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Photosensitizing thiophenes from the Tageteae Downum, Kelsey 1981

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PHOTOSENSITIZING THIOPHENES FROM THE "TAGETEAE by KELSEY DOWNUM B.S., Colorado State University, Fort C o l l i n s , Colorado, 1975 M.S., Colorado State University, Fort C o l l i n s , Colorado, 1978 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSHOPHY in THE FACULTY OF GRADUATE STUDIES (Department of Botany) We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA 13 August 1981 c) Kelsey Downum, 1981 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e a n d s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e h e a d o f my d e p a r t m e n t o r by h i s o r h e r r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p a r t m e n t o f The U n i v e r s i t y o f B r i t i s h C o l u m b i a 2075 W e s b r o o k P l a c e V a n c o u v e r , C a n a d a V6T 1W5 Date /Q C4^^,^T/9R/ i i ABSTRACT Two s e p a r a t e a s p e c t s i n v o l v i n g t h e t h i o p h e n e s o f t h e T a g e t e a e ( A s t e r a c e a e ) were i n v e s t i g a t e d . The f i r s t c o n c e r n e d t h e d i s t r i b u t i o n of f o u r t h i o p h e n e s i n T a g e t e s p a t u l a L. w h i c h were examined by h i g h p r e s s u r e l i q u i d c h r o m a t o g r a p h y (HPLC). The d e r i v a t i v e s were f o u n d t o be d i f f e r e n t i a l l y d i s t r i b u t e d t h r o u g h o u t h y d r o p o n i c a l l y grown p l a n t s . The p r e d o m i n a n t t h i o p h e n e s i n r o o t s were 5 - ( 4 - a c e t o x y - 1 - b u t e n y l ) - 2 , 2 ' - b i t h i e n y l (BBT-OAc) and 5 - ( b u t e n - 3 - y n y l ) - 2 , 2 ' - b i t h i e n y l ( B B T ) . BBT-OAc was t h e main d e r i v a t i v e i n s h o o t s , whereas 2,2':5',2''-t e r t h i e n y l ( a l p h a - T ) was t h e major compound i n f l o w e r p e t a l s . BBT and one u n i d e n t i f i e d compound were f o u n d t o o c c u r i n l e a f g l a n d s . The l e v e l s o f BBT-OAc i n s h o o t s and BBT-OAc and BBT i n r o o t s i n c r e a s e d o v e r t h e l i f e of t h e p l a n t and r e a c h e d a p l a t e a u f o l l o w i n g f l o w e r i n g . A l p h a - T i n r o o t s and b o t h a l p h a -T and BBT i n s h o o t s r e m a i n e d a t low l e v e l s o v e r t h e l i f e of t h e p l a n t w h i l e 5 - ( 4 - h y d r o x y - 1 - b u t e n y l ) - 2 , 2 ' - b i t h i e n y l (BBT-OH) was f o u n d t o be an minor component of r o o t s o r s h o o t s . F o u r t e e n s p e c i e s f r o m f o u r g e n e r a of t h e t r i b e T a g e t e a e were a l s o s c r e e n e d f o r t h e p r e s e n c e o f t h i o p h e n e s by HPLC. R e p r e s e n t a t i v e s of D y s s o d i a , P o r o p h y l l u m , and T a g e t e s a l l c o n t a i n e d t h i o p h e n e s , b ut none were d e t e c t e d i n s p e c i e s o f P e c t i s . The s e c o n d p a r t o f t h i s s t u d y c o n c e r n e d t h e p h o t o b i o c i d a l e f f e c t s of i s o l a t e d t h i o p h e n e s on E s c h e r i c h i a c o l i B w h i c h was u s e d a s a model b i o l o g i c a l s y s t e m . A l p h a - t e r t h i e n y l ( a l p h a -T ) , i n t h e p r e s e n c e o f UV-A i r r a d i a t i o n (320nm-400nm), was found to be a Type II photosensitizer which required oxygen for the expression of b i o l o g i c a l a c t i v i t y . Scavenger studies with sodium azide and BHT suggested that both singlet oxygen and superoxide were generated by the photosensitized reaction. C e l l u l a r inactivation by alpha-T was sensitive to temperature and studies with recombination d e f i c i e n t mutants of E. c o l i K-12 did not indicate that damage to c e l l u l a r DNA occurred. Proteins were found to be substantially affected by the photoactivated reaction. SDS-gel electrophoresis revealed that both cytoplasmic and membrane-associated proteins might be. crosslinked following treatment with alpha-T and UV-A. iv TABLE OF CONTENTS ABSTRACT 1 LIST OF TABLES 1 LIST OF FIGURES ! ACKNOWLEDGEMENTS 1 PREFACE 1 SECTION I. PHYTOCHEMICAL ASPECTS OF THIOPHENES 2 INTRODUCTION ; .---3 MATERIALS AND METHODS 9 Chemicals 9 Plant Material 10 Dried Plant Specimens 10 Hydroponics 11 Extraction of Thiophenes 12 Id e n t i f i c a t i o n of Thiophenes 13 Chromatography 14 TLC 14 Column Chromatography . 16 High Pressure Liquid Chromatography 16 Qualitative HPLC Studies 17 Quantitative HPLC Studies 17 Phototoxicity of Thiophene Standards 19 RESULTS AND DISCUSSION ' 19 Structural V e r i f i c a t i o n of Standards 21 Chromatography 21 Qualitative Analysis of Thiophenes 25 Quantification of Thiophenes 29 Dis t r i b u t i o n of Thiophenes in the Tageteae 38 Bi o l o g i c a l A c t i v i t y of Thiophene Standards 41 CONCLUSION 42 SECTION I I . PHOTOBIOLOGICAL ASPECTS OF THIOPHENES 44 INTRODUCTION 45 MATERIALS AND METHODS 50 Irradi a t i o n Sources 50 Survival Curves 51 Bacte r i a l Preparation 54 Assay Procedure 54 Alpha-Terthienyl Action Spectrum 55 Rec Mutant Assay 57 Scavenger Studies 57 Temperature Studies 58 Crosslinking of E. c o l i Proteins 59 SDS-Polyacrylamide Gel Electrophoresis 60 RESULTS AND DISCUSSION 61 Survival Curves 61 Action Spectrum 65 Studies on Repair Deficient Mutants of E. c o l i 67 Aerobic-Anaerobic Studies 69 Effect of Scavengers on Alpha-T Phototoxicity 74 Temperature Studies 78 Effe c t s of Alpha-T on C e l l u l a r Proteins 84 CONCLUSION 88' v i APPENDIX I 90 LITERATURE CITED 92 v i i L I S T OF TABLES T a b l e 1. O c c u r r e n c e o f t h i o p h e n e s i n t h e A s t e r a c e a e 5 T a b l e 2. C o m p o s i t i o n of h y d r o p o n i c g r o w t h medium 12 T a b l e 3. T h i n l a y e r c h r o m a t o g r a p h i c d a t a o f t h e t h i o p h e n e s . 23 T a b l e 4. H i g h p r e s s u r e l i q u i d c h r o m a t o g r a p h i c d a t a of t h i o p h e n e d e r i v a t i v e s 24 T a b l e 5. T h i o p h e n e c o n c e n t r a t i o n s i n c r u d e T. p a t u l a e x t r a c t s 36 T a b l e 6. D i s t r i b u t i o n of t h i o p h e n e s w i t h i n t h e t r i b e T a g e t e a e 39 T a b l e 7. P h o t o t o x i c i t y of t h i o p h e n e s t o m i c r o o r g a n i s m s ... 41 T a b l e 8. D o s i m e t r i c p a r a m e t e r s u s e d t o d e t e r m i n e t h e a c t i o n s p e c t r u m o f a l p h a - T 53 T a b l e 9. S t r a i n s o f E. c o l i K-12 m u t a n t s and t h e i r r e l e v a n t c h a r a c t e r i s t i c s 58 T a b l e 10. Resp o n s e of v a r i o u s E. c o l i r e c m u t a n t s t o a l p h a - T , BBT, b l e o m y c i n and 8-MOP 68 t v i i a LIST OF FIGURES Figure 1. The thiophenes isolated and i d e n t i f i e d from Tagetes patula L 4 Figure 2. A proposed biosynthetic pathway for the formation of thiophenes from the precursor, the "penta-yne-ene" 6 Figure 3. Chemical structures of the thiophenes used in these studies 20 Figure 4. HPLC trace of the UV absorbing components from the roots of 8 week old hydroponically grown T. patula. 26 Figure 5. HPLC trace of the UV absorbing components from the shoots of 8 week old hydroponically grown T. patula. 27 Figure 7. HPLC trace of the UV absorbing components from the leaf glands of 8 week old hydroponically grown T. patula 27 Figure 6. HPLC trace of the UV absorbing components from flowers of 8 week old hydroponically grown T. patula. . 28 Figure 8. HPLC standard concentration curve for BBT-OH (VII) 31 Figure 9. HPLC standard concentration curve for BBT-OAc (VIII) 32 Figure 10. HPLC standard concentration curve for BBT (IX). F i g u r e 11. HPLC s t a n d a r d c o n c e n t r a t i o n c u r v e f o r a l p h a - T (X) . .. 34 F i g u r e 12. The a c c u m u l a t i o n o f t h i o p h e n e s i n r o o t s o v e r t h e l i f e o f h y d r o p o n i c a l l y grown T. p a t u l a 35 F i g u r e 13. The a c c u m u l a t i o n o f t h i o p h e n e s i n s h o o t s o v e r t h e l i f e o f h y d r o p o n i c a l l y grown T. p a t u l a 37 F i g u r e 14. I r r a d i a n c e (mW/cm2) v e r s u s w a v e l e n g t h f o r t h e f l u o r e s c e n t s o u r c e s u s e d i n t h e s e s t u d i e s 52 F i g u r e 15. S u r v i v a l c u r v e s of E. c o l i i n r e s p o n s e t o v a r i o u s d o s e s of a l p h a - T and UV-A i r r a d i a t i o n 62 F i g u r e 16. S u r v i v a l c u r v e s o f E . c o l i i n r e s p o n s e t o v a r i o u s d o s e s of BBT and UV-A i r r a d i a t i o n 64 F i g u r e 17. A c t i o n s p e c t r u m o f t h e b a c t e r i c i d a l a c t i v i t y of a l p h a - T v e r s u s t h e a b s o r p t i o n f o r a l p h a - T between 320nm and 400nm 66 F i g u r e 18. Response of E. c o l i B t o a l p h a - T and UV-A i r r a d i a t i o n under a e r o b i c c o n d i t i o n s 71 F i g u r e 19. Response of E. c o l i B t o a l p h a - T and UV-A i r r a d i a t i o n under a n a e r o b i c c o n d i t i o n s 72 F i g u r e 20. Response of Pseudomonas a e r u g i n o s a t o a l p h a - T and UV-A i r r a d i a t i o n u n d e r a e r o b i c and a n a e r o b i c c o n d i t i o n s 73 F i g u r e 21. S u r v i v a l o f E. c o l i B i n t h e p r e s e n c e of a l p h a -T and UV-A i r r a d i a t i o n w i t h 20 mM sodium a z i d e 75 F i g u r e 22. S u r v i v a l o f E. c o l i B i n t h e p r e s e n c e of a l p h a -T and UV-A i r r a d i a t i o n w i t h 50 uM BHT 77 F i g u r e 23. S u r v i v a l o f E. c o l i B i n t h e p r e s e n c e of a l p h a -X T and UV-A i r r a d i a t i o n with 20 mM sodium azide and 50 uM BHT 79 Figure 24. Survival of E. c o l i B in the presence of alpha-T and UV-A i r r a d i a t i o n at various i r r a d i a t i o n temperatures .. 80 Figure 25. Arrhenius plot of the data presented in Figure 24 82 Figure 26. The effect of incubation temperature on the survival of E. c o l i in the presence of alpha-T and UV-A i r r a d i a t i o n 85 Figure 27. SDS-gel electropherogram of the proteins of E. c o l i B following treatment with alpha-T and UV-A i r r a d i a t i o n . 86 x i ACKNOWLEDGEMENTS I w i s h t o thank t h e members of my r e s e a r c h c o m m i t t e e , D r s . A.D.M. G l a s s , P . J . H a r r i s o n , R.E.W. Hancock and G.H.N. Towers f o r t h e i r h e l p f u l s u g g e s t i o n s t h r o u g h o u t my r e s e a r c h a nd f o r t h e i r c a r e f u l r e a d i n g and c r i t i c i s m of t h i s m a n u s c r i p t . S p e c i a l t h a n k s , t o D r . R.E.W. Hancock f o r h i s g e n e r o u s a s s i s t a n c e , g u i d a n c e , i n t e r e s t , and e n t h u s i a s m and t o t h e p e o p l e o f h i s l a b o r a t o r y whose k i n d h e l p , a c c e p t a n c e , and u n d e r s t a n d i n g made my work t h e r e e n j o y a b l e . I am e s p e c i a l l y g r a t e f u l — t o - — D r . G.H.N. Towers f o r my i n t r o d u c t i o n i n t o t h e f i e l d o f p h o t o b i o l o g y a nd f o r h i s a d v i c e , e n c o u r a g e m e n t , and c o n t i n u e d s u p p o r t o v e r my t i m e i n h i s l a b o r a t o r y . I would a l s o l i k e t o thank D r s . T. A r n a s o n , E . Camm, F. G a r c i a , M. T e p f e r , and R. S u e t f e l d f o r many h e l p f u l s u g g e s t i o n s and d i s c u s s i o n s and D r s . G a r c i a and S u e t f e l d f o r p r o v i d i n g r e f e r e n c e compounds. Thank s t o Ms. Z. Abramowski and Ms. E.A. Graham f o r t e c h n i c a l a s s i s t a n c e a nd t o D r . D a v i d Z i t t i n i n t h e B i o s c i e n c e s D a t a C e n t e r f o r a s s i s t a n c e i n p r e p a r i n g t h i s m a n u s c r i p t . I c o u l d n e v e r s a y enough a b o u t t h e p a t i e n c e , u n d e r s t a n d i n g and s u p p o r t w h i c h my w i f e , J u l i e , has o f f e r e d t h r o u g h i t a l l . Thank y o u . 