting factor (cells brought to stationary phase by nitrogen limitation retain v iab i l i ty longer than when ca rbohydra te - s ta rved)° . When a stationary phase cell suspension is subcultured, the cells in succession pass through a lag phase, a short l ived period of exponential growth, a period of declining relative growth rate and then again enter stationary phase (Figure 6). Time Figure 6. Model curve relating cell number per unit volume of culture to time in a batch-grown plant cell suspension culture. Phases of the growth cycle are labelled. Tradi t iona l ly at each subculture, cultures are ini t iated by a relatively high cell density inoculum and the cells therefore accomplish only a very l imited number of divisions (cell number doublings) before again entering stationary phase. The basic pattern of increase in cell number per unit volume in batch culture is illustrated in Figure 6^ .^ Dur ing lag phase the stationary phase cells of the inoculum embark upon a massive synthesis of new cytoplasm and associated organelles, replicate their D N A and then begin to divide. Then for a strictly l imited period of time, cell division rate is constant and maximal. During this period the mean cell volume declines sharply. Fol lowing this the growth begins to decline (slowly at first and later at an ever-increasing rate), mean cell volume increases, and the cells move into a stationary phase. There is evidence that subsequent differentiation may be determined by conditions operating during the period of cell mult ipl icat ion (meristematic phase)^'', and that the production of many secondary plant products normally occurs in non-dividing ceils, often in non-dividing ceils which have embarked upon a special pathway of differentiation. Thus, during the progress of batch culture, cultured cells pass through a series of contrasted physiological states which encompass cells in transition to a meristematic state; cells expressing high meristematic activity; cells undergoing expansion and becoming either metabolically quiescent or geared to express certain restricted metabolic pathways. The production of secondary products by cell cultures has been shown to be most active during a restricted phase of the growth cycle. Secondary plant product biosynthesis wi thin whole plants range from products widely distributed in the plant kingdom and whose synthesis is not confined to specialized cells or cell groups, to those characteristic of particular families, genera or species, and whose synthesis takes place in localized and highly specialized cells or cell groups. As might be expected, compounds of the first group (e.g. coumarins, particular flavonoids, phytosterols) either identical with or chemically related to those of the whole plant, have been frequently detected in plant tissue and cell cultures^^. In contrast, compounds at the other end of the range have, with few i f any exceptions, only been detected in tissue cultures in which a high level of organization (cytodifferentiation or organ initiation) has developed^^. This suggests that progress toward inducing particular specialized patterns of cytodifferentiation in cell cultures wi l l need to be achieved before they can be util ized for the study of the synthesis of volatile oils, resins, latex constituents, and so on. Certainly the most promising immediate application of cell cultures in the synthesis of secondary plant products would be expected to come from work with appropriately highly dispersed cell suspensions in which the desired class of compounds can be demonstrated even i f currently at very low yield. There are now a number of instances where not only primary metabolites (e.g. amino acids, nucleotides) but also secondary products produced by cell suspension cultures, have been shown to be released in significant amounts into their culture medium. As examples may be mentioned lignans^*^ and diterpene derivatives^^ In such cases a maintenance culture in a closed continuous system should enable the chemical product to be continuously harvested from a fixed culture biomass. This would discount the objection sometimes raised with respect to plant cell cultures as potential commercial biosynthetic systems in that they are very slow growing compared with microorganisms. The premise on which the present work is based is that i f a large biomass could function to release, over a long period, a secondary plant product or a desired derivative from a supplied substrate, then the time required to grow up the biomass would be of small economic consequence. Further, the abi l i ty in the closed continuous system of constantly removing the released metabolite could mimic a biological "sink", preventing any feedback inhibi t ion of synthesis. It would also stop the product from exerting any "stalling" effect (toxicity due to the action of the product at the exposed surfaces of the cells) by preventing its accumulation. P R O D U C T I O N O F P O D O P H Y L L O T O X I N S B Y P L A N T T I S S U E C U L T U R E S Podophyllotoxins as mentioned previously, have been isolated from several plant species. However, isolation of the target compounds by extraction from the plant can be associated with various problems, seasonal variation being one of them. Detailed analysis of P. peltatum plants raised in the United Kingdom and also in the United States of America , has confirmed variation in lignan pattern between indiv idual plants, but in general, dormant plants contain podophyllotoxin (4), Q-peltatin (58), and yS-peltatin (59), with ;3-peltatin usually predominating^* (Figure 7). Subsequent analysis of root specimens excised from plants during the growth season (March-August) show the peltatin content rapidly disappearing, and by May only traces can be detected. Podophyllotoxin is now the main constituent. Towards the end of July, some peltatin can again be observed in many plants, coinciding with 4 58 59 Figure 7. Some of the lignans of P. peltatum. the onset of senescence. Dormant plants are then shown to have regained the normal lignan pattern. The advantages afforded by direct production of podophyllotoxin by plant cell culture, together with the possible isolation of new metabolites, led to an interest in the culturing of Podophyllum species. A n article by van Uden et a r in 1989 described the first isolation of podophyllotoxin from cell suspension cultures. The cell suspension culture was initiated from a callus culture of Podophyllum hexandrum. It was reported that the callus was very d i f f icu l t to initiate. The yield of podophyllotoxin was typically between 0 and 0.1% based on dry weight. Ear l ier work in our laboratory led to the development of a cell suspension culture of Podophyllum peltatum. Leaf, stem, and root expiants of P. peltatum were initiated on one of Kadkade's^** variations of Murashige-Skoog^^ (MS) medium that contained 2,4-D (O.I mg/L) , kinetin (0.2 mg/L) , casamino acids (Difco: 500 mg/L) and agar (Difco Bacto: 8 g/L) . As in the case of the Dutch workers, it was extremely d i f f icu l t to propagate the cal l i due to severe fungal contamination, especially in root material, and to lethal tissue browning. Although many stem and 3 root-derived cal l i survived, extreme sensitivity to the effects of tissue browning persisted and required scrupulous removal of any dark tissue at each transfer. Currently ca l l i are maintained on one-half MS medium supplemented with naphthalene acetic acid (1.0 mg/L) , kinetin (0.2 mg/L) , and casamino acids (100 mg/L) . Shake flask studies for growth improvement of suspension cultures led to media with low auxin content in which root-derived cell line R3 showed signs of organogenesis. Repeated manual selections of root structures from filtered culture provided inocula that eventually gave healthy, differentiated suspensions. Subsequent removal of hormones, optimization of sucrose content (15 g /L ) and rebalancing of the medium to one half standard salts concentration gave a healthy habituated cell line that has been perpetuated in hormone free one-half MS broth (init ial pH = 5.8) since September of 1988. Subculture requires f i l t rat ion to obtain the cell aggregates free of spent medium and inoculation of fresh broth at a rate of 15 ml aggregates per 300 ml medium. Biomass doubles once (from 4- 8.5 g /L) and stationary phase is reached in 14-16 days. Inoculum from shake flasks transferred into Microferm (New Brunswick Scientific) bioreactors grows successfully (5.5 - 15 L) and provides sufficient material for characterization of the metabolites produced. In a 5.5 L bioreactor culture, the following yields were obtained: podophyllotoxin (49 mg), 4'-demethylpodophyllotoxin (36 mg), and 17 mg of an inseparable mixture of deoxypodophyllotoxin and podophyllotoxone. The dry biomass of mature suspension cultures has been determined to be 7.5-8.5 g / L and on this basis the yield of podophyllotoxin in this bioreactor study was estimated at 0.32-0.36%^^. In conclusion, it is clear that the developed cell culture of Podophyllum peltatum is not only an excellent potential source of the desired podophyllotoxins, but can also serve as a means for the biotransformation of appropriate synthetic substrate to the desired end products. O B J E C T I V E S O F T H I S I N V E S T I G A T I O N Based on the previous information, we set the objectives of this worlc as follows: 1. Synthesis of substrate dibenzylbutyrolactones 56 and 57 and use of the developed differentiated Podophyllum peltatum cell cultures in biotransformation experiments to produce the a ry l tetralin products 60 and 61 respectively. 2. Explore the use of a semi-continuous biotransformation process to produce the above mentioned aryl tetralin products, and, 3. Investigate the relationship between cell suspension age and biotransformation abilities to determine the best biotransformation conditions. R E S U L T S A N D D I S C U S S I O N Synthesis of Trans 2-(4-hydroxy-3,5-dimethoxybenzyl)-3-(3-hydroxy-4-isopropoxy-a-hydroxybenzyl)butanolide (56). As mentioned, previous work on the synthesis of compounds such as 54 has already been done in our group. The sequences described in this thesis are modified versions of these previous synthetic routes, both in terms of substrate substitution and reaction conditions, part icularly in the tandem Michael addition-aldol condensation step and subsequent functional group deprotections. We anticipated no surprises during the synthesis of precursor 56 and, in fact, were able to perform all of the following reactions on gram scale. The synthetic route is shown in Scheme 12. The sequence starts with the selective protection of 3,4-dihydroxybenzaldehyde (62). Reaction of this cathecol with 1.3 equivalents of isopropyl iodide in DMSO using potassium carbonate as a base produced a mixture of isopropyl ethers with the 3-hydroxy-4-isopropoxy derivative 63 being the predominant isomer (70% isolated yield). The position of the isopropyl side chain can be confirmed by N M R spectroscopy, since a positive Nuclear Overhauser Effect (NOE) is found for the signal at 6 6.95 (H5) when the signal attributed to the methine heptet (6 4.75) is irradiated. Benzylation of this phenol with benzyl chloride in ethanol using potassium carbonate as a base and a catalytic amount of sodium iodide proceeded in 70% yield. Reaction of this protected benzaldehyde with thiophenol catalyzed by boron trif luoride etherate afforded the corresponding thioketal in 56% isolated yield. The reaction has to be carried out under reduced temperature (-40° C) to avoid ether hydrolysis. It was found that i f the reaction time exceeded 30 minutes the yield was reduced, presumably due to benzyl ether and/or isopropyl ether cleavage. The other fragment (67) needed for the construction of dibenzyl butyrolactone 56 was prepared from commercially available syringaldehyde by benzylation (benzyl HO HO XX 5 62 a) i-Prl. K2CO3 b) BnCI, Nal R2O CHO PhSH 63: Ri=H.R2«i.Pr 63a: Ri=R2= i-Pr 63b: Ri= i-Pr, R2- H 64: Ri= Bn, R2» i-Pr SPh BF3Et20 ; ^ o ^ 65 a) n-BuU b) r V = < SPh C) .Br CHaO^ '^y^OCHa OBn 68 r l OBn OCH, 67 HgO, BF3 Et20 THF- H2O a) NaBH, b) Pd/C, H2 PhS. SPh CH3O' y 'c>CH3 OBn 69 0CH3 70: Ri=R2= Bn 71:Ri=Bn, R2=0H 56: Ri=R2= OH Scheme 12. The synthesis of substrate 56 from 3,4-ciihydroxybenzaldehyde (62). chloride, K 2 C O 3 , catalytic amount of N a l in ethanol, 85% yield), aldehyde reduction (NaBH4 in ethanol, 97% yield) and reaction with phosphorus tribromide in ether (76% yield). The key step of the synthetic plan, i.e. the Michae l addition of the anion of thioketal 65 to 7-crotonolactone and subsequent a lkyla t ion of the intermediate anion with aryl bromide 67, deserves some attention. This reaction is of special importance because it allows one to synthesize a great variety of l ignan precursors depending on the aromatic substitution pattern. Metallated thioketals show, in general, a high preference for non-conjugate 1,2-addition to a,^-unsaturated carbonyl compounds. It was found additionally that anion-stabilizing groups enhance the tendency of metallated dithio-acetals towards conjugate addi t ion. In fact, the first published example of a facile 1,4-addition of a l i thio-dithiane to an unsaturated carbonyl compound (7-crotonolactone) is that leading to 72^^. On the other hand, Ishibashi et a l^^ reported the addit ion of a protected cyanohydrin to 7-crotonolactone leading to 73 while the dithio-diphenyl thioketal fa i led to give the desired conjugate addition product. In our hands, the addition of metallated dithio-diphenyl thioketals proved to be a sensitive reaction with dramatic yield variations i f the conditions were not carefully controlled. However, we succeeded eventually in achieving yields consistently in the order of 75%. Dithianes are in most cases converted to the l i th io derivatives by treatment with «-butyl l i th ium^^ at temperatures between -30° and -10° C. Lower temperatures can be used i f additional activation (for instance, aryl groups) is present. In these cases, other metallating reagents and metals other than l i th ium are applicable. The origin of the acidity of hydrogen atoms adjacent to divalent sulfur has been the OQ subject of some resea rch°° . The best results were obtained when thioketal 65 was reacted wi th 1.1 equivalents of w-butyl l i th ium at -70° C for 25 min. Stringent temperature control was required as well as a very slow rate of 7-crotonolactone addition to the metallated thioketal solution. After stirring this mixture for 40 min, the benzyl bromide was introduced (again at a slow rate) and the temperature allowed to rise slowly to room temperature. Quenching with water and the usual work-up afforded the desired dibenzylbutyrolactone 68. The IR spectrum of this compound showed the carbonyl absorption at 1770 cm" ' corresponding to a 7-lactone, both electron impact and high resolution mass spectroscopy showed the fragment corresponding to M'''-2SPh but elemental analysis agreed favorably for the empirical formula C49H49O7S2. The ' H nmr spectrum of the product showed a doublet for the methyl groups of the isopropyl side chain at S 1.38 ppm and the corresponding methine heptet situated at 6 4.59 ppm. The methylene protons of CI" appear as an A B X system at S 2.7 (dd, J = 5, 14 Hz) and 5 3.10 (dd, J = 4, 14 Hz) due to their coupling with the proton located at C2. This proton appears as a multiplet at S 2.83-2.90, while the corresponding multiplet for the proton at position 3 is located at 6 3.2-3.25 ppm. The A B X system for the methylene protons of C4 shows clearly at S 3.35 (dd, J = 8, 11 Hz) and 6 4.21 (dd, J = 3, 11 Hz). The fact that the latter component of this A B X system shows a coupling constant of 3 Hz, suggests that this signal belongs to the proton cis to the proton at position 3. A singlet integrating for 6 protons located at 6 3.68 was assigned to the methoxy groups present on ring D, while two additional singlets integrating for two protons each at S 4.97 amd 5.03 ppm were attributed to the methylene protons of the benzyl protecting groups. The aromatic proton signals are visible as a singlet for two protons at S 6.19 corresponding to the protons on ring D , a doublet at S 6.80 (J = 8 Hz) for the proton at position 5' on r ing A and a doublet of doublets at S 7.03 (J = 2, 8 Hz) for the proton located at C 6 ' of the same ring. A multiplet at S 7.15-7.50 integrated for the remaining 21 protons present in the molecule. The trans relationship between H2 and H3 was assumed on a thermodynamic basis and could be confirmed at a later stage of the synthesis. The next step was the hydrolysis of the dithio-ketal derivative to the corresponding ketone. This step was a crucial one since it is known that this class of compounds does not hydrolyze easily. The problem is that the equi l ibr ium shown below lies far to the left. Therefore, only irreversible removal of the thiol or of the ketone can push it to the right. The irreversible removal of the thiol can be done by one of the following methods: dis t i l l ing low molecular weight thiols, transacetallization, formation of a transition metal thiolate (Ag, C d , Hg), oxidation to a higher oxidation state of sulfur and alkylation to a sulfide. Most of these procedures go back to Fischer^^ who used 5% H C l , HgCl2 , A g N 0 3 , Br2, and H N O 2 on his sugar ethyl dithio-acetals. Jan Palaty^^ in our group found al l too well these diff icul t ies , but fortunately a relatively recent procedure^^ which uses mercury (H) oxide and boron trif luoride etherate proved to be suitable for our purposes. Care had to be taken in controlling the temperature at 0° C to avoid benzyl ether hydrolysis by the boron tr if luoride during the in i t i a l stage of the reaction. A rapid f i l t ra t ion through silica gel removed easily the biphenyl disulphide byproduct of the reaction affording the pure ketone 69 in excellent yield. This compound could be crystallized from ethanol and showed in the IR spectrum absorptions at 1775 (lactone) and 1663 (ketone) cm"* respectively. The *H nmr spectrum of 69 when compared to that of dithioketal 68, showed a reduction in the number of phenyl protons to 10, and a considerable downfield shift of the H3 multiplet to 6 3.57 ppm. Mass spectroscopy showed the molecular ion at m/z 610, and the only significant fragments at m/z 519 (loss of benzyl) and m/z 91 (tropilium ion), the latter being the base peak. Reduction of the ketone 69 wi th sodium borohydride gave the corresponding alcohol as a mixture of epimers in 83% yield. The IR spectrum showed a band at 3492 cm'^ characteristic of alcohols, the band of the lactone carbonyl at 1767 cm"^ and the disappearance of the ketone band at 1663 cm"^ In the nmr spectrum, the signals for the proton at C 7 ' of both epimers can be seen at 6 4.2 and 4.5 ppm. The presence of the mixture of epimers at C 7 ' complicates the assignment of the signals for the other protons that now present closer chemical shifts. The doublet for the methyl groups of the isopropyl side chain is located at S 1.45 ppm and the protons at positions 2, 3 and 7" give rise to a multiplet at S 2.50-3.2 ppm. The multiplet corresponding to the methylene protons of C4, is centered at S 3.70 ppm, and the singlet for the methoxy groups on ring D is at £ 3.78 ppm. The isopropyl methine heptet is located at S 4.50 and the signals corresponding to the methylene protons from the benzyl protecting groups appear at S 4.98 and 5.10 ppm. Two singlets attributed to the aromatic protons of ring D, are located at S 6.26 and 6.40 ppm, and their integration reflects the same epimeric ratio than the signals for the C 7 ' proton. The aromatic protons of ring A (positions 2 ' , 5' and 6 ' ) appear as a multiplet at S 6.7-6.9 and the phenyl protons from the benzyl protecting group produce a multiplet at S 7.2-7.5 ppm. The major isomer was assigned as the /3-alcohol based on the relative accessibility of the carbonyl faces in the most favorable configuration of the starting material as shown by Dreiding models. Other closely related dibenzylbutanolides also have been shown to produce mainly the ^-hydroxy epimer upon reduction of the q 1 ketone . This assignment was not entirely crucial since both isomers can be used to produce the glycoside without the need of isolation. In the reaction conditions of glycosidation, only the C-4 /9-glycoside is obtained'*^. Since sodium borohydride is an inexpensive reagent, and the reactions proceeded in good y ie ld , we did not investigate the improvement of the epimer ratio by, for example, using sterically hindered hydride donors. The alcohol (70) was then subjected to hydrogenolysis using P d / C (10%) as catalyst. The reaction proceeded at a fast rate using I atm. of hydrogen and producing 56 in 74% isolated y ie ld (Scheme 13). If the reaction was left for a longer period, however, over-hydrogenolysis occurred to give product 54 (84%) in which the C-7 alcohol has been lost. The spectroscopic data of this compound proved to be OH OH Scheme 13. The hydrogenolysis of compound 70. identical with a sample prepared by Terry Jarvis via the desulfurization-hydrogenolysis of dithioketal 68 with Raney-Nickel . On the other hand, too short reaction times afforded mixtures of mono- and didebenzylatcd products 71 and 56. The * H nmr spectrum of the monobenzylated product 71 showed a reduction in the number of phenyl protons to 5, and the disappearance of one of the benzylic methylene signals due to a benzyl group and present in the starting material (5 4.98 ppm), while the other (5 5.10 ppm) remained. In general, the position and multiplets of the rest of the signals were consistent with the remaining benzyl ether being at position 3' . The mass spectrum of compound 71 showed the molecular ion at m/z = 522, being the fragment at m/z = 91, the base peak. We found that upon running the hydrogenolysis reaction for 50 min, yields in the order of 75% for compound 56 could be achieved. The product presents in the IR spectrum absorptions at 3459 (alcohol) and 1757 (lactone) cm'* respectively. In the *H nmr spectrum, the doublet for the isopropyl methyl is located at S 1.4 ppm. The protons at positions 2 and 3 produce multiplets centered at S 2.98 and 2.64 ppm respectively. The A B X system characteristic of the C 7 " methylene protons is situated at S 2.85 (dd,d J = 5.4, 13.6 Hz) and 3.10 (dd, J = 4.9, 13.6 Hz) while the signal due to the protons at C4 appears together with the ring D methoxy signals as a multiplet at S 3.8-3.95 ppm. The isopropyl methine, together with the proton at C 7 ' produce a multiplet at 6 4.52 - 4.62. The aromatic protons are evident as a singlet at S 6.4 for ring D and a multiplet at 6 6.65-6.86 ppm for r ing A . Three one-proton signals located at S 1.7, 5.4 and 5.75 ppm disappeared upon addition of deuterated water. Mass spectrometry showed the molecular ion at m/z 432 and a fragmention pattern that is characteristic of dibenzyl butyrolactones of this k ind , giving rise to a base peak with m/z 167 corresponding to the fragmentation of the C7"-C2 bond. 56 Having thus developed a short and efficient synthesis of precursor 56, we turned our attention to the biotransformation experiments, with the intention of performing the oxidative coupling to the aryl tetralin 60 (Scheme 14). Scheme 14. The proposed biotransformation of substrate 56. The Biotransformation of Trans-2-(4-hydroxy-3,5-dimethoxybenzyl)-3-(3-hydroxy-4-isopropoxy-a-hydroxybenzyl)butanolide (56) with Podophyllum peltatum cell suspension cultures. We felt that precursor 56 incorporated many of the characteristics previously shown to be important for successful biotransformation. It has a hydroxy group at position 4" and a sufficiently nucleophilic ring A to make the cyclization of the hypothetical quinone methide intermediate possible (Scheme 15). Scheme 15. Hypothetical mechanism for the ring closure of substrate 56 to aryl tetralin 60. It also has a protective group (isopropyl) which can be cleaved'-^ i n the presence of the methoxy groups of ring D to give an entry to the methylenedioxy system'^-' and the hydroxyl group already in position 7 ' . If the enzyme's active site is reached, this would clearly provide an efficient entry to the podophyllotoxins. Isolation and Structure Elucidation of the Major Biotransformation Products of 56 with Podophyllum peltatum Ce l l Suspension Culture. In order to isolate sufficient quantities of extracts (and later, metabolites) from the biotransformation of substrate 56 with P. peltatum cells, a large scale biotransformation was set up, using cells grown in a Microferm reactor for 2 days (450 mL drained cells, 18 days old, in 3 L 1/2 MS - 1.5% sucrose medium) and 3.0 g of substrate dissolved in ethanol (70 mL). Biotransformation was allowed to proceed while the consumption of the precursor was monitored by chromatographic analysis of broth samples. We were pleased to f ind