1 PREFACE P l a n t s p r o d u c e many d i v e r s e c h e m i c a l c o n s t i t u e n t s w h i c h e l i c i t i n t e r e s t i n g b i o l o g i c a l a c t i v i t i e s i n i s o l a t e d o r p u r i f i e d f o r m . The t h i o p h e n e s a r e one s u c h g r o u p o f compounds s y n t h e s i z e d by members of t h e p l a n t f a m i l y A s t e r a c e a e and a r e c h a r a c t e r i s t i c o f t h e t r i b e T a g e t e a e . T h e s e d e r i v a t i v e s a r e t o x i c t o w a r d a wide v a r i e t y of b i o l o g i c a l o r g a n i s m s when e x c i t e d .by longwave u l t r a v i o l e t (UV-A) i r r a d i a t i o n . T h e i r f u n c t i o n w i t h i n t h e p l a n t i s s t i l l unknown and r e m a i n s u n s t u d i e d , but t h e i r p h o t o t o x i c p r o p e r t i e s s u g g e s t t h a t t h i o p h e n e s may p l a y a p r o t e c t i v e r o l e i_n s i t u . The work t o be p r e s e n t e d was a p r e l i m i n a r y i n v e s t i g a t i o n i n t o two s e p a r a t e a r e a s i n v o l v i n g t h i o p h e n e s . T h i o p h e n e p h y t o c h e m i s t r y was t h e f i r s t a r e a t o be s t u d i e d . The o c c u r r e n c e , d i s t r i b u t i o n and l e v e l s o f t h e s e b i o l o g i c a l l y a c t i v e compounds i n one p l a n t , T a g e t e s p a t u l a L. were e s t a b l i s h e d . The s e c o n d a r e a c o n c e r n e d t h e p h o t o t o x i c mechanism o f a c t i o n i n v o k e d p r i m a r i l y by one t h i o p h e n e , a l p h a - T . The u l t i m a t e g o a l of t h i s work was t o d e t e r m i n e b a s e l i n e i n f o r m a t i o n w h i c h m i g h t be u s e d f o r f u t u r e r e s e a r c h i n t o some o f t h e more i n t e r e s t i n g a s p e c t s o f t h e s e n a t u r a l p l a n t p r o d u c t s . What a r e t h e i r _in s i t u f u n c t i o n s ? A r e t h e y p r o t e c t i v e o r do t h e y have some o t h e r p h y s i o l o g i c a l o r m e t a b o l i c f u n c t i o n ? How do p l a n t s p r o t e c t t h e m s e l v e s f r o m t h e e f f e c t s o f t h e s e p h o t o a c t i v e d e r i v a t i v e s ? T h e s e a r e o n l y a few of t h e p o t e n t i a l r e s e a r c h a r e a s where t h i s d a t a c o u l d be u s e f u l . ' SECTION I . PHYTOCHEMICAL ASPECTS OF THIOPHENES 3 INTRODUCTION T h i o p h e n e s c o m p r i s e a d i s t i n c t c l a s s o f n a t u r a l p r o d u c t s w h i c h a r e c h a r a c t e r i s t i c o f t h e A s t e r a c e a e , t h e l a r g e s t f l o w e r i n g p l a n t f a m i l y . T h e s e s u l f u r c o n t a i n i n g compounds were i n i t i a l l y i s o l a t e d f r o m p e t a l s o f t h e " A f r i c a n " v a r i e t y o f t h e common m a r i g o l d ( T a g e t e s e r e c t a L.; T r i b e T a g e t e a e ) ( S e a s e and Z e c h m e i s t e r , 1947). A l p h a - t e r t h i e n y l ( a l p h a - T ) ( F i g u r e 1) was t h e f i r s t n a t u r a l t h i o p h e n e t o be i d e n t i f i e d ( Z e c h m e i s t e r and S e a s e , 1947; U h l e n b r o e k and B i j l o o , 1958). Ten y e a r s l a t e r a s e c o n d t h i o p h e n e , 5 - ( b u t - 3 - e n ) - 2 , 2 ' - b i t h i e n y l (BBT) was i s o l a t e d ( U h l e n b r o e k and B i j l o o , 1959). A d d i t i o n a l t h i o p h e n e d e r i v a t i v e s have been r e p o r t e d from t w e l v e s p e c i e s o f T a g e t e s (Bohlmann and H e r b s t , 1962; A t k i n s o n e_t a_l. , 1965; Bohlmann <et a l . , 1973; Bohlmann and Z d e r o , 1979) as w e l l as o t h e r g e n e r a i n t h e t r i b e T a g e t e a e i n c l u d i n g two s p e c i e s o f D y s s o d i a (Bohlmann and Z d e r o , 1976), and one s p e c i e s o f P o r o p h y l l u m (Bohlmann e t a l . , 1973). T h i o p h e n e s have been i s o l a t e d f r o m n i n e t r i b e s i n a d d i t i o n t o t h e T a g e t e a e , b u t t h e y have n o t been r e p o r t e d f r o m t h e A s t e r e a e , C a l e n d u l e a e , C i c h o r i e a e , o r M u t i s i e a e nor have t h e y been r e p o r t e d from any o t h e r h i g h e r p l a n t f a m i l y (Bohlmann e t a l . , 1 9 7 3 ) . T a b l e I shows t h e o c c u r r e n c e o f t h i o p h e n e s i n o t h e r t r i b e s i n t h e f a m i l y ( S o r e n s o n , 1977). The n a t u r a l t h i o p h e n e s p o s s e s s one, two o r t h r e e a r o m a t i c , f i v e - m e m b e r e d , s u l f u r c o n t a i n i n g r i n g s p e r m o l e c u l e ( F i g u r e 2) and a r e b i o s y n t h e t i c a l l y d e r i v e d from o l e i c a c i d (Bohlmann e t a l . , 1973). P o l y a c e t y l e n i c i n t e r m e d i a t e s , w h i c h a r e l o n g c h a i n m o l e c u l e s w i t h c o n j u g a t e d t r i p l e bonds (Bohlmann, 1973), a r e 4 EC-CH,-CHjOH S-fa-hydroxy-A-butenyl)-2,2-bithienyl [BBT-Oh] C£C-CH r CH f O-COCH, 5- (4-acefoxy- 1-bufeny/)-2,2-bfthienyl [BBT-OAc] ^ C S C - C H = C H , s s 5-(3-bt/fen- ^ynyl)-2,i-bithienyl {BBT) 2,2':5',2-f»rt/i/eny/ («T) F i g u r e 1. The t h i o p h e n e s which mere i s o l a t e d and i d e n t i f i e d from Taaetes p a t u l a L. 5 T a b l e 1 . The o c c u r r e n c e o f t h i o p h e n e s w i t h i n t h e A s t e r a c e a e ( a f t e r S o r e n s o n , 1977). M o n o t h i o p h e n e s D i t h i o p h e n e s T e r t h i o p h e n e s T r i b e A n t h e m i d e a e + -A r c t o t i d e a e + + A s t e r e a e - -C a l e n d u l e a e - -C i c h o r i e a e - -C y n a r e a e + + E u p a t o r i e a e + • -H e l e n i e a e + + H e l i a n t h e a e + + I n u l e a e + + M u t i s i e a e - -Senec i o n e a e + . + T a g e t e a e + + V e r n o n i e a e + — formed by o x i d a t i o n of t h i s e i g h t e e n c a r b o n f a t t y a c i d . D e c a r b o x y l a t i o n o f s p e c i f i c p o l y a c e t y l e n e s has been p r o p o s e d t o l e a d t o a t w e l v e c a r b o n i n t e r m e d i a t e , t h e " p e n t a - y n e - e n e " , w h i c h may be t h e b i o s y n t h e t i c p r e c u r s o r of t h i o p h e n e s i n t h e A s t e r a c e a e . S u l f i d e a d d i t i o n ( v i a an unknown d o n o r ) t o t h i s i n t e r m e d i a t e , f o l l o w e d by c y c l i z a t i o n ( s ) , r e s u l t s i n t h e v a r i o u s mono, d i and t e r t h i e n y l d e r i v a t i v e s (Bohlmann e t a l . , 1973). T h i s a s p e c t o f m e t a b o l i s m , however, has n o t been c o n f i r m e d by b i o s y n t h e t i c s t u d i e s . 6 PROPOSED BIOSYNTHESIS OF TKIOPHTKES Q-terthier.yl (after Bohlmann et a l . , 1973) F i g u r e 2. A proposed b i o s y n t h e t i c pathway l e a d i n g to the f o r m a t i o n o f t h i o p h e n e s from the p o l y a c e t y l e n i c p r e c u r s o r , the "penta-yne-ene". 7 D i s c o v e r y of t h e p o t e n t n e m a t i c i d a l p r o p e r t i e s of a l p h a - T and BBT l e d t o t h e o r i g i n a l i n t e r e s t i n t h e s e d e r i v a t i v e s o f T. e r e c t a (TJhlenbroek and B i j l o o , 1958 and 1959). Gommers (1972) and Gommers and G e e r l i n g s (1973) s u b s e q u e n t l y showed t h a t t h e n e m a t i c i d a l a b i l i t y of a l p h a - T was e n h a n c e d i n t h e p r e s e n c e o f d a y l i g h t and s p e c i f i c a l l y t h e UV-A r e g i o n of t h e s p e c t r u m (320 nm t o 400. nm). Chan e_t a_l. (1975) r e p o r t e d t h a t b o t h a l p h a - T and BBT were p h o t o t o x i c o r b a c t e r i c i d i a l i n UV-A, but c a u s e d no l e t h a l e f f e c t i n t h e d a r k . The p h o t o b i o c i d a l o r p h o t o t o x i c e f f e c t s o f a l p h a - T have s u b s e q u e n t l y been shown t o w a r d a wide v a r i e t y o f b i o l o g i c a l o r g a n i s m s i n a d d i t i o n t o nematodes and i n c l u d e b a c t e r i a (Chan e t a l . , 1975), f u n g i ( D a n i e l s , 1965), a l g a e ( A r n a s o n e t a l . , 1981), i n s e c t s ( A r n a s o n e t a l . , 1980; Wat e_t a_l. , i n p r e s s ) and v a r i o u s o t h e r o r g a n i s m s (Towers, 1980). The p h o t o t o x i c i t y of BBT has t h u s f a r o n l y been d e m o n s t r a t e d w i t h b a c t e r i a (Chan e t a_l. , 1975) (a d e t a i l e d a c c o u n t of t h e p h o t o i n d u c e d t o x i c mechanism of a c t i o n o f a l p h a -T and BBT t o w a r d b a c t e r i a i s p r e s e n t e d i n S e c t i o n I I . P h o t o b i o l o g i c a l A s p e c t s o f T h i o p h e n e s ) . The t o x i c i t y o f i s o l a t e d t h i o p h e n e s i n t h e p r e s e n c e o f l i g h t l e a d s one t o q u e s t i o n t h e i r _in s i t u s i g n i f i c a n c e . Do t h i o p h e n e s a f f o r d p l a n t s some p r o t e c t i v e a d v a n t a g e as o p p o s e d to' p l a n t s l a c k i n g them? C o n v e r s e l y , i f t h e s e compounds a r e so t o x i c , how do p l a n t s p r o t e c t t h e m s e l v e s from t h e p h o t o i n d u c e d e f f e c t s o f t h e s e t o x i c compounds? Answers t o t h e s e q u e s t i o n s were n o t s o u g h t i n t h i s s t u d y , b u t i n i t i a l i n f o r m a t i o n w h i c h c o u l d p e r h a p s g i v e c l u e s t o p o s s i b l e answers was examined. 8 The d i s t r i b u t i o n o f t h i o p h e n e s i n members of t h e T a g e t e a e has been t h o u g h t t o i n c l u d e o n l y s p e c i f i c p a r t s o f t h e p l a n t (Towers and Wat, 1978). R o o t s were f o u n d t o be t h e p r i m a r y l o c a t i o n o f a l p h a - T and BBT, b u t f l o w e r p e t a l s ( Z e c h m e i s t e r and S e a s e , 1947) and t h e pappus o f a c h e n e s (Chan e t a _ l . , 1975) were a l s o shown t o p o s s e s s low c o n c e n t r a t i o n s o f t h e s e compounds. I s o l a t i o n of t h e s e compounds g e n e r a l l y i n v o l v e d e i t h e r column or t h i n l a y e r c h r o m a t o g r a p h y ( C u r t i s and P h i l l i p s , 1962; A t k i n s o n e_t a l . , 1965). The compounds were d e t e c t e d by f l u o r e s c e n c e under longwave UV i r r a d i a t i o n o r by t h e use o f a c o n c e n t r a t e d s u l f u r i c a c i d s o l u t i o n o f i s a t i n w h i c h i n d u c e d c o l o r r e a c t i o n s w i t h t h e v a r i o u s t h i o p h e n e d e r i v a t i v e s . I s o l a t i o n r e q u i r e d l a r g e amounts o f p l a n t m a t e r i a l and y i e l d s were g e n e r a l l y low due t o t h e low c o n c e n t r a t i o n s i n t h e p l a n t and due t o i n s t a b i l i t y o f t h e compounds (Bohlmann e_t a l •, 1 973). L a c k o f a s e n s i t i v e q u a n t i t a t i v e method has h i t h e r t o p r e v e n t e d s t u d i e s i n v o l v i n g t h e d i s t r i b u t i o n and a c c u m u l a t i o n of t h i o p h e n e s . B e f o r e i_n s i t u s t u d i e s c a n be c o n d u c t e d , i t i s n e c e s s a r y t o know t h e d i s t r i b u t i o n and l e v e l s of t h e v a r i o u s d e r i v a t i v e s i n t h e p l a n t as w e l l as t h e r e l a t i v e t o x i c i t i e s o f e a c h component. T h i s s t u d y was u n d e r t a k e n t o d e t e r m i n e : 1) t h e c o m p o s i t i o n o f t h i o p h e n e s i n T. p a t u l a ; 2) t h e i r d i s t r i b u t i o n t h r o u g h o u t t h e p l a n t ; 3) whether s p e c i f i c compounds a r e a c c u m u l a t e d i n p a r t i c u l a r t i s s u e s ; o r 4) whether t h e l e v e l s p r e s e n t a r e s u f f i c i e n t t o a c c o u n t f o r p r o t e c t i o n . T h e s e s t u d i e s were d i f f i c u l t t o c a r r y o u t i n i t i a l l y , b e c a u s e o f t h e l a c k o f a 9 s u i t a b l e s y s t e m f o r j t h e i r d e t e c t i o n and q u a n t i f i c a t i o n . T h e r e f o r e a s e n s i t i v e , a n a l y t i c a l method was d e v e l o p e d w h i c h u s e d h i g h p r e s s u r e l i q u i d c h r o m a t o g r a p h y (HPLC). W i t h t h i s new t e c h n i q u e i t was p o s s i b l e t o s t u d y t h i o p h e n e d e r i v a t i v e s i n r e l a t i v e l y c r u d e e x t r a c t s o f T. p a t u l a . Q u a l i t a t i v e d i s t r i b u t i o n and q u a n t i t a t i v e a c c u m u l a t i o n , of t h i o p h e n e s o v e r t h e l i f e o f t h e p l a n t were t h e n e x a m i n e d . MATERIALS AND METHODS C h e m i c a l s N - b r o m o s u c c i n i m i d e , 2 - b r o m o t h i o p h e n e ( I I ) , 2-m e t h y l t h i o p h e n e ( I I I ) , 2 , 5 - d i b r o m o t h i o p h e n e ( V ) , and t e t r a m e t h y l a m m o n i u m c h l o r i d e (TMA) were p u r c h a s e d from A l d r i c h C h e m i c a l Company. T h i o p h e n e ( I ) and 2 - a c e t y l t h i o p h e n e (IV) ( m e t h y l 2 - t h i e n y l k e t o n e ) were o b t a i n e d from M a t h e s o n , C o l e and B e l l C h e m i c a l s and Eastman Kodak. F l u o r e s c e i n was p u r c h a s e d from Sigma w h i l e i s a t i n and v a n i l l i n were from N u t r i t i o n a l B i o c h e m i c a l s and Baker C h e m i c a l Company, r e s p e c t i v e l y . A l p h a - t e r t h i e n y l was s y n t h e s i z e d by me and l a t e r by D r . F. G a r c i a , C h e m i s t r y D e p a r t m e n t , N a t i o n a l U n i v e r s i t y o f M e x i c o , u s i n g t h e method of Kooreman and Wynberg ( 1 9 6 7 ) . The s t a n d a r d o f 5 - ( b u t e n - 3 - y n y l ) - 2 , 2 ' - b i t h i e n y l was i s o l a t e d f r o m e i t h e r T. p a t u l a , T. e r e c t a , o r D y s s o d i a p a p p o s a p l a n t m a t e r i a l . R e f e r e n c e s o l u t i o n s of 5 - ( 4 - h y d r o x y - 1 - b u t y n y l ) - 2 , 2 ' - b i t h i e n y l ( V I I ) and 5 - ( a c e t o x y - 1 - b u t y n y l ) - 2 , 2 ' - b i t h i e n y l ( V I I I ) were k i n d l y s u p p l i e d by D r . R. S u e t f e l d , U n i v e r s i t y o f M u n s t e r , 10 d u r i n g t h e l a t e r s t a g e s o f t h i s s t u d y . The compound 2,2'-d i t h i o p h e n e (VI) was s y n t h e s i z e d by t h e method o f Sease and Z e c h m e i s t e r ( 1 9 4 7 ) . A l l o t h e r c h e m i c a l s and s o l v e n t s were f r o m l a b o r a t o r y s t o c k s ( A . C . S ) . HPLC s o l v e n t s f r o m F i s h e r S c i e n t i f i c were u s e d f o r HPLC a n a l y s e s . Sephadex LH-20 (25-1OOu, P h a r m a c i a ) was u s e d f o r g e l f i l t r a t i o n c h r o m a t o g r a p h y . P l a n t M a t e r i a l T a g e t e s p a t u l a L. ( f r o m J . L . Hudson, Redwood C i t y , CA) p l a n t s were u s e d f o r a l l e x p e r i m e n t s . P l a n t s were grown g e n e r a l l y i n t h e f i e l d between t h e months o f May and September. A l t e r n a t i v e l y , p l a n t s were grown i n growth chambers ( C o v i r o n , C o n t r o l l e d E n v i r o n m e n t s LTD., W i n n i p e g , M a n n i t o b a , Canada) a t 25°C, 100% R.H. and a p h o t o p e r i o d of 16 h o u r s l i g h t / 8 h o u r s d a r k . The p l a n t s r e q u i r e d 6 t o 9 weeks f o r f l o w e r i n g under e i t h e r c o n d i t i o n . D r i e d P l a n t S p e c i m e n s D r i e d p l a n t s were u s e d f o r t h e e x a m i n a t i o n o f t h i o p h e n e s i n t h e t r i b e T a g e t e a e . P e c t i s f i 1 i p e s v a r . subnuda (K13230), P. i m b e r b i s (13559, 13566), P. l o n q i p e s ( d i p l o i d ; K11375, 13500, 13508 and t e t r a p l o i d ; K11368, 13529, 13535), P. p a p p o s a v a r . p a p p o s a (K13149), and P. p r o s t r a t a (K13279) were s u p p l i e d by D r . D a v i d J . K e i l , C a l i f o r n i a P o l y t e c h n i c U n i v e r s i t y , San L u i s O b i s p o , CA. T a g e t e s m u l t i f l o r a (389a,b) and T. e l l i p t i c u m (112) were c o l l e c t e d by Timmothy J o h n s , U n i v e r s i t y of M i c h i g a n , 11 Ann A r b o r , MI. D y s s o d i a a n t h e m i d i f o l i a , D. d e c i p l e n s ( 5 8 ) , D. p a p p o s a , P o r o p h y l l u m g r a c i l e , T a g e t e s c o r o n o p i f o l i a ( 1 0 5 ) , T. f i l i f o l i a ( 7 9 ) , T. l u n u l a t a , T. l u c i d a (1-11), and T. t e n u i f o l i a (106) were s u p p l i e d by D r . G.H.N. Towers, U n i v e r s i t y o f B r i t i s h C o l u m b i a , V a n c o u v e r , B.C. T a g e t e s  l e m m o n i i ( U n i v e r s i t y o f C a l i f o r n i a B o t a n i c a l Garden 74.251) was s u p p l i e d by D r . John S t r o t h e r , H e r b a r i u m , U n i v e r s i t y o f C a l i f o r n i a , B e r k e l e y , CA. T a g e t e s l u n u l a t a , T. p a t u l a , and T. e r e c t a were grown i n t h e f i e l d f r o m s e e d . D u p l i c a t e v o u c h e r s p e c i m e n s were i d e n t i f i e d by D r . John S t r o t h e r . V o u c h e r s p e c i m e n s were d e p o s i t e d i n t h e B o t a n y Department H e r b a r i u m , U.B.C. H y d r o p o n i c s A c h e n e s of T. p a t u l a were g e r m i n a t e d on two l a y e r s of m o i s t e n e d Whatman #1 f i l t e r p a p e r i n g l a s s p e t r i d i s h e s . The d i s h e s were c o v e r e d and l e f t on a l a b o r a t o r y b e nch t o p f o r f o u r t o f i v e d a y s . U n i f o r m s e e d l i n g s a p p r o x i m a t e l y f i v e c e n t i m e t e r s l o n g were s e l e c t e d a t t h i s t i m e and t r a n s f e r r e d t o n u t r i e n t s o l u t i o n f o l l o w i n g t h e method o f D r . A.D.M. G l a s s , D epartment of B o t a n y , U.B.C. ( p e r s o n a l c o m m u n i c a t i o n ) . The. n u t r i e n t s o l u t i o n ( T a b l e 2) was c h a n g e d c o m p l e t e l y on a w e e k l y b a s i s . P l a n t s were kept i n g r o w t h chambers under t h e p r e v i o u s l y d e s c r i b e d c o n d i t i o n s . 12 Table 2. Composition of hydroponic growth medium. A l l concentrations are micromolar (uM). Potassium n i t r a t e 600 uM Calcium n i t r a t e 400 Ammonium phosphate 200 Magnesium sulfate 100 Potassium chloride 5.0 Borate 2.5 Manganese sulfate 0.2 Zinc sulfate 0.2 Copper(II) sulfate 0.05 Molybdate 0.05 Fe-EDTA 2.0 Extraction of Thiophenes T. patula tissue was extracted by three methods. The f i r s t method was used for q u a l i t a t i v e analyses and involved the extraction of fresh plant material. Tissue was ground in methanol in a Waring blendor (three times) and the mixture f i l t e r e d on a Buchner funnel using Whatman #1 f i l t e r paper. The f i l t r a t e was dil u t e d 1:1 with d i s t i l l e d water and extracted three times with equal volumes of petroleum ether (PE) (30-60°C) in a separatory funnel. The combined PE fractions were dried with anhydrous sodium sulfate and stored at -20°C. Al t e r n a t i v e l y , the PE fr a c t i o n was evaporated to dryness on a rotary evaporator. Following evaporation, the residue was resuspended in HPLC grade methanol and stored at -20°C u n t i l 13 HPLC a n a l y s i s . A l l p r o c e d u r e s were c o n d u c t e d u n d e r dimmed room l i g h t t o a v o i d p h o t o d e g r a d a t i o n of t h e e x t r a c t e d compounds. The s e c o n d method was u s e d f o r t h e i s o l a t i o n o f r e f e r e n c e compounds and i n v o l v e d t h e b u l k e x t r a c t i o n o f f r e e z e d r i e d p l a n t t i s s u e . F r e s h p l a n t s were f i r s t f r o z e n a t -80°C and t h e n l y o p h i l i z e d . The d r i e d m a t e r i a l was s e p a r a t e d i n t o s h o o t s o r r o o t s and g r o u n d t o a f i n e powder i n a W i l e y m i l l . The powdered p l a n t m a t e r i a l was e x t r a c t e d i n a S o x h l e t a p p a r a t u s f o r 24 h o u r s w i t h PE ( 3 0 - 6 0 ° C ) . The PE was d r i e d o v e r s o d ium s u l f a t e and e i t h e r e v a p o r a t e d t o d r y n e s s and r e s u s p e n d e d i n m e t h a n o l f o r HPLC a n a l y s i s o r l e f t i n PE f o r TLC or column c h r o m a t o g r a p h y . The t h i r d t e c h n i q u e was u s e d f o r t h e e x t r a c t i o n o f t h i o p h e n e s from d r i e d h e r b a r i u m s p e c i m e n s . One t o f i v e grams o f d r i e d m a t e r i a l was c r u s h e d t o a f i n e powder and p l a c e d i n t o 20 ml of 95% e t h a n o l . The m a t e r i a l was a l l o w e d t o e x t r a c t f o r 3 t o 4 weeks a t w h i c h t i m e t h e powdered p l a n t m a t e r i a l was removed by f i l t r a t i o n . The e t h a n o l i c e x t r a c t was t h e n c o n c e n t r a t e d by r o t a r y e v a p o r a t i o n t o a p p r o x i m a t e l y 1 t o 2 ml and examined by HPLC. T h i s method was u s e d b e c a u s e i t was s i m p l e and a l l o w e d t h e s i m u l t a n e o u s e x t r a c t i o n o f a l l s a m p l e s . T h i o p h e n e s were e x t r a c t e d s u c c e s s f u l l y f r o m s e v e n y e a r o l d d r i e d t i s s u e . I d e n t i f i c a t i o n of T h i o p h e n e s The t h i o p h e n e s i s o l a t e d f r o m T. p a t u l a were a n a l y z e d by v a r i o u s s p e c t r o s c o p i c methods. U l t r a v i o l e t a b s o r p t i o n s p e c t r a and mass s p e c t r a were u s e d f o r c o m p a r i s o n w i t h p u b l i s h e d d a t a 14 for s t r u c t u r a l v e r i f i c a t i o n . UV spectra were recorded in spectral grade methanol using either a Pye-Unicam SP8-100 UV/Vis or a Unicam SP-800A UV/Vis spectrophotometer. Emission and excitation spectra were determined in either methanol or ethanol on a Perkin-Elmer 650-1Os fluorescence spectrophotometer with a Perkin-Elmer 150 Xenon power supply. Infra-red spectroscopy was conducted on a Unicam SP-200G grating spectrophotometer using either chloroform or carbon d i s u l f i d e . Mass spectral analyses were recorded with a Hewlett Packard Model 5985B GLC-MS by the Chemistry Department at Simon Fraser University. Chromatographic separation of compounds was c a r r i e d out with a c a p i l l a r y SE-30 column and a temperature program from 80°C to 220°C at l5°C/min. Chromatography TLC Thin layer chromatography was used for q u a l i t a t i v e analysis and small scale i s o l a t i o n of compounds. Separations were carr i e d out on unactivated s i l i c a gel with and without fluorescent indicator. A n a l y t i c a l sheets (purchased from Eastman Kodak) and S i l i c a Gel G (type 60) (Brinkmann Instruments, Canada) spread to a wet thickness of 0.5 mm on glass plates were used for a n a l y t i c a l and preparative TLC, respectively. The chromatograms were developed in one dimension in rectangular chambers without e q u i l i b r a t i o n using one of three 15 solvent systems: A. PE (30-60°C):Acetone (99:1) B. PE C. PE:Diethyl Ether (9:1) Solvent system A was used mainly. Thiophenes were detected by their c h a r a c t e r i s t i c fluorescence under longwave UV or by reaction with three spray reagents. The spray reagents used included: 1) V a n i l l i n Spray (Pieman et a l . , 1980) 0.5g v a n i l l i n 9 ml 95% ethanol 0.5 ml cone, s u l f u r i c acid 3 drops g l a c i a l acetic acid 2) I satin Spray (Curtis and P h i l l i p s , 1962) 0.4% i s a t i n in cone, s u l f u r i c acid 3) Succinimide-Fluorescein Spray ( s p e c i f i c for sulfur compounds) spray solution I: 0.35 g N-bromosuccinimide 100 ml methyl chloroform spray solution I I : a. 0.33 g fluorescein 100 ml 0:1 N NaOH 16 b. 3 ml o f a . 