that both starting material and products were present in the culture medium. The analysis of the broth samples also indicated a fair ly rapid biotransformation; substrate consumption was >50% after 48 h, and only a trace remained after 4 days of incubation. At this point, the Microferm was harvested, and cells and broth were extracted separately. Extract ion of the cells was carried out using ethyl acetate and methanol (Figure 8). The cells were homogenized in ethyl acetate, followed by f i l t rat ion through Celite 545, and the cell residue was washed with ethyl acetate. The aqueous filtrate was separated, extracted with ethyl acetate, and the organic layers were combined. Solvent removal yielded the ethyl acetate extract. Sonication of the cell residue in methanol, followed by f i l t rat ion through Celite 545, washing with methanol and solvent removal constituted the methanol extract. Cells homogenization in EtOAc; filtration residue t filtrate sonication with MeOH; filtration and solvent removal EtOAc layer t Aq. layer Methanol Extract 2.4 g extraction with EtOAc EtOAc layer Aq. layer t Discard solvent removal EtOAc extract 3.74 g Figure 8. Extraction procedure for the P. peltatum cells. The broth was saturated with sodium chloride and extracted with ethyl acetate (Figure 9). A methylene chloride extract was also obtained. Chromatographic analysis of these extracts showed lignan-type compounds only in the ethyl acetate extracts of broth and cells. The methanol and methylene chloride extracts were not investigated further. The ethyl acetate extracts were subjected to chromatographic separation (Figure 10). EtOAc layer Broth 3L NaQ addition; filtradon EtOAc extraction t Aq. layer EtOAc layer Aq. layer EtOAc layer Aq. layer solvent removal t CH2CI2 layer Aq. layer solvent removal EtOAc extract 3.11 g CH2CI2 extract 8.7 mg t Discard Figure 9. Extraction procedure for P. peltatum spent medium. Part ial separation of the broth extract was carried out by a quick f i l t rat ion through sil ica gel, eluting with chloroform:methanol 25:1. The column was also Broth «xtract 3.11g fraction 1 ; 870 mg column chromatography C H C I 3 : MeOH 25:1 MsOHIOO Styrène 74; 92 mg fraction 2; 930 mg column chromatography CH2CI2 : MeOH 25:1 Aryl tetralin 75 301 mg column chromatography CHCI3 : MeOH 25:1 MeOH fraction 3: 440 mg fraction 4; (methanol wash) 850 mg column chromatography CHCig: MeOH 25:1 C H C I 3 : MeOH 10:1 Hydroxy aryl tetralin 76; 155 mg fractbn A; 60 mg fraction B; 667 mg fraction C; 96 mg TLC CHCI3: MeOH 19:1 Styrène 74; 15 mg column chromato-graphy CHCI3: MeOH 19:1 Aryl tetralin 75; 583 mg fraction D; (methanol wash) 67 mg column chroma-tography CHCI3: MeOH 25:1 Hydroxy aryl tetralin 76; 35 mg TLC CHCI3: MeOH 19:1 Hydroxy aryl tetralin Methoxy aryl tetralin 77; 12 mg 78; 28 mg Figure 10. Chromatographic separation of the broth extract. flushed with methanol to remove the polar material. Four crude fractions containing complex mixtures of metabolites were collected. Fraction 1 contained mostly non-polar cell produced material (870 mg). Fractions 2 (930 mg) and 3 (440 mg) contained lignans and were orange-brown solids. Fraction 4 (850 mg) was a dark brown solid containing the polar material. Fraction 1 yielded upon chromatographic separation (chloroform:methanol 25:1) 92.0 mg of 74 and 301 mg of 75. Fraction 2 was st i l l very complex and required a partial purif icat ion by chromatography eluting with chloroform:methanol 25:1, and a f inal wash with methanol to remove the polar material. Again 4 fractions were obtained. Fraction A (60.2 mg) contained mostly cell material but when applied to preparative thin-layer chromatography afforded 15.0 mg of compound 74. Fraction B (667.8 mg) contained the main metabolic product, and was further purif ied by flash column chromatography (chloroform:methanol 19:1) to yield 583.8 mg of compound 75. Fraction C (98.2 mg) was a mixture of three compounds possessing very similar retention times on T L C , and after a column chromatography (chloroform:methanol 25:1) and preparative T L C (chloroform:methanol 19:1), afforded 35.5 mg of 76, 12.7 mg of 77, and 28.0 mg of 78. Fraction D (67.6 mg) was a dark solid containing polar material. Fraction 3 consisted mostly of compound 76 and indigenous metabolites, and was purif ied by flash column chromatography (chloroform:methanol 25:1) to obtain an additional 155.5 mg of compound 76 as a white solid. Evidence that led to the structure elucidation of these compounds follows (Figure 11). Figure II . Biotransformation products from substrate 56 with P. peltatum cells. Compound 74 was isolated as a light yellow solid with a molecular formula of C23H260g (high resolution mass spectrometry). Its IR spectrum shows the carbonyl band at 1720 c m ' ^ well below the value of 1770 cm'^ observed for substrate 56. The U V spectrum showed a bathochromic shift with a maximum at 324 nm relative to 281 nm for compound 56, consistent with a cinnamic ester. The product presented in the ' H nmr spectrum the signals characteristic for a r ing opened compound, part icularly the aromatic region in which ortho and meta coupling gives rise to mult ipl ic i ty . The absence for the A B X signal for the protons of C 7 ' ' and of the multiplet for the proton at C2 indicated the presence of unsaturation at these positions. Accordingly, a new singlet was located at 6 7.6 ppm and assigned to the v iny l i c proton at C7" . The rest of the signals are down-field with respect to those of compound 56. The proton at C3 presents a multiplet centered at S 4.08 ppm and the signals for the C4 methylene protons are located at 6 4.28 and 4.57 ppm. The methoxyl signal is at 6 3.93 and the proton at C 7 ' presents a doublet centered at S 5.02 ppm. The aromatic protons of r ing D produce a singlet at S 7.10 while those of r ing A give rise to a multiplet at 6 6.64-6.82 ppm. The stereochemistry of the double bond is believed to be E based on the chemical shift of the proton at position 7". It has been shown in similar lignans^^ that when the geometry of the olefin is Z , the v iny l i c proton signal appears at chemical shifts around S 6.6 ppm, while in the E configuration, the deshielding effect of the neighboring carbonyl group shifts this signal down-field to about 6 7.5 ppm. The chemical shift of S 7.6 ppm for the proton at C 7 " in our case suggests an E configuration for the double bond. Compounds 75 to 78 showed in the nmr spectrum an aromatic proton signal pattern consisting exclusively of singlets, implying substitution at C 6 ' of compound 56, and therefore, that ring closure was achieved. The appearance of only one signal for both H 2 ' and H 6 ' indicated that ring D had essentially complete freedom of rotation. The main metabolite was identif ied as aryl tetralin 75. This product showed an IR absorption at 1774 cm'* which is consistent with the presence of the lactone carbonyl group. The mass spectrum gave a molecular ion at m/z 430 which is 2 mass units less than the molecular ion peak of the substrate 56 and, in contrast to the fragmentation pattern presented by 56, the product on hand showed at base peak at m/z 154, i.e. loss of ring D, and a fragment with m/z 388 (loss of iso-propyl). These fragments are characteristic of r ing closed compounds. 75 75a As mentioned earlier, the *H nmr spectrum of compound 75 shows no ortho or meta coupling between the aromatic protons. The protons at position 2 ' and 6' gave a singlet at S 6.46, the protons at positions 5 and 8 occurred as one proton singlets at 5 6.38 and 6.88 ppm respectively. The trans stereochemistry of the lactone was confirmed by analysis of the signals produced by the al iphatic protons. H2 appears as a doublet of doublets at S 3.19 ppm with coupling constants of 11.4 and 14 Hz , suggesting also a trans relationship with H I whose signal shows as a doublet at 5 3.96 ppm with coupling constant of 11.4 Hz . These values are in good agreement with the reported coupling constants of H2 in compound 75a of 11 and 14 Hz. The product 75a was prepared by Jan Palaty^^ via the peroxidase catalyzed cycl izat ion of the corresponding dibenzylbutyrolactone using Catharanthus roseus cell free extracts, and the stereochemistry was unambiguously determined by X - r a y analysis. Also in the H nmr spectrum of 75, the isopropyl methyl signals appear now as two doublets at S 1.14 and 1.28 ppm. The signal corresponding to the isopropyl methine, appears together with the multiplet for the C3a methylene protons at S 4.25-4.47 ppm. The multiplet for H3 is located at S 2.68, while the doublet for H4 is at 6 4.85 ppm (J = 2.7 Hz). The methoxy groups singlet is located at S 3.86 and three signals integrating for one proton each at 6 1.55, 5.5 and 5.7 ppm exchanged with heavy water. The U V spectrum showed maxima at 283 and 215 nm. Compound 76 was isolated as a white powder with molecular formula C23H26O9 (high resolution mass spectrometry). The presence of a strong M'*'-18 peak in the electron impact mass spectrum suggested that the extra oxygen formed part of a hydroxyl group. The product showed an IR band at 3484 cm'^ characteristic of hydroxy groups and a band at 1746 cm*^ consistent with the lactone carbonyl. The U V spectrum showed maxima at 290 and 206 nm consistent with the aromatic rings of the aryl tetralin. In the ^ H nmr spectrum, the aromatic singlets can be seen at S 7.08, 6.61 and 6.21 ppm for H8, H5 and H 2 ' , H 6 ' respectively. A n additional one proton singlet is found at 6 5.22 ppm assigned to H I , while the signal for H2 is missing. The system for the aliphatic protons is clearly visible. H4 shows as a doublet with J = 7.8 Hz at 6 4.03 ppm, H3 occurs together with the methoxy signal at S 3.62 ppm, and the A B X system for the methylene protons is located at 5 4.21 (dd, J = 8.3, 9.6 Hz) and 4.37 (dd, J = 9.6, 9.6 Hz) ppm. A positive N O E between H3 and H4 indicates that they have a cis relationship and, therefore, the hydroxyl group at C4 must be beta-oriented. The stereochemistry of the lactone ring appears to be cis according to the similarity of the coupling constants of H3 with both protons at C3a, since a r igid trans fusion would be l ike ly to produce very different dihedral angles between these two positions (3 and 3a). Such a situation would translate into much different J values for this system. N O E irradiation experiments failed to provide conclusive evidence to assign the orientation of the aromatic r ing attached to C I , and attempts to crystallize the material for X- ray analysis were unsuccessful. Compound 77 shared many of the characteristics of 76. The high resolution mass spectrum indicated the same molecular formula and similar fragmentation pattern; it had IR absorptions at 3424 (OH) and 1709 (lactone) cm'*, and U V maxima at 289 and 210 nm. The * H nmr spectrum showed again only singlets for the aromatic protons and an additional one proton singlet at 6 5.10 ppm assigned to H I . The system for the aliphatic protons is again clearly visible. The proton at H3 is seen as a multiplet at 6 3.40 ppm, the A B X system for the C3a methylene protons is located at 6 4.04 (dd, J = 8, 8 Hz) and 4.22 (dd, J = 8, 9.6 Hz) ppm, and the doublet for H4 at S 4.62 ppm with J = 6.0 Hz. These assignments were confirmed by multiple irradiation experiments. For example, upon irradiation at 5 3.4 ppm, the signals due to the protons at C3a (5 4.04, 4.22 ppm) collapse to doublets with J = 8 Hz , while the doublet for the proton at C4 (5 4.62 ppm) turns into a singlet. Again the stereochemistry of the lactone ring fusion is most probably cis as suggested by the similarity of the coupling constants of H3 with both protons at C3a, but this evidence is not conclusive. F ina l ly , compound 78 presented a very similar * H nmr spectrum when compared to aryl tetralin 75, except that an additional methyl group singlet resonance at 6 3.84 ppm was noted, and now only two protons could be exchanged with heavy water (6 5.47, 5.68 ppm). High resolution mass spectrometry indicated the molecular formula C24H28O8 (14 mass units more than compound 75), and the electron impact spectrum showed the characteristic fragmentation pattern with a strong molecular ion peak and the fragment at m/z = 154 (ring E) as in the case of compound 75 confirming a ring-closed structure. Chromatographic purif icat ion of the ethyl acetate extract obtained from the cells was done in a similar way. The majority of the material, by weight, was cell produced material, but an additional 124.4 mg of r ing closed product 75 were obtained. In summary, this experiment afforded 5 biotransformation products in the fol lowing yields: styrene 74: 107 mg (3.5%) ; aryl tetralin 75: 1.00 g (33.5%); hydroxy aryl tetralin 76: 191 mg (6.18%); hydroxy aryl tetralin 77: 12.7 mg (0.4%) and O-methyl-aryl tetralin 78: 28.0 mg (0.9%). The total recovery was 1.34 g (44.6%). We concluded that the long biotransformation time of this experiment had been detrimental in the f inal recovery, the biotransformation proceeded possibly to give water soluble compounds (e.g. glycosides), or the starting precursor was catabolized or otherwise irreversibly bound to cellular components. However, we felt encouraged to investigate the possibility of a semi-continuous process to exploit the same biomass to effect several biotransformation experiments. Since only a small fraction of product was obtained from the cell extract, harvesting of the spent medium under aseptic conditions should be a practical way of adding and removing substrate and biotransformation products. The Semi-continuous Biotransformation of Substrate 56 with Podophyllum peltatum Ce l l Suspension Culture. The ideal state for a cell suspension culture is that of morphological, biochemical and genetic homogeneity, grown in a fu l ly controllable environment. The majority of investigations involving plant cell suspension cultures have been performed under batch culture conditions. Under these conditions, the cells grow in a l imited amount of medium and multiply to form a population in which succeeding generations progressively modify their environment. This situation produces a sequence of changes in the culture that is referred to as the culture cycle. Under these conditions, the metabolism of the cells is also altered and it is extremely d i f f icu l t , i f not impossible, to determine any factor responsible for these fluctuations. In continuous culture, medium is added into a constant volume of growing culture producing a steady-state condition for growth The environment imposes a constant growth rate on the cells so that the doubling time and overall metabolism of the cells remain constant and thus characteristic of the steady-state. It must be noted, however, that the cells divide randomly. The ideal conditions would include synchronicity of the cells so that instead of an average condition as is obtained in steady-state conditions from the randomly d iv id ing population of cells, a synchronous pattern of change, which coincides with the cell cycle and repeats itself with each successive doubling of the cell population, would be produced. This would have the advantage that enzymes or metabolites occurring only at certain stages of the cell cycle, could be obtained at maximal yields. In our experiments, we envisaged a semi-continuous process in which the cells would be grown for a f ixed period of time corresponding to the lag, growth or stationary phase of the culture cycle, and resuspend them in diluted medium (with just enough nutrients to maintain the live cells) containing the substrate (Figure 12). In this way, we would minimize the time required to prepare biomass. This "draw and f i l l " process would result in a series of partial batch cultures that would have abbreviated lag phases and would probably not reach stationary phase provided resuspension is done quickly. Once this draw and f i l l process is initiated, the physiology of the culture would be that which is created by this process, somewhat between batch and continuous culture. The age of the ini t iat ing culture may sti l l play a role in the biotransformation outcome, in that it may be related to the production of the relevant enzymes. We first attempted this type of study with 7-day old cells (growth phase). The Microferm reactor was fitted with a peristaltic pump and a filter that would enable Substrate addition t Grow cells in bioreactor Incuba te Remove broth containing the product Cell aggregates Add new medium containing substrate Add new medium containing substrate Incuba te Remove broth InCUbate containing the product 12. Schematic representation of the semi-continuous biotransformation process. the removal of the broth and resuspension of the cells in situ. The reactor was inoculated wi th 180 mL drained cells (RI - 1.3330, p H - 4.5, inoculum, 15 days old) and grown for a fu l l cycle (18 days), after which the spent medium was removed and the cells were resuspended in fresh medium. Seven days after resuspension, the cells were healthy and uncontaminated (pH = 4.43, R I - 1.3342) indicating that the peristaltic pump worked satisfactorily. The cells, now in growth phase, were resuspended in 1/10 MS - 0.3% sucrose medium supplemented with 2.5 m M M E S as buffer and the p H was adjusted to 6.5 which was considered the optimum for the ring closure to occur according to data available from Jan Palaty^^. Substrate 56 (168.3 mg) was added, dissolved in ethanol (5 mL), and biotransformation was allowed to proceed for 22 h. After this time, the broth was removed and replaced with 1/10 MS -0.3% sucrose medium containing 200 mg of precursor 56 in 7.5 mL ethanol and 2.5 m M M E S as buffer. Aga in pH was automatically controlled to 6.5 and the Microferm was harvested (cells and broth) after 22 h of incubation. The results are shown in Table 1. Substrate added (me) Extract obtained (mz) Recovered substrate % Yield of 74 % Yield of 75 % Yield of 76 % Recovery % 168.3 281.7 0.3 1.1 29.9 11.0 42.2 200.0 513.0 11.5 1.8 51.3 12.8 69.9 ceUs 2190.0 12.5 12.5 Table 1. Semi-continuous biotransformation of substrate 56 with 7-day old P. peltatum cells. Incubation time was 22 h, pH = 6.5. As can be seen, similar yields to the ones obtained in the previous biotransformation were obtained in the first experiment. However, in entry #2, the overall recovery and ring closure yields are higher. This could be explained in several ways. F i rs t ly , the cells may be accumulating products or starting material from the first biotransformation, and are being released in the second experiment; but the starting material recovered from the cell extract (24.9 mg) indicates the contrary. Cells do not retain either products or starting material. Another explanation would be that the cells are able to respond better to biotransformation after they have been exposed to the substrate. We wanted to know i f this trend of increasing biotransformation abi l i ty would be maintained throughout a larger number of biotransformations. We also wanted to check i f the cells retained the biotransformation abi l i ty after repeated resuspension, and to what extent the stress to which they were subjected affected the biotransformation outcome. To answer these questions, another set of experiments was done. Stationary phase cells would be assessed under the same experimental conditions, but a large number of biotransformations was planned. To this effect, a Microferm was started (180 mL drained cells, RI = 1.3335, pH = 4.63, inoculum 19 days old) and the cells were grown for 17 days (RI = 1.3334, pH = 4.8). The spent medium was removed and the cells were resuspended in 1/10 MS - 0.3% sucrose medium containing substrate 56 (200 mg in 5 mL ethanol) and 2.5 m M MES buffer. The p H was automatically controlled to 6.5, and biotransformation was allowed to proceed for 24 h. After this time the broth was removed and extracted, and the cells were resuspended in broth containing substrate 56 under the same conditions. The operation was repeated three times (entries 1-4, Table 2 ) after which time it was found that some cells were dying, presumably due to lack of nutrients. It was decided to do the fol lowing biotransformations at a higher sucrose concentration, and 1/5 MS - 0.6% sucrose medium was arbi trar i ly chosen (entries 5 and on). Also during these biotransformations (entries 5 and 6), the pH control system failed leading us to believe that this was not a relevant parameter for our biotransformations since conversion occurred apparently at the same rate when the pH was left to drift . Problems during the extraction of these experiments in particular, forced us to resuspend the cells in fu l l strength (1/2 MS - 1.5% sucrose) medium for one day to give them a chance to recover (entry 7). Three more experiments were done after this time using 1/5 MS - 0.6% sucrose medium (entries 8-10), before resuspension in 1/2 MS - 1.5% sucrose medium and normal growth conditions were established (entries 11-14). After one month of normal culture conditions, the cell suspension was found to be able to effect biotransformation (entries 15 and 16) even though many cells had died. The results are shown in Table 2. The same trend as with the 7-day old experiments was observed, namely, that the second biotransformation of a given series afforded best results (Entries #1 vs. 2, and #15 vs. 16). In these cases, the recoveries are highest as are the ring closure yields, but in any subsequent biotransformation the percentage of ring-closed compounds decreases and tends toward stabilization at about 23% for aryl tetralin 75. In entry #8, the recovery is good, but there is a poor biotransformation since a significant amount of substrate was not transformed indicating that the cells had not recovered from the previous experiments. In general, we thought that enzymatic modification of the substrate and/or products gave water soluble compounds which would remain in the aqueous phase since the cell extract did not reveal the presence of these metabolites. In fact, glycosidation processes (amongst others) might be in effect in the cell suspension culture. It is also worth mentioning the high yield of compound 76 in entry #16 (40%), possibly indicat ing that longer incubation times favored the production of this compound, but results are d i f f icu l t to interpret because of the low recoveries. However, this was an exciting experiment on the one hand, in that enzymatic activity was retained throughout a large series of experiments. Entry Substrate added(mg) medium strength time (h) Extract obtained (mz) Substrate recovery % Yield of 74 % Yield of 75 % Yield of 76 % Recovery % 1 200 1/10 24.5 331.8 26.2 1.1 32.2 5.9 55.2 2 200 1/10 26.8 251.4 37.2 9.2 47.0 9.5 78.5 3 200 1/10 45.0 243.7 17.5 26.7 1.4 40.8 4 200 1/5 43.2 230 10.9 21.2 9.9 38.7 5 200 1/5 55.8 286 6 200 1/5 46.1 242.9 7 1/2 23.7 97.8 8 200 1/5 23.7 152 52.3 1.9 26.9 12.8 72.2 9 200 1/5 25.8 246 5.0 18.3 11.0 32.9 10 200 1/5 23.5 244 13.3 25.7 7.0 41.7 11 _ 1/2 167.0 271 12 1/2 192.2 299 13 1/2 240.6 667 14 1/2 96.1 350 15 500 1/5 50.7 434 3.1 12.9 12.6 27.8 16 814 1/2 182.8 1632 22.3 40.1 62.3 cells • • _ 6241 • _ _ Table 2. Semi-continuous biotransformation of substrate 56 with 17-day old P. peltatum cells. Entries 1-5 with pH controlled to 6.5. We thought next of assessing the influence of pH drif t in the biotransformations since it could be important for better yields and recoveries. With the experience gained from this bioreactor, we returned to younger ceils st i l l i n their growth phase (7 days) and performed seven consecutive biotransformation experiments. In these studies, 1/5 MS - 0.6% sucrose medium was used and the p H was allowed to drif t . A much better reproducibili ty was obtained, and normal cell growth could be continued after the biotransformation experiments were finished, the cells being healthy and normal in a l l respects. The results are shown in Table 3. Entry Substrate a(ided(mg) Extract obtained (TOR) Substrate recovery % Yield of 74 % Yield of 75 % Yield of 76 % Recovery % 1 200.0 375 5.4 2.1 32.6 1.1 45.6 2 200.0 229 7.0 3.9 25.7 6.9 41.0 3 200.0 237 7.2 1.8 27.1 14.7 47.8 4 200.0 220 9.3 3.8 28.8 10.3 48.2 5 200.0 223 5.8 2.5 27.2 9.9 43.2 6 200.0 192 2.7 0.3 20.1 11.6 33.9 7 200.0 208 4.0 0.9 22.6 10.3 36.5 Table 3. Semi-continuous biotransformation of substrate 56 wi th 7-day old P. peltatum cells. P H was not controlled. We found that there were no more abrupt variations in yields, but only half of the material was accounted for, again glycosidation or degradation of the precursor and/or products being the suspected cause. With the data gathered from al l of these experiments, we concluded that the enzymes were more dependent on pH (6.5 gave better yields and recoveries) than on cell age. The biotransformation products can be explained using a common intermediate, namely, the hypothetical quinone