100 ml o f 95% e t h a n o l Column C h r o m a t o g r a p h y Column c h r o m a t o g r a p h y on s i l i c a g e l was i n i t i a l l y u s e d t o i s o l a t e s t a n d a r d compounds f r o m c r u d e p l a n t e x t r a c t s a n d t o p u r i f y p r o d u c t s f r o m o r g a n i c s y n t h e s e s . C o l u m n s were d e v e l o p e d w i t h PE (30-60°C) f o l l o w e d by i n c r e a s i n g a mounts o f a c e t o n e t o e l u t e t h e more p o l a r t h i o p h e n e s . I s o l a t e d compounds were c h e c k e d f o r p u r i t y by HPLC. H i g h P r e s s u r e L i q u i d C h r o m a t o g r a p h y Q u a l i t a t i v e a nd q u a n t i t a t i v e a n a l y s e s o f t h i o p h e n e s were c a r r i e d o u t on a V a r i a n S e r i e s 5000 LC. A V a r i s c a n 634 S s p e c t r o p h o t o m e t e r s e t t o 350 nm was u s e d t o d e t e c t e l u t e d compounds. V a r i a n M i c r o P a k MCH-10 (4 mm x 30 cm) a n d MCH-10' (8 mm x 50 cm) o c t a d e c y l s i l a n e r e v e r s e - p h a s e c o l u m n s were u s e d t o s e p a r a t e d e r i v a t i v e s i n a n a l y t i c a l and p r e p a r a t i v e modes, r e s p e c t i v e l y . An i s o c r a t i c s o l v e n t s y s t e m c o n s i s t i n g o f a c e t o n i t r i l e and w a t e r ( 7 2 : 2 8 ) c o n t a i n i n g 10 mM e a c h o f p o t a s s i u m p h o s p h a t e b u f f e r (pH 3.2 t o 3.5) a n d TMA was u s e d t o e l u t e t h i o p h e n e s f o l l o w i n g t h e m ethod o f P h i l l i p s a n d T o w e r s ( 1 9 8 1 ) . A l l l i q u i d c h r o m a t o g r a p h y was c o n d u c t e d a t room t e m p e r a t u r e u s i n g f l o w r a t e s o f 1 m l / m i n f o r a n a l y t i c a l work 1 7 a n d 3 t o 5 m l / m i n f o r p r e p a r a t i v e w o r k. Q u a l i t a t i v e HPLC S t u d i e s The d i s t r i b u t i o n o f t h i o p h e n e s i n h y d r o p o n i c a l l y grown T. p a t u l a was e x a m i n e d by HPLC. The r o o t s , s h o o t s a n d f l o w e r s f r o m m a t u r e f l o w e r i n g p l a n t s were e x t r a c t e d by t h e f i r s t m ethod d e s c r i b e d ( s e e E x t r a c t i o n o f T h i o p h e n e s ) . L e a f g l a n d s were r u p t u r e d w i t h c a p i l l a r y p i p e t t e s . The o i l was c o l l e c t e d by c a p i l l a r y a c t i o n a n d t h e n t r a n s f e r r e d i n t o HPLC g r a d e m e t h a n o l by f o r c i n g a i r t h r o u g h t h e p i p e t t e s . A p p r o x i m a t e l y 800 t o 1000 g l a n d s were s a m p l e d f r o m s e v e r a l p l a n t s . T h i s o i l , t h e s h o o t , r o o t a n d f l o w e r e x t r a c t s were e x a m i n e d i m m e d i a t e l y . Q u a n t i t a t i v e HPLC S t u d i e s Q u a n t i t a t i o n o f t h i o p h e n e d e r i v a t i v e s i n c r u d e p l a n t e x t r a c t s was c a r r i e d o u t by r e v e r s e - p h a s e HPLC w i t h UV d e t e c t o r s e t t o 350 nm. S t o c k s o l u t i o n s o f e a c h t h i o p h e n e s t a n d a r d were p r e p a r e d by d i l u t i o n i n t o 9 5 % e t h a n o l . C o n c e n t r a t i o n c u r v e s were d e t e r m i n e d by f i l l i n g t h e 20 u l i n j e c t i o n l o o p w i t h t h e v a r i o u s d i l u t e d s t a n d a r d s a n d m e a s u r i n g r e s u l t i n g peak a r e a s f r o m t h e c h a r t r e c o r d e r . A t l e a s t f i v e i n j e c t i o n s o f e a c h d i l u t i o n were made. Peak a r e a s were d e t e r m i n e d by m u l t i p l y i n g t h e peak h e i g h t (H) i n cm by t h e w i d t h a t h a l f h e i g h t (W1/2) i n cm. A l l v a l u e s were t h e n m u l t i p l i e d by t h e r e c o r d e r e x p a n s i o n f a c t o r (S) t o c o r r e c t f o r r e c o r d e r s e n s i t i v i t y . 18 P e a k A r e a = H x W 1 / 2 x S T h e d a t a w a s a n a l y z e d b y l i n e a r r e g r e s s i o n a n d t h e l i n e o f b e s t f i t u s e d f o r c o n c e n t r a t i o n d e t e r m i n a t i o n s f r o m c r u d e e x t r a c t s . A l l c o n c e n t r a t i o n c u r v e s h a d c o e f f i c i e n t o f d e t e r m i n a t i o n v a l u e s ( r 2 ) o f 0 . 9 9 o r b e t t e r . T w o s t u d i e s i n v o l v e d q u a n t i t a t i v e H P L C . T h e f i r s t w a s t h e d e t e r m i n a t i o n o f t h i o p h e n e l e v e l s i n m a t u r e , f l o w e r i n g T . p a t u l a p l a n t s g r o w n h y d r o p o n i c a l l y . C o n c e n t r a t i o n s o f t h e k n o w n t h i o p h e n e s w e r e d e t e r m i n e d f o r r o o t s , s h o o t s ( s t e m s a n d l e a v e s ) , i m m a t u r e l e a v e s , m a t u r e l e a v e s , a n d f l o w e r s . T i s s u e w a s e x t r a c t e d b y t h e f i r s t m e t h o d d e s c r i b e d ( s e e E x t r a c t i o n o f T h i o p h e n e s ) . T h i o p h e n e l e v e l s w e r e d e t e r m i n e d i m m e d i a t e l y f o l l o w i n g e x t r a c t i o n . I n t h e s e c o n d s t u d y , d e t e r m i n a t i o n o f t h i o p h e n e c o n c e n t r a t i o n s f r o m e a r l y s e e d l i n g s t a g e t h r o u g h f l o w e r i n g w a s i n v e s t i g a t e d . F o r t h i s , a p p r o x i m a t e l y 4 5 T . p a t u l a s e e d l i n g s w e r e g r o w n u n d e r h y d r o p o n i c c o n d i t i o n s . F i v e p l a n t s , s e l e c t e d r a n d o m l y , w e r e h a r v e s t e d a t 2 t o 3 w e e k i n t e r v a l s . T h e p l a n t s w e r e p o o l e d a n d f r o z e n a t - 8 0 ° C , l y o p h i l i z e d , a n d e x t r a c t e d b y S o x h l e t e x t r a c t i o n . T h e e x t r a c t w a s i m m e d i a t e l y a n a l y z e d b y H P L C a n d t h i o p h e n e c o n c e n t r a t i o n s d e t e r m i n e d b y c o m p a r i s o n o f p e a k a r e a s w i t h t h e c o n c e n t r a t i o n c u r v e s o f t h e a p p r o p r i a t e s t a n d a r d . S t a n d a r d d e v i a t i o n s a s s o c i a t e d w i t h H P L C i n j e c t i o n g e n e r a l l y v a r i e d b y l e s s t h a n 5 % . 19 Phototoxicity of Thiophene Standards The b i o l o g i c a l a c t i v i t i e s of BBT-OH (VII), BBT-OAc (VIII), BBT (IX) and alpha-T (X) were examined by the method of Chan et a l . (1975). E. c o l i , Pseudomonas aeruginosa, and Saccharomyces  cerevisiae were tested. E. c o l i was grown in nutrient broth (Difco). P. aeruginosa was grown in PP2 (Difco) medium while S. cerevisiae was grown in Sabouraud dextrose broth (Difco). Cultures were grown overnight at either 30°C (yeast) or 37°C (bacteria) with shaking at 250 rpm. The overnight cultures were di l u t e d (1:100) in fresh media and 0.1 ml spread onto agar plates with appropriate nutrients. Whatman #1 f i l t e r paper discs (7 mm in diameter) loaded with 5 ug to 10 ug of each standard were placed on the lawns and the plates were incubated for 30 min. Following incubation, the plates were irradiated for one hour by four Sylvania F20T12/BLB lamps (see Irradiation Sources in Section I I ) , incubated overnight and then scored for inh i b i t o r y zones. Unirradiated control plates were also prepared for comparison. RESULTS AND DISCUSSION Figure 3 l i s t s the structures of thiophenes used in t h i s study. I to VI do not occur naturally in members of the Tageteae, but were used for comparative purposes and in synthetic work. VII, VIII, IX and X were isolated from Tagetes  patula L. 20 /) R = H ; R '=H //) R=Br; R = H R<C ///) R = C H , ; R=H S IV) R = C O C H „ R=H V) R= Br; R=Br VI) R = H ; R '=H VII) R = H ; R'= C 5 C - C H r C H , O H S S Vlll) R = H ; R= C = C - C H 2 - C H 2 0 - C O C H , IX) R=H; R'= C E C - C H = C H 2 F i g u r e 3. Chemical s t r u c t u r e s of the t h i o p h e n e s used i n these s t u d i e s . Compounds I to VI a r e not found n a t u r a l l y whereas compounds V I I t o X were i s o l a t e d from Tagetes p a t u l a L. 21 S t r u c t u r a l V e r i f i c a t i o n o f S t a n d a r d s S t r u c t u r a l v e r i f i c a t i o n o f t h e n a t u r a l l y o c c u r r i n g t h i o p h e n e s i n v o l v e d s p e c t r o s c o p i c methods. A p p e n d i x I l i s t s t h e d a t a o b t a i n e d from r e f e r e n c e compounds i s o l a t e d f r o m T. p a t u l a o r s y n t h e s i z e d c h e m i c a l l y . UV, IR, f l u o r e s c e n c e , and MS s p e c t r a f o r most compounds a r e g i v e n . UV a b s o r p t i o n maxima a r e f o l l o w e d by p u b l i s h e d e x t i n c t i o n c o e f f i c i e n t s i n p a r e n t h e s e s . Mass s p e c t r a s h owing r e l a t i v e i n t e n s i t i e s o f m/e p e a k s (£10%) a r e a l s o g i v e n . F l u o r e s c e n c e d a t a showing e x c i t a t i o n maxima a r e f o l l o w e d by e m i s s i o n maxima ( f l u o r e s c e n c e ) i n p a r e n t h e s e s . V e r i f i c a t i o n of t h e s t r u c t u r e s of V I I , V I I I , IX and X were b a s e d on c o m p a r i s o n of s p e c t r a l d a t a w i t h p r e v i o u s l y p u b l i s h e d r e s u l t s . The s t r u c t u r e of a l p h a - T (X) was a l s o c o n f i r m e d by c h e m i c a l s y n t h e s i s . C h r o m a t o g r a p h y S e p a r a t i o n o f t h i o p h e n e d e r i v a t i v e s f r o m T. p a t u l a was a c c o m p l i s h e d by f o u r c h r o m a t o g r a p h i c methods. T h i n l a y e r (TLC) and column c h r o m a t o g r a p h y were u s e d f o r q u a l i t a t i v e a n a l y s i s and p r e p a r a t i v e i s o l a t i o n o f t h i o p h e n e s f r o m c r u d e p l a n t e x t r a c t s . T a b l e 3 l i s t s TLC d a t a f o r q u a l i t a t i v e s e p a r a t i o n of v a r i o u s t h i o p h e n e s . Compounds V I I , V I I I , IX, and X were f o u n d t o f l u o r e s c e under longwave UV i r r a d i a t i o n (320 nm t o 400 nm) w h i l e t h e t h i o p h e n e s (I t h r u V I ) d i d n o t . Of t h e two r e a g e n t s u s e d f o r v i s u a l i z a t i o n , v a n i l l i n y i e l d e d t h e most v i v i d and c h a r a c t e r i s t i c c o l o r r e a c t i o n s . V a n i l l i n a l s o seemed t o be more 22 sensitive than i s a t i n for detection of BBT-OH (VII) and BBT-OAc (VIII). Neither compound gave color reactions with i s a t i n but did react with v a n i l l i n at the same concentrations. Presumably the compounds were below the l e v e l of d e t e c t a b i l i t y of i s a t i n although Pieman et a l . (1980) found the two reagents to be equally sensitive for detection of alpha-T (0.05 ug). BBT (IX) and alpha-T (X) were e a s i l y distinguished by color reaction with either v a n i l l i n or i s a t i n . A l l derivatives reacted with the succinimide-fluorescein spray which i s s p e c i f i c for sulfur containing compounds. Reverse-phase high pressure l i q u i d chromatography (HPLC) was used for q u a l i t a t i v e and quantitative analyses of the thiophenes in crude plants extracts as well as for preparative work. Coupled with a UV detector, HPLC was the most useful and v e r s a t i l e chromatographic method for thiophenes. Table 4 l i s t s uncorrected retention times (R(t)), corrected retention times (R(t')= R(t) - to), r e l a t i v e retention (a), and resolution values (R) of the four naturally derived thiophene reference compounds. Elution of standards was in the order of decreasing p o l a r i t y . BBT-OH (VII), the most polar compound, was retained least (R(t')= 3.5 min) and was eluted before the more non-polar thiophenes BBT-OAc (VIII) (R(t')= 5.0 min), BBT (IX) (R(t')= 7.2 min) and alpha-T (X) (R(t')= 8.9 min), respectively. Thiophenes isolated from crude extracts of T. patula by preparative TLC, column chromatography or preparative HPLC were submitted to combined GLC-MS analysis for structural elucidation. 23 T a b l e 3. C o l o r r e a c t i o n s o f t h i o p h e n e s w i t h v a n i l l i n and i s a t i n s p r a y r e a g e n t s , UV f l u o r e s c e n c e and Rf v a l u e s ( i n S o l v e n t S ystem A ) . Compound t h i o p h e n e ( I ) Reagent UV V a n i l l i n I s a t i n F l u o r e s c e n c e v i o l e t 2 - b r o m o t h i o p h e n e t u r q u o i s e ( I I ) 2 - m e t h y l t h i o p h e n e g r e e n ( I I I ) 2 - a c e t o t h i o p h e n e r e d t o (I V ) o r a n g e 2 , 5 - d i b r o m o - . p u r p l e t h i o p h e n e (V) 2 , 2 ' - d i t h i o p h e n e b l u e • ( V I ) BBT-OH ( V I I ) v i o l e t g r a y g r a y brown p u r p l e NT BBT-OAc ( V I I I ) b l u e b l u e b l u e Rf 0.11 0.93 0.11 0.74 0.90 0.75 0.00 0.09 BBT (IX) A l p h a - T (X) g r e e n i s h b l u e o l i ve g r e e n g r e e n b l u e b l u e b l u e 0.67 0.61 no r e s p o n s e NT - Not t e s t e d T h i n l a y e r and column c h r o m a t o g r a p h y have been u s e d e x t e n s i v e l y f o r t h e s e p a r a t i o n o f t h i o p h e n e s f r o m c r u d e p l a n t e x t r a c t s ( Z e c h m e i s t e r and S e a s e , 1947; U h l e n b r o e k and B i j l o o , 1958, 1959; Chan e t a l . , 1975; S e l v a e t a l . , 1978) and f o r t h e p u r i f i c a t i o n of s y n t h e t i c t h i o p h e n e s ( S e a s e and Z e c h m e i s t e r , 24 T a b l e 4. R e t e n t i o n t i m e s ( R ( t ) ) , c o r r e c t e d r e t e n t i o n t i m e s ( R ( t ' ) ) , r e l a t i v e r e t e n t i o n ( a ) , and r e s o l u t i o n (R) o f t h i o p h e n e s t a n d a r d s s e p a r a t e d by HPLC ( t o = 1.9 m i n ) . T h i o p h e n e S t a n d a r d R ( t ) R ( t ' ) BBT-OH ( V I I ) 5.4 min 3.5 min 1.43 3.53-BBT-OAc ( V I I I ) 6.9 5.0 1.44 4.00 BBT (IX) 9.1 7.2 1.24 3.09 A l p h a - T (X) 10.8 8.9 HPLC p a r a m e t e r s a r e l i s t e d i n M a t e r i a l s and Methods. 1947; U h l e n b r o e k and B i j l o o , 1960; A t k i n s o n e t a l . , 1965; P a t r i c k and Honegger, 1974). T h e s e t e c h n i q u e s were u s e f u l f o r t h e i n i t i a l l o c a t i o n and i s o l a t i o n of t h i o p h e n e s from p l a n t s when c o u p l e d w i t h UV f l u o r e s c e n c e and c o l o r r e a c t i o n w i t h v a n i l l i n (Pieman e t a_l. , 1980), but were n o t a c c e p t a b l e f o r q u a n t i t a t i v e a n a l y s e s of compounds. R e v e r s e - p h a s e HPLC p r o v e d t o be t h e b e s t c h r o m a t o g r a p h i c t e c h n i q u e f o r q u a n t i t a t i o n . 25 Q u a l i t a t i v e A n a l y s i s of T h i o p h e n e s The d i s t r i b u t i o n of t h i o p h e n e s i n d i f f e r e n t p a r t s o f h y d r o p o n i c a l l y grown T. p a t u l a p l a n t s was i n v e s t i g a t e d by HPLC and f l u o r e s c e n c e m i c r o s c o p y . The UV a b s o r b i n g compounds e x t r a c t e d f r o m t h e r o o t s o f e i g h t week o l d p l a n t s a r e shown i n F i g u r e 4. By c o m p a r i s o n w i t h t h e c o r r e c t e d r e t e n t i o n t i m e s ( R ( t ' ) ) o f s t a n d a r d s , BBT-OH ( V I I ) , BBT-OAc ( V I I I ) , BBT ( I X ) , and a l p h a - T (X) were a l l f o u n d t o be p r e s e n t a l o n g w i t h s e v e r a l o t h e r u n i d e n t i f i e d compounds. BBT-OAc ( V I I I ) and BBT were t h e p r e d o m i n a n t d e r i v a t i v e s i n r o o t s . S h o o t , m a t e r i a l ( l e a v e s and stems) c o n t a i n e d t h e same t h i o p h e n e d e r i v a t i v e s as r o o t s ( F i g u r e 5) b u t i n q u i t e d i f f e r e n t p r o p o r t i o n s . BBT-OAc ( V I I I ) was s t i l l p r e v a l e n t i n s h o o t s , but BBT was a much l e s s i m p o r t a n t d e r i v a t i v e . An u n i d e n t i f i e d compound w i t h a R ( t ' ) o f 6.5 m i n u t e s was f o u n d t o be a m a j o r s h o o t component. F l o w e r s ( F i g u r e 6) a l s o c o n t a i n e d V I I , V I I I , IX and X. In a d d i t i o n , two u n i d e n t i f i e d components w i t h R ( t ' ) ' s o f 6.5 and 10.1 m i n u t e s were p r e s e n t i n s u b s t a n t i a l amounts. Of t h e known t h i o p h e n e s , a l p h a - T (X) was t h e m a j o r d e r i v a t i v e i n f l o w e r s , a l t h o u g h BBT-OAc ( V I I I ) was w e l l r e p r e s e n t e d . I t s h o u l d be m e n t i o n e d t h a t t h e u n i d e n t i f i e d compounds ( R ( t ' ) 6.5 and 10.1 minutes.) d i s p l a y e d s i m i l a r UV a b s o r p t i o n s p e c t r a t o o t h e r t h i o p h e n i c d e r i v a t i v e s ( s e e A p p e n d i x I) and a r e p r o b a b l y a l s o t h i o p h e n e s . The l e a f g l a n d s of m a r i g o l d s have been s t u d i e d f o r t h e i r v o l a t i l e , i n s e c t r e p e l l i n g components (de M u c c i a r e l l i and M o n t e s , 1970; de V i l l i e r s e t a l . , 1971; O k o t h , 1973). They have been shown t o c o n t a i n a number of m o n o t e r p e n e s ( e . g . t a g e t o n e 26 IX 0 1 2 3 4 ~~5 6 7 8 9 10 11 Time (min) Figure 4. HPLC trace of the UV absorbing compounds which were extracted from the roots of 8 week old hydroponically grown T. patula. Time (min) Figure 5. HPLC trace of the UV absorbing compounds which were extracted from the shoots of 8 week old hydroponically grown T. patula t 0.01 a u i— — 1 • " «• J_ i i i i i i , 0 1 2 3 4 5 6 7 8 9 10 11 12 Time (min) Figure 7. HPLC trace of the UV absorbing compounds in the o i l of leaf glands from 8 week old hydroponically grown T. patula. Figure 6. HPLC trace of the UV absorbing compounds were extracted from flower petals of hydroponically T. patula. wh i c h grown 29 and t a g e t e n o n e ) , but t h i o p h e n e s were not f o u n d . BBT and one u n i d e n t i f i e d component ( R ( t ' ) 6.5 m i n u t e s ) , however, were d e t e c t a b l e by HPLC ( F i g u r e 7 ) . A n a t o m i c a l l y , l e a f g l a n d s a r e a s s o c i a t e d w i t h r e s i n c a n a l s . The p r e s e n c e o f t h i o p h e n e s i n l e a f g l a n d s m ight a l s o i n d i c a t e t h e i r p r e s e n c e i n t h e r e s i n c a n a l s a s w e l l . The r e s i n c a n a l s y s t e m c o u l d o f f e r a c o n v e n i e n t t r a n s p o r t s y s t e m i n t h e p l a n t f o r t h e s e n o n - p o l a r compounds. F l u o r e s c e n c e m i c r o s c o p y of f r e s h T. p a t u l a t i s s u e was c o n d u c t e d t o d e t e r m i n e whether c o n c e n t r a t i o n s o f t h i o p h e n e s c o u l d be d e t e c t e d v i s u a l l y . T h e i r i n t e n s e b l u e t o v i o l e t f l u o r e s c e n c e under longwave UV was s u g g e s t i v e t h a t t h i s t e c h n i q u e m i g h t be u s e f u l f o r d e t e c t i o n o f t h i o p h e n e s a t t h e c e l l u l a r o r t i s s u e l e v e l . R oot, stem, l e a f and f l o w e r s e c t i o n s were examined, however no f l u o r e s c e n c e was f o u n d w h i c h would s p e c i f i c a l l y i n d i c a t e t h e i r l o c a t i o n i n c e l l s o r t i s s u e s . The a b s e n c e o f f l u o r e s c e n c e i n t i s s u e e x c i t e d by longwave UV c o u l d i n d i c a t e t h a t e i t h e r t h i o p h e n i c d e r i v a t i v e s a r e n o t c o n c e n t r a t e d i n s p e c i f i c r e g i o n s of t h e p l a n t or t h a t t h e i r f l u o r e s c e n c e i s q u e n c h e d _in s i t u , p r e v e n t i n g t h e i r d e t e c t i o n by t h i s method. Q u a n t i f i c a t i o n of T h i o p h e n e s T h i o p h e n e l e v e l s i n p l a n t s were measured by HPLC. Q u a n t i f i c a t i o n by t h i s new t e c h n i q u e i n v o l v e d t h e use o f s t a n d a r d c o n c e n t r a t i o n c u r v e s w h i c h were d e t e r m i n e d s e p a r a t e l y f o r e a c h r e f e r e n c e compound. F i g u r e s 8, 9, 10 and 11 g i v e t h e r e l a t i o n s h i p s between d e t e c t o r r e s p o n s e ( r e c o r d e d as peak a r e a ) 30 and amount of standard injected for each reference compound. It was necessary to determine standard curves for each compound in d i v i d u a l l y since the extinction c o e f f i c i e n t s d i f f e r e d at the monitoring wavelength (350 nm). This technique was s e n s i t i v e enough to quantify thiophenes in the nanomolar (10" 9 M) range which allowed analyses of as l i t t l e as 1 g of fresh tissue. Crude T. patula extracts from d i f f e r e n t regions of plants (75 days old) were determined quantitatively (Table 5; page 36). Roots showed the highest concentrations of VIII and IX. Alpha-T (X) was prevalent in flowers and BBT-OH was a minor component throughout the plant. Analysis of immature and mature leaf tissue revealed that growing leaves contained considerably higher levels of thiophenes than mature tissue. Concentration differences between older and younger leaf tissue may be a result of the loss of compounds by: 1) degradation in older tissue r e s u l t i n g from either biochemical or photo-induced mechanisms; 2) transport from older tissue to younger tissue; 3) v o l a t i l i z a t i o n from glands; or 4) d i l u t i o n in older t i s s u e . Conversely, younger tissue may a c t i v e l y synthesize thiophenes which perhaps could account for higher concentrations in younger tissue. The fate of thiophenes over the l i f e of the plant was also investigated q u a n t i t a t i v e l y . Figure 12 shows the lev e l s of VIII, IX and X from root extracts of hydroponically grown plants. BBT-OAc (VIII) and BBT (IX) were the main thiophene components in crude root extracts during early seedling growth. Both derivatives increased in concentration for the f i r s t 80 31 200 r 150 h ft ec 4 100 h 20 30 [BBT-OH] (nanomoles) 40 50 Figure B. HPLC concentration curve for BBT-OH (VII) which re l a t e s peak area (detector response) and amount of standard injected (nanomoles). 