methide which could tautomerize to give styrene 74 or undergo nucleophil ic attack by ring A to produce the ring-closed compound 75 (Scheme 16). CH3O J OCH3 OH 75 Scheme 16. The intermediacy of a hypothetical quinone methide in the formation of products 74 and 75. The a ry l tetralin 75 could then undergo 0-methylation to 78 or enzymatic hydroxylat ion to 76. This latter process might involve multiple steps in nature, for example, conversion of 75 to a styrene, that is, dehydrogenation at C1-C2, epoxidation followed by epoxide opening or perhaps direct hydroxylat ion at the activated C2 carbon atom. No evidence concerning these mechanistic speculations is available from our studies. It should also be emphasized that an alternative mechanism involving di radical coupling of 56, and involving peroxidase enzymes, cannot be excluded from the present studies (see later). Biotransformation Reproducibility. Summary of the Semi-continuous Biotransformation Experiments. The average yields for the semi-continuous biotransformation of substrate 56 with P. peltatum cells, in the experiments discussed above, are presented in Table 4. CeUAge Recovered substrate % Yield of 74 % Yield of 75 % Yield of 76 % Recovery % 7-day (table 1) 8.1 1.4 40.6 11.9 41.5 17-day (table 2) 18.3 1.3 25.9 12.2 50.0 7-day (tables) 5.9 2.1 26.3 10.2 42.3 Table 4. Average yields for the semi-continuous biotransformation of substrate 56 with P. peltatum cells. As can be seen from this table, the average biotransformation results fa l l wi thin experimental error. It is worth mentioning the yie ld of 40.6% for the ring closed product 75 when the biotransformation is done under controlled pH (6.5, entry 1) versus not controlled conditions (entry 3). These experiments illustrate also the reproducibil i ty of the biotransformation outcome between different batches of cell culture. For example, entries 2 and 3 compare the average yields obtained over the space of several months. The experiment summarized in Table 2 required 69 days to completion while that of Table 3, 14 days. Clearly, the abi l i ty of the cells to biotransform the supplied substrate is maintained for relatively long periods of time without significant yield variations. The Biotransformation of l-(3,5-Dlmethoxy-4-hydroxy-phenyI)-6,4-dlhydroxy-3-hydroxymethyI-7-lsopropoxy-l,2,3,4-tetrahydro-2-naphthoic acid 7-lactone (75) with Podophyllum peltatum Cell Suspension Cultures. To check the stability of aryl tetralin 75 under the biotransformation conditions, a series of experiments were done and 3, 11, 17, and 21-day old shake flask cultures were assessed. A r y l tetralin 75 in 3 mL ethanol was added to each flask containing 300 m L of cell suspension culture. A series of control flasks were set up adding only 3 mL ethanol and reaction progress was followed by withdrawing 2.5 mL aliquots. H P L C (Rad-Pak C i g Waters, H 2 0 - M e O H 55:45 with 0.1% A c O H , U V detector) and T L C analysis (CHCl3 -MeOH 9:1) of the ethyl acetate extracts were done after 0, 3, 6, 9 and 24 h, after which the flasks were harvested by homogenizing together cells and spent medium prior to ethyl acetate extraction. The analytical conditions used are suitable for the detection of compounds 75-78. The data (Figure 13) indicate a fair stability for the aryl tetralin 75 under the biotransformation conditions. In one case (21-day old cells experiment) the concentration of aryl tetralin 75 dropped by more than 50% after 3 h of incubation, but a repeat of this experiment revealed a similar behavior to the rest of the experiments suggesting that this particular case was exceptional and l ike ly due to the inevitable variation between different shake flasks; particularly when a differentiated culture like ours is used. The fact that the hydroxylated compound 76 was not detected in every case and that the rate of consumption of product 75 was relatively slow (89-91% of the product was recovered unchanged after 24 h) suggests Time (hours) Figure 13. Time-course study to check stability of aryl tetralin 75 under the biotransformation conditions. that the process going from 75 to 76 is l ike ly to require long biotransformation times increasing the possibility of 75 being biotransformed further along the way, but not exclusively to product 76, thereby providing for a more complex situation. Perhaps C F E technology would be a better option for these studies since variations between cell batches are eliminated and experiments are run on a much shorter time scale. Such studies w i l l be undertaken by other coworkers in our laboratory. We were prompted next to investigate the biotransformation of a more closely related analogue of podophyllotoxin, and compound 57 seemed appropriate (Scheme 17). The fact that 57 incorporates the methylenedioxy group, would make it presumably more stable towards oxidations or degradations during biotransformation, and i f ring closure could be achieved, a more direct way into the podophyllotoxins would be on hand. Should the ring closure produce the wrong stereochemistry at C ( l ) , we intended to employ a synthetic pathway involving the formation of a double bond (see 80), followed by directed catalytic hydrogéna t ion to give the desired rra«s- l ,2-aryl tetralin system (Scheme 18). Very similar hydrogéna t ions have been reported in the literature^^. C H g O ^ y ^ O C H a C H a O ' ] ! OCH, OH OH 57 61 Scheme 17. The proposed biotransformation of substrate 57. Scheme 18. Proposed C ( l ) epimerization sequence from compound 79 to epipodophyllotoxin (6). Synthesis of Trans-2-(4-hydroxy-3,5-diniethoxybenzyl)-3-(3,4rmethylenedioxy)-7-hydroxybenzyl)butanolide (57). The synthesis of substrate 57 was done along the same lines as that of substrate 56, and is outlined in Scheme 19. The sequence starts with readily available piperonal (81) which upon treatment with thiophenol and boron t r i f luor ide etherate at -40° C for 15 min afforded thioketal 82 (98% yield) as a yellow o i l . Michae l addition of the anion of 82 generated by the addit ion of «-butyl l i th ium, to 7-crotonolactone followed by in situ a lkylat ion with the aryl bromide 67, produced the desired dibenzylbutyrolactone 83 in 55% yield. The PhSH 81 BFa.EtaO PhS. SPh 82 2) TKsrotonolactone CHjO^r^OCH, OBn 6 7 1)NaBH4 2) Pd/C. H2 0CH3 OBn 83 HgO, BFa-EtgO THF-H2O C H 3 O ' ^ " 0 C H 3 OBn 5 7 Scheme 19. The synthesis of substrate 57 from piperonal (81). product could be crystallized from acetone and showed a fragment corresponding to M'^'-PhS on electron impact mass spectroscopy. However, both H R M S and elemental analysis were in agreement wi th the molecular formula C40H36O7S2. The IR spectrum of this compound showed the carbonyl band at 1770 cm'^ corresponding to a 7-lactone. In the ^ H nmr spectrum, the signal for the methylenedioxy group was present at 6 6.05 ppm, H2 produced a multiplet at 6 2.85-2.92 ppm while H3 produced another multiplet at S 3.29-3.34 ppm. Aga in , the A B X system for the lactone methylene was clearly visible at 5 3.50 and 4.45 ppm. The coupling constants of the lower f ield component of this system were 3 and 10 Hz , indicating that the proton responsible for this component is most l ikely cis to H3. Thioketal hydrolysis was carried out in 85.5% yield by treatment with red mercuric oxide and boron tr if luoride etherate to produce ketone 84 as white crystals when crystallized from ethyl ether. The IR spectrum showed two bands for carbonyl groups at 1770 (lactone) and 1670 c m ' ' (ketone) while the ' H nmr spectrum showed a reduction in the aromatic protons to 5 and a downfield shift for H3 compared to that of compound 83 which appeared now as a doublet of doublets of doublets at 6 4.00 ppm. The signal for H2 is seen as a multiplet at 5 3.58 ppm. The protons at position 7" are located at S 3.0 (dd, J = 6, 14 Hz) and 3.08 (dd, J = 5, 14 Hz), the singlet for the methoxy groups on ring D is situated at S 3.7. The A B X system for the methylene protons of C4 is located at S 4.10 (dd, J = 8, 9 Hz) and 4.39 (dd, J = 8, 9 Hz) ppm, and the methylene singlet from the benzyl protecting group is seen at 5 4.92 ppm. The aromatic protons appear as a two proton singlet at S 6.29 (H2", 6"), a one proton doublet at 6 6.80 ( H 5 ' , J = 8 Hz) and a seven proton multiplet at S 7.22-7.48 ppm. Electron impact mass spectroscopy showed the molecular ion at m/z = 488 with a base peak at m/z 91 arising from the loss of the benzyl protecting group. Treatment of this compound with sodium borohydride (1.4 equiv.) in methanol at 0° C produced alcohol 85 in 95% yield as a white foam. The product was present as a mixture of cpimers at C 7 ' and the major isomer was assumed to be the /9-alcohol on the same grounds as those presented earlier for compound 70. The ' H nmr spectrum showed the new proton at CI' as a doublet with J = 6.7 H z at 5 4.60 ppm, a new broad signal that could be exchanged with heavy water at 6 1.70, the singlet for the methoxy groups at S 3.80 and the multiplet for the methylene protons of C4 centered at 5 3.9 ppm. The protons at positions 2, 3 and 7" create a multiplet at S 2.50-3.10 ppm, while the protons of r ing A (5 ' , 6 ' , and 2 ' ) appear as a multplet at 6 6.6-6.8 ppm. The rest of the aromatic protons are under a singlet at S 6.4 (H2", 6") and a multiplet at S 7.24-7.50 (5H from benzyl ether) ppm. The IR spectrum shows absorptions at 3600 (alcohol) and 1770 (lactone) c m ' ' and mass spectrometry showed the molecular ion at m/z 490. F ina l ly , catalytic hydrogéna t ion of compound 85 under 1 atm of hydrogen for 6 h with P d / C 10% as catalyst afforded substrate 57 in 86.6% yield. The loss of the benzyl protecting group was evidenced by the disappearance in the ' H nmr spectrum of the signals at S 5.0 and 7.24-7.50 ppm, and the molecular ion at m/z 402 shown in the mass spectrum of 57. We had now a 5-step synthetic route for substrate 57 starting with piperonal and could turn once again to biotransformation experiments. The Biotransformation of Trans-2-(4-hydroxy-3,5-dimethoxybenzyl)-3-(3,4-methyIenedioxy)-7-hydroxybenzyI)butanoIlde (57) with Podophyllum peltatum Cel l Suspension Cultures. Biotransformation experiments with substrate 57 were carried out using P. peltatum cells at various ages and different incubation periods, while maintaining the pH at 6.5. In a l l cases, biotransformation was observed to produce a major, more polar metabolite, which after extensive chromatographic pur i f icat ion of the ethyl acetate extract from the harvested bioreactors, was isolated as a pale yellow solid with mp = 222-225° C (EtOAc) . High resolution mass spectrometry indicated a molecular formula of C21H20O8 (two hydrogen atoms had been lost) but the high degree of fragmentation observed in the electron impact spectrum suggested a ring-opened type compound. The product showed an IR band at 1715 cm"^ for a cinnamic ester carbonyl band and the U V spectrum presented maxima at 335, 240 and 208 nm supporting the presence of a strong chromophore. The ' H nmr spectrum demonstrated clearly that the compound in hand was the 0—V styrene 86. / \ The aromatic region presented multiplets similar to the precursor compound 57; however, a new singlet whose integration corresponded to one proton was present at 6 7.40 ppm, and was assigned to the olefinic proton H7". The system of the aliphatic protons was again clearly visible as in the case of compound 74 and the assignments CH3O 86 were made making use of multiple irradiation experiments. The methoxy groups' singlet appears at 6 3.85 ppm while H3 presents a broad signal centered at S 4.10 ppm. The A B X system for the methylene protons of C4 is seen as a broad triplet centered at 8 4.24 and a broad doublet at 4.40 ppm. The proton at H 7 ' is located at S 4.95 ppm, and the aromatic protons are present at 6 6.6 ( H 6 ' ) . 6.75 ( H 5 ' and 2 ' ) and 7.10 ppm (H2" and 6"). As mentioned earlier, 117" is located at 6 7.4 ppm which permits the assignment of an E configuration for the double bond on the same grounds as with compound 74. The yield of 86 varied according to incubation times and oscillated around 60% for 20 h. It was observed that shorter biotransformation times lead to higher yields based on recovered starting material. However, extensive research on this area by us 97 and other members of the group failed to produce any ring-closed compound. We concluded, therefore, that the presence of a free hydroxyl group in ring B was essential for this process to occur. Considering this fact and the excellent parallel of the present studies, involving the cyclization of 56, with those of earlier experiments concerning a similar biotransformation of the closely related dibenzylbutanolide to 75a by J. Palaty^^, it seems l ikely that a free radical process in the conversion, 56 to 75, is involved. It is pertinent to note that Palaty's experiments were pursued with crude enzyme preparations (CFE) derived from the cell culture of C. roseus, an indole alkaloid producing culture containing significant levels of peroxidase enzyme. In those studies, Palaty was able to relate the yield of 75a versus the number of peroxidase units involved. Since the present studies were performed with cells of P. peltatum, the levels of "peroxidases" wi thin the growing cells versus yield of 75 were not evaluated. However, such studies with crude enzyme preparations from P. peltatum and biotransformation yields of 56 to 75 are presently underway in our laboratory. In conclusion, the mechanistic pathway shown in Scheme 20 is presented to provide a rationale for the results obtained. Scheme 20. Proposed mechanistic pathway for the formation of products 74 and 75 in the biotransformation of substrate 56 by P. peltatum cell suspension cultures. P H A R M A C O L O G I C A L S C R E E N I N G O F T H E C E L L P R O D U C E D M E T A B O L I T E S , A N D S Y N T H E T I C S U B S T R A T E S . A number of samples from both synthetic and plant cell derived metabolites was forwarded for pharmacological screening at C I B A - G E I G Y , Basel, Switzerland. In order to best characterize the biological act ivi ty of these new derivatives in comparison with etoposide (VP-16), an assessment of their activity in some cell lines and particularly on VP-16 resistant cells was proposed. In view of the known toxicity of etoposide and podophyllotoxin, it was thought important to ascertain the toxicological profiles of the preparations at this early stage of research, i f possible, in cell systems. The compounds were tested for inhibi t ion of cell proliferation in vitro. Two cell lines were used, the human KB31 cell and a derivative thereof, KB8511 which was selected for resistance toward colchicine and expresses the mdr-1 (multi-drug resistance) phenotype. The results obtained indicated that some of the compounds possessing the "wrong" stereochemistry at C I , do lack cross-resistance and are apoarentlv more notent than etoposide. However, since these products are l ike ly to be subjected to patent protection, the details of these studies w i l l not be published unti l later. In conclusion, the biotransformation of the synthetic substrate by the cell cultures of P. peltatum to the "wrong" stereochemistry, for example, §6 to 7 i , may turn out to be highly desirable in producing a novel family of pharmacologically active compounds. S U M M A R Y The present studies have provided an efficient and versatile route to a series of dibenzylbutanolides to be util ized in biotransformation experiments involving whole cells of P. peltatum and/or crude enzyme preparations derived therefrom. The use of the semi-continuous fermentation process applied in these studies is novel and has an exciting potential in the future even for large scale commercial processes. The shortcomings in the use of plant cell cultures as expressed by various practitioners in the f ie ld , such as long term periods for metabolite production, are clearly eliminated by such methodology. We have shown that "enzymatic activity" in converting, for example, the dibenzylbutanolide 56 to 75, can be maintained for several months, wi th in a given batch of P. peltatum cells. Although, additional studies are required to optimize conditions, this "biological factory" is certainly of interest for future experiments. F ina l ly , it should be noted that the conversion of 56 to 75, although providing, at present, the "wrong" stereochemistry in terms of intermediates for etoposide synthesis, does afford the opportunity to obtain novel podophyllotoxin analogues which may exhibit interesting pharmacological properties. F U T U R E W O R K These studies provide an excellent basis for the direction of future research. The cell line has been shown to produce the relevant enzymes for r ing closure but the fact that the configuration at C ( l ) of the biotransformation product is epimeric with that of the natural product gives room for speculation. There are many parameters influencing the outcome of the reaction under study, but in the light of our results, it is unlikely that factors such as cell age, medium composition, and biotransformation time or pH are responsible for the observed stereochemistry. One can speculate that i f the precursor is reaching the enzyme's active site, something is preventing it from adopting the appropriate conformation for ring closure, resulting in the obtained product. Since both phenolic groups on rings A and D appear to be cr i t ica l for enzymatic r ing closure to occur, one can put forward the hypothesis that the enzyme uses these particular groups as "handles" for the transformation, in a manner such that the substrate "sits" in the enzymatic active site in a way that determines the stereochemical outcome of the process. The fact that matairesinol (47) which possesses the phenolic hydroxyl groups at other centers, has been shown to be incorporated into podophyllotoxin (4) when fed to P. hexandrum plants^^ provides a possible support for this hypothesis (Scheme 21) although clearly the relationship between the enzymes wi th in our cell culture and those wi th in the plant has not been established. OH OCH3 P . hexandrum OCH, Matairesinol (47) podophyllotoxin (4) Scheme 21. The incorporation of matairesinol (47) into podophyllotoxin (4) by P. hexandrum plants. A possible experiment to test this hypothesis could involve the biotransformation of a precursor such as 87 (Scheme 22). Scheme 22. Proposed biotransformation for substrate 87. The synthesis of this precursor could be achieved in a similar way to that of substrate 56, wi th the only essential change in the overall sequence being an inversion of the in i t i a l step involv ing introduction of the isopropyl and benzyl groups in the starting aldehyde. The protection of 3,4-dihydroxy benzaldehyde (62), as outlined in Scheme 23, would produce a regio isomer of 64 that would eventually be elaborated into precursor 87. Studies along this line have been ini t ia ted. HO, N ^ ' ^ c ^ o BnCI Nal HO. XHO HO l-Prl K2CO3 OHO BnO' 62 89 90 Scheme 23. Proposed synthesis of 3-isopropoxy-4-benzyloxy-benzaldehyde (90) from 3,4-dihydroxybenzaldehyde (62). Alternat ively, the glycosidation of the biotransformation substrate at position 7' could have some effect in the biotransformation outcome, since a more soluble product would be more readily available for enzymatic transformation in the cell suspension culture. Also, the incorporation of a bulky substituent in this position may change the conformation adopted for ring closure and therefore, the stereochemical outcome of the reaction. We were thus prompted to explore biotransformations of podophyllotoxin (4) and related compounds aiming to identify a whole cell or enzymatic system capable of glucosylating regioselectively these compounds. This would have the added advantage of obviating the need for the protecting groups employed in the chemical transformation. Work along this line has been started in collaboration with the group of Professor Bruno Botta in Italy to whom we have forwarded some of our synthetic precursors. Work is being considered in our group to attempt this transformation using microbial transglucosylation technology'". The synthetic sequences described in this work provide access to the racemic modification of the dibenzylbutenolides. However, the synthesis of homochiral 56 is being considered. The chiral synthesis of 56 would make use of the chiral synthon 4-Q1 menthyloxybutenolide as described in a recent paper by Ward et a r (1992) which follows also the tandem Michael addition-alkylation approach used in our synthesis. Advances in this direction w i l l certainly be part of the future work in our group. C H A P T E R 2. T H E T R I P D I O L I D E F A M I L Y . I N T R O D U C T I O N . T r i p d i ù l i d c (5) and triptolide (91) are the first recognized naturally occurring diterpene triepoxides, and represent the first reported natural products containing the 18(4 3)ûèeo-abietane skeleton. These compounds were first isolated from extracts of go Tripterygium wilfordii Hook by Kupchan and co-workers i n 1972' (Figure 14). tripdiolide (5) triptolide (91) Figure 14. The structures of tr ipdiolide (5) and triptolide (91). The plant Tripterygium is a vine indigenous to Ch ina belonging to the family Celastraceae. There are three species. Tripterygium wilfordii grows in the mountainous areas of south-east and southern China , Tripterygium hypoglaucum Level occurs in south-western China, and Tripterygium regelii Sprague et Takeda is found in the north-east of China. The most common species is T. wilfordii, and it has been the most studied. The plant has a long history of being used as a herbal medicine in the treatment of rheumatoid arthritis, chronic hepatitis, and various skin disorders with more or less promising results. The chemistry of the plant has been studied for half a century and several alkaloids, diterpenoids, triterpenoids and sesquiterpenes, have been isolated and characterized. Interest in the plant was renewed when the contraceptive properties of a "refined extract" from the root xylem were discovered'^' ' . Prel iminary results from tests on the effects of this extract in male rats and in men showed a strong male anti-fert i l i ty effect as well as some anti-inflammatory and immunosuppressive activities. A n alcoholic extract of the plant showed significant activity in vivo against mice L-1210 and P-388 leukemias and in vitro against K B cells from human carcinoma of the nasopharynx'^^. The male anti-fert i l i ty effect attracted our attention since the crude extract induced reversible infer t i l i ty in men without significant side effects, and with the levels of testosterone in serum, potency and l ibido apparently unaffected. There is a great deal of research aimed at developing a safe, fully reversible method for family planning for men. At present, the male partner has only a narrow choice i f he wishes to participate in family planning, namely, the choice between the condom, a vasectomy, or withdrawal. No acceptable anti-fert i l i ty drug for men has yet been produced despite major research efforts by several agencies. A method based on chemical anti-fert i l i ty agents would be highly desirable, and in view of the interesting act ivi ty of the plant extracts, efforts have been directed towards the isolation, characterization, and mode of action of the chemical responsible for the anti-fert i l i ty effect of Tripterygium wilfordii. It is believed that T. wilfordii extracts damage the epidydimal spermatozoa, and to a lesser extent, the spermatogenic cells. A t present, it is d i f f i cu l t to comment on the events underlying the reduction of sperm density without apparent testicular damage. The inhibi t ion of spermatogenesis by one of the components of the extract may serve as a possible explanation. The data gathered so far indicated that spermatogenic cell types most sensitive to the action of the extract are the spermatids and the spermatocytes. To understand this, it is necessary to consider some basic facts about spermatogenesis and its chemical impairment. C H E M I C A L I M P A I R M E N T O F S P E R M A T O G E N E S I S . A normal fertile adult male produces in excess of 100 mil l ion new sperm every day'^-^. However, sperm are not produced wi th in a day. Each takes ten weeks to generate'^^ and during this process, there arc many complex changes in shape and function that are needed to transform the stem germ cells (spermatogonia) into the characteristic s p e r m ' T h e s e include the formation of a long tail for swimming, massive condensation of the cell's D N A so that it can be packaged inside the sperm's nucleus, and the formation of an acrosome or cap over the head of the sperm to store important enzymes used by the sperm to penetrate the protective layer which surrounds the female egg. A l l these changes occur in an orderly and precisely timed sequence, and while it is the germ cells which are undergoing these changes, it is the Sertoli cells (sometimes called "nurse" cells) which orchestrate and control these changes'*^^. The Sertoli cells do so by secreting many different proteins, each of which has a different function and may target a particular germ cell type. The details on this control network are sti l l very poorly understood, but research is gradually putting together the story by identifying the "messenger" proteins and their relevant functions. Chemicals that impair normal sperm production probably do so by disrupting the normal protein messenger system in a highly specific way, which w i l l differ between chemicals. O f a l l the components of the extract of T. wilfordii, the diterpenoid epoxides, triptolide and tripdiolide, are the most l ikely to be responsible for this effect. Detailed toxicity studies of tripdiolide in mice and dogs have been reported '^ ' . Structural data from X- ray crystallographic studies and from spectroscopic studies clearly indicate, as can be seen wi th molecular models, that the C14 ;9-hydroxyl group go of this compound is hydrogen bonded to the C 9 , l l epoxide oxygen atom'''^. Both compounds suffer selective nucleophilic attack by propanethiol at C9, while C14 epitriptolide is recovered unchanged under the same reaction condit ions '^^ (Figure 15). A proposal relating to the hydrogen bonding observed in 5 and 91 to their chemical reactivity and biological act ivi ty has been presented'^^. Figure 15. A lky la t i on of triptolide (91) wi th propane thiol. O T H E R C L I N I C A L A N D P H A R M A C E U T I C A L USES O F T H E P L A N T . The past two decades have seen increasing c l in ica l use, by Chinese physicians, of extracts from the plant. The preparations which have been used are extracts from the dried root xylem. Various disorders have been treated ranging from rheumatoid arthritis and ankylosing spondi l i t i s '^^^ through a variety of skin disorders. The extract used in these treatments, is found to be more potent than the conventional non-steroidal antirheumatic agents such as salicylates, indomethacin, and phenylbutazone, and can be substituted for corticosteroids in some skin diseases and in some patients who are steroid-dependent or who have contraindications to steroids. Its therapeutic effectiveness is believed to be related to its anti-inflammatory and immunosuppressive effects. A comparison of the pharmacological effects of different preparations of the plant available in Chinese markets, showed that a refined extract, prepared by extraction of the root xylem with water and chloroform followed by column chromatography'^^, contained the main anti-inflammatory constituent of Tripterygium wilfordii^^^^. Pcntacyclic triterpencs and diterpenes arc present in this preparation, as well as other constituents (possibly glycosides) '^^. In view of this, research in the area of the diterpene biosynthesis in Tripterygium sp. could provide novel analogues with interesting activities in the area of auto-immune disorders such as rheumatoid arthritis. T O T A L S Y N T H E S E S , A number of approaches have been adopted in the total synthesis of tr ipdiolide and triptolide. The major obstacles that have to be overcome are the construction of the triepoxide system in ring C and the butenolide in ring A . The trans A / B ring junction is stable in aqueous solution at neutral pH while in alkaline conditions, a complete conversion to the cis junction takes place'^^. Alka l ine or acidic equilibration through the extended enolate must therefore be avoided. The approach adopted by Berchtold et a l ' ^ ' * was to synthesize the dihydronaphthalenone 93, as a starting material, providing the B / C ring fragment of the abietane skeleton (Scheme 24). Construction of ring A via annulation of the napththalenone provided a suitable functionalized tr icycl ic intermediate, 96, for the construction of the ring C triepoxide system and the butenolide in r ing A . Annulat ion was achieved v ia alkylation of 93 with the iodobutyrolactone 94. Opening of the lactone gave 95 which yielded 96 after aldol condensation. Reduction of the aldehyde, acidic hydroysis and rearrangement of the double bond completed the synthesis of the lactone ring. a) NaH, DMF b) MeaNH c) CrOa, Pyr. CXÎH3 OCH3 a) NaBH4 EtOH, 2N HCI b) mCPBA c) EtgN. OCH, d) 2,4.6-trimethyl-pyridine, MeSOgCI e) H2. Pd/C. EtOAc (CH3)2N neutral AI2O3 EtOAc a) CrOa. AcOH b) BBrs, CHaCIa C) NaBH^ . BOH Nal04. MeOH 98 R^-CHa, R^-R^-O 99 R ' - H , R^-R?-0 100 R' -R^-H . R^-OH mCPBA erne 24. Synthesis of triptolide (91) by Berchtold et al. Hydroxylat ion at C7 and periodate oxidation produced the epoxy dienone 101. Further epoxidation gave racemic triptonide (102), while reduction gave a 3:1 mixture of racemic 1 ,^ epitriptolide (103) and triptolide (91) rcspectivley. This and most other syntheses lead to a C14-phenol product which facilitates the synthetic incorporation of the epoxide system. A synthesis of (±)-tr iptol ide, involving fewer steps, comprised construction of ring C onto an appropriate A B fragment (Scheme 25), derived from decalone 104. In this synthesis Garver and van Tamelen' '^^ make use of the alkene intermediate 105, to construct the butenolide 97, via introduction of a hydroxyl group at C3, rearrangement with thionyl chloride to afford the a l ly l ic halide 107 and f inal ly conversion of the latter to the a l ly l ic alcohol 108. Addi t ion of dimethylformamide dimethylacetal to this alcohol, was followed by a carbene [2,3]-sigmatropic rearrangement to 110. Further elaboration yielded the key intermediate 97. Tokoroyama et al devised alternative routes to the C r ing system (Scheme 26) from laevopimaric acid'*^^ (112) and to the butenolide ring of triptolide (Scheme 27) and tr ipdiolide from dehydroabietic a c i d ' ^ ^ (122). The ring C construction shown in Scheme 26 involves the endoperoxide formation and rearrangement to the diepoxide 113. Double bond manipulations and further epoxidations and reductions yielded the desired triepoxide system 121, and the epimeric triepoxides 119 and 120. Scheme 27 shows Tokoroyama's construction of the butenolide ring from dehydroabietic acid (122). This is a multistep sequence through the exocyclic olefin 123, Claisen rearrangement to give 127, and f inal ly S N ^ ' attack on 130 by chloride and closure to the butenolide ring 132. Synthesis of the tr ipdiolide analogue 136 consisted of an elimination process via hydroperoxide to give the diene 134 and ring closure of the C2 hydroxylated intermediate 135 yielding the desired tripdiolide analogue 136. Cl' d5 T H 104 l ^ ' ^ ' ^ Y ^ O C H a H 105 r - ' ^ r V ^ o c H , SOCIa/pyr B20,0». 2h HO' a) KOAc, DMSO 75», 24h, b) NaOCHa. CH3OH 25». 2h a) mCPBA. CH2CI2 25». 6h. b) LDA. THF 25», 24h, r H p ^ y ^ o c H o 106 \ OCH3 N - I - O C H 3 xylene, reflux 4A sieves (CH3OH) 3d. O C H , O C H , O C H , a) mCPBA, CH2CI2 25», 30h. b) U N(Si(CH3)3)2 THF. 0»-25». 2h O C H , 1MHCI (aq),THF 25». 10 min OCH3 Scheme 25. Synthesis of 97 by Garver and van Tamelen. Scheme 26. Triepoxide construction from laevopimaric acid (112) by Tokoroyama et al. Oxidative decarboxylation dehydroabietic acid 122 SOCI2 Et20. 25= PhSH / NaOEt DMSO, 25» PhSCH2CI/t-BuOK DME,-10° *SPh 126 (continued) 123 Se02 EtOH / H2O 124 127 N-chlorosuccinimide AgNOs CH3CN / H2O 128 Scheme 27. Synthesis of the butenolide ring of t r ipdiol ide and triptolide from dehydroabietic acid (122) by Tokoroyama et al. (A) a) PCC, CH2CI2 b) CH2N2, Et20 .0» 129 a) LDA. THF, -78° b) Mo05«pyr«HMPA SOCI2, Et20 25» NaOH . EtOH / H2O 132 Scheme 27 - Continued. (B) a) t-BuOK . THF , 0» b) AcCI AcO, O2 / rose bengal UV light, 25» acetone / methanol 134 N-bromosuccinimide H2O/DMSO 25» NaCI02, dioxane / H2O A synthesis of racemic butenolide 97 by van Tamelen and L e i d e n ^ * is one of the most efficient yet designed (Scheme 28). It results in 15% yield after 12 steps with only four purifications required. It is also a biogenetic type synthesis modeled on the biosynthetic cyclizat ion of geranylgeraniol. The key step is the cyclization of intermediate 142 which forms both A and B rings with the correct trans junction. A chira l synthesis passing through the same intermediate 143 was carried out by van Tamelen et a r " % using dehydroabietic acid (122) as starting material. The starting material 145 (Scheme 28) is available from 122 via the sequence shown in Scheme 37 (see later). Hydrolysis of 189 in the latter scheme affords 145. Malcolm R o b e r t s ' ' ^ in our group, developed a synthetic route to dehydroisoabietanolide (132) from dehydroabietic acid (122). In his synthesis (Scheme 30), oxidative decarboxylation of dehydroabietic acid led in one step to the exocyclic olefin (150). Ozonolysis and condensation with carbon disulphide followed by methyl iodide quenching afforded ketene thioketal 152 in good yield. Dimethyl sulphonium methylide addition followed by acidic hydrolysis produced dehydroisoabietanolide (132) in a one pot reaction. This latter reaction involves intermediate epoxide 153 and provides for a short and efficient synthesis. a) Ba(0H)2 Et20/H20, 90' 17h b) LlAIH4.Et20, 0» MeS02Cl/Et 3N CH2CI2, O" 97 Scheme 28. Biogenetic-type synthesis of 97 by van Tamelen and Leiden. tO^ 145 O 148 CH3U a) SOCI2. Bz/DMF. 50' b) NaNa. HaO/acetone. 0° c) toluene, 100" d) LiAIH* . THF, reflux e) HC02hl/H2CO(aq). reflux f) (i) mCPQA, CHCI3, - 20» (ii) EtaN, -20° to reflux a) i-Pfa N Li b) H2CO (g) THF. -78» "OH 146 Os04/Nal04 AcOH/dioxane/HaO 147 oxidize to aldehyde and continue as in scheme 27 149 Scheme 29. C h i r a l synthesis of 97 by van Tamelen et al . Scheme 30. Ch i r a l synthesis of dehydroisoabietanoiide (132) by Roberts. A B I E T A N E A N D ûéeo-ABIETANE B I O S Y N T H E S I S . The biosynthesis of this class of diterpenes is believed to occur via geranylgeraniol (154) cycl izat ion to the abietane type skeleton (Scheme 31). While most research has been directed to the understanding of the early stages of diterpene biosynthesis, it is the last transformations (butenolide and triepoxide formation) that are most relevant to our study. The structures of the aèeo-ab ie tane butenolides and abietanes isolated from l iv ing plants of Tripterygium sp. are shown in Figure 16. Figure 16a shows the diterpenes isolated from T. wilfordii cell cultures. These compounds vary only in the abietanes Scheme 31. Biosynthetic generation of the abietane skeleton. 164 R= C H 3 triptophenolide methyl ether I OH 166 neotriptophenolide o 167 isoneotriptophenolide Figure 16. Diterpenes isolated from Tripterygium plants. Figure 16 - Continued. 122 (l)-dehydroabietic acid RsCHs. ieOa R = H, 160 Figure 16a. Diterpenes isolated from Tripterygium wilfordii tissue cultures. degree of oxidation and can provide some clues as to the order of events leading to tr ipdiolide (5). The isolation of dehydroabietic acid (122) and the hydroxy acid 160 from the cell cultures of T. wilfordii led to proposals of the biosynthetic pathway to tr ipdiolide (5)^' ' , in which dehydroabietic acid 122 and the acid 168 were implicated (Scheme 32). dehydroabietic acid (122) 168 Tripdiolide (5) dehydroisoabietanolide (132) Scheme 32. Proposed biosynthetic pathway to t r ipdiol ide (5) via dehydroabietic acid (122). Previously, chemically related precursors possessing an abietane-type structure have been synthesized in our laboratory in an effort to investigate the biosynthetic pathway subsequent to dehydroabietic acid (122). Biotransformation studies of dehydroabietic acid, as well as of the synthetic precursors 132 and 168-174, were carried out using Tripterygium wilfordii cell cu l tures ' ' ^ (Figure 17). The most promising results were obtained by Malco lm Roberts using precursor 132^'^, which showed considerable uti l ization by the cells to yield C2 and C7 oxidation products (Scheme 33). Figure 17. Synthetic precursors ut i l ized in previous biotransformation studies in Prof. Kutney's group. 33. Biotransformation of dehydroisoabietanoiide (132) with Tripterygium cells. In light of these results, a new biosynthetic pathway was proposed from pimaradiene (179) ' ' ^ (Scheme 34). In this scheme, hydroxylat ion of ring C is triptolide 91 tripdiolide 5 Scheme 34. Biosynthetic pathway to the diterpene triepoxides proposed by Roberts. presumed as well as lactone ring formation before oxidat ion at C2 or C7 can occur. The hydroxylat ion of the aromatic ring seemed a requisite for the formation of the epoxide system. However, direct epoxidation of aromatic rings have been observed in some microorganisms (e.g. Pseudomonas piitida)^^^ to give the corresponding arene oxides (Scheme 35). Eulcaryotic organisms (fungi, yeasts, and higher organisms) utilize mono-oxygenases such as cytochrome P-450 to give these arene epoxides which can be hydrolyzed to a trans diol . Oxidat ion of this diol yields a cathecol, while loss of water results in the effective hydroxylat ion of the original aromatic ring. In contrast. Scheme 35. Oxidat ion of benzene by prokaryotic and eukaryotic organisms. prokaryotic organisms (bacteria) hydroxylate aromatic compounds by dioxygenase enzymes which catalyze a cycloaddit ion reaction with molecular oxygen to yield a dioxetane. This can be reduced to a c /5-diol and/or cathecol. Hydroxyla t ion is often the in i t i a l step in the degradation of aromatic compounds by microorganisms in the environment, and by the liver. If a similar enzymatic system is present in the T. wilfordii cell culture, dehydroisoabietanoiide (132) would s t i l l be considered as a strong candidate for biotransformation studies. Although in Malcolm Roberts' work no incorporation into the diterpene triepoxide was obtained, changes in experimental parameters such as time of addition of the precursor, length of incubation, culture medium used, etc., might also yield the desired biotransformations. Production of Tripdiolide (5) and Triptolide (91) by Plant Ceil Tissue Cultures. As part of the tissue culture program of our group, a cell suspension culture of Tripterygium wilfordii has been established'' '*. Stem and leaf expiants were obtained from T. wilfordii plants maintained under normal greenhouse conditions. Expiants were placed on B5 and P R L - 4 media (standard tissue culture medium developed by Gamborg and Eveleigh) solidified with Bacto-agar (8 g /L) and supplemented with numerous combinations of 2,4-dichlorophenoxyacetic acid (D), kinetin (K) , 1-naphthaIeneacetic acid ( N A ) , indole-3-acetic acid (I), 6-benzylaminopurine (B), 4-aminobenzoic acid (P), and coconut milk (Co). The expiants and resulting cal l i were incubated at room temperature in darkness. Many cal l i grew and were transferred to fresh media of the same or different composition. Prel iminary selection of promising cell lines was based on growth vigor as well a qualitative thin-layer chromatography and cytotoxic activity analyses"'*. The cell line designated TRP4a was selected for further investigation after these screenings. This cell line was initiated as a leaf expiant on PRl2Co]oo (PRL-4 medium supplemented with indole-3-acetic acid (2 mg/L) and coconut milk (100 mL/L) ) , transferred to PRD2Co]oo agar (PRL-4 medium supplemented with 2,4-dichlorophenoxyacetic acid (2 mg/L) and coconut milk (100 m L / L ) ) , and maintained in this medium. Suspension cultures of T R P 4 a were generated in PRD2C0100 medium and were maintained as stock cultures by regular subculture at 3-week intervals. A rapid T L C assay of tr ipdiolide using fluorimetric detection that was accurate for t r ipdiolide concentrations of 0.2 to 3.6 pg was deve loped ' ' ^ and a detailed investigation in terms of tr ipdiolide production versus variations in growth conditions was done ' ' ^ . It was found advantageous to maintain TRP4a stock cultures in the r ich PRD2C0100 medium and encourage tripdiolide production by transferring into MSNA0.5K0.5 medium (MS medium supplemented with naphthaleneacetic acid (0.5 mg/L) and kinet in (0.5 mg/L)). Tr ipdiol ide production was found to be at a level of 4.0 mg /L , or 36 times greater than that reported for the plant by Kupchan et a l^^. More recent investigations on the isolation and characterization of natural products from T. wilfordii plants led to the discovery of nine novel di terpenoids ' '^ . A review that summarizes the earlier studies with the TRP4a cell line of T. wilfordii is available while more recent and detailed studies with TRP4a derived metabolites is described in the Ph.D. thesis of M . Roberts and in a recent p u b l i c a t i o n " ^ . In order to produce large amounts of pharmacologically active diterpene and 118 triterpene natural products, a method was developed by Mijo Samija in our group, where elicitation with a strain of the fungus Botrytis stimulated the production of oleanane and friedelane triterpene acids. A n excellent review of the chemistry of the plant is also available in his thesis"^ . In order to derive additional information about the biosynthesis and production of tr ipdiolide and triptolide, a number of biotransformation experiments were envisaged. Such experiments would involve substrates possessing the abietane skeleton, at a lower oxidation level, and their incubation with the T R P 4 a cell line to evaluate their role, i f any, in the production of the target compounds. It is in this area that the present investigation was directed. O B J E C T I V E S O F T H I S I N V E S T I G A T I O N . 1. Biotransformation of dehydroisoabietanolide (132) wi th cell free extracts ( C F E , crude enzyme preparations) from the TRP4a cell l ine to provide a more direct entry into the C7 and/or C2 oxidation products, and i f possible, an entry to the triepoxide system. 2. In the event that the crude enzyme preparations were incapable of significant biotransformation, synthesize a r ing C activated precursor and evaluate its biotransformation by the T R P 4 a cell line. A l i ke ly candidate for this study would be the C12 or C14 hydroxy derivatives of 132. R E S U L T S A N D D I S C U S S I O N Synthesis of Dehydroisoabietanoiide (132). A t the time the present worlc was being done, studies in our laboratories were directed towards a short synthesis of dehydroisoabietanoiide (132) to be used on our biotransformation studies. Shortly after my arr ival at the Universi ty of Bri t ish Columbia, Malcolm Roberts completed his synthesis of 132 and it was decided to use this sequence for our own investigations. Minor changes to the original synthetic plan were done in order to optimize the yield of the sequence or to facilitate its repetition. The starting material, dehydroabietic acid (122) is available in unlimited quantity at insignificant cost, is optically active, and incorporates most of the carbon skeleton of tr ipdiolide (5). The only disadvantage to starting with dehydroabietic acid is that the range of synthetic approaches is l imited by the chemistry of the starting material rather than by one's imagination. This became more apparent when ring C functionalization was attempted {vide infra). Consequently, the strategy for the synthesis was one of opportunism: to f ind out what would work, then attempt to exploit the results. In the original synthesis of Roberts (Scheme 30), the exo-olefin 150 was produced by oxidative decarboxylation of 122 with lead tetraacetate yielding a mixture of the endo-, and exo-olefins, which requires careful chromatography on silver nitrate treated silica gel for the f inal purif icat ion (Figure 18). In our studies we followed a reported approach ' , involving the concerted elimination of an amine N-oxide in order to avoid the production of the isomeric olefins (Scheme 36). Thus, treatment of dehydroabietic acid with excess thionyl chloride and a catalytic amount of D M F in benzene, afforded the acid chloride. This crude product was treated with sodium azide in acetone and subsequent heating of the resulting acyl azide in toluene effected the Curt ius rearrangement to 183. A l l of these reactions were conveniently monitored by IR spectroscopy. Reduct ion of the isocyanate wi th l i th ium Figure 18. The isomeric olefins from the oxidative decarboxylation of dehydroabietic acid. Scheme 36. Production of the exo-olefin 150 from dehydroabietic acid (122). aluminum hydride proceeded in good yield to the secondary methyl amine, and the latter was then subjected to Eschweiler-Clarke mcthylation wi th aqueous formaldehyde in ref luxing formic acid. The resulting tertiary amine was converted to the corresponding amine oxide by treatment with /wera-chloroperoxybenzoic acid at -20° C. After quenching the excess peracid with triethyl amine the reaction mixture was brought to reflux, at which point the elimination of dimethyl hydroxyamine took place. The isomerically pure product was readily isolated by column chromatography upon elution with hexanes. The overall yield was typically in the order of 70%. The direct production of the isocyanate 183 from dehydroabietic acid was also attempted. Treatment of 122 with diphenylphosphoryl az ide '^^ afforded poor yields of the isocyanate, and in view of the excellent results obtained with the Curtius sequence, this route was not investigated further. The next step in the sequence was the ozonolysis of the exo-olefin to afford ketone 151. This step was carried out using the same conditions reported by Rober t s "^ , that is, treatment of 150 with ozone in a 5:1 mixture of methanol:methylene chloride to afford an average yield of 90% of 151. The ketone 151 is stable to mild acid, but is rapidly epimerized by base to a 3:1 mixture of the A/B-cis and A/B-trans ketones. The two ketones are inseparable by chromatography but can be readily distinguished by ' H nmr spectroscopy. In general, it has been f o u n d ' ^ ' that there is a downfield shift of the CIO angular methyl signal of a l l A/B-cis compounds of this class, relative to the corresponding A/B-irans compounds. Typica l ly , epimerization at C5 resulted in a 0.2 to 0.3 ppm downfield shift for the angular methyl signal. The sensitivity of the C5 stereochemistry in 151 required certain precautions in handling the ketone (Scheme 37). The product, however, was sufficiently stable to be stored as a solid in the refrigerator for several weeks. Scheme 37. The epimerization of C5 in Icetone 151. The attachment of a C3 substituent (later to become CI8) was thoroughly 109 explored by van Tamelen ' "^ ; the ketone failed to add carbon dioxide, ethyl-, methyl-, or phenyl formates. Malcolm Rober ts" '^ found that the alkylat ion with carbon disulphide followed by methyl iodide quenching resulted in a near quantitative yie ld of the a-oxo ketone dithioketal 152. The synthetic possibilities of this class of compounds were first foreseen by Corey ' ^^ who also used the l i th ium salt of 4-methyl-2,6-di-/-butyIphenol as a sterically shielded base which would not attack an electrophile, but would deprotonate the substrate. In our case, the base did not epimerize C5 as shown from the angular methyl position of 6 1.1 ppm in the ' H nmr spectrum of the ketene-dithioketal 152 when compared to 5 1.06 ppm for the corresponding signal in the /ra/is-fused ketone 151. Product 152 could be crystall ized from isopropyl alcohol. The f ina l transformation (Scheme 38) involved a one pot conversion of the dithioketal 152 to the lactone 132 via the epoxide 153. Treatment of 152 with dimethylsulphonium methylide in T H F presumably gave a mixture of epoxides, which was hydrolysed without pur i f ica t ion to give directly dehydroisoabietanolide (132). The yield of this reaction tended to vary and after an investigation of different hydrolytic conditions an optimized yield of 73% could be achieved. The lactone was an o i l which slowly sol idif ied to a white mass and could be crystallized from ethanol (Mp = 97-99° C). The product presented in the > = C H 2 .THF. -20» H3C Scheme 38. The synthesis of dehydroisoabietanoiide (132) from ketone 151. IR spectrum, absorptions at 1757 (lactone) and 1678 (unsaturation) cm" ' . In the ' H nmr spectrum, the signal for the angular methyl is located at S 1.02 ppm, while the lactone methylene (C19) protons are positioned at S 4.78 ppm. The aromatic protons appear at S 6.99 (brs, H14), 7.06 (brd, H12), and 7.27 ppm (d, H l l ) . The lactone presents an absorption maximum at 221 nm in the U V spectrum and a molecular ion at m/z 296 in electron impact mass spectroscopy. Hav ing this precursor in hand, we turned our attention to biotransformation experiments. Dehydroisoabietanoiide (132) Biotransformation with Cel l Free Extracts (CFE) derived from the T R P 4 a Ceil Line. From the work of Malcolm Roberts, we knew that the TRP4a cells would oxidize the lactone 132 at positions 7 and 2, when incubated for a long period of time. We now wished to establish a C F E capable of effecting the same transformation, and i f possible, evaluate