32 Figure 9. HPLC concentration curve for BBT-OAc (VIII) which r e l a t e s peak area (detector response) and amount of standard injected (nanomoles). 33 Figure 10. HPLC rel a t e s peak area injected (nanomoles) concentration curve (detector response) for BBT (IX) which and amount of standard 34 200 r 1 1__ • 10 20 30 40 50 [Alpha -7] (nanomoles) Figure 1 1 . HPLC concentration curve for alpha-T (X) which re l a t e s peak area (detector response) and amount of standard injected (nanomoles). 35 Flowering I >° IX Time (days after germination) F i g u r e 12. The c o n c e n t r a t i o n (umole/g f r wt) of BBT-OAc ( V I I I ) (A A ) , BBT (IX) (• •) and alpha-T (X) <o O) i n the r o o t s of h y d r o p o n i c a l l y grown T. p a t u l a over time. 36 T a b l e 5. T h i o p h e n e l e v e l s i n c r u d e e x t r a c t s of h y d r o p o n -i c a l l y grown p l a n t s . S t a n d a r d d e v i a t i o n o f t h r e e i n j e c t i o n s a r e l i s t e d i n p a r e n t h e s e s . A l l c o n c e n t r a t i o n s a r e i n m i c r o m o l e s p e r gram f r e s h w e i g h t (uM/g f r e s h w t ) . Compound Root S h o o t Immature L e a f M a t u r e L e a f F l o w e r BBT-OH ( V I I ) 0.2(0.0) 0.2(0.0) 0.3(0.1) BBT-OAc ( V I I I ) 12.3(0.3) 6.4(0.2) 1.5(0.3) 0.6(0.1) 0.5(0.1) BBT (IX) 26.7(1.1) 0.6(0.1) 0.4(0.0) A l p h a - T (X) 0.5(0.2) 0.5(0.1) 0.5(0.1) - below q u a n t i f i a b l e l e v e l s •0.4(0.1 ) 1 .3(0.2) d a y s a f t e r g e r m i n a t i o n b u t r e a c h e d a p l a t e a u a f t e r t h a t t i m e . The c o n c e n t r a t i o n o f a l p h a - T (X) c h a n g e d v e r y l i t t l e o v e r t h e e x p e r i m e n t a l p e r i o d . BBT-OAc ( V I I I ) was t h e dom i n a n t t h i o p h e n e i n s h o o t s ( F i g u r e 1 3 ) . BBT (IX) and a l p h a - T (X) were e q u a l l y c o n c e n t r a t e d and d i d n o t i n c r e a s e a p p r e c i a b l y o v e r t i m e . BBT-OAc ( V I I I ) , on t h e o t h e r hand, i n c r e a s e d u n t i l a r o u n d 75-80 d a y s when an e q u i l i b r i u m c o n c e n t r a t i o n was r e a c h e d . The l e v e l i n g o f BBT-OAc ( V I I I ) i n t h e s h o o t and BBT-OAc ( V I I I ) and BBT (IX) i n t h e r o o t s f o l l o w e d f l o w e r i n g q u i t e c l o s e l y . The same t i m e d e p e n d e n t t r e n d s were a l s o o b s e r v e d d u r i n g r e p l i c a t i o n s of t h e s e e x p e r i m e n t s . . Figure 13. The concentration (umole/g f r wt) of BBT-OAc (VIII) <* BBT (IX) (• • > and alpha-T (X) (O O) in shoots of hydroponically grown T. patula over time. 38 D i s t r i b u t i o n o f T h i o p h e n e s i n t h e T a g e t e a e The T a g e t e a e c o m p r i s e s 16 t o 18 g e n e r a d i s t r i b u t e d g e n e r a l l y i n t h e warmer a r e a s o f N o r t h and S o u t h A m e r i c a ( K e i l , 1975; S t r o t h e r , 1977). C h e m i c a l l y , t h e t r i b e i s c h a r a c t e r i z e d by t h e l a c k of s e s q u i t e r p e n e l a c t o n e s o r p y r r o l i z i d i n e a l k a l o i d s , c h a r a c t e r i s t i c of many o t h e r t r i b e s of t h e A s t e r a c e a e , and by t h e p r e s e n c e of t h i o p h e n i c and s p e c i f i c m o n o t e r p e n o i d d e r i v a t i v e s ( R o d r i g u e z and Mabry, 1977). F i f t e e n s p e c i e s w h i c h r e p r e s e n t t h e f o u r l a r g e s t g e n e r a o f t h e t r i b e ( D y s s o d i a , P e c t i s , P o r o p h y l l u m , and T a g e t e s ) were examined f o r t h e p r e s e n c e of BBT-OH ( V I I ) , BBT-OAc ( V I I I ) , BBT (IX) and a l p h a - T ( X ) . T a b l e 6 l i s t s t h e r e s u l t s of t h i s s u r v e y . B i t h i e n y l and t e r t h i e n y l d e r i v a t i v e s were common i n t h e g e n e r a D y s s o d i a , P o r o p h y l l u m and T a g e t e s . T h i o p h e n e s , however, were t o t a l l y l a c k i n g i n s p e c i e s o f P e c t i s . B a s e d on m o r p h o l o g i c a l c h a r a c t e r s , P e c t i s has no c l o s e r e l a t i v e s among o t h e r g e n e r a o f t h e t r i b e ( K e i l , 1975; S t r o t h e r , 1977).. The c h e m i c a l u n i q u e n e s s of t h e genus i s r e f l e c t e d by t h e f a c t t h a t 20 o u t o f a p p r o x i m a t e l y 80 s p e c i e s t h u s f a r e x a m i n e d d i s p l a y t h e - K r a n z syndrome o r C4 m e t a b o l i s m ( S m i t h and T u r n e r , 1975). The l a c k o f t h i o p h e n e s f o u n d i n t h i s s t u d y may be f u r t h e r e v i d e n c e t h a t P e c t i s , t h e l a r g e s t genus i n t h e T a g e t e a e , o c c u p i e s a more d i s t a n t r e l a t i o n s h i p w i t h o t h e r members of t h e t r i b e . 39 T a b l e 6. D i s t r i b u t i o n of t h i o p h e n e s w i t h i n t h e t r i b e T a g e t e a e . THIOPHENES P l a n t S p e c i e s V I I V I I I IX X D y s s o d i a a n t h e m i d i f o l i a B e n t h . + + D. d e c i p i e n s B a r t l . + + + + — D. p a p p o s a ( V e n t . ) H i t c h c . + + + + P e c t i s f i l i p e s H a r v . & G r a y P. i m b e r b i s A. G r a y P. l o n g i p e s A. G r a y d i p l o i d t e t r a p l o i d P. p a p p o s a H a r v . & Gray P. p r o s t r a t a Cav. P o r o p h y l l u m g r a c i l e B e n t h . + + • + + P. r u d e r a l e (+) (+) (+) (+)e P. l a n c e o l a t u m DC. (+) (+)c T a g e t e s c o r o n o p i f o l i a W i l l d . + + + + T. e l l i p t i c a Sm. (+) (+)b,c T. e r e c t a L. + + + + 40 T a b l e 6. ( c o n t i n u e d ) P l a n t S p e c i e s V I I V I I I IX X T a g e t e s f i l i f o l i a L a q . + ( + ) ( + )b T. g l a n d u l i f e r a S c h r a n k . ( + ) (+) ( + ) b T. g r a c i l i s DC. ( + ) (+) ( + ) ( + )d T. i n d i c a ( + ) ( + ) ( + ) ( + )b T. l e m m o n i i A. Gr a y + + + + T. l u n u l a t a O r t . + + + + T. l u c i d a Cav. ( + ) ( + ) ( + ) ( + )b T. m i n u t a L. ( + ) + + + a T. m u l t i f l o r a HBK. + + T. p a t u l a L. + + + + T. s i g n a t a B a r t l . (+) ( + ) ( + )b T. t e n u i f o l i a Cav. + (+) + + b T. t e r n i f l o r a HBK. ( + ) (+) ( + ) ( + )d T. z y p a q u i r e n s i s H.and B. ( + ) d a . A t k i n s o n e t a l . , 1965 - b. Bohlmann e t a l . , 1973 c . Chan e t a l . , 1979 d. Bohlmann and Zdero,1979 e. Bohlmann e t a l . , 1980 41 B i o l o g i c a l A c t i v i t y o f T h i o p h e n e S t a n d a r d s Compounds V I I , V I I I , IX and X were a l l f o u n d t o g i v e p o s i t i v e p h o t o t o x i c b i o a s s a y s ( T a b l e 7) u s i n g E . c o l i B, P. a e r u g i n o s a and S. c e r e v i s i a e . A l p h a - T and BBT have p r e v i o u s l y been shown t o k i l l b a c t e r i a i n t h e p r e s e n c e of UV-A i r r a d i a t i o n (Chan e t a l . , ( 1 9 7 5 ) ; a l s o see S e c t i o n I I ) . BBT-OH ( V I I ) and BBT-OAc ( V I I I ) , however, had n e v e r been examined b e f o r e . The u n i d e n t i f i e d s h o o t and f l o w e r component w i t h R ( t ' ) of 6.5 m i n u t e s was a l s o f o u n d t o be p h o t o t o x i c t o E . c o l i and S. c e r e v i s i a e . The mechanism of t h i o p h e n e p h o t o t o x i c i t y was e x p l o r e d i n more d e t a i l i n S e c t i o n I I ( s e e P h o t o b i o l o g i c a l A s p e c t s o f T h i o p h e n e s ) . T a b l e 7. P h o t o t o x i c i t y of t h i o p h e n e s t o m i c r o o r g a n i s m s . T e s t O r g a n i s m s BBT-OH BBT-OAc BBT A l p h a - T B a c t e r i a E s c h e r i c h i a c o l i B + + + + Pseudomonas a e r u g i n o s a + + + + Y e a s t S a c c h a r o m y c e s c e r e v i s i a e + + + + 42 CONCLUSION T h i s s t u d y was a i n i t i a l i n v e s t i g a t i o n i n t o t h e d i s t r i b u t i o n o f t h i o p h e n e s i n members of t h e t r i b e T a g e t e a e . A new method o f s e p a r a t i n g and d e t e c t i n g t h e s e d e r i v a t i v e s was d e v e l o p e d u s i n g h i g h p r e s s u r e l i q u i d c h r o m a t o g r a p h y w i t h UV d e t e c t i o n s y s t e m . T h i s t e c h n i q u e was u s e d t o examine t h e t h i o p h e n i c p r o f i l e o f one s p e c i e s , T a g e t e s p a t u l a L . S t a n d a r d r e f e r e n c e compounds o f BBT-OH, BBT-OAc, BBT and a l p h a - T were f i r s t i s o l a t e d and i d e n t i f i e d , and t h e n t h e i r d i s t r i b u t i o n was e x a m i n e d . The c o n s t i t u e n t s i n r o o t s were f o u n d t o be d i f f e r e n t f r o m t h o s e o f s h o o t s . BBT-OAc and BBT were t h e major components of r o o t s w h i l e BBT-OAc was t h e dominant compound i n s h o o t s . A l p h a - T was t h e main d e r i v a t i v e i n f l o w e r p e t a l s and BBT i n l e a f o i l g l a n d s . The c o n c e n t r a t i o n o f BBT-OAc and BBT i n r o o t s i n c r e a s e d o v e r t h e l i f e o f t h e p l a n t u n t i l f l o w e r i n g when t h e l e v e l s s t a b i l i z e d . BBT-OAc a l s o i n c r e a s e d i n c o n c e n t r a t i o n i n s h o o t s o v e r t i m e . A l p h a - T i n r o o t s and b o t h a l p h a - T and BBT i n s h o o t s r e m a i n e d a t f a i r l y low l e v e l s o v e r t h e l i f e t i m e o f t h e p l a n t . HPLC was a l s o u s e d t o s c r e e n 14 s p e c i e s from t h e t r i b e T a g e t e a e f o r t h i o p h e n e d e r i v a t i v e s . Members from f o u r g e n e r a were examined f o r t h e p r e s e n c e of t h e r e f e r e n c e t h i o p h e n e s common t o T. p a t u l a . A l l o f t h e s p e c i e s of D y s s o d i a , P o r o p h y l l u m , and T a g e t e s c o n t a i n e d some of t h e r e f e r e n c e t h i o p h e n e s , but s p e c i e s of P e c t i s showed no t h i o p h e n e s i n d e t e c t a b l e c o n c e n t r a t i o n s . The p h o t o t o x i c e f f e c t s o f i s o l a t e d a l p h a - T and BBT have 43 been d e m o n s t r a t e d t o w a r d a wide s p e c t r u m o f b i o l o g i c a l o r g a n i s m s . BBT-OH and BBT-OAc were t h e r e f o r e a l s o examined f o r p h o t o b i o c i d a l p r o p e r t i e s . The p h o t o t o x i c i t i e s o f BBT-OH and BBT-OAc were shown u s i n g E . c o l i , P. a e r u g i n o s a and S. c e r e v i s i a e a s a s s a y o r g a n i s m s . I f t h e p h o t o t o x i c p r o p e r t i e s o f t h e s e compounds c a n be e x t r a p o l a t e d t o o t h e r b i o l o g i c a l s y s t e m s , a s have been w i t h a l p h a - T , t h e n i t seems p o s s i b l e t h a t t h e s e compounds c o u l d p l a y an i m p o r t a n t p r o t e c t i v e r o l e i n s i t u . SECTION I I . PHOTOBIOLOGICAL ASPECTS OF THIOPHENES 45 INTRODUCTION T h i o p h e n e s were f i r s t i s o l a t e d and c h a r a c t e r i z e d f r o m p e t a l e