the oxidation of r ing C. A cell free extract is prepared after cell disintegration, by removing, v ia centrifugation, insoluble material. Before centrifuging, the mixture is described as an homogenate; after centrifugation as much as possible of the enzyme should be present in the supernatant. There are many methods for cellular disintegration, and there are many types of cells. Most cells have particular characteristics which need special attention during disintegration. Plant cells are generally more d i f f icu l t to disrupt than animal cells because of the cellulosic cell wal l . Plant cells are highly compartmentalized; in most cases, there is a large vacuolar space which can be f i l led with proteases or other compounds, and the chloroplasts, starch granules and other organelles occupy much of the cytoplasmic space. On occasion, the desired enzyme is present in an organelle, and in such instances one can either isolate the organelle or do a complete tissue disruption. If the cells are not broken properly, an otherwise excellent source might be overlooked because much of the act ivi ty was not released but removed with the residue while preparing the extract. The particular problem of marked changes in enzyme composition during different growth phases must be carefully studied. Preliminary studies on the cell culture should be carried out to determine what physiological state contains the highest concentration of the enzyme required. In this work, Tripterygium wilfordii cell suspension cultures were grown in shake flasks in the appropriate medium for the required period of time. A flask was harvested after every two days of culture, and pH and R I were measured, and the microscopic puri ty was evaluated. The contents of the flasks were filtered through Mira-cloth in a Buchner funnel attached to a water aspirator to allow thorough draining and the resultant cell mass was washed with distilled water. The cells were taken into the cold room (4° C), mixed in a plastic container with 180 m L of 0.02 M phosphate buffer (pH = 6.4), homogenized with an Ul t ra Turrax T-25 disperser for 30 sec, allowed to stand for an additional 30 sec, and homogenized again for 30 sec. The homogenization was repeated three times, the resulting homogenate was transferred to plastic centrifuge flasks and centrifuged at 10,000 g for 30 min (T = 4° C). The supernatant (CFE) thus obtained was decanted, and peroxidase act ivi ty and protein content were measured. For these determinations, the Bio-Rad protein assay was used for the measurement of protein concentration and peroxidase activity was evaluated by the pyrogallol-purpurogallin assay method. (For details, see Experimental.) Biomass (dry) was also determined for every harvested flask. A number of biotransformation experiments using cells at different ages were performed (Scheme 39). For each experiment, a number of flasks were set adding 22 peroxidase units of the C F E to a mixture of the lactone 132 (10 mg) dissolved in ethanol (2 mL), 15 mL of distilled water, 35 mL of phosphate buffer (pH = 6.4), and 2.6 equivalents of a 0.5% solution of hydrogen peroxide. Experiments were also carried out to assess membrane bound enzymes and cofactors (hydrogen peroxide, flavine mononucleotide, manganese dichloride). Biotransformations using the cell homogenate, the resuspended pellet, and the cell free extract were carried out with and without the cofactors. A control flask was set with everything except the C F E . The flasks were stirred at room temperature for different periods of time (typically 30 min, 2 h, and overnight), and harvested adding 25 mL of ethyl acetate. The resulting emulsion was quickly filtered through a short pad of Celite 545 in a fritted glass fil ter funnel and the Celite was washed with more ethyl acetate (25 mL). The filtrate was extracted twice with ethyl acetate (25 mL each time) and the Celite was sonicated for 20 min with enough ethyl acetate to cover it Cell Culture 1) RItration 2) wash with distilled water 3) horTK>genization in buffer (pH 6.4) Cell Homogenate 1) Substrate administration with cofactors 2) Incubation Work-up Cell pellet • Addition of buffer Resuspended pellet 1) Substrate administration with cofactors 2) Incubation Centrifugation (10,000 g, 30 min.) t Supematant (CFE) 1) Substrate administration with cofactors 2) Incubation Work-up t Work-up 1) Substrate administration without cofactors 2) Incubation Work-up Scheme 39. Preparation of the cell homogenate, resuspended pellet and C F E , and biotransformation. completely, then filtered and rinsed. The combined organic extracts were dried, filtered and rotary evaporated to dryness. T L C (toluenerethyl acetate 4:1 and chloroform:methanol:acctic acid 95:5:1) and H P L C (reverse phase, water:methanol 25:75) analyses were carried out on each sample. The results indicated no oxidation of the lactone 132, and the substrate was recovered in essentially quantitative yield (97-98% recovery). The lack of oxidation products suggested either that aromatic r ing activation is necessary prior to biotransformation, or that the relevant enzymes are extracellular or are destroyed during the C F E preparation. Several experiments fol lowing the podophyllotoxin line of research made by Jan Palaty and using the A C 3 cell line of Catharanthus roseus for biotransformation of 132 were done. In other studies, this cell line had demonstrated high peroxidase activity and the capacity to oxidize structurally diverse substrates, but biotransformation was not detected in our case. Dehydroisoabietanoiide (132) Biotransformation with T R P 4 a Cells. Dur ing preparation of C F E , some enzymatic act ivi ty may have been lost so experiments were also conducted using whole cells of the TRP4a cell line to verify the abil i ty, i f any, to biotransform 132. Experiments with cells of different ages (0, 7, and 10 days old) and different incubation periods (24, 48, 72 and 92 h) were qualitatively analyzed by T L C . The results indicated that, although the enzymes responsible for C7 oxidation, that is, conversion of 132 to 175 (Scheme 40), are present in the early stage of the culture, older cells and longer incubation times give better yields (i.e. a higher biomass:precursor ratio favours the conversion). Experiments with C F E prepared from the same batch of cells resulted in the recovery of only the substrate, therefore, indicating that indeed, "hydroxylase" activity is being lost during C F E preparation. 132 175 Scheme 40. The biotransformation of dehydroisoabietanolide 132 into 175 by T. wilfordii cell suspension culture. It was clear at this point that C F E technology would not be useful for our purposes and, since whole cells produced relatively uninteresting results in the above studies, our attention was directed into functionalization of other sites of the substrate molecule (see later). Dehydroisoabietanolide (132) Oxidat ion with Mortierella isabellina. While the above studies were underway, consideration was given to the possibility of obtaining C2 hydroxylated analogues of 132 by means of microbial transformation. If success could be achieved, such substrates may prove of interest in future biotransformation studies with T R P 4 a , part icularly since tripdiolide possesses a C2-hydroxyl function. The fungus, Mortierella isabellina, was selected after screening various microorganisms, since it consistently and selectively hydroxylates resin acids at C2 under appropriate condi t ions '^^ . A n experiment with this fungus was carried out using five Erlenmeyer flasks, each containing 50 mL of D Y E (Dextro yeast extract) medium and 5 mg of the lactone 132 in 0.5 mL of ethanol. Add i t ion was made at time zero (i.e. at inoculation time) and the cultures grown in a rotary shaker at 30° C and 200 rpm for 4, 8, 24, 48, and 56 h. The cultures were harvested to obtain samples for analysis. The results indicated the production of a new metabolite, more polar than the precursor, as well as almost complete consumption of the starting material after 48 h of incubation. The n.'-.w spot on T L C was not present in a culture grown under the same conditions. Microscopic inspection of the culture treated with the substrate, showed significant growth inhibi t ion , where practically a l l cells remained in the lag phase. The product was isolated in 37% yield and characterized as the C7-;9-hydroxyl derivative 175. A l l spectroscopic data were identical to those reported previously by M . Rober t s "^ . No attempt to improve this yield was pursued. A l l these results made clear to us that further "activation" of the aromatic ring in 132 was required. Perhaps, a ring C hydroxylated compound would be an interesting candidate based on the previously proposed biosynthetic pathway (see, for example, 163 in Scheme 34). The Synthesis of a Ring C-activated Substrate. A search of the literature revealed only two reactions which proceeded efficiently on the C ring of dehydroabietic acid: acetylation at C12^^^ and dinitration at C12 and C 1 4 ' ^ ^ We did not enjoy the luxury of f lex ib i l i ty in the substitution pattern of the aromatic nucleus, and this loss of synthetic f lexibi l i ty caused considerable d i f f icu l ty in this portion of the project. It was felt that the ideal precursor would be the C14 hydroxy derivative triptophenolide (163) since the plant cell probably effects the biological equivalent to Alder 's oxidation of or/Ao-hydroxymethyl phenols with periodate as uti l ized by Berchtold'^'* in his synthesis of triptolide (Scheme 24, vide supra). Published routes to 14-hydroxy-dehydroabietic acid are lengthy and based on dinitrat ion, reduction and diazotizations reactions (Scheme 4 1 ) ' ^ ' . We speculated that the butenolide functionality may not endure the reaction conditions involved in this process. Problems were also anticipated for the synthesis of the lactone from 14-'COjH 112 COjH Zn, AcOH HNO3 H2SO4 1)TFA, NaN02 2) KI COjH 188 1) TFA, NaN02 2) SOCI2 3) MeOH, Pyr 4) AC2O, Pyr CO2H 186 CO2H ^89 Scheme 41. Synthesis of 14-acetoxydehydroabietic acid (189) by Demers. hydroxy-dehydroabictic acid and, therefore, it was decided to proceed in i t ia l ly with a synthesis of the C I 2 hydroxy derivative, isotriptophenolide (190). We hoped that this precursor would be transformed by the cell culture derived enzyme system into the corresponding epoxy-dienone (191) via benzylic hydroxylat ion and subsequent 1,4-addition (Scheme 42), or even further along the biosynthetic pathway of tr iptolide to generate novel analogues of the triepoxide system. The latter compounds could be very useful in providing a "family" of diterpene epoxides for pharmacological screening wi th in the areas of contraception, immunosuppression, etc. For this reason an efficient synthesis of the hydroxy lactone 190 was pursued. Scheme 42. The proposed biotransformation of substate 190. The synthesis of substrate 190 involved Friedel-Crafts acylation of dehydroisoabietanoiide (132), followed by Baeyer-Vil l iger oxidation and hydrolysis of the resulting ester to the desired phenol (Scheme 43). Dehydroisoabietanoiide (132) and acetyl chloride in carbon disulphide were added to a luminum chloride in anhydrous carbon disulphide as solvent. After overnight ref lux, the solvent was removed and the residue treated with a cold (ice) aqueous solution of hydrochloric acid. Workup of the hydrolyzed mixture afforded the 12-acetyl derivative 192 and none of the isomeric 14-acetyl derivative. This compound could be purif ied by crystallization from methanol (68% yield) to give the pure product as white crystals. The product showed two carbonyl bands in the IR spectrum at 1751 and 1669 c m ' , and in the H nmr spectrum, a new methyl singlet appeared at 5 2.58 ppm. The C14 and C l l aromatic protons were present as singlets at S 7.15 and 7.47 ppm respectively. The Baeyer-Vil l iger oxidation was performed under the conditions reported by Chamberl in and Canan K o c h in 1989'^^. They reported the use of me/û-chloroperoxybenzoic acid catalyzed by trifluoroacetic acid as a simple and effective means of effecting the reaction. In our case, the reaction proceeded in almost quantitative yield. The reaction took usually 20 h to completion, and was best monitored by T L C ut i l iz ing isopropyl ether as eluting solvent. Also this was the only solvent found capable of resolving the starting material from the product. In the IR spectrum of 193, the carbonyl band for the acetate appears at 1680 c m ' ' , and in the ' H nmr spectrum, the methyl singlet is centered at S 2.33 ppm mCPBA. cat. TFA OH OAc Scheme 43. Synthesis of substrate 190 from dehydroisoabietanolide (132). while the singlets for the aromatic protons at positions C14 and C l l have moved upfield to S 6.96 and 7.06 ppm respectively. The f ina l hydrolysis proved to be sensitive to basic conditions since coloured impurities were noted, and was thus best performed under acidic conditions. A reported selective hydrolysis of aryl acetates wi th p-toluencsulfonic acid adsorbed on silica g e l ' ^ ' ' proved ineffective but when the acetate was treated with concentrated hydrochloric acid in methanol, the phenol 190 could be isolated in 95% yield after crystallization from acetone-water. The product showed IR bands at 3320 (hydroxyl) and 1748 (lactone carbonyl) c m ' ' , and in ' H nmr spectrum, the aromatic protons were present as singlets at S 6.72 (C14) and 6.91 ( C l l ) ppm. We were now in a position to test the biotransformation of this "activated" substrate with T. wilfordii cell cultures. Isotriptophenolide (190) Biotransformation with T R P 4 a Cells. Prel iminary studies on the biotransformation of isotriptophenolide (190) were done using whole cells since the l ikelihood of success is greater in this type of experiment. Several experiments were carried out using the TRP4a cell line. A n in i t i a l experiment of incubating 190 with TRP4a cells grown in MSNA0.5K0.5 medium for 7 days was performed. A solution of the phenol 190 (50 mg) in ethanol was added to the cultures (1 L ) in shake flasks and the culture was incubated for 7 days. Even though analytical T L C of the reaction mixture sti l l showed a considerable quantity of starting material remaining, further incubation was not considered in order to avoid low recovery of products - a situation encountered in the podophyllotoxin experiments, where overoxidation of the substrate was consistently observed. The flasks were harvested by homogenizing cells and broth together, and extracting the mixture with ethyl acetate in accord with the general procedure developed in the first part of this thesis. T L C analysis of the crude extract did not sliow a significant difference between the control flask and the biotransformation experiment except for a very faint spot slightly less polar than the starting material . The crude extract was pur i f ied by column chromatography with gradient elution (hexanes:ethyl acetate 8:2 - to hexanes: ethyl acetate 2:8) and the polar material was f ina l ly removed with ethyl acetatcrmethanol 1:1. The fractions containing the new spot and the recovered substrate were further pur i f ied by preparative thin layer chromatography eluting with hexanes:ethyl acetate 7:3 to afford 38.0 mg (76%) of recovered substrate and 4.3 mg (8%) of a new metabolite, the latter obtained as a yellowish solid. The new product was characterized as the C12 methyl ether (194). The ' H nmr spectrum was very similar to the phenol 190 but the signal for the additional methyl appears at S 3.84 ppm. In the mass spectrometry, the molecular ion peak was noted at m/z 326, with a base peak at m/z 311 (M^-15). No C7 or C2 oxidation products were detected. isotriptophenolide (190) methyl isotriptophenolide (194) Scheme 44. The biotransformation of isotriptophenolide (190) by T. wilfordii whole cells. In an attempt to force the cells to metabolize the supplied substrate, two experiments with "starved" cells (0% sucrose and 0.2% sucrose M S N A 0 5 K 0 5 respectively) and adding the substrate at inoculation time, were carried out. In the first experiment (0% sucrose) the cells died and the substrate was recovered unchanged, whereas with the other culture only a trace of the methyl ether could be detected along with significant amounts of recovered substrate. C O N C L U S I O N S . While a more thorough investigation on the biotransformation of isotriptophenolide (190) was not possible wi th in the constraints of this research effort, Mr. Kang Han in our group has been continuously studying this problem. Interestingly enough, he has found that T. wilfordii cell suspension cultures are indeed capable of oxidizing this substrate to the epoxy dienone 191 under appropriate conditions. This appears to indicate that the biosynthesis of the diterpene triepoxides may well proceed through an intermediate such as triptophenolide (163) via the biological analogue of Alder 's oxidation of or//io-hydroxymethyl phenols and subsequent epoxidations (Scheme 45). It is also most probable that the introduction of the hydroxyl group at C14 occurs prior to ring C aromatization since no aromatic hydroxylat ion could be demonstrated. Future research in this area should involve the biotransformation of triptophenolide (163) to clarify this situation. Such studies are currently underway in our laboratory. Scheme 45. The proposed biogenesis of the triepoxide system. E X P E R I M E N T A L S E C T I O N E X P E R I M E N T A L A l l experiments involving moisture-sensitive reagents were carried out under a positive pressure of nitrogen or argon gas, as indicated. Solvents were commercial reagent grade except that technical grade ether, methylene chloride, and ethyl acetate were used for extractions in most instances. Unless otherwise noted, anhydrous magnesium sulphate was used to dry organic solutions prior to solvent removal. Tetrahydrofuran, ether, benzene, and toluene were freshly distilled from sodium and benzophenone ketyl under argon prior to use i f strictly anhydrous solvents were required; otherwise, reagent grade anhydrous ethers, benzene, and toluene were used. Column chromatography was carried out using Merck sil ica gel 60, 230-400 À mesh, while analytical and preparative T L C was performed using Merck pre-coated silica gel 60 F254 T L C plates. A l l solvents for column chromatography were reagent grade and received no additional purification or drying prior to use. Synthetic samples were visualized on analytical T L C plates by U V and by spraying with a 5% solution of ammonium molybdate in 10% sulphuric acid, followed by heating at 125° C unt i l blue spots developed. Tissue culture extracts were visualized on T L C by spraying first with a 30% solution of concentrated sulphuric acid in glacial acetic acid, then with a 5% solution of anisaldehyde in isopropanol, followed by heating at 125° C for approximately 10 minutes. Lignan-type compounds were developed by spraying with a 10:3 mixture of glacial acetic acid and concentrated ni t r ic acid followed by heating at 125° C for 5 min, giving typically red or brown spots. ' H nmr spectra were run in CDCI3 at 400 M H z using a Bruker WH 400 spectrometer, unless otherwise quoted. Tetramethylsilane was used as the internal standard and al l peaks were recorded in ppm (5) relative to TMS (5 0.00 ppm). Low resolution mass spectra were recorded using Kratos MS 50 and MS 80 mass spectrometers. H igh resolution (HRMS) mass spectra were run on a Kratos MS 50 mass spectrometer. Chemical ionization mass spectra were recorded on a Delsi-Nermag RIO-lOC mass spectrometer using isobutanc as the carrier gas. IR spectra were recorded on a Perkin Elmer 71 OB infrared spectrophotometer, and Fourier transform IR spectra were recorded on a Perkin Elmer 1710 infrared Fourier transform spectrophotometer. U V spectra were recorded on a Perkin Elmer Lambda 4B U V / V I S spectrophotometer, using quartz cells of 1 cm path length. A l l melting points were recorded on a Reichert melting point apparatus and are uncorrected. Elemental analyses were carried out by Mr . P. Borda, of the Microanalyt ical Laboratory, Universi ty of Bri t ish Columbia, Vancouver. Plant cell tissue culture production was carried out by Gary Hewitt , David Chen, Fay Hutton, Radka Milanova, Nikolay Stoynov, and myself at the Biological Services Laboratories, Chemistry Department, Universi ty of Brit ish Columbia. The cultures were grown in shake flasks and in glass a i r l i f t or mechanically stirred fermentors (Labroferm, New Brunswick Scientific or Microferm, New Brunswick Scientific, respectively). Cel l culture methods were developed by Gary Hewitt of the Biological Services section of our Department. Cultures were raised in the dark at 26° C using an appropriate l iquid medium (see individual culturing methods). Growth was monitored through the refractive index (Galileo refractometer, 25° C) and pH and microscopic purity were also determined at the end of the growth cycle. For the fungal experiments, a culture of Mortierella isabellina was grown on 10 ml of P D A in test tubes at 25° C or in a Roux bottle with 15 ml of P D A at 25° C for 10 days by Ms. Elizabeth Bugante who also provided technical assistance for these experiments. Cel l free extracts were prepared according to the methods described in this Experimental Part by Ms. Radka Milanova. Hydrogen peroxide was prepared from a stock solution (30% w/v) . The solutions were standardized by iodimetric titration with sodium thiosulphate. A'-Butyli thium solutions were standardized by tritration against diphenyl acetic acid in anhydrous T H F . High pressure l iqu id chromatography was performed using a Waters C j g "Radial Pack" l iqu id chromatography cartridge, a Waters 440 Absorbance Detector set at 280 and 254 nm, and a methanol/water eluent, containing 0.1% of acetic acid. Gas chromatography analyses were carried out with an H P 5840A gas chromatograph fitted with a Hewlett Packard HP-1 column (100% dimethylpoly-siloxane, non-polar fused sil ica capillary column) 25 m x 0.2 mm x 0.5 nm f i lm thickness. Ozone was generated in a Welsbach model T-23 laboratory ozonator. 3 - H Y D R O X Y - 4 - I S O P R O P O X Y B E N Z A L D E H Y D E (63): To a solution of 3,4-dihydroxybenzaldehyde (62) (5.0 g, 36.2 mmol) in DMSO (18 mL) was added anhydrous potassium carbonate (5.6 g, 3.12 equiv.) and 4.8 mL (8.2 g, 1.33 equiv.) of distilled isopropyl iodide, and the solution was stirred overnight in the dark. The reaction was quenched by pouring into brine (50 mL), followed by slow acidif icat ion with 1 N H C l (114 mL). The mixture was extracted with dichloromethane (3 x 50 mL), the combined organic extracts were washed with acidic brine (50 mL), dried, filtered and evaporated in vacuo to yield 6.39 g of the crude product. Column chromatography was carried out using the flash technique (4 cm diameter column, 6" silica gel), collecting 10-mL fractions, and eluting with benzene: ethyl acetate:acetic acid 9:0.5:0.5 (500 mL). The fol lowing compounds were isolated and characterized in order of elution: 0.40 g, 5%. IR (NaCl neat): u^nzx 1'705 cm'* (aldehyde). ' H nmr S: 1.41 (12H, m, CH(CH3)2), 4.53 ( I H , heptet, CH(CH3)2, J = 6 Hz), 4.64 ( I H , heptet, CH(CH3)2, J = 6 Hz), 6.98 ( I H , d, H5, J = 8 Hz), 7.44 (2H, m, H2, H6), 9.83 ( I H , s, CHO) . U V (MeOH) A ^ a x 0o8 «): 303 (4.00), 278 (4.11), 232 (4.13), 213 (4.09). MS m/z: 222 (M+), 180, 138. H R M S calc. for C i 3 H i g 0 3 : 222.1256; found: 222.1258. Ana l , calcd. for C13H18O3: C 70.25, H 8.15; found: C 70.15, H 8.23. Previously unpublished U V and elemental analysis data. 4 - H Y D R O X Y . 3 - I S O P R O P O X Y B E N Z A L D E H Y D E (63b):^ '* 0.26 g, 4%. IR (NaCl neat) i/niax^ '^05 cm'* (aldehyde); ' H nmr S: 1.38 (6H, d, CH(CH3)2, J = 6 Hz), 4.75 ( I H , heptet, CH(CH3)2, J = 6 Hz) , 6.35 ( I H , brs, OH) , 7.05 ( I H , d, H5, J = 8 Hz), 7.42 (2H, m, H2 H6), 9.83 ( IH , S, C H O ) . MS m/z: 180 (M+), 137, 109. H R M S calc. for CJ0H12O3: 180.0786, found: 180.0783. Anal , calcd. for C10H12O3: C 66.66, H 6.70; found: C 66.40, H 6.84. Previously unpublished U V and elemental analysis data. 3 , 4 - D I I S O P R O P O X Y B E N Z A L D E H Y D E (63a): ,.74 3 - H Y D R O X Y - 4 - I S O P R O P O X Y B E N Z A L D E H Y D E (63): HO^ J s ^ ^CHO In i t ia l ly 5.6 g (70%) as an o i l that crystallized to white needles. M p - 63-65° C (EtOAc-hcxanes). IR ( K B r pellet): i /^ax 1^05 cm" ' (aldehyde); ' H nmr S: 1.40 (6H, d, CH(CH3)2, J = 6 Hz), 4.75 ( I H , heptet, CH(CH3)2), J = 6 Hz), 5.80 ( I H . brs, OH) , 6.95 ( I H , d, H5, J - 8 Hz), 7.43 (2H, m, H2, H6), 9.84 ( I H , s, CHO) . U V (methanol) Ajiiax (log O ' 311 (3.12), 276 (4.00), 229 (4.00). MS m/z: 180 (M*), 137, 109. H R M S calc. for C10H12O3: 180.0786, found: 180.0783. A n a l , calcd. for C10H12O3: C 66.66, H 6.70; found: C 66.74, H 6.75. Previously unpublished U V and elemental analysis data. 3 - B E N Z Y L O X Y - 4 - I S O P R O P O X Y B E N Z A L D E H Y D E (64)J4 4-isopropoxy-3-hydroxybenzaldehyde (3.47 g, 0.0192 mol) was dissolved in ethanol (25 mL) . Potassium carabonate (3.0 g), sodium iodide (0.1 g) and benzyl chloride (3.0 g, 2.72 mL, 0.0237 mol, 1.23 equiv.) were added. The mixture was refluxed protected from moisture for 4 h, when the reaction was complete. The reaction was cooled and water (10 mL) was added. Af ter evaporation of the ethanol in vacuo, the residue was diluted with water (25 mL) and extracted with dichloromethane ( 3 x 1 0 mL). The combined organic extracts were washed with 1 N N a O H (10 mL) and with water (10 mL) before drying, f i l t rat ion, and rotary evaporation. The crude product (5.0 g) was purified by flash column chromatography (4.0 cm diameter, 5.5" sil ica gel) eluting with 1 L of petroleum ether:ethyl acetate 9:1, and collecting 20-mL fractions, to give 3.68 g (70.78%) of tlie product as a colorless o i l . I R (neat) J^max-1686 cm" ' (aldehyde), ' H nmr S: 1.40 (6H, d, CH(CH3)2. J = 6 Hz), 4.65 (1 H , heptet, CH(CH3)2. J = 6 Hz), 5.22 (2H, s, Ph -CH2-0), 7.0 ( I H , d, H5, J = 8 Hz), 7.30-7.50 (7H, m, aromatic), 9.82 ( I H , s, C H O ) . U V (methanol) Ajnax ('08 «): 305 (4.11), 275 (4.28), 230 (4.38), 207 (4.48). M S m/z: 270 (M"^). 228 (1.2), 179 (6.5), 91 (100). H R M S calcd. for C17H18O3: 270.1256, found: 270.1249. Previously unpublished U V and elemental analysis data. 3 - B E N Z Y L O X Y - l - B I S ( P H E N Y L T H I O ) M E T H Y L - 4 - I S O P R O P O X Y B E N Z E N E (65):'^'* 3-benzyloxy-4-isopropoxybenzaldehyde (3.68 g, 13.6 mmol) was dissolved under nitrogen in dry chloroform (30 mL). The solution was cooled to -40° C with an acetonitrile-dry ice bath and 2.94 mol (2.1 equiv.) of thiophenol were slowly added maintaining the inner temperature of -40° C, followed by the addition of 4 mL (2.3 equiv.) of boron tr if luoride etherate maintaining good s t i r r ing and temperature of -40° C. The resulting brown mixture was stirred at -40° C for 20 min, and then quenched by pouring it over ice cold water (40 mL). The resulting mixture was extracted with dichloromethane (3 x 30 mL) and the combined organic extracts were washed successively wi th K O H (50 mL), water (50 mL), and brine (50 mL). Dry ing , f i l t rat ion and rotary evaporation afforded 4.82 g of the crude product as a yellow-brownish o i l , which was pur i f ied by flash column chromatography (4.5 cm diameter, 5.5" silica gel) eluting with 5% ethyl acetate in petroleum ether. 20-mL fractions were collected, to afford 3.58 g (55.63%) of the product as a clear oil which slowly solidifies to a white solid. IR (neat) i^max' 2895 (aromatic), 1480 c m ' ' . ' H nmr 6: 1.30 (6H, d. PhS ,SPh CH(CH3)2, J - 6 Hz), 4.45 ( I H , heptet, CH(CH3)2, J - 6 Hz) . 5.0 (2H, s, CH2 -Ph) , 5.33 ( I H , s, CH(SPh)2), 6.78 (2H, m, H 2 ' , H 6 ' ) , 6.98 ( I H , d, H 5 ' , J= 2 Hz), 7.20-7.50 (15H, m, aromatic). U V (methanol) A ^ a x (log «): 216 (4.56). MS m/z: 363 (M"^- SPH) (100), 321 (33.1), 91 (38.5). H R M S calcd. for C29H28S2O2: 472.1531, found: 472.1525. A n a l , calcd. for C29H28S2O2: C 73.70, H 5.96, S 13.56; found: C 73.68, H 6.02, S 13.45. Previously unpublished U V data. 4 - B E N Z Y L O X Y - 3 , 5 - D I M E T H O X Y B E N Z A L D E H Y D E (67a): Syringaldehyde (5.0 g, 0.027 mol) was dissolved in ethanol (50 mL) and anhydrous potassium carbonate (5.0 g), sodium iodide (0.5 g), and benzyl chloride (4.4 g, 4 mL, 1.28 equiv.) were added. The mixture was refluxed protected from moisture for 3 h, when the reaction was complete. After cooling the mixture, water (50 mL) was added and the ethanol was removed in vacuo. The residue was diluted with water (50 mL) and extracted with dichloromethane (3 x 25 mL) . The combined organic extracts were successively washed with 1 N N a O H (25 mL), and water (25 mL). Dry ing , f i l t ra t ion and rotary evaporation afforded 7.88 g of crude product which was purif ied by flash column chromatography (4.5 cm diameter, 5.5" si l ica gel) eluting with 1.5 L of a 20% solution of ethyl acetate in petroleum ether and collecting 20-mL fractions. Rotary evaporation yielded 6.357 g (85.05%) of the pure product as an oi l which slowly crystallized to off-white crystals. Mp = 52-54° C (EtOAc-hexanes). IR (KBr ) i/max-' 1685 (aldehyde) cm" ' , ' H nmr S: 3.90 (6H, s , - O C H 3 ) , 5.23 (2H, s, C H 2 -Ph), 7.12 (2H, s, H2 , H6), 7.24-7.5 (5H, m, aromatic), 9.86 ( I H , s, C H O ) . U V (methanol) Amax (log 0 : 287 (4.05), 223 (4.13), 220 (4.13). MS m/z: 272 (M"^, 26.9), 181 (10.8), CHO OBn 125 (22.0), 110 (22.4), 91 (100). H R M S calcd. for C16HJ6O4: 272.1049; found: 272.1048. A n a l , calcd. for C ] 6 H i 6 0 4 : C 70.58, H 5.91; found: C 70.58, H 5.92. 4 - B E N Z Y L O X Y - 3 , 5 - D I M E T H O X Y B E N Z Y L A L C O H O L (67b): 4-benzyl-syringaldehyde (6.35 g, 0.023 mol) was dissolved in ethanol (100 mL), at room temperature. Sodium borohydride (635 mg, 2.9 equiv.) was added in one portion and the mixture was stirred at room temperature for 1 h. Ana ly t ica l T L C on 30% ethyl acetate in petroleum ether showed reaction completion. The reaction was quenched by the addition of 1 N HCI (20 mL) (the mixture turns clear at pH = 2.0) and the ethanol was removed in vacuo. The residue was diluted with water (25 mL) and extracted with dichloromethane (3 x 25 mL). The combined organic extracts were dried, filtered, and rotary evaporated to yield 6.47 g of crude product as a light yellow oi l . The crude was purif ied by flash column chromatography (4.0 cm diameter column, 5.5" s i l ica gel) eluting with 2 L of 30% solution of ethyl acetate in petroleum ether and collecting 20-mL fractions. Rotary evaporation yielded 6.18 g (96.7%) of the pure product as a colorless o i l . IR (neat) t^max- 3436 (alcohol), 1127 (ether) cm" ' , ' H nmr 5: 1.75 (IH, brs, D2O+, OH) , 3.83 (6H, s, -OCH3), 4.62 (2H, d, C H 2 - O H , J = 4 Hz), 5.0 (2H, s, CH2 -Ph) , 6.60 (2H, s, H2, H6), 7.24-7.5 (5H, m, aromatic). U V (methanol) ^max (log «): 210 (4.91). MS m/z: 274 (M+, 16.9), 183 (46.9), 155 (24.2), 127 (36.3), 123 (12.9), 95 (14.4), 91 (100). H R M S calcd. for C16H18O4: 274.1205, found: 274.1205. Ana l , calcd. for C16H18O4: C 70.07, H 6.60; found: C 70.23, H 6.76. CHjOH OBn 4 - B E N Z Y L O X Y - 3 , 5 - D I M E T H O X Y B E N Z Y L B R O M I D E (67): ÇHjBr C H a O ^ Y ^ O C H a OBn 6.18 g (22.5 mmol) of 4-benzyloxy-3,5-dimcthoxybenzyl alcohol were dissolved in anhydrous ether (40 mL) under argon. To this solution were slowly added (over 20 min) 2.47 g (1.2 equiv.) of phosphorus tribromide dissolved anhydrous ether (10 mL) maintaining the reaction temperature at 25-30° C (water bath). A t the end of the addition, the reaction mixture was stirred at room temperature unti l control T L C (s.s. hexanes-EtOAc 1:1) showed reaction completion. The mixture was then poured into a separatory funnel and washed with brine (3 x 25 mL) , then with saturated sodium carbonate solution (25 mL) and brine (25 mL). The organic phase was dried, filtered and concentrated in vacuo to yield 6.44 g of the crude product as a light yellow oi l . The aryl bromide was purif ied by flash column chromatography (4.5 cm diameter, 6" silica gel) eluting with 2 L of petroleum ether:ethyl acetate 9:1 and collecting 20-mL fractions. Rotary evaporation afforded 5.76 g (75.9%) of a clear oi l that solidif ied upon cooling to a white mass. IR (neat) I'max- 3003, 2938, 2838, 1593 c m ' ' , ' H nmr 5: 3.85 (6H, s, -OCH3), 4.49 (2H, s, CH2-Br), 5.0 (2H, s, CH2-Ph), 6.62 (2H, s, H2, H6), 7.30-7.53 (5H, m, aromatic). U V (methanol) A^ax Cog «): 260 (3.45). MS m/z: 336 (M+, 3.4), 338 (M"^, 3.4), 257 (10.6), 91 (100). H R M S calcd. for C i o H p O s B r : 338.0342, 336.0362; found: 338.0357,336.0358. Ana l , calcd. for Ci6Hi703Br: C 56.99, H 5.07, Br 23.69; found: C 57.13, H 5.17, Br 23.53. T R A N S - 2 - ( 4 - B E N Z Y L O X Y - 3 , 5 - D I M E T H O X Y B E N Z Y L ) - 3 - ( 3 - B E N Z Y L O X Y - 4 . I S O P R O P O X Y - a , a - B I S ( P H E N Y L T H I O ) B E N Z Y L ) B U T A N O L I D E (68): ,.74 PhS SPh BnO. V C H g O ' ^ y ^ O C H a OBn 6.05 g (0.0128 mol) of the phenyl thioketal 65 were dissolved in dry T H F (60 mL) and the solution was cooled under nitrogen to -70° C (acetone-dry ice bath). 8.83 mL (l . I equiv.) of a 1.6 M solution of n-butyl l i th ium in hexanes were slowly added via the addit ion funnel, keeping the reaction temperature < -68° C. After the addition was finished, the funnel was rinsed with dry T H F (3 mL) and the resulting deep-yellow solution was stirred at -70° C for 25 min. Af t e r this time, a solution of butenolide (1.21 g, 1.1 equiv.) in dry T H F (10 mL) was added very slowly (dropwise, over a 1 h period) via the addit ion funnel while keeping the reaction temperature < -68° C. Af ter the addition was completed, the mixture was stirred at -70° C for 40 min and then, a solution of the aryl bromide 67 (6.5 g, 1.5 equiv.) in T H F (13 mL) was slowly added via the addition funnel keeping the temperature < -68° C at all times. After the addit ion was finished, the reaction mixture was stirred at -70° C for 1 h, then slowly allowed to reach room temperature, and stirred for a further hour at this temperature. The reaction was quenched adding water (90 mL) and removing most of the T H F by rotary evaporation. The resulting suspension was extracted with ethyl acetate (3 x 100 mL) and the combined organic extracts were dried, filtered and concentrated to dryness to yield 15.09 g of crude product as an orange oi l . The product was purif ied by flash chromatography using a 4.5 cm diameter column (6" si l ica gel) and eluting with 1.6 L of hexanes:ethyl acetate 3:1 collecting 20-ml fractions. Solvent removal afforded 7.91 g (76%) of the pure product as a white foam. IR ( K B r ) ujaax- 1^70 (lactone) c m ' ' , ' H nmr S: 1.38 (6H, d, CH(CH3)2, J - 6 Hz) , 2.7 ( I H , dd, H7". J = 5, 14 Hz), 2.83-2.90 ( I H , m, H2), 3.10 ( I H , dd, H7", J - 4, 14 Hz), 3.2-3.25 ( I H , m, H3), 3.35 ( I H , dd, H4, J = 8, 11 Hz), 3.68 (6H, s, OCH3), 4.21 ( I H , dd, H4, J = 3, 11 Hz) , 4.55 ( I H , septet, CH(CH3)2, J = 6 Hz) , 4.97 (2H, s, CH2-Ph), 5.03 (2H, s, CH2 -Ph) , 6.19 (2H, s, H2", H6"), 6.80 ( I H , d, H 5 ' , J - 8 Hz) , 7.03 ( IH , dd, H6', J = 2, 8 Hz) , 7.15-7.50 (21H, m, aromatic). U V (methanol) Amax dog «): 219 (4.70). MS m/z: 595 (M+-2SPh). H R M S calcd. for C37H39O7 (M+-2SPh): 595.2696, found: 595.2699. Ana l , calcd. for C49H48O7S2: C 71.97, H 6.04, S 8.0; found: C 72.18, H 5.95, S 7.83. Previously unpublished U V data. T R A N S - 2 - ( 4 - B E N Z Y L O X Y - 3 , 5 - D I M E T H O X Y B E N Z Y L ) - 3 - ( 3 - B E N Z Y L O X Y - 4 -I S O P R O P O X Y - o - O X O - B E N Z Y L ) B U T A N O L I D E (69): O OBn 2.31 g (1.8 equiv.) of red mercuric oxide were suspended in 15% aqueous T H F (36 mL) under nitrogen, and the suspension was stirred at -5° C (mcthanol-ice bath). Boron t r i f luor ide etherate (12.5 mL, 17 equiv., recently distil led) was added dropwise via addi t ion funnel keeping the temperature < 0° C. After the addition was complete, the reaction mixture was stirred for 10 min at -5° C and the thioketal 68 (4.9 g, 0.0059 mol) in dry T H F (833.5 mL) was added dropwise maintaining the temperature at 0° C. The addition funnel was washed with T H F (20 mL) and the washing added to the mixture. The orange suspension was allowed to reach room temperature and stirred for 2 h at this temperature. During this time the mixture was clear. After this time, the reaction was heated to 35° C for 1 h after which the control T L C (s.s. hexanesrethyl acetate 4:2) showed reaction completion. The reaction was quenched by adding saturated sodium bicarbonate solution (230 mL) , shaking well and separating the organic layer. This was then diluted with dichloromethane (250 mL) and washed with sodium bicarbonate solution (3 x 120 mL). The combined aqueous washings were extracted with dichloromethane (120 mL) and the mixed organic extracts dried, filtered and rotary evaporated to yield 7.25 g of crude product. This was purif ied by flash column chromatography (4.5 cm diameter, 6" si l ica gel) packed in i t i a l ly with dichloromethane. The crude product was dissolved in dichloromethane (146 mL) (with warming) and applied to the column top. The column was run with 1.5 L of dichloromethane collecting 75-ml fractions and then the solvent was changed to hexanes:ethyl acetate 4:2 (1.2 L) and continued the elution collecting 20-mL fractions. Rotary evaporation of the solvent yielded 3.22 g (88.2%) of pure product as white crystals. Mp = 111-113° C (EtOH). IR ( K B r ) fmax- 1^ 75 (lactone), 1663 (ketone) cm" ' , ' H nmr 6: 1.40 (6H, d, CH(CH3)2, J = 6 Hz), 3.0 (2H, d, H7", J = 6.1 Hz), 3.62 (6H, s, -OCH3), 3.57 ( I H , m, H3), 3.99 ( I H , dd, H4, J = 8, 9 Hz), 4.01 ( I H , dd, H4, J = 9, 9 Hz), 4.3 ( I H , m, H2), 4.65 ( I H , septet, CH(CH3)2, J = 6 Hz), 4.90 (2H, s, CH2 -Ph) , 5.12 (2H, 5, CH2-Ph) . 6.26 (2H, s, H2", H6"), 6.82 ( I H , d, H 5 ' , J = 8 Hz), 7.2-7.50 (12H, m, aromatic). U V (methanol) Xj^ax (log e): 310 (4.37), 280 (3.44), 231 (4.76), 211 (5.03). MS m/z: 610 (M+, 0.4), 91 (100). H R M S calcd. for C37H38O8: 610.2567; found: 610.2568. Anal , calcd. for C37H38O8: C 72.78, H 6.26; found: C 73.00, H 6.29. T R A N S - 2 - ( 4 - B E N Z Y L O X Y - 3 , 5 - D I M E T H O X Y B E N Z Y L ) - 3 - ( 3 - B E N Z Y L O X Y - 4 -I S O P R O P O X Y - 7 ' ^ - H Y D R O X Y B E N Z Y L ) B U T A N O L I D E (70): OH The ketone 69 (4.48 g, 7.3 mmol) was suspended in methanol (530 mL), and the suspension cooled to 0-5° C (ice-water bath). 417 mg (6.0 equiv.) of sodium borohydride were added in one portion, and the mixture was stirred under nitrogen at 0-5° C for 4 h when the control T L C (s.s. hexanes:ethyl acetate 1:1) showed reaction completion. The reduction was quenched adding enough 1 N H C l solution to obtain acid pH (50 mL) and most of the methanol was removed in vacuo. The resulting suspension was diluted with brine (55 mL) and extracted wi th dichloromethane (3 x 200 mL). The organic extracts were washed with water (140 mL) dried, filtered and rotary evaporated to afford 4.19 g of crude product as a white foam. Flash column chromatography (4.5 cm diameter, 5.5" silica gel) eluting with 1.5 L of hexanes:ethyl acetate 6:4, and collecting 20-mL fractions afforded 3.76 g (83.65%) of the pure alcohol, which appeared as one spot on T L C . IR ( K B r ) vrnax- 3492 (OH), 1767 (lactone) cm" ' , ' H nmr 5: the product consists of approximately 20% Q-OH and 80% 7'/3-OH epimers. 1.45 (m, CH(CH3)2), 1.58 (brs, D2O+, OH) , 2.50-3.2 (m, H2, H3, H4, H7"), 3.70 (m, H4^), 3.78 (s, OCH3), 4.2 (m, H 7 ' a ) , 4.3 (m, H4a), 4.50 (m, H 7 ' ^ , CH(CH3)2), 4.98 (s, CH2 -Ph) , 5.10 (s, CH2 -Ph) , 6.26 (s, H2"a ) , 6.40 (s, H2"^), 6.7-6.9 (m, H 2 ' , H 5 ' , H6'), 7.2-7.5 (m, aromatic). U V (methanol) A ^ g x ('«g 0 : 278 (3.74), 214 (4.72). MS m/z: 612 (M"^, 2.8), 594 (M'*'-H20, 1.6), 91 (100). H R M S calcd. for C37H40O8: 612.2723; found: 612.2716. Anal , calcd. for C37H40O8: C 72.54, H 6.57; found: C 72.70, H 6.66. T R A N S - 2 - ( 4 - H Y D R O X Y - 3 , 5 - D I M E T H O X Y B E N Z Y L ) - 3 - ( 3 - H Y D R O X Y - 4 -I S O P R O P O X Y - 7 ' ) 9 - H Y D R O X Y B E N Z Y L ) B U T A N O L I D E (56): In a 500 mL R B flask, 2.54 g of 10% Palladium on charcoal were suspended in ethanol (75 mL). The vessel was purged with vacuum - hydrogen three times, pressurized to 1 atm of hydrogen, and stirred for 1 h at room temperature. A solution of 3.76 g (6.1 mmol) of the benzyl ether 70 in reagent grade ethyl acetate (75 mL) was added to the suspension via canule with hydrogen pressure. The reaction was stirred under 1 atm. of hydrogen unt i l analytical T L C of a sample (s.s. hexanes-ethyl acetate 2:3) showed reaction completion (approx. 50 min. Over-hydrogenation must be avoided). The mixture was then filtered in a Schott packed with Celite 545 and the Celite and flask were washed with ethanol (75 mL) and ethyl acetate (75 mL). The filtrate was rotary evaporated to dryness to afford 2.71 g of crude poduct which was purif ied by flash column chromatography (4.5 cm diameter column, 5.5" sil ica gel) eluting wi th 1.5 L of hexancs:ethyl acetate 1:1, collecting 20-mL fractions. Removal of the solvent in vacuo yielded 1.97 g (74.3%) of the product (which consisted of approximately 8% a-OH and 92% 7 ' ^ - O H epimers), as a white foam. IR (KBr) J^max-3459 (OH), 1757 (lactone). ' H nmr 6: 1.4 (6H, d, CH(CH3)2 , J = 6 Hz), 2.64 ( I H , m, H3), 2.85 ( I H , dd, H 7 " , J = 5.4, 13.6 Hz), 2.98 ( IH , m, H2), 3.10 ( IH , dd, H7" , J = 4.9, OH OH 13.6 Hz), 3.8-3.95 (8H, m, H4, OCH3), 4.41 (d, H 7 ' a , J » 7 Hz) , 4.52-4.62 (2H, m, H 7 ' ^ , CH(CH3)2), 1.7 ( I H , brs, D2O+, -OH), 5.4 ( I H , brs, D2O+, -OH) , 5.75 ( I H , brs, D2O+, OH), 6.30 (s, . H 2 ' ' , 6"a), 6.40 (2H, s, H 2 " , 6"^), 6.65-6.86 (3H, m, aromatic). U V (methanol) X ^ a x Oog 0 : 281 (3.75), 213 (4.47). MS m/z: 432 (M+, 0.3). 414 (M+-H2O, 0.3), 373 (44.3), 262 (11.7), 224 (11.1), 205 (14.6), 189 (14.2). 181 (14.2), 178 (28.3). 175 (12.4), 167 (100). H R M S calcd. for C23H28O8: 432.1784; found: 432.1778. Ana l , calcd. for C23H28O8: C 63.89, H 6.52; found: C 63.06, H 6.77. T R A N S - 2 - ( 4 - H Y D R O X Y - 3 , 5 - D I M E T H O X Y B E N Z Y L ) - 3 - ( 3 - B E N Z Y L O X Y - 4 -I S O P R O P O X Y - 7 ' y 3 - H Y D R O X Y B E N Z Y L ) B U T A N O L I D E (71): This product is found when the hydrogenolysis reaction is not complete and can easily be hydrogenolysed to the tr ihydroxy compound. White foam: IR (KBr ) i^max-3471 (OH), 1762 (lactone), ' H nmr 5: 1.35 (6H, d, CH(CH3)2, J = 6 Hz) , 1.51 ( IH , brs, D2O+, OH) , 2.5-3.15 (4H, m, H2, H3, H7"), 3.70 (2H, m, H4), 3.85 (6H, s, OCH3), 4.54 (2H, m, CH(CH3)2, H 7 0 , 5.10 (2H, m, CH2 -Ph) , 5.40 ( I H , brs, D2O+, OH) , 6.41 (2H, s, H2", H6"), 6.7-6.9 (3H, m, H 2 ' , H 5 ' , H 6 ' ) , 7.25-7.42 (5H, m, aromatic). U V (methanol) ^max (log 0: 280 (0.47), 209 (4.65). MS m/z: 522 (M+, 9.2), 504 (M"^-H20, 2.5), 432 (12.2), 207 (12.5), 167 (49.2), 149 (10.6), 139 (13.5), 91 (100). H R M S calcd. for C30H34O8: 522.2253; found: 522.2256. Anal , calcd. for C30H34O8: C 68.96, H 6.55; found: C 68.11, H 6.61. T R A N S - 2 - ( 4 - H Y D R O X Y - 3 , 5 - D I M E T H O X Y B E N Z Y L ) - 3 - ( 3 - H Y D R O X Y - 4 -I S O P R O P O X Y B E N Z Y D B U T A N O L I D E (54):'^ '* Subproduct of hydrogenolysis. If the reaction is carried out for 5 h, an isolated yie ld of 84% can be achieved. White foam: IR (KBr) : 3540 (OH), 1775 (lactone), ' H nmr 5: 1.35 (6H, d, CH(CH^)2, J = 6 Hz) , 2.48-2.65 (4H, m, H2, H3, H7"), 2.87 (IH, dd, H 7 ' , J = 5, 14 Hz), 2.95 (IH, dd, H 7 ' , J = 6, 14 Hz), 3.82-3.95 (7H, m, H4, OCH3), 4.11 (IH, dd, H4, J = 6, 8 Hz), 4.52 (IH, heptet, CH(CH3)2, J = 6 Hz), 5.40 (IH, brs, D2O+, OH), 5.68 (IH, brs, D2O+, OH) , 6.40 (2H, s, H2 ' ' , H6' ' ) , 6.47 (IH, dd, H 6 ' , J = 2, 8.5 Hz), 6.62 (IH, d, H 2 ' , J = 2 Hz), 6.74 (IH, d, H5 ' , J = 8.5 Hz). U V (methanol) Ajnax (log £): 281 (3.62), 216 (4.20). MS m/z: 416 (M+, 21.9), 414 (11.2), 374 (24.5), 372 (18.6), 209 (40.5), 167 (100). H R M S calcd. for C23H28O7: 416.1835, found: 416.1829. Ana l , calcd. for C23H28O7: C 63.58, H 6.96; found: C 63.58, H 6.49. l - B I S ( P H E N Y L T H I O ) M E T H Y L - 3 , 4 - M E T H Y L E N E D I O X Y B E N Z E N E (82)J^ Piperonal (1.0 g, 6.7 mmol) was dissolved in dry chloroform (10 mL) and the solution cooled under nitrogen to -40° C. 1.4 mL (2.0 equiv.) of thiophenol and 2.5 equiv. of boron tr if luoride etherate were added via syringe and the resulting mixture was stirred for 15 min at -40° C. The reaction was quenched by pouring into ice cold water (10 mL) and extracting wi th chloroform (3 x 10 mL) . The combined organic extracts were washed with 10% K O H solution (2 x 10 mL) , water (10 mL), and brine (10 mL). D r y i n g and rotary evaporation afforded the product (2.3 g, 98%) which was not pur i f ied further, as a yellow o i l homogeneous by T L C . I R (neat) I'max- ^890 (C stretch), ' H nmr 6: 5.37 ( I H , s, CH(SPh)2), 5.96 (2H, s, -O -CH2 -O- ) , 6.66 ( I H , d, H5, J = 4 Hz), 6.77 ( I H , dd, H6, J = 2, 4 Hz), 6.98 ( IH , d, H2, J = 2 Hz), 7.22-7.44 ( lOH, m, aromatic). U V (methanol) A^ax (log 0 : 248 (4.14), 282 (3.88). MS m/z , 352 (M+). H R M S calcd. for C20H16O2S2: 352.0592; found: 352.0598. AnaL calcd. for C20H16O2S2: C 68.15, H 4.58; found: C 68.11, H 4.63. Sulfur not determined. T R A N S - 2 - ( 4 - B E N Z Y L O X Y - 3 , 5 - D I M E T H O X Y B E N Z Y L ) - 3 - I 3 , 4 - M E T H Y L E N E D I O X Y -a , Q - B I S ( P H E N Y L T H I O ) B E N Z Y L I B U T A N O L I D E (83):*^ ^ . PhS^ SPh OBn Piperonal diphenyl dithioketal 82 (2.0 g, 5.7 mmol) was dissolved in anhydrous T H F (10 mL) under nitrogen and cooled to -78° C. To this were added 3.6 mL (1.0 equiv.) of a 1.6 M solution of n-butylli thium in hexanes controlling the reaction temperature at -70° C. The resulting solution was stirred for 2.5 h at -70° C, and a solution of 480 mg (1 equiv.) of the butenolide in anhydrous T H F (8 mL) was slowly added via syringe. Af ter this, the mixture was stirred at -70° C for another 2 h, and a solution of 2.06 g (1.07 equiv.) of the benzyl bromide 67 in T H F (10 mL) was introduced. The reaction mixture was allowed to warm to room temperature, and quenched with water (25 mL) and extracted with ethyl acetate (3 x 50 mL). The combined organic extracts were washed with water (25 mL) , dried, and evaporated in vacuo to give 4.48 g of crude mixture which from flash column chromatography (4.5 cm diameter, 5.5" sil ica gel ) eluting with dichloromethane, yielded the pure produce as a yellow solid (2.16 g, 55%). Mp = 55-57° C (acetone). IR ( K B r ) u^^^^: 1770 (lactone). ' H nmr S: 2.79 ( IH , dd, HI", J = 5, 14 Hz), 2.85-2,92 ( I H , m, H2), 3.12 ( I H , dd, H 7 " , J = 4, 14 Hz) , 3.29-3.34 ( I H , m, H3), 3.50 ( I H , dd, H4, J - 8, 10 Hz), 3.70 (6H, s, -OCH3), 4.45 ( I H , dd, H4, J = 3, 10 Hz) , 5.02 (2H, s, CH2-Ph), 6.05 (2H, d, O-CH2 -O) , 6.18 (2H, s, H 2 " , H6") , 6.73 ( I H , d, H 5 ' , J « 8 Hz), 7.12 ( I H , dd, H 6 ' , J = 2, 8 Hz), 7.20-7.47 (14H, m, aromatic). MS m/z: 583 ( M ^ , -PhS). U V (methanol) A ^ a x (log c): 279 (3.82), 286 (3.80). H R M S calcd. for C34H31O7S: 583.1790; found: 583.1792. Anal , calcd. for C40H36O7S2: C 69.34, H 5.24, S 9.25; found: C 69.18, H 5.42, S 9.23. T R A N S - 2 - ( 4 - B E N Z Y L O X Y - 3 , 5 - D I M E T H O X Y B E N Z Y L ) - 3 - ( 3 , 4 - M E T H Y L E N E D I O X Y - a -O X O - B E N Z Y L ) B U T A N O L I D E (84):*^^ Red mercuric oxide (2.48 g 11.4 mmol) was suspended in 15% aqueous T H F (40 mL) and cooled under nitrogen to 0-5° C (ice-water bath). 12.92 m L of recently disti l led boron tr i f luoride etherate were added keeping the temperature at about 0° C. After st irring the suspension for 10 min at -5-0° C, a solution of the thioketal 83 (4.0 g, 6.3 mmol) in anhydrous T H F (80 mL) was slowly added maintaining the inner temperature at 0 ° C. After the addition was complete, the mixture was allowed to reach room temperature, stirred for 2 h at this temperature, and then heated to 30-35° C (warm water bath) for a further hour. Once the reaction was judged complete by analytical T L C (hexanesrethyl acetate 4:2), the mixture was cooled to room temperature, and diluted with dichloromethane (400 mL) and saturated aqueous sodium carbonate (400 mL). The organic layer was separated and washed with water (200 mL), dried, filtered, and concentrated in vacuo to afford 4.8 g of a creamy solid. Purif icat ion of the crude mixture by flash column chromatography (4.0 cm diameter, 6.0" silica gel) packed in i t ia l ly with dichloromethane (dissolving the sample in warm dichloromethane (100 mL) and forerunning with 1 L ofthe same solvent followed by 700 mL of hexanes:ethyl acetate 4:2 collecting 20-mL fractions yielded 2.67 g (85.57%) of the pure product as a white solid. Mp = 43-44° C (ethyl ether). IR (KBr ) i/max" 1770 (lactone), 1670 (ketone), ' H nmr S: 3.00 ( IH , dd, H 7 " , J = 6, 14 Hz), 3.08 ( IH , dd, H7" , J = 5, 14 Hz), 3.58 ( I H , m, H2), 3.70 (6H, s, OCH3), 4.00 ( IH , ddd, H3, J = 8, 8, 8 Hz), 4.10 ( I H , dd, H4, J = 8, 9 Hz), 4.39 ( IH , dd, H4, J = 8, 9 Hz), 4.92 (2H, s, CH2-Ph), 6.01 ( I H , d, -O -CH2 -O- , J = 1 Hz), 6.04 ( IH , d, O - C H 2 - O , J = 1 Hz), 6.29 (2H, s, H2" , H6"), 6.80 ( I H , d, H 5 ' , J = 8 Hz), 7.22-7.48 (7H, m, aromatic). U V (methanol) ^max (Jog e): 275 (3.82), 314 (3.90). MS m/z: 488 (M+), 91 (100). T R A N S - 2 - ( 4 - B E N Z Y L O X Y - 3 , 5 - D I M E T H O X Y B E N Z Y L ) - 3 - ( 3 , 4 - M E T H Y L E N E D I O X Y . 7 ' / 3 - H Y D R O X Y B E N Z Y L ) B U T A N O L I D E (85): OH OBn The ketone 84 (3.0 g, 6.1 mmol) was suspended in methanol (430 mL) and the suspension cooled in an ice water bath. Sodium borohydride (335 mg, 1.45 equiv.) was added and the mixture was stirred at 0° C for 5 h. When the reaction was judged complete by T L C analysis (s.s. hexanes:ethyl acetate 1:1) 53 m L of 1 N HCI were added to quench it. The mixture was concentrated in vacuo to remove most of the methanol, and the resulting suspension was diluted with brine (40 mL) , and extracted with dichloromethane (3 x 460 mL). The combined organic extracts were washed with water (110 ml), dried, filtered and rotary evaporated to yield 2.88 g (95.68%) of the product as a white foam homogeneous by T L C . IR ( K B r ) ^max- 3600 (OH), 1770 (lactone), ' H nmr S: 1.70 ( I H , brs, D2O+, OH), 2.50-3.10 (4H, m, H2, H3, H7"), 3.80 (6H, s, OCH3), 3.9 (2H, m, H4), 4.60 ( I H , d, H 7 ' , J = 6.7 Hz) , 5.0 (2H, s, CH2 -Ph) , 5.98 (2H, d, O - C H 2 - O , J = 1 Hz), 6.40 (2H, s, H2", H6"), 6.6-6.8 (3H, m, H 5 ' , H 6 ' , H 2 ' ) , 7.24-7.50 (5H, m, aromatic). Also the 7 a - 0 H isomer is present in 18%. U V (methanol) ^max (log 0 : 282 (3.60). MS m/z: 490 (M+). H R M S calcd. for C24H26O8: 490.1627; found: 490.1627. Ana l , calcd. for C24H26O8: C 68.56, H 5.34; found: C 68.78, H 5.56. T R A N S - 2 - ( 4 - H Y D R O X Y - 3 , 5 - D I M E T H O X Y B E N Z Y L ) - 3 - ( 3 , 4 - M E T H Y L E N E D I O X Y - 7 ' ^ -H Y D R O X Y B E N Z Y L ) B U T A N O L I D E (57):''^ OH Pal ladium on charcoal (10%, 550 mg) was suspended in ethanol (26 mL) in a hydrogéna t ion flask. The vessel was purged evaporating wi th vacuum and breaking it with hydrogen (3 times). The flask was pressured to 1 atm of hydrogen and the suspension stirred for 1 h at room temperature. A solution of the benzylether (1.298 g, 2.6 mmol) in ethyl acetate (26 mL) was added via canule with hydrogen pressure, and the reaction mixture was stirred under 1 atm of hydrogen for a further 6 h when control T L C (s.s. hexanes:ethyl acetate 2:3) showed reaction completion. The suspension was filtered through a small pad of Celite 545, washing the bed with ethanol (50 mL). Evaporation of the filtrate produced 1.0496 g of a light green foam which upon flash chromatography purif icat ion (4 cm diameter, 6.0" si l ica gel), eluting with hexanes:ethyl acetate 2:3 (1.5 L) and collecting 15-mL fractions afforded 918.5 mg (86.6%) of the pure product as a white foam. I R ( K B r ) i/max- 3.449 (OH), 1762 (lactone), ' H nmr 5: 1.52 ( I H , brs, D2O+, OH), 2.52-3.16 (4H, m, H2, H3, H7"), 3.87 (6H, s, OCH3), 3.94 (2H, m, H4), 4.65 ( I H , d, H 7 ' , J = 6.8 Hz), 5.98 (2H, m, O-CH2 -O) , 6.40 (2H, s, H 2 " , H6"), 6.52-6.78 (3H, m, aromatic). Approximately 14% of the C7 epimer could be observed. U V (methanol) A^ax ('og «): 214 (4.39), 283 (3.79). MS m/z: 402 (M^). H R M S calcd. for C21H20O8: 402.1314; found: 402.1302. Anal , calcd. for C21H20O8: C 62.69, H 5.50; found: C 62.39, H 5.70. OH G E N E R A L P R O C E D U R E F O R S U B C U L T U R E O F P L A N T S U S P E N S I O N C U L T U R E S . Stringent precautions arc needed for the protection of cell line purity. Information on medium, inoculum age and volume, and usual subculture interval for each stock culture is provided under the indiv idual plant species headings. The suspensions to be subcultured were assessed visually (usual colour?, usual degree of aggregation?, usual cell density?, gross contamination?) and microscopically (at 100 and 400X for obvious contaminants such as filamentous fungi or motile bacteria). A small sample (10-25 mL) was filtered and p H and R.I. measured. For transfer to new media, only cultures that appeared normal in a l l aspects were used. Transfer routine: Using aseptic technique, in the clean air station, the stipulated aliquots of inocula were transferred into fresh media. Absolute accuracy of inocula volumes is not needed, but a measuring device should be used. More than one flask of inoculum was used for each new set of flasks to ensure that a l l new stock cultures came from more than a single source and a note was made as to which flasks were derived from which flasks. Whenever possible, not al l flasks of a set of stock cultures were used or discarded. One or two flasks remained on the shaker in case the new culture behaved abnormally, became contaminated, or it was discovered that there was a mistake in the preparation of the new media. From each flask of inoculum, some culture was plated onto nutrient agar (1 mL streaked on the agar surface), incubated at 25° C for 72 h, and examined for bacterial or fungal growth. A n y contamination positives were not used for experiments or for future stock culture transfers. Some culture was also plated on agar-solidified stock culture broth as an additional safeguard to perpetuate each stock culture in callus form. These cultures were kept at room temperature, in the dark, for at least one month after subculture. A t approximately monthly intervals, ca l l i maintained as stocks for each cell line were transferred to fresh agar media. Old flasks of stock cultures were discarded when it was obvious that their respective progeny was healthy. Podophyllum peltatum cell line 128-B. Medium: MS (1/2) - 1.5% sucrose, vessel size: 1 L , medium volume: 300 mL, inoculum volume: 25 mL of drained cells, normal period between transfers: 17 days. Tripterygium wilfordii cell line T R P 4 a - B . Medium: PRD2C010O1 vessel size: 1 L , medium volume: 500 mL, inoculum volume: 50 mL, normal period between transfers: 14 days. Media Preparations. (a) MS medium for P. peltatum and T. wilfordii (production) Stock Solution Use NH4NO3 66.0 g/L MACRO KNO3 76.0 g/L 25 mL/L KH2P04-H20 6.8 g/L MgS04-7H20 14.8 g/L Final Concentration 1650 mg/L 1900 mg/L 170 mg/L 370 mg/L H3BO3 1.24 g/L 6.2 mg/L MICRO MnS04-H20 4.46 g/L 5 mL/L 22.3 mg/L ZnS04-7H20 1.72 g/L 8.6 mg/L Stock Solution Use Final Concentration Kl 0.166 g/L 0.83 mg/L TRACE Na2Mo04-2H20 0.05 g/L 5 mL/L 0.25 mg/L CoCl2-6H20 0.005 g/L 0.025 mg/L CUS04-5H20 0.005 g/L 0.025 mg/L CaCl2-2H20 40.0 g/L 11 mL 440.0 mg/L Na2EDTA 4.0 g/L 9.3 mL 37.35 mg/L FeS04-7H20 3.0 g/L 27.85 mg/L Thiamine-HCl 0.01 g/L 0.1 mg/L VITAMINS Nicotinic acid 0.05 g/L 0.5 mg/L Pyridoxine HCl 0.05 g/L 10 mL 0.5 mg/L Glycine 0.2 g/L 2.0 mg/L Myo inositol 10.0 g/L 100.0 mg/L Sucrose 20 g/L 20 g/L 2% NAA 0.05 mg/mL 1 mL/L 0.5 mg/L Kinetin 0.5 mg/mL 1 mL/L 0.5 mg/L For MSN(o.5 mg/L)K-(0.5 mg/L) . "se as indicated, f inal pH=5.8. For MS (1/2) - 1.5%, use half of everything but leave out the hormones ( N A A and kinetin), and use 15 g / L of sucrose so that the f inal concentration is 1.5%. Adjust the pH to 5.8 once the f inal volume has been reached, using 1 M N a O H or 1 M H C l as required. To prepare agar, add 7-8 g of Difco agar per liter of medium. (b) PRDCo mediiun f o r T. wilfordii (maintenance) Stock S o l u t i o n Use F i n a l Concen t r a t i on (NH4)2S04 40 .0 g / L 200 mg/L NaH2P04-H20 18.0 g / L 90 mg/L Na2HP04 6.0 g / L 30 mg/L K l 0.15 g / L 5 mL/L 75 mg/L H3BO3 0.6 g / L 0.30 mg/L Na2Mo04-2H20 0.05 g / L 0.25 mg/L CoCl2-6H20 0.05 g / L 0.25 mg/L KNO3 100.0 g / L 10 mL/L 1000 mg/L K C l 30.0 g / L 300 mg/L MgSG4-7H20 50.0 g / L 250 mg/L MnS04-4H20 2.64 g / L 5 m l / L 13.2 mg/L ZnS04-7H20 0.6 g / L 30 mg/L CuS04-5H20 0.05 g / L 0.25 mg/L CaCl2-2H20 40 .0 g / L 3.75 mL/L 150 mg/L Na2EDTA 4 .0 g / L 9.3 mL/L 37.2 mg/L FeS04-7H20 3.0 g / L 27.9 mg/L Case in h y d r o l y s a t e 2 .0 g / L 0.2% Sucrose 20.0 g / L 2% Coconut m i l k 100 mL/L 10% 2,4-D 4 mL/L 2 mg/L Stock S o l u t i o n Use F i n a l Concen t r a t i on n i c o t i n i c a c i d 0.1 g / L 1 mg/L th i amine -HCl 1.0 g / L 10 mL/L 10 mg/L p y r i d o x i n e ' t H C l 0.1 g / L 1 mg/L myo i n o s i t o l 10.0 g / L 100 mg/L Fina l pH adjusted to 6.2 with 1 M N a O H or 1 M H C l as required. B I O T R A N S F O R M A T I O N O F S U B S T R A T E 56 W I T H P. PELTATUM C E L L S U S P E N S I O N C U L T U R E . P. peltatum cells were grown in a Microferm for 2 days (450 mL drained cells, R.I. = 1.3333, pH = 4.8, inoculum 18 days old) in 3 L of 1/2 MS - 1.5% sucrose medium at T = 26° C, 300-400 m L / m i n L air and stirring at 440 rpm with two impellers. After this period, no apparent contamination was found (microscopic inspection and N A plate), the pH was 4.9 and R.I. = 1.3349. Substrate 56 (3.0 g) dissolved in ethanol (70 mL) was injected into the Microferm while f i l tering with a sterile H V (0.2 ^) membrane to keep aseptic conditions. The broth turned mi lky and biotransformation was allowed to proceed withdrawing samples to check reaction progress after 2 days (48 h, 40 min), 4 days (89 h, 30 min) and 5 days (111 h, 40 min). Analy t ica l data indicated that the disappearance of substrate 56 was >50% after two days and only a trace of it remained after 4 days. The Microferm was harvested (pH = 4.35, R.I. = 1.3352, no apparent contamination shown by microscopic inspection) and the broth and cells were extracted separately. The cells were soaked overnight in 700 mL E t O A c , drained and homogenized in E t O A c (500 mL) using an Ul t r a Turrax T25 blender. The mass was filtered through a short pad of Celite 545 and the filtrate was mixed with the first E t O A c extract, dried, filtered and evaporated to dryness to yield 3.74 g of extract. The cell debris and Celite were sonicated wi th methanol (700 mL) for 30 min , fil tered, dried and concentrated to yield 2.4 g of methanolic cell extract. The broth was extracted as follows: ethyl acetate (800 mL) were added and the mixture shaken vigorously for 15 min. Then salt was added to saturate the aqueous layer and the mixture was stirred vigorously for another 15 min. Some protein separated forming a gelatinous layer that was filtered off with the aid of Celite (30 g). The cake was washed with ethyl acetate (100 mL) and the filtrate transferred to a separatory funnel. The aqueous layer was drained and extracted with ethyl acetate (2 X 800 mL). The organic extracts were combined, dried, fi l tered, and rotary evaporated to give 3.1 g of extract as a brown foam. A second extraction of the broth was made using dichloromethane (1.5 L) to give 8.7 mg of an orangey extract. Analysis of the extracts indicated the presence of lignan-type compounds in the ethyl acetate extracts, and these were purified by repeated column chromatography using chloroform-methanol mixtures as described in the discussion. F ina l purifications were made by preparative T L C using the same eluant. The following metabolites were identified in order of elution: E - 2 - ( 3 , 5 - D I M E T H O X Y - 4 - H Y D R O X Y B E N Z Y L I D E N E ) - 3 - ( 3 , 7 - D I H Y D R O X Y - 4 -I S O P R O P O X Y B E N Z Y L ) B U T A N O L I D E (74): i-PrO 107 mg (3.5%). I R ( K B r ) uj^a^: 1720 cm"' (carbonyl). ' H nmr (methanol-d 4) 5: 1.3 (6H, d, CH(CH3)2) , 4.08 ( I H , m, H3), 4.28 ( I H , m, H4), 4.49 ( I H , septet, CH(CH3 )2) , 4.57 ( I H , m, H4), 5.02 (d, I H , H 7 ' ) , 3.93 (6H, s, OCH3), 6.64-6.82 (3H, m, aromatic), 7.10 (2H, s, H2", H6"), 7.50 ( I H , s, H7"). U V (methanol) A^ax : 324 (3.77), 203 (4.19). M S m/z: 430 (M+, 18.0), 412 (M+-H2O, 7.8), 372 (11.4), 248 (28.1), 220 (15.8), 211 (14.9), 191 (52.7), 181 (43.6), 154 (85.8), 137 (100). H R M S calcd. for C23H26O8: 430.1628; found: 430.1621. The product failed to give satisfactory elemental analysis. l - ( 3 , 5 - D I M E T H O X Y - 4 - H Y D R O X Y P H E N Y L ) - 6 , 4 ^ - D I H Y D R O X Y - 3 -H Y D R O X Y M E T H Y L 7 - I S O P R O P O X Y - l , 2 , 3 , 4 . T E T R A H Y D R O - 2 - N A P H T H O I C A C I D 7 L A C T O N E (75) : The data presented are for an inseparable mixture of 75 and the 4a-isomer (ratio of 75 to 4Q-isomer 10:1). Nmr data as cited below allowed the analysis of the isomer ratio. 1.00 g (33.5%). IR ( K B r ) Uj^^x- 3495 (OH), 1774 (lactone) cm" ' , ' H nmr 5: 1.14 (3H, d, CH(CH3)2), 1.28 (3H, d, CH(CH3)2), 1.55 ( I H , brs, D2O+, OH) , 2.58 (m, H3Q), 2.68 ( IH , m, H3^), 3.19 ( I H , dd, H2, J = 11.4, 14 Hz), 3.86 (6H, s, OCH3), 3.96 ( IH , d, H I , J = 11.4 Hz), 4.25-4.47 (3H, m, H3a, CH(CH3)2), 4.85 ( I H , d, H4;9, J = 2.7 Hz), 4.9 (d, H4Q, J = 12.3 Hz), 5.5 ( IH , brs, D2O+, OH), 5.7 ( I H , brs, D2O+. OH), 6.30 (s, H5a), 6.38 ( IH , s, H5^), 6.42 (s, H 2 " , 6"Q), 6.46 (2H, s, H 2 " , H/96"), 6.88 ( I H , s, H8). U V (methanol) A^ a x ('og 0 : 283 (3.68), 215 (4.53). MS m/z: 430 ( M ^ , 35.3), 388 (44.0), 154 (100). H R M S calcd. for C23H26O8: 430.1628; found: 430.1622. The product failed to give satisfactory elemental analysis. Attempts to crystallize the material afforded only a sticky solid. l - ( 3 , 5 - D I M E T H O X Y - 4 - H Y D R O X Y P H E N Y L ) - 2 , 4 / 5 , 6 - T R I H Y D R O X Y - 3 -H Y D R O X Y M E T H Y L - 7 - I S O P R O P O X Y - l , 2 , 3 , 4 - T E T R A H Y D R O - 2 - N A P H T H O I C A C I D L A C T O N E (76): OH OH 191 mg (6.18%). IR (KBr) uj^^^: 3484 (CH), 1746 (lactone). ' H nmr S: 1.33 (3H, d, CH(CH3)2), iM (3H, d, CH(CH3)2), 3.51-3.62 (7H, m, H3, OCH3) , 4.03 (d, I H , H4, J = 7.2 Hz), 4.21 (dd, I H , one of lactone, J = 8.3, 9.6 Hz), 4.37 (dd, I H , one of lactone, J = 9.6, 9.6 Hz), 4.66 ( IH , septet, CH(CH3)2), 5.22 ( IH , s, HI ) , 6.21 (2H, s, H 2 ' , H 6 ' ) , 6.61 ( I H , s, H5), 7.08 ( I H , s, H8). U V (methanol) A ^ a x (log e): 290 (3.56), 206 (4.40). MS m/z: 446 (M+, 0.9), 428 (M+-H2O, 2.1), 386 (2.1), 286 (81.6), 264 (100). H R M S calcd. for C23H26O9: 446.1577; found: 446.1583. No satisfactory elemental analysis could be obtained. Attempts to crystallize the material produced only a white mass. l - ( 3 , 5 - D I M E T H O X Y - 4 - H Y D R O X Y P H E N Y L ) - 2 , 4 a , 6 - T R I H Y D R O X Y - 3 -H Y D R O X Y M E T H Y L - 7 - I S O P R O P O X Y - l , 2 , 3 , 4 - T E T R A H Y D R O - 2 - N A P H T H O I C A C I D L A C T O N E (77): 12.7 mg (0.4%). IR (neat): 3424 (OH), 1709 (lactone) c m ' ' . ' H nmr S: 1.32 (two doublets, 6 H , CH(CH3)2), 3.39 ( I H , m, H3), 3.72 (6H, s, OCH3), 4.04 (dd, I H , one of lactone, J = 8, 8 Hz) , 4.24 (dd, I H , one of lactone, J = 8, 9.6 Hz), 4.56 ( I H , septet, CH(CH3)2), 4.61 ( I H , d, H4, J = 6 Hz), 5.08 ( IH , s, H I ) , 6.49 (2H, s, H 2 ' , H 6 ' ) , 6.78 ( I H , s, H5), 7.0 ( I H , s, H8). U V (methanol) A ^ a x (log «): 289 (3.68), 210 (4.53). MS m/z: 446 (M+, 0.7), 428 (M+-H2O, 10.2), 386 (10.0), 264 (100). H R M S calcd. for C23H26O9: 446.1577; found: 446.1585. No satisfactory elemental analysis could be obtained. Attempts to crystallize the product produced a sticky solid. l - ( 3 , 5 - D I M E T H O X Y - 4 - H Y D R O X Y P H E N Y L ) - 6 - H Y D R O X Y - 3 - H Y D R O X Y M E T H Y L - 7 . I S O P R O P O X Y - 4 ) 8 - M E T H O X Y - l , 2 , 3 , 4 - T E T R A H Y D R O - 2 - N A P H T H O I C A C I D L A C T O N E (78): OCH, HO i-PrO C H 3 0 - " Y ^ O C H 3 OH 28.0 mg (0.9%). IR (CHCI3) ujaax'. nmr S: 1.4 (3H, d, CH(CH3)2), 1.26 (3H, d, CH(CH3)2). 1.61 ( I H , brs, D2O+. OH) , 2.68 ( IH , m, H3), 3.18 ( I H , dd, H2, J = 11.5, 14 Hz), 3.84, 3.86 (two singlets, 9 H , OCH3). 3.96 ( I H , d, H I , J = 11.5 Hz), 4.25-4.47 (3H, m, H3a, CH(CH3)2), 4.85 ( I H , d, H4, J = 2.6 Hz), 5.48 ( I H , brs, D2O+, OH) , 5.68 ( I H , brs, D2O+, OH) , 6.38 ( I H , s, H5), 6.47 (2H, s, H 2 ' , H 6 " ) , 6.88 ( I H , s, H8). MS m/z: 444 (M+, 18.8), 402 (15.5), 154 (42), 40.0 (100). H R M S calcd. for C24H28O8: 444.1784; found: 444.1791. This thick o i l could not be crystall ized. No satisfactory elemental analysis could be obtained. A R Y L T E T R A L I N 75. S T A B I L I T Y S T U D I E S . P. peltatum cells were grown in shake flasks for 3, 11, 17, and 21 days and eight experiments with one flask each (containing 300 mL of cell suspension) were started. A r y l tetralin 75, dissolved in 3 mL of ethanol was added under aseptic conditions to four flasks (one of each age), and a control was set adding 3 mL ethanol to the remaining flasks. A l l flasks were incubated in the rotary shaker and reaction progress was followed by withdrawal of 2.5 mL aliquots and reverse phase H P L C analysis every hour. After 5 h of incubation, the flasks were incubated overnight and harvested the next morning. For harvesting, the cells and spent medium were homogenized together using an Ul t ra Turrax T-25 disperser and filtered through Celite 545. The filtrate was saturated with N a C l and extracted with E t O A c (3 x 150 mL). The cell debris and Celite were sonicated for 30 min in E t O A c (150 mL), filtered and washed with more E t O A c (50 mL). The combined organic extracts (from spent medium and cell debris) were dried, fi l tered, and rotary evaporated to dryness. The ethyl acetate extracts (usually c.a. 250 mg) were purif ied separately by flash column chromatography using a 3 cm diameter column, packed with 7.0" si l ica gel using a 19:1 mixture of chloroform:methanol (600 mL) collecting 10-mL fractions. The pure recovered product, typically fractions 12-24, was pooled together and rotary evaporated, affording yields in the range of 72-94.1%. B I O T R A N S F O R M A T I O N O F S U B S T R A T E 57 W I T H P. PELTATUM C E L L S U S P E N S I O N C U L T U R E . P. peltatum cells were grown in a bioreactor for 7 days. After this period, no contamination was apparent upon microscopic inspection (pH = 4.5, R.I. = 1.3340). The pH was adjusted to 6.5 with 0.2 M K O H solution, and 1 mL of polypropylen glycol was added as anti-foam agent. A solution of substrate 57 (491 mg) in ethanol (10 mL) was added to the cell culture f i l ter ing through a sterile H V (0.2 p) membrane, and biotransformation was allowed to proceed for 5 h. T L C analysis of a sample indicated partial conversion to a more polar compound with strong U V absorption. The broth was filtered out under aspectic conditions, and the cells were resuspended in 1/10 MS medium (3.0 L) containing a sterile solution of substrate 57 (501.7 mg) in ethanol (10 mL). Biotransformation was allowed to proceed for 20 h. After this time, the broth was again removed and the cells resuspended in 1/10 MS medium (3.0 L) containing a solution of the substrate 57 (488.2 mg) in ethanol (10 ml). Biotransformation proceeded for 20 h before the broth was removed and the cells resuspended in fresh 1/2 MS - 1.5% sucrose medium (3.0 L) . The three broths from this experiment were extracted immediately following the same procedure as with the previous biotransformations (vide supra) affording 1.66 g, 469 mg, and 324 mg, respectively. A typical purif icat ion procedure is exemplified with the first broth extract: The extract was dissolved in ethyl acetate and applied to a 4 cm diameter column packed with the flash technique (6" si l ica gel) using hexane:EtOAc 2:3 (2 L) . The column was run collecting 72, 10-mL fractions and then 25-mL fractions until the solvent was finished. The column was then eluted successively with E t O A c (500 mL) collecting 25-mL fractions, 10% methanol in E t O A c (500 mL) collecting 25-mL fractions and methanol (500 mL) collecting 50-mL fractions. The fractions were spotted on T L C and those which had similar composition were grouped together and rotary evaporated to dryness. Fraction 1 (from 1 to 31) contained non polar material (40.4 mg), fraction 2 (from 32-72) contained recovered starting material (136.4 mg), fraction 3 (from 73-112) contained a mixture of recovered starting material and the styrene 86 (102.5 mg). Fraction 4 (from 113 to 124) contained the product 86 (243.9 mg), fraction 5 (from 125 + 132) contained indigenous metabolites (186.0 mg), fraction 6 (from 132-158) consisted of recovered polypropylen glycol (822.6 mg) and fraction 7 (methanol wash) contained the polar material as a brown solid (120 mg). The total recovery was 1.65 g (98.9%). Y i e l d of styrene 86 based on recovered starting material = 273.9 mg (90.3%) (50 mg recovered starting material + 30 mg styrene from fraction 3). 2 - ( 3 , 5 - D I M E T H O X Y - 4 - H Y D R O X Y B E N Z I L I D E N E - 3 - ( 3 , 4 - M E T H Y L E N E D I O X Y - 7 -H Y D R O X Y B E N Z Y D B U T A N O L I D E (86): IR ( K B r ) i / m a v 3357 (OH), 1715 (lactone). ' H nmr (DMSO-d 6) S: 3.85 (6H, s, OCH3), 4.10 ( I H , brs, H3), 4.24 ( I H , brt, H4), 4.40 ( I H , brd, H4), 4.95 ( I H , brs, H7') . 5.80 ( I H , brs, D2O+, OH) , 5.94 (2H, brd, O-CH2 -O) , 6.6 ( I H , d, H6), 6.75 (2H, m, H 5 ' , H 2 ' ) , 7.10 (2H, s, H 2 ^ H6"), 7.40 ( I H , s, H7''), 9.07 ( I H , brs, D2O+, OH). U V (methanol) X^nax (log 0 : 208 (4.34), 240 (4.24), 335 (4.28). MS m/z: 400 (M+, 100), 353 (36), 312 (11.6), 311 (52), 287 (31.6), 286 (96.8), 285 (28.9), 258 (11.8), 251 (16.2), 250 (87.6). H R M S calcd. for C21H20O8: 400.1158; found: 400.1161. Despite several attempts aimed to crystallize the product, no satisfactory elemental analysis could be obtained. D E H Y D R O A B I E T I C A C I D (122) P U R I F I C A T I O N . Technical grade dehydroabietic acid ( ICN, 200.0 g) was dissolved in warm ethanol (488 mL) in a 1.5 L capacity l iquid- l iquid extractor. Ethanolamine (40.0 mL) and water (488 mL) were added while stirring, and the resulting mixture extracted continuously with petroleum ether (60-80° C) for 20 h, while being kept at 50-55° C . The resulting aqueous solution, thus freed of neutral material, was boiled briefly to drive out any dissolved petroleum ether. Cooling this solution to 0-5° C overnight, resulted in a semi-solid mass, which was broken up manually. Fi l t rat ion provided the ethanolamine salt of dehydroabietic acid, which was stirred with 50% aqueous ethanol (132 mL) at 4 ° C and vacuum filtered. The moist salt was dissolved in hot ethanol (334 mL), acetic acid (33.2 mL) was added, and water (160 mL) was gradually added to the boiling solution unti l the cloud point was reached. The hot solution was filtered, and cooled to room temperature. The acid that crystallized was isolated by f i l t rat ion, rinsed with 50% aqueous ethanol (40 mL), and air-dried to provide 59.8 g of dehydroabietic acid, 88.7% pure by G.C. analysis. M P = 160-161° C (Lit . = 171-172° C). D E H Y D R O A B I E T I C A C I D A N A L Y S I S B Y G A S C H R O M A T O G R A P H Y . This method analyzes acids as methyl esters and is comparable to diazomethane treatment. Sample preparation: dissolve approximately 5.0 mg of sample in 1 mL of methanol. A d d one drop of 1% w/v phenolphtalein in methanol solution, and then dropwise 10% w / v tetramethyl ammonium hydroxide in methanol solution mixing well unti l the pink colour remains for 5 min without change. Instrument conditions: Init ial temperature: 220° C, f inal temperature: 245° C, stop time: 20 min; rate: 0.7° C / m i n , At tn: 1; Detector temp. : 300° C, Injector/temp: 300 ° C. With these parameters the retention time for methyldehydro abietate is 15 min. 1 8 - N O R A B I E T A - 4 ( 1 9 ) , 8 , l l , 1 3 - T E T R A E N E (150):^^^ Dehydroabietic acid (44.2 g, 0.147 mol) was dissolved in benzene (250 ml). Th iony l chloride (13 mL, 0.18 mol) and D M F (0.5 mL) were added, and the solution stirred for 1 h at room temperature. Af ter this time, the mixture was warmed to 68-73° C and this range of temperature was maintained for 30 min. The solution was then refluxed for 15 min, then cooled and rotary evaporated. The crude acid chloride (IR (neat) i^max 1^86 cm*') was taken up in reagent grade acetone (300 mL), and the solution cooled to -5-0° C in a methanol-ice bath. A solution of sodium azide (12 g, 0.18 mol) in water (40 mL) was added dropwise, and wi th vigorous stirring. Toluene (100 mL) was then added, and the organic layer decanted, dried, fi l tered, and the acetone was removed by rotary evaporation. The solution of acyl azide (IR (toluene) i/niax- 2120 (azide), 1692 (carbonyl) cm"') was brought to 300 mL with additional toluene and heated slowly to reflux. Af ter refluxing for 30 min, an IR of a sample of the solution showed complete reaction. The solvent was then removed in vacuo. The crude isocyanate (183) (IR (neat) i^max- 2250 (NCO) cm"') was added to a suspension of L A H (7 g, 0.18 mmol) in anhydrous T H F (600 mL), and the mixture refluxed with st irr ing under nitrogen overnight. Successive dropwise addition of acetone (10 mL) , water (7 mL), 15% aqueous sodium hydroxide (7 mL), and more water (20 mL) produced a thick white suspension which was filtered and triturated with hot T H F (200 mL) . The combined organic solutions were rotary evaporated to give the crude monomethyl amine. This was refluxed with formic acid (100 mL) and 35% aqueous formaldehyde (50 mL) for 3 h, and the solvents removed in vacuo. The resulting tarry mass was shaken with ether (300 mL) and 4 N N a O H (200 mL) unt i l dissolved. The organic layer was drained, dried, fi l tered, and evaporated. The crude dimethylamine (184) was obtained as a golden syrup. To a solution of 40.9 g (0.13 mol) of this product in chloroform (680 mL), cooled to -40° C, was added 85% M C P B A (34.8 g, 0.17 mol) in small portions over a 20 min period. Once the addition was complete, the reaction was stirred 10 min at -40° C. Triethylamine (8.2 mL, 82 mmol) was added, and the solution brought to reflux. After 30 min, the reaction mixture was cooled and the solvent removed in vacuo. The residue was taken up in ether (550 mL) and washed with 10% sulfuric acid (500 mL), 10% potassium carbonate (2 x 500 mL), dried, filtered, and rotary evaporated. The crude exo-olefin (31.8 g) was purif ied by gravity column chromatography (200 g of sil ica gel) eluting with hexanes to afford the pure exo-olefin as a colourless oi l (20.8 g, 55.8% overall). IR (neat) t/max: 3050 (C=CH2). 2925 (CH), 1650 (C=C). ' H nmr S: 1.02 (3H, s, CIO, CH3), 1.25 (6H, d, CH(CH3)2, J = 6 Hz), 1.51-2.92 (m, aliphatic H), 4.61 ( I H , d, H4, J = 2 Hz), 4.86 ( I H , d, H4, J = 2 Hz), 6.94 ( I H , brs, H14), 7.01 ( IH , brd, H12), 7.22 ( I H , d, H l l ) . MS m/z: 254 (M+, 42.9), 239 (92.9), 211 (4.5), 197 (100). H R M S calcd. for C19H26: 254.2034; found: 254.2030. U V (methanol) Aniax (log t): 207 (4.19). Ana l , calcd. for C19H26: C 89.70, H 10.30; found: C 89.90, H 10.22. 1 8 , 1 9 - D I N O R A B I E T A - 8 , l l , 1 3 - T R I E N - 4 . 0 N E (151): A stock solution of the olefin 150 (20.8 g, 0.081 mol) in methanol-methylene chloride 5:1 (945 mL) was divided in 4 portions of 240 m L each. Ozone was passed into the stirred solutions at -78° C unti l they turned a pale blue colour (approximately 1 h per flask at 2.2 psi of oxygen, 90 volts, and a flow of 0.015 L /min ) . The reaction mixtures were stirred for a further 30 min at -70° C without the ozone stream. Ana ly t i ca l T L C on hexanes of the reaction media showed reaction completion. Dimethyl sulphide (2 m L , 26.5 mmol) was added to each flask and the reaction mixtures were stirred at room temperature for 20 h. A n a l y t i c a l T L C (s.s. hexanes:ethyl acetate 9:1) showed that al l the reactions were al ike, and they were mixed at this point, and the solvent removed in vacuo. The residue was dissolved in hexanes:ethyl ether 2:1 (690 mL) and washed with water (3 x 86 mL) and brine (86 mL). The aqueous layer was extracted back with ether (130 mL) and the combined organic layers were dried, fi l tered, and rotary evaporated. The yellow residue was separated by flash column chromatography (4.5 cm diameter, 6" s i l ica gel) using hexanes:ethyl acetate 9:1 to yield 18.0 g (90.6%) of the ketone 151 as a white solid and 2.0 g (9.04%) of diketone 151b as a white solid. Mp = 40-42° C. IR (CHCI3) i/max-" 2950 (CH) , 1710 (carbonyl). ' H nmr 5: 1.06 (3H, s, C I O CH3), 1.24 (6H, d, CH(CH3)2), 1.76-2.94 (m, aliphatic), 6.95 ( I H , brs, H14), 7.04 ( I H , brd, H12), 7.22 ( I H , d, H I 1). U V (methanol) A^ax (log e): 214(3.98). 1 8 , 1 9 - D I N O R A B I E T A - 8 , l l , 1 3 - T R I E N - 4 - O N E (151): ,110 O MS m/z: 256 (33.3, M"^), 241 (100). H R M S calcd. for C18H24O: 256.1827; found: 256.1826. A n a l , calcd. for C18H24O: C 84.33, H 9.43; found: C 84.30, H 9.21. 1 8 , 1 9 - D I N O R A B I E T A . 8 , l l , 1 3 - T R I E N - 4 , 7 - D I O N E (151b):**° 12 M p = 107-108° C. IR (CHCI3) j/max: 2950 (CH) , 1715 (carbonyl), 1680 (carbonyl). ' H nmr 6: 1.17 (3H, s, CIO, C H 3 , J = 6 Hz) , 1.27 (6H, d, CH(CH3)2), 1.98-3.30 (m, aliphatic), 7.38 ( I H , d, H l l , J - 8 Hz), 7.45 ( I H , dd, H12, J - 3, 8 Hz), 7.95 ( I H , d, H I 4 , J = 3 Hz). U V (methanol) Amax: ( loge): 254 (3.56), 204 (4.09). MS m/z: 270 ( M ^ 35.6), 255 (100). H R M S calcd. for C18H22O2: 270.1620; found: 270.1613. A n a l , calcd. for C18H22O2: C 79.96, H 8.20; found: C 80.18, H 8.23. 3 - D I M E T H Y L T H I O M E T H Y L E N E - 1 8 , 1 9 - D I N O R A B I E T A - 8 , l l , 1 3 - T R I E N - 4 - O N E (152) :"0 A^-Butyllithium (1.6 M in hexanes, 41.5 mL, 66.4 mmol) was added to a stirred solution of 4-methyl-2,6-di-/-butylphenol (14.7 g, 66.6 mmol) in anhydrous T H F (348 mL) at 0-5° C under argon. Carbon disulphide (13.4 mL, 0.22 mmol) was added, and the resulting light yellow solution was allowed to warm to room temperature. A solution of the ketone 151 (6.6 g, 25.7 mmol) in dry T H F (52 mL) was added, and the flask that contained the ketone solution was washed wi th 18 mL dry T H F and this washing was added to the reaction mixture. S t i r r ing was continued at room temperature for 48 h. Ana ly t i ca l T L C of a sample (s.s. hexanes:ethyl acetate 9:1) showed no remaining starting material. Methyl iodide (8.94 mL, 142.01 mmol) was then added and the reaction mixture was stirred (wrapped in aluminum foil) for a further 20 h. The solvent was evaporated and the residue dissolved in ether (910 mL) , washed with water (3 x 280 mL) and brine (280 mL) , dr ied, filtered, and concentrated in vacuo. The orange residue (26.3 g) was purif ied by gravi ty column chromatography (263 g of s i l ica gel) using 1.3 L of hexanes as eluent, to recover the phenol and followed by hexanes:ethyl acetate 9:1 (2.1 L ) to yie ld 8.44 g (91.37%) of the ketone thioketal as an orange oi l which solidifies upon cooling. M p = 68-70° C ( i -PrOH). IR ( K B r ) j/jnax: 2956 (CH), 1714, 1680 (C=0). ' H nmr S: 1.1 (3H, s, CIO CH3), 1.23 (6H, d, CH(CH3)2) , 1.76-3.42 (m, aliphatic, H) , 2.37 (3H, s, CH3 -S) , 2.39 (3H, s, CH3 -S) , 6.95 ( I H , brs, H14), 7.04 ( I H , brd, H12), 7.22 ( I H , d, H l l ) . U V (methanol) A^ax (log «): 211 (4.23), 314 (3.81). MS m/z: 360 (M+, 15.7), 345 (27.1), 313 (10.3), 256 (24.7), 253 (71.1), 241 (62.4), 220 (20.8), 213 (19.0), 205 (81.8), 141 (22.5), 1129 (29.3), 115 (22.1), 91 (100). H R M S calc. for C21H28OS2: 360.1583, found: 360.1584. Ana l , calcd. for C21H28OS2: C 69.99, H 7.77, S 17.79; found: C 70.19, H 7.87, S 17.94. 1 9 - H Y D R O X Y - 1 8 ( 4 -* 3)ABE0ABIETA-3,SA1,12-TETRAEN-1S-01C A C I D L A C T O N E (132): .110 O, iV-butyll i thium (1.6 M in hexanes, 5.3 m L , 8.48 mmol) was added to a stirred suspension of trimethyl sulphonium iodide (1.92 g, 9.39 mmol) in dry T H F (40 mL) at -70° C under argon, and the reaction mixture was allowed to warm to -10° C over 30 min. The reaction mixture was cooled again to -70° C and a solution of the ketene thioketal 152 (2.26 g, 6.26 mmol) in T H F (12 mL) was added slowly via canule. Some precipitate appeared. The flask that contained the thioketal was washed with 1.0 mL dry T H F and the washing was added to the reaction flask. The mixture was stirred at -70° C for 30 min, and then allowed to slowly reach room temperature (in approximately 45 min). After st irring at room temperature for 2 h, control T L C (s.s. hexanesrethyl acetate 9:1) showed no remaining starting material. The solvent was evaporated at room temperture, and the residue was dissolved in ethyl ether (160 mL) and washed with water (2 x 20 mL). Removal of the solvent left a residue which was dissolved in acetonitrile (13.5 mL) and the methanol (40 mL) , and the solution was cooled in an ice bath. Concentrated H C l (4.8 mL) was added while stirring and the cooling bath was removed immediately after the addition. The reaction mixture was stirred at room temperature for 40 h, and the methanol and acetonitrile were evaporated. The residual suspension was extracted into ether (160 mL) and the organic solution was washed with saturated sodium bicarbonate (3 x 20 mL) and water (20 mL), dried, filtered, and rotary evaporated to afford an orangey-brown residue which was purif ied by gravity column chromatograph (100.0 g of silica) using 1 L of hexanes:ethyl acetate 8:2 as eluent to give 1.374 g (73.9%) of the pure lactone 132 as an oi l which slowly solidifies to give a white solid. Mp = 97-99° C (EtOH). IR (KBr ) Umax- 2959 (CH), 1757 (C=0), 1678 (C=C). ' H nmr S: 1.02 (3H, s, CIO CH3), 1.24 (6H, d, CH(CH3)2), 1.71-2.60 (m, aliphatic), 2.73 ( IH , m, H5), 2.86 ( I H , septet, H15), 3.02 (2H, m, H7), 4.78 (2H, m, H19), 6.99 ( I H , brs, H14), 7.06 ( I H , brd, H12), 7.27 ( I H , d, H l l ) . U V (methanol) Amax (log «): 221 (4.00). MS m/z: 296 (M+, 28.8), 281 (100). H R M S calcd. for C20H24O2: 296.1776; found: 296.1776. A n a l , calcd. for C20H24O2: C 81.05, H 8.15; found: C 81.06, H 8.20. 1 2 - A C E T Y L - 1 9 - H Y D R O X Y - 1 8 ( 4 - 3 M 5 £ 0 ^ 5 / £ 7 ' / < - 3 , 8 , l l , 1 3 - T E T R A E N - 1 8 - O I C A C I D L A C T O N E (192): Anhydrous aluminum trichloride (2.93 g, 22 mmol) was suspended in carbon disulphide (150 mL) , and a solution of the lactone (2.07 g, 6.9 mmol) and acetyl chloride (2.34 mL, 33 mmol) in carbon disulphide (40 mL) was added under efficient stirring. A brown precipitate appeared and the mixture was refluxed under argon with good st i rr ing overnight. The carbon disulphide was evaporated at room temperature and iced H C l (112 mL) (3:10) was added to the residue. The mixture was stirred for 15 min in the ice-water bath and then 30 min at room temperature. Af ter al l the complex had been destroyed, the product was extracted into ethyl ether (225 mL), washed wi th water (2 x 75 mL), saturated sodium bicarbonate solution (3 x 75 mL), more water (75 mL), and f inal ly with brine (75 mL) . The organic solution was dried, fi l tered, and rotary evaporated to yield the crude product which was crystallized from methanol (30 mL) and vacuum dried at 50° C for 1 h to yield 1.61 g (68.36%) of the pure product as white crystals. Mp = 161-163° C (MeOH). IR ( K B r ) i/max: 2952 (CH), 1751 (C=0), 1669 (C=0). ' H nmr S: 1.05 (3H, s, CIO CH3), 1.23 (6H, dd, CH(CH3)2), 1.70-2.63 (m, aliphatic), 2.58 (3H, s, CH3C=0) , 2.73 ( IH , m, H5), 3.07 (2H, brt, H7), 3.48 ( I H , septet, H15), 4.80 (2H, m, H19), 7.15 ( I H , s, H14), 7.47 ( I H , s, H l l ) . U V (methanol) Amax (log e): 253 (3.89), 217 (4.39). MS m/z: 338 (M+, 35.4), 323 (100). H R M S calcd. for C22H26O3: 338.1882; found: 338.1882. Ana l , calcd. for C22H26O3: C 78.08, H 7.73; found: C 78.14, H 7.60. 1 2 - A C E T O X Y - 1 9 - H Y D R O X Y - 1 8 ( 4 -* 3)ABEOABIETA-3,S,n,13-TEtRAEfi-lS-OlC A C I D L A C T O N E (193): To a solution of the acetyl lactone 192 (250 mg, 0.735 mmol) in dichloromethane (1.5 mL) was added m-chloroperoxybenzoic acid (330 mg, 2.6 equiv.) and the resulting suspension was cooled to -5-0° C (ice-methanol bath) under argon. Trif luoroacetic acid (60 fiL) was added as catalyst, and the mixture was allowed to reach room temperature protected from light, and stirred overnight, unt i l analytical T L C showed reaction completion (s.s. i-propyl ether, developed twice). The reaction took usually 20 h to completion, then it was diluted wi th ethyl acetate (10 mL) and washed once each with 10% Na2S03 (1.5 mL), saturated K H C O 3 (1.5 mL) and brine (1.5 mL), the organic layer was dried, filtered and concentrated in vacuo to yie ld the essentially pure compound as a white foam (261.3 mg, 99.8%). The ester can be used as is or purif ied by flash column chromatography (1.5 cm diameter, 5.5" s i l ica gel) eluting with 200 mL of di isopropyl ether and collecting 5-mL fractions to y ie ld 188.2 mg (71.8%) of the pure acetoxy lactone. IR (neat): 2950 (CH), 1750 (C=0), 1680 (C=0). ' H nmr S: 1.04 (3H, s, CIO CH3), 1.21 (6H, dd, CH(CH3)2) , 1.70-2.57 (m, aliphatic), 2.33 (3H, s, OAc) , 2.73 ( I H , brm, H5), 2.91-3.07 (3H, m, H7, H15), 4.80 (2H, brd, H19), 6.96 ( I H , s, H14), 7.06 ( I H , s, H l l ) . U V (methanol) Ajnax ('og 0: 218 (4.21). MS m/z: 354 (M+, 2.8), 312 OAc (36), 297 (25.6), 149 (31.9), 110 (100). HRMS calcd. for C22H26O4: 354.1831; found: 354.1830. 12,19-DIHYDROXY-18(4 ^ 3)/45£C>^5/£r/l-3,8,ll ,13-TETRAEN-18-OIC ACID LACTONE (190): To a solution of the acetoxy lactone 193 (159.6 mg, 0.45 mmol) in methanol (10 mL), was added 1 mL of concentrated H C l at room temperature. The mixture was stirred at room temperature unt i l T L C showed reaction completion (i-propyl ether developed twice, approximately 5 h), the reaction mixture was then diluted with water (10 mL) and the bulk of methanol was removed in vacuo. The resulting suspension was extracted with ethyl acetate (3 x 10 mL) and the combined organic extracts were washed once with water (10 mL), 5% sodium bicarbonate (10 mL), water (10 mL), and brine (10 mL) , dried, f i l tered, and rotary evaporated. The resulting solid (156.4 mg) was crystall ized from acetone:water (1.5:1 mixture, charcoal treatment included) to yield 133.7 mg (95%) of the pure product as off-white crystals. Mp = 194-197° C (decomposition). IR ( K B r ) u^ax- 3320 (OH), 2965 (CH), 1748 (C=0). ' H nmr S: 1.00 (3H, s, CIO CH3), 1.23 (6H, dd, CH(CH3)2). 1.70 ( I H , m, H I ) , 1.90 (2H, m, H6), 2.32-2.59 (3H, m, H I , H2), 2.72 ( I H , brm, H5), 2.97 (2H, m, H7), 3.16 ( I H , septet, H15), 4.67 ( I H , brs, D2O+, OH) , 4.78 (2H, brd, H19), 6.72 ( I H , s, H14), 6.91 ( I H , s, H l l ) . U V (methanol) Xj^ax (log 0 : 283 (3.57). MS m/z: 312 (M"*", 100), 297 (53.3), 149 (42.8), 115 (14.7), 91 (15). H R M S calcd. for C20H24O3: 312.1726; found: 312.1724. Anal , calcd. for C20H24O3: C 76.9, H 7.73; found: C 77.0, H 7.83. D E H Y D R O I S O A B I E T A N O L I D E B I O T R A N S F O R M A T I O N : T R F 4 a C E L L F R E E E X T R A C T E X P E R I M E N T S , A N D C U L T U R E C H A R A C T E R I Z A T I O N . T. wilfordii cell suspension cultures were grown in P R D C o or M S N A K media for the required period. The cells were harvested f i l ter ing through Mira-cloth. The R.I. and p H of the filtrate were measured and a sample of the cells was taken for dry weight determination after weighing the wet cell mass. The cells were rinsed wi th distilled water (500 mL) and taken into the cold room where the remainder of the preparation was done (T = 0-4° C). Phosphate buffer (0.02 M , pH = 6.6, 180 mL) was added to the cells in a plastic container and the mixture was homogenized with an Ul t ra Turrax T-25 disperser for 30 sec, then allowed to cool for another 30 sec. The homogenization was repeated three times and the homogenate was transferred to plastic centrifuge flasks and centrifuged at 10,000 g for 30 min (T = 4° C). The supernatant was decanted and peroxidase activity (pyrogallol) and protein content (Bio Rad) were determined. For each cell age, a minimum of 3 Erlenmeyer flasks were set mixing phosphate buffer (pH = 6.6, 35 mL), lactone 132 (10 mg dissolved in 2 mL ethanol), disti l led water (15 mL) , 0.5% hydrogen peroxide (1 mL, 2.6 equiv.) and the equivalent of 22 peroxidase units of C F E . The flasks were stirred at room temperature for the required period of time (usually 30 min, 2 h, and overnight), and quenched adding 25 mL of ethyl acetate. The mixture was quickly filtered through a short pad of Celite 545 in a fritted glass fil ter funnel under vacuum and the Celite was washed with ethyl acetate (25 mL). The filtrate was extracted with ethyl acetate (2 x 25 mL) while the Celite was sonicated in ethyl acetate (20 mL) for 20 min, filtered, and the filtrate was mixed with the organic extracts, dried, fi l tered, and concentrated in vacuo. H P L C analysis was done on each sample (reverse phase, C j g cartridge, methanokwater 75:25 with 0.1% acetic acid) and T L C in toluene:ethyl acetate 4:1 and chloroform:methanol:acetic acid 95:5:1. The lactone was recovered by flash column chromatography of the crude extracts eluting with an 8:2 mixture of hexanes:ethyl acetate, yielding typically 93-97% recovered product. M E A S U R E M E N T O F P R O T E I N C O N C E N T R A T I O N : B I O - R A D P R O T E I N A S S A Y One part of the dye reagent (Bio-Rad Protein Assay Dye Reagent Concentrate) was diluted with four parts of disti l led water. The diluted dye reagent solution (5 mL) was then added to a test tube containing C F E (0.1 mL) and the solution was mixed thoroughly. After using a reference sample (prepared by mixing phosphate buffer (0.1 mL, 0.1 M , p H 6.6) and the diluted dye solution (5 mL) to adjust the reading of the U V spectrometer at 595 nm to zero, the absorbance of the C F E was then measured at the same wavelength. The protein concentration can be calculated from the standard curve which was produced by dissolving known amounts of bovine serum albumin (BSA) powder in the same buffer to produce a set of standard solutions (0.1 mg/mL to 0.01 mg/mL), adding aliquots (0.1 mL) of these solutions to the diluted dye (5 mL) and measuring absorbances at 595 nm. M E A S U R E M E N T O F P E R O X I D A S E A C T I V I T Y : P Y R O G A L L O L - P U R P U R O G A L L I N A S S A Y . C F E (1 mL) was added to a 50 mL Erlenmeyer flask containing 5% aqueous pyrogallol solution (2 mL) , 0.1 M phosphate buffer (2 mL, p H 6.6), freshly-prepared 0.5% hydrogen peroxide solution (1 mL) and distilled water (14 mL) at 20° C. This mixture was allowed to stand for 20 seconds at 20° C, then 2 M sulphuric acid (I mL) was added to quench the reaction and the solution was then extracted with ether (2 x 25 mL). After the reading of the U V spectrometer was adjusted to zero to 420 nm by a reference sample which was an ether extract (2 x 25 mL) from a mixture of 5% pyrogallol solution (2 mL), 0.1 M phosphate buffer (3 mL, pH 6.6), freshly prepared 0.5% hydrogen peroxide solution (1 mL) and distilled water (14 mL) , the absorbance of the organic extract from the C F E reaction was then recorded at the same wavelenth. The standard curve can be obtained by measuring absorbance at 420 nm of a set of standard solutions prepared by dissolving purpurogallin (0.5 to 3.5 mg) in ether (50 ml). D E H Y D R O I S O A B I E T A N O L I D E B I O T R A N S F O R M A T I O N : A S S E S S M E N T O F M E M B R A N E - B O U N D E N Z Y M E S A N D C O F A C T O R S . T. wilfordii cells were grown in P R D C o medium for 14 days and the cells were harvested and homogenized in phosphate buffer (pH = 6.6). One half of the cell homogenate was set aside for biotransformation experiments and the other half was centrifuged at 10,000 g. The supernatant (CFE) was decanted and the pellet was resuspended in pH 6.6 buffer. The following experiments were done: a) Cell homogenate. (3.86 units of peroxidase/mL, 0.804 mg of protein/mL). In two 500 mL Erlenmeyer flasks were mixed distilled water (75 mL), phosphate buffer (pH 6.6, 175 mL), hydrogen peroxide solution (0.5%, 5 mL), lactone 132 (50 mg in 10 mL ethanol) and the equivalent amount of 125 units of peroxidase of cell homogenate. The flasks were incubated for 3 and 24 h respectively, and quenched by the addition of ethyl acetate (125 mL). Sodium chloride was added to saturation and the mixture was filtered using some Celite 545 as a filter aid. The filtrate was extracted with ethyl acetate (2 x 100 mL) and the Celite and cell debris were extracted by sonication in ethyl acetate (100 mL) for 30 min. The mixed organic extracts were dried, filtered, and concentrated in vacuo. b) Resuspended pellet. (2.5 units of peroxidase/mL, 0.80 mg protein/mL). In two 500-mL Erlenmeyer flasks were mixed distilled water (75 mL), phosphate buffer (pH 6.6, 175 mL), hydrogen peroxide solution (0.5%, 5 mL, 2.5 equiv.), lactone 132 (50 mg in 10 mL of ethanol), and the equivalent amount of 275 peroxidase units of resuspended pellet. The flasks were incubated for 24 and 48 h, and worked up as with the cell homogenate. c) Ce l l free extract plus cofactors. (4.72 peroxidase uni ts /mL, 0.636 mg protein/mL). In two 500-mL Erlenmeyer flasks were mixed dist i l led water (75 mL), phosphate buffer (pH 6.6, 175 mL) , hydrogen peroxide solution (0.5%, 2.5 equiv., 5 mL), lactone 132 (50 mg in 10 ml ethanol), manganese chloride tetrahydrate (16.6 mg, 0.5 equiv.). Flavine mononucleotide monohydrate (50.8 mg, 0.5 equiv.) and the equivalent of 125 peroxidase units of cell free extract. The flasks were incubated for 3 and 24 h, and worked up as before. The crude extracts were analyzed by H P L C and the lactone recovered by flash column chromatography in hexanes:ethyl acetate 8:2. D E H Y D R O I S O A B I E T A N O L I D E B I O T R A N S F O R M A T I O N : T R P 4 a W H O L E C E L L E X P E R I M E N T S . T. wilfordii cells were grown in M S N A K shake flasks for 0, 7 and 10 days. Lactone 132 (8.8 mg in 1 mL ethanol) was added to the cultures and the flasks were incubated for 48, 72 and 92 h. A control flask without precursor was also run for every experiment. One flask containing the lactone 132 (12.0 mg in 1 mL ethanol) in MSNA0.5K0.5 medium (125 mL) was also incubated without cells for 48 h. The samples were harvested homogenizing the cells and broth with an Ul t ra Turrax T-25 disperser and f i l ter ing through Celite. The filtrate was saturated with sodium chloride and extracted with ethyl acetate (2 x 100 mL). The cell debris was sonicated with ethyl acetate (50 mL) for 30 min, filtered through Celite, and the Celite was washed with ethyl acetate. The organic layers (from the spent medium and the cell debris) were combined, dried, filtered, and the solvent was evaporated. T L C analysis of the extracts (hexane:ethyl acetate 6:4) showed that oxidation had occurred, with the C7 alcohol 175 and C7 ketone 176 being the main products. Their concentration increased in the longer time/older cell experiments. The blank experiment had only unchanged lactone 132 after incubation on M S N A K medium. D E H Y D R O I S O A B I E T A N O L I D E B I O T R A N S F O R M A T I O N : MORTIERELLA ISABELLINA E X P E R I M E N T S . A culture of M. isabellina was grown in a Roux bottle with 15 mL of P D A at room temperature for 10 days. Inoculum was prepared by washing the surface of this culture with sterile D Y E medium (10 mL). The spore suspension was diluted to 25 m L with D Y E medium. The resulting suspension contained approximately 9.84 x 10 spores per m L (as determined using a Howard Mold counting chamber) and was inoculated into 550 mL of D Y E medium (i.e. a volumetric ratio of 0.45:10) so as to give 1.96 X 10^ spores per mL. 50 mL of this mixture were transferred into each of 11 500-mL Erlenmeyer flasks. A blank flask contained only medium and lactone but no fungus. A l l cultures used for the biotransformation contained 5.0 mg of lactone 132 dissolved in 0.5 mL ethanol. The cultures were incubated at 30° C and 200 rpm in a rotary shaker for 48 h. Microscopic inspection of the cultures showed that the fungus was sti l l on the lag phase (only some spores had germinated). The flasks were harvested combining the filtrates (after Whatman No. 1 paper) and rinsing well both flasks and spores with ethyl acetate in 25 mL portions (275 m L total). The broth was extracted with ethyl acetate (2 x 275 mL), the organic fractions were combined, washed with brine (250 mL), dried, filtered, and concentrated in vacuo, to yield 94.0 mg of a yellow thick o i l . T L C analysis of the extract showed consumption of the starting material and the appearance of the C7 alcohol 175. The crude was purif ied by flash column chromatography (1.5 cm diameter column, 5.5" silica gel) eluting with hexanesiethyl acetate 6:4 (250 mL) and collecting 5-mL fractions. After this, the column was flushed with ethyl acetate (100 mL). The product was a sticky o i l (23.4 mg, 37%). Spectroscopic data was identical wi th the reported values of M . R o b e r t s " ^ . I S O T R I P T O P H E N O L I D E B I O T R A N S F O R M A T I O N W I T H T. WILFORDII C E L L S . T. wilfordii cells were grown in MSNAQ 5K0.5 medium in shake flasks for 7 days. A solution of isotriptophenolide (190) (50 mg) in ethanol (2 mL) was added to 1 L of the suspension culture in 500 mL batches. A control experiment was conducted in which only ethanol was incubated wi th 500 mL of culture. The flasks were incubated unt i l T L C analysis showed partial consumption of the precursor (7 days) to avoid overoxidation and were harvested (pH = 4.7, R.I. = 1.3352). The cells and spent medium were homogenized and extracted with ethyl acetate as in the other experiments {vide supra) to yield a brown solid extract (448.6 mg for the experiment and 279.2 mg for the control). The crude extract was pur i f ied by column chromatography (4.5 cm diameter, 6" sil ica gel) with the fol lowing eluants: hexanesrethyl acetate 8:2 (1 L ) , hexanes:ethyl acetate 6:4 (1 L ) , hexanes:ethyl acetate 2:8 (500 mL) and ethyl acetate:methanol 1:1 (500 mL). The fractions that contained the recovered precursor and the new metabolite were f ina l ly purif ied by preparative thin layer chromatography eluting twice with hexanes:ethyl acetate 7:3 to yield 38.0 mg (76%) of recovered isotriptophenolide and 4.3 mg (8.23%) of the methyl ether 194. I R ( C H C l 3 ) ./max i^^'^Y 2944 (CH), 1748 (C=0). ' H nmr 5: 1.60 (3H, s, CIO CH3), 1.21 (6H, dd, CH(CH3)2), 1.76 ( I H , m, HI ) , 1.92 (2H, m, H6), 2.42 ( I H , m, HI ) , 2.52 (2H, m, H2), 2.35 ( I H , brm, H5), 2.97 (2H, m, H7), 3.26 ( IH , septet, H15), 3.84 (3H, s, OCH3), 4.78 (2H, brd, C19), 6.81 ( I H , s, H14), 6.93 ( I H , s, H l l ) . MS m/z: 326 (M+, 80.6), 311 (100), 163 (73.4). 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Chemistry
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Studies on plant cell cultures of Podophyllum Peltatum and Tripterygium Wilfordii for biosynthesis of biologically active compounds
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