x t r a c t s o f " A f r i c a n m a r i g o l d " , T a g e t e s e r e c t a L. ( Z e c h m e i s t e r and S e a s e , 1947). The m a j o r t h i o p h e n e s i d e n t i f i e d , a l p h a - t e r t h i e n y l ( a l p h a - T ) and 5 - ( b u t e n - 1 - y n y l ) - 2 , 2 ' - b i t h i e n y l ( B B T ) , were s c r e e n e d f o r a n t i b i o t i c a c t i v i t y a g a i n s t s e v e r a l b a c t e r i a i n c l u d i n g E s c h e r i c h i a c o l i and Pseudomonas o v a l i , b u t no b i o l o g i c a l r e s p o n s e was f o u n d . E i g h t e e n y e a r s l a t e r , D a n i e l s (1965) o b s e r v e d t h a t m a r i g o l d s e e d s ( a c h e n e s ) c a u s e d a " p h o t o t o x i c " r e s p o n s e when p l a c e d on lawns o f C a n d i d a a l b i c a n s and i r r a d i a t e d w i t h longwave ( n o n - g e r m i c i d a l ) UV l i g h t , b u t t h a t no h a l o of g r o w th i n h i b i t i o n o c c u r r e d i n t h e d a r k . The compounds l a t e r f o u n d r e s p o n s i b l e f o r t h i s p h o t o a c t i v a t e d a n t i b i o t i c e f f e c t were i d e n t i f i e d a s a l p h a - T and BBT (Chan e t a l . , 1975). S i n c e t h e s e i n i t i a l f i n d i n g s t h i o p h e n e s i n t h e p r e s e n c e o f UV-A i r r a d i a t i o n {320-400 nm) have been shown t o be t o x i c t o b a c t e r i a and y e a s t s (Chan e t a_l. , 1975) as w e l l as a l g a e ( A r n a s o n e_t §_1. , 1981), t r e m a t o d e c e r c a r i a l a r v a e (Graham e t a l . , 1980), nematodes (Gommers and G e e r l i n g s , 1973; Gommers et a l . , 1980), m o s q u i t o and b l a c k f l y l a r v a e ( A r n a s o n e t al., 1980; Wat e t a l . , i n p r e s s ) and f i s h (Towers, 1980). A l p h a - T has a l s o been shown t o e l i c i t s e v e r e e r y t h e m a and p r o l o n g e d h y p e r p i g m e n t a t i o n i n human and g u i n e a p i g s k i n i r r a d i a t e d w i t h UV-A (Chan e t a l . , 1977; Towers e t a l . , 1979). .The p h o t o b i o c i d a l e f f e c t s of t h e t h i o p h e n e s have s t i m u l a t e d i n t e r e s t i n t h e i r i r i v i v o mechanism of a c t i o n . P h o t o s e n s i t i z e r s have been d e f i n e d as c h e m i c a l s w h i c h c a u s e 46 o t h e r components i n a s y s t e m t o r e a c t when e x c i t e d by r a d i a t i o n ( T u r r o and L a m o l a , 1977). P h o t o s e n s i t i z e d r e a c t i o n s o c c u r p r i m a r i l y a s one of two t y p e s as d i s c u s s e d by K r i n s k y (1977) and S p i k e s ( 1 9 7 7 ) . I n i t i a l l y , a g r o u n d s t a t e s e n s i t i z e r m o l e c u l e (°S) i s e x c i t e d t o a s i n g l e t e x c i t e d s t a t e ('S) by t h e a b s o r p t i o n o f a p h o t o n (hv) ( 1 ) . The 1S s t a t e i s s h o r t l i v e d ( 1 0 ~ 9 t o 1 0 " 6 S ) and c a n u n d e r g o i n t e r s y s t e m c r o s s i n g o v e r t o a l o n g e r l i v e d ( 1 0 ~ 3 t o 10s) t r i p l e t - e x c i t e d s t a t e ( 3 S ) . hv °S > 1S > 3S (1 ) The 3 S s t a t e , b e i n g l o n g e r l i v e d , has t h e c a p a c i t y t o i n i t i a t e T ype I o r Type II r e a c t i o n s . Type I r e a c t i o n s (2) p r o c e e d v i a r a d i c a l o r redox mechanisms g e n e r a l l y i n t h e a b s e n c e of m o l e c u l a r o x y g e n . Type I 3 S + A > S- + At (2) Type I I o r p h o t o d y n a m i c r e a c t i o n s (3) i n v o l v e e n e r g y t r a n s f e r between m o l e c u l a r oxygen and 3 S i n a s p i n c o n s e r v e d r e a c t i o n . Type I I 3 S >°S + 102 (3) T h i s mechanism a c c o u n t s f o r n e a r l y a l l o f t h e q u e n c h i n g of 3 S by m o l e c u l a r oxygen and g e n e r a t e s °S and s i n g l e t oxygen ('02) w h i c h i s r e s p o n s i b l e f o r t h e p h o t o o x i d a t i o n o f many b i o l o g i c a l m o l e c u l e s . A l e s s e f f i c i e n t r e a c t i o n between 3 S and m o l e c u l a r 47 oxygen i n v o l v e s e l e c t r o n t r a n s f e r and i s a s p e c i a l c a s e of a Type I mechanism ( 4 ) . 3 S >St + 02-- (4) T h i s pathway l e a d s t o t h e f o r m a t i o n o f s u p e r o x i d e r a d i c a l (02-) and a s e m i - o x i d i z e d f o r m of t h e s e n s i t i z e r ( S i ) . B o t h of t h e s e r a d i c a l s may r e a c t w i t h v a r i o u s b i o l o g i c a l m o l e c u l e s . F u r o c o u m a r i n s o r p s o r a l e n s a r e u n d o u b t e d l y t h e most e x t e n s i v e l y s t u d i e d g r o u p o f n o n - p h o t o d y n a m i c o r Type I p h o t o s e n s i t i z e r s . I_n v i v o , 8 - m e t h o x y p s o r a l e n (8-MOP) k i l l s m i c r o o r g a n i s m s by p h o t o i n d u c e d monoadduct f o r m a t i o n and i n t e r s t r a n d c r o s s l i n k a g e s w i t h DNA (Song and T a p l e y , 1979). The p h o t o b i n d i n g i s t e m p e r a t u r e i n d e p e n d e n t and d o e s n o t r e q u i r e oxygen ( O g i n s k y e t a l . , 1959); M u s a j o and R o d i g h i e r o , 1972). On t h e o t h e r hand, t h e rn v i v o p h o t o s e n s i t i z a t i o n by t h e p o l y a c e t y l e n e , p h e n y l h e p t a t r i y n e (PHT), r e c e n t l y i n v e s t i g a t e d by A r n a s o n e_t al. (1980) was f o u n d t o i n a c t i v a t e E . c o l i more e f f i c i e n t l y i n t h e a b s e n c e o f o x y g e n . U n l i k e 8-MOP, PHT does not c r o s s l i n k DNA (Wat e_t a l . , 1977) and t h u s . r e p r e s e n t s a new c l a s s o f Type I p h o t o s e n s i t i z e r . More t h a n 400 p h o t o d y n a m i c compounds a r e known w h i c h s e n s i t i z e b i o l o g i c a l m o l e c u l e s by a Type I I or s i n g l e t oxygen mechanism ( S a n t a m a r i a and P r i n o , 1972). The i_n v i v o t a r g e t s o f t h e s e s e n s i t i z e r s a r e q u i t e v a r i a b l e and a r e b e l i e v e d t o depend on t h e c e l l u l a r l o c a t i o n of t h e s e n s i t i z e r , t h e s i n g l e t oxygen g e n e r a t i n g e f f i c i e n c y o f t h e s e n s i t i z e r d u r i n g i r r a d i a t i o n and 48 t h e s u s c e p t i b i l i t y o f t h e t a r g e t m o l e c u l e a n d / o r s t r u c t u r e t o p h o t o o x i d a t i o n by s i n g l e t o x y g e n . The l o c a t i o n o f t h e s e n s i t i z e r i s d i c t a t e d by i t s p e n e t r a b i l i t y i n t o t h e c e l l . I t o (1978) has s u g g e s t e d t h a t p h o t o d y n a m i c compounds may i n a c t i v a t e c e l l s by r e a c t i n g e i t h e r o u t s i d e t h e c e l l i f t h e s e n s i t i z e r i s e x c l u d e d o r f r o m w i t h i n i f t h e compound c a n p a s s t h e c y t o p l a s m i c membrane. He a l s o p o i n t s o u t t h a t a u n i q u e f e a t u r e of p h o t o d y n a m i c a c t i o n i s t h a t t h e g e n e r a t i o n s i t e o f s i n g l e t oxygen and t h e r e a c t i o n s i t e may be d i f f e r e n t . In f a c t , i n an aqueous e n v i r o n m e n t , s i n g l e t oxygen may d i f f u s e and r e a c t up t o 100 nm f r o m t h e g e n e r a t i o n s i t e (Wagner e t a_l. , 1980). T h i s i s q u i t e d i s t i n c t from Type I s e n s i t i z e r s w h i c h r e a c t t h r o u g h r a d i c a l mechanisms i n w h i c h t h e s e n s i t i z e r and s u b s t r a t e must be i n c l o s e p r o x i m i t y a t t h e t i m e of r e a c t i o n . In v i t r o and in v i v o s t u d i e s w i t h v a r i o u s Type I I p h o t o s e n s i t i z e r s have shown t h a t many b i o l o g i c a l l y i m p o r t a n t m o l e c u l e s a r e s u s c e p t i b l e t o p h o t o o x i d a t i o n by s i n g l e t o x y g e n . F r e e amino a c i d s , e.g. t r y p t o p h a n , a n d amino a c i d r e s i d u e s i n p r o t e i n s (Dubbelman e t a l . , 1980), u n s a t u r a t e d f a t t y a c i d r e s i d u e s i n l i p i d s (Rawls and Van S a n t e n , 1970), c h o l e s t e r o l (Teng an d S m i t h , 1 973; Suwa e_t a l . , 1978), and n u c l e i c a c i d s (Webb e t al., 1 9 7 9 ) have a l l been shown t o r e a c t w i t h s i n g l e t o x y g e n . T h i s damage t o b i o l o g i c a l m o l e c u l e s _in v i v o i s e x p r e s s e d by l o s s of membrane i n t e g r i t y r e s u l t i n g from l i p i d p e r o x i d a t i o n and c r o s s l i n k i n g or d e s t r u c t i o n of membrane p r o t e i n s (Dubbelman e t a l . , 1980; Lamola and D o l e i d e n , 1980), 49 i n a c t i v a t i o n o f c y t o p l a s m i c enzymes (Yamamoto e t a_l. , 1979) or g e n e t i c damage (Webb e t a l . , 1 9 7 9 ) , r e s p e c t i v e l y . The _in v i v o mechanisms o f a c t i o n o f d i f f e r e n t t h i o p h e n e s a r e i n t e r e s t i n g and may have s i g n i f i c a n t ir\ s i t u r a m i f i c a t i o n s . A l p h a - T i s t h e o n l y n a t u r a l l y d e r i v e d t h i o p h e n e t o be examined t h u s f a r , but d i f f e r e n c e s o f o p i n i o n r e g a r d i n g i t s mechanism of a c t i o n a l r e a d y e x i s t . A Type I or r a d i c a l mechanism was p r o p o s e d by Kagan e t a _ l . (1980) who r e p o r t e d : 1) t h a t p h o t o s e n s i t i z a t i o n o f E. c o l i by a l p h a - T was not oxygen d e p e n d e n t ; 2) i n t h e p r e s e n c e of UV-A, l a b e l l e d a l p h a - T c o v a l e n t l y bonded t o c a l f thymus DNA and C a n d i d a u t i l i s DNA; and 3) t h a t c o v a l e n t b o n d i n g d i d n o t i n v o l v e c r o s s l i n k i n g of d o u b l e s t r a n d e d DNA as w i t h 8-MOP. However, t h e Type I I or p h o t o d y n a m i c n a t u r e o f a l p h a - T has been d e m o n s t r a t e d i n s e v e r a l s t u d i e s u s i n g v a r i o u s t e c h n i q u e s . Bakker e_t a l . ( 1979) showed t h a t t h e in v i t r o i n a c t i v a t i o n o f g l u c o s e - 6 - p h o s p h a t e d e h y d r o g e n a s e was oxygen d e p e n d e n t . A c e t y l c h o l i n e s t e r a s e i n a c t i v a t i o n and p h o t o h e m o l y s i s o f human e r y t h r o c y t e s as w e l l as c e l l u l a r i n a c t i v a t i o n o f E. c o l i were a l l e n h a n c e d under a e r o b i c c o n d i t i o n s (Wat e_t al. , 1980; A r n a s o n e_t a_l. , 1981). A s i n g l e t oxygen mechanism has been i n d i c a t e d by t h e work of b o t h B a k k e r ' s g r o u p and Towers' g r o u p ( A r n a s o n e_t a_l. , 1981). Damage t o c y t o p l a s m i c membranes of human e r y t h r o c y t e s has been p o s t u l a t e d as a p o s s i b l e s i t e o f a l p h a - T p h o t o s e n s i t i z e d a t t a c k (Yamamoto e t a l . , 1979; Wat e t a l . , 1980). The i_n v i v o mechanism o f a c t i o n of two t h i o p h e n e s were examined i n t h i s s t u d y . A l p h a - T was t h e p r i m a r y s e n s i t i z e r 50 s t u d i e d , a l t h o u g h s e v e r a l i m p o r t a n t a s p e c t s o f BBT p h o t o s e n s i t i z a t i o n were a l s o i n v e s t i g a t e d . D a t a w i l l be p r e s e n t e d w h i c h show t h a t t h e p h o t o t o x i c i t y of a l p h a - T t o w a r d E. c o l i : 1) i s due t o a l p h a - T and not t o a p h o t o p r o d u c t ; 2) a p p e a r s t o f o l l o w m u l t i p l e - h i t i n a c t i v a t i o n k i n e t i c s ; 3) i s oxygen d e p e n d e n t ; 4) may i n v o l v e s i n g l e t oxygen and p o s s i b l y o t h e r e x c i t e d s p e c i e s of oxygen; 5) i s not a f f e c t e d by t h e i n a b i l i t y o f v a r i o u s m u t a n t s t o p e r f o r m g e n e t i c r e c o m b i n a t i o n o r p o s t - r e p l i c a t i o n r e p a i r ; 6) i s s e n s i t i v e t o i r r a d i a t i o n t e m p e r a t u r e ; and 7) p r o b a b l y i n v o l v e s p h o t o d y n a m i c a t t a c k of t h e c y t o p l a s m i c membrane w h i c h i s r e f l e c t e d i n c r o s s l i n k i n g o f membrane p r o t e i n s . BBT p h o t o s e n s i t i z a t i o n , on t h e o t h e r hand, a p p e a r s t o f o l l o w s i n g l e - h i t i n a c t i v a t i o n k i n e t i c s of E. c o l i and i s a f f e c t e d by t h e a b i l i t y o f m u t a n t s t o p e r f o r m g e n e t i c r e c o m b i n a t i o n and p o s t - r e p l i c a t i o n r e p a i r , w h i c h s u g g e s t s t h e i n v o l v e m e n t o f c e l l u l a r DNA. MATERIALS AND METHODS I r r a d i a t i o n S o u r c e s T h r e e s o u r c e s o f UV-A i r r a d i a t i o n (320 nm t o 400 nm) were commonly u s e d d u r i n g t h e s e s t u d i e s ( t h e p a r t i c u l a r s o u r c e i s s p e c i f i e d i n e a c h e x p e r i m e n t a l s e c t i o n ) . F l u o r e s c e n t s o u r c e s c o n s i s t e d o f e i t h e r f o u r h o r i z o n t a l S y l v a n i a F20T12/BLB lamps or two v e r t i c a l S y l v a n i a F15T8/BL lamps. A 1000 w a t t xenon a r c lamp ( O r i o n C o r p o r a t i o n ) was u s e d t o e s t a b l i s h t h e a c t i o n s p e c t r u m o f a l p h a - T . W a v e l e n g t h bands were s e l e c t e d a t 10 nm 51 i n t e r v a l s between 320 nm and 400 nm by i n t e r f e r e n c e f i l t e r s ( C o r i o n C o r p o r a t i o n , h a l f - b a n d w i d t h 10 nm). I r r a d i a n c e o f t h e f l u o r e s c e n t UV-A s o u r c e s a s w e l l as t h e w a v e l e n g t h s p e c i f i c i t y and i r r a d i a n c e of t h e i n t e r f e r e n c e f i l t e r s u s e d i n c o n j u n c t i o n w i t h t h e xenon lamp were measured w i t h a R e s e a r c h R a d i o m e t e r ( I n t e r n a t i o n a l L i g h t I n c . ) . I r r a d i a n c e v e r s u s w a v e l e n g t h f o r t h e f l u o r e s c e n t lamps a r e shown g r a p h i c a l l y i n F i g u r e 14. The i r r a d i a n c e d e t e r m i n e d a t t h e maximum w a v e l e n g t h o f e a c h i n t e r f e r e n c e f i l t e r a l o n g w i t h s e v e r a l p e r t i n e n t d o s i m e t r i c p a r a m e t e r s u s e d i n p r e p a r i n g t h e a c t i o n s p e c t r u m a r e l i s t e d i n T a b l e 8. S u r v i v a l C u r v e s The methods u s e d t o d e t e r m i n e t h e s u r v i v a l o f E. c o l i i n r e s p o n s e t o v a r y i n g c o n c e n t r a t i o n s o f a l p h a - T and BBT i r r a d i a t e d w i t h UV-A were e s s e n t i a l l y t h o s e o f A r n a s o n e t a l . ( 1 9 8 0 ) . O v e r n i g h t c u l t u r e s o f E . c o l i B-23(+) ( M i c r o b i o l o g y D e p a r t m e n t , UBC) were grown i n n u t r i e n t b r o t h a t 37°C w i t h s h a k i n g . The c e l l s were c e n t r i f u g e d t w i c e i n s t e r i l e p h y s i o l o g i c a l s a l i n e (PSS, 0.87% N a C l ) a t 1000 x g f o r 10 m i n u t e s and r e s u s p e n d e d i n s t e r i l e PSS t o an o p t i c a l d e n s i t y of 0.1 a t 520 nm u s i n g a S p e c t r o n i c 20 p h o t o m e t e r . T h r e e ml a l i q u o t s o f t h e r e s u l t i n g b a c t e r i a l s u s p e n s i o n were a s e p t i c a l l y p i p e t t e d i n t o q u a r t z c u v e t t e s and a p p r o p r i a t e amounts of e i t h e r a l p h a - T o r BBT were ad d e d i n 20 u l o f 95% e t h a n o l . The c u v e t t e s were i n c u b a t e d w i t h s h a k i n g f o r 30 m i n u t e s a t 37°C f o l l o w e d by i r r a d i a t i o n w i t h S y l v a n i a F20T12/BLB lamps a t a d i s t a n c e of 15 52 2.8 r W i i i • * * 300 320 340 360 380 400 420 Wavelength [nm) Figure 14. Radient emittance (mW/cm2, versus wavelength of the fluorescent sources used throughout these studies. Measurements were taken 10 cm from the F20T12/BLB lamps and 20 cm from the F15T8/BL lamps. 53 T a b l e 8 . I r r a d i a n c e a t t h e w a v e l e n g t h m a x i m u m o f e a c h i n t e r f e r -e n c e f i l t e r w i t h s e v e r a l p e r t i n e n t d o s i m e t r i c p a r a -m e t e r s u s e d t o d e t e r m i n e t h e a c t i o n s p e c t r u m o f a l p h a - T W A V E L E N G T H I R R A D I A N C E E N E R G Y F L U E N C E E N E R G Y / h v h v F L U E N C E ( n m ) (mW x I 0 4 c m " 1 ) ( J x 1 0 2 c m " 1 ) (J x 1 O ^ h v 1 ) (# h v c m 2 ) 3 1 0 6 . 9 8 . 2 6 . 4 1 . 3 3 2 0 7 . 1 8 . 5 6 . 2 1 . 4 3 3 0 7 . 0 8 . 4 6 . 0 1 . 4 3 4 0 7 . 0 8 . 4 5 . 9 1 . 4 3 5 0 7 . 0 8 . 4 5 . 7 1 . 5 3 6 0 7 . 0 8 . 4 5 . 5 1*5 3 7 0 7 . 0 8 . 4 5 . 4 1 . 6 3 8 0 7 . 3 . 8 . 8 5 . 2 1 . 7 3 9 0 7 . 0 8 . 4 5 . 1 1 . 7 4 0 0 7 . 0 8 . 4 5 . 0 1 . 7 c m ( s e e I r r a d i a t i o n S o u r c e s ) . S a m p l e s o f 0 . 1 m l w e r e r e m o v e d a t a p p r o p r i a t e t i m e s d u r i n g i r r a d i a t i o n ; t h i s n e c e s s i t a t e d s u c c e s s i v e r e m o v a l a n d s u b s e q u e n t r e p l a c e m e n t o f c u v e t t e s u n d e r t h e U V - A l a m p s . C o n t r o l s c o n t a i n i n g 1 u g / m l o f a l p h a - T a n d B B T w e r e a s s a y e d s i m u l t a n e o u s l y i n t h e d a r k a s w e l l a s c e l l s w h i c h w e r e i r r a d i a t e d w i t h o u t p h o t o s e n s i t i z e r . S a m p l e s w e r e d i l u t e d i n P S S a n d p l a t e d o n n u t r i e n t a g a r . A l l e x p e r i m e n t s w e r e r e p e a t e d a t l e a s t t h r e e t i m e s a n d t h e d a t a p r e s e n t e d a r e r e p r e s e n t a t i v e . 54 B a c t e r i a l P r e p a r a t i o n L i q u i d c u l t u r e s o f E. c o l i B and Pseudomonas a e r u g i n o s a were grown t o s t a t i o n a r y p h a s e a t 3 7 ° C . ' A e r o b i c c u l t u r e s were shaken a t 250 rpm w h i l e a n a e r o b i c c u l t u r e s were grown i n a BBL GasPak A n a e r o b i c S ystem w i t h GasPak 02 c o m b u s t e r and C02 g e n e r a t o r p a c k s t o c r e a t e and m a i n t a i n a n a e r o b i c c o n d i t i o n s . An i n o r g a n i c s a l t medium ( H i n d e n n a c h and H e n n i n g , 1975) was u s e d t o c u l t u r e c e l l s and e i t h e r 0.4% g l u c o s e or s u c c i n a t e was a d d e d as t h e c a r b o n s o u r c e f o r E. c o l i and P. a e r u g i n o s a , r e s p e c t i v e l y . C u l t u r e s o f P. a e r u g i n o s a a l s o r e c e i v e d p o t a s s i u m n i t r a t e as a. t e r m i n a l e l e c t r o n a c c e p t o r f o r a n a e r o b i c r e s p i r a t i o n . S u b c u l t u r e s i n f r e s h medium w i t h a f i n a l d i l u t i o n of 1 i n 100 were us e d f o r e x p e r i m e n t s u n l e s s i n d i c a t e d o t h e r w i s e . A s s a y P r o c e d u r e A 3-necked, b o r o s i l i c a t e r e a c t i o n f l a s k ( M u l t i - p u r p o s e j a c k e t e d r e a c t o r from P i e r c e C h e m i c a l Co.) w i t h a 10ml c a p a c i t y , e q u i p p e d w i t h s t i r b a r , gas i n l e t , and l e u r - l o k s a m p l i n g p o r t was u s e d f o r a l l i r r a d i a t i o n e x p e r i m e n t s u n l e s s o t h e r w i s e i n d i c a t e d . I n i t i a l l y , 9 ml o f BM2 medium w i t h a p p r o p r i a t e s u p p l e m e n t s was added t o t h e s t e r i l i z e d r e a c t i o n f l a s k . E i t h e r m e d i c a l a i r ( L i n d e , Type I ) , He ( L i n d e , 9 9 . 9 9 5 % ) , or He/02 (4:1) was p a s s e d t h r o u g h a m i l l i p o r e f i l t e r (Type HA, 0.45 urn) t h e n b u b b l e d t h r o u g h t h e g r o w t h medium f o r 15 m i n u t e s p r i o r t o a d d i t i o n o f c e l l s . A f t e r f l u s h i n g w i t h t h e a p p r o p r i a t e 55 g a s , 1 ml of 1 i n 10 d i l u t e d c e l l s was a d d e d t o t h e r e a c t i o n f l a s k . The c e l l s were i n c u b a t e d f o r one hour i n a i r , 3 h o u r s i n He and 30 m i n u t e s i n He/02. A l p h a - T was t h e n added i n 95% e t h a n o l and t h e c u l t u r e s were i n c u b a t e d f o r 30 m i n u t e s i n t h e d a r k and t h e n i r r a d i a t e d w i t h c o n t i n u o u s gas b u b b l i n g t h r o u g h t h e medium. Samples of 0.5 ml were w i t h d r a w n u s i n g a s t e r i l e 1 ml s y r i n g e a t v a r i o u s t i m e s d u r i n g i n c u b a t i o n and i r r a d i a t i o n . V i a b l e c e l l numbers were d e t e r m i n e d by d i l u t i n g and p l a t i n g f o l l o w e d by c o l o n y c o u n t i n g a f t e r 24 h o u r s i n c u b a t i o n a t 37°C. A l p h a - T e r t h i e n y l A c t i o n S p e c t r u m E. c o l i B was u s e d t o d e t e r m i n e t h e a c t i o n s p e c t r u m of a l p h a - T between 320 nm and 400 nm. A f t e r c e l l s u r v i v a l was d e t e r m i n e d f o r a l p h a - T ( s e e S u r v i v a l C u r v e s ) a c o n c e n t r a t i o n was c h o s e n w h i c h would g i v e e f f e c t i v e c e l l u l a r i n a c t i v a t i o n . The c o n c e n t r a t i o n of a l p h a - T c h o s e n was 'lug/ml. T h e s e s t u d i e s were c a r r i e d o u t i n s t e r i l e m i c r o t i t r a t i o n p l a t e s ( G i b c o S c i e n t i f i c ) h a v i n g 96 w e l l s p e r p l a t e . B a s a l m i n e r a l s a l t s medium c o n t a i n i n g 0.4% g l u c o s e (150 u l ) was added t o e a c h o f t h e w e l l s . T h i s was f o l l o w e d by t h e a d d i t i o n of a p p r o x i m a t e l y 1000 c e l l s s u s p e n d e d i n BM2 (100 u l ) . The b a c t e r i a l s u s p e n s i o n was p r e p a r e d by d i l u t i n g an o v e r n i g h t c u l t u r e i n t o f r e s h BM2 (1 i n 1 0 5 ) . The s e n s i t i z e r was t h e n added i n 5 u l of 95% e t h a n o l t o g i v e a f i n a l c o n c e n t r a t i o n p e r w e l l o f 1 ug/ml. The p l a t e ' s were i n c u b a t e d a t 37°C f o r 30 m i n u t e s and t h e n i r r a d i a t e d . The i r r a d i a t i o n s o u r c e was a 1000 watt xenon a r c lamp ( s e e 56 I r r a d i a t i o n S o u r c e s ) . The d i s t a n c e f r o m t h e s o u r c e was a d j u s t e d t o g i v e 0.7 W/m2 a t e a c h of t h e wave b a n d s . F o l l o w i n g c a l i b r a t i o n , p l a t e s were i r r a d i a t e d a t e a c h w a v e l e n g t h f o r two m i n u t e s a t room t e m p e r a t u r e f o l l o w e d by i n c u b a t i o n a t 37°C f o r 18 h o u r s . A p l a t e c o n t a i n i n g t h e s e n s i t i z e r and b a c t e r i a , but u n i r r a d i a t e d , was a l s o p r e p a r e d as a d a r k c o n t r o l . B a c t e r i a l g r o w t h ( t u r b i d i t y ) was measured s p e c t r o p h o t o m e t r i c a l l y a t 620 nm u s i n g a T i t e r t e k M u l t i s c a n P h o t o m e t e r (Flow L a b o r a t o r i e s ) . The o p t i c a l d e n s i t y o f t h e w e l l s on e a c h i r r a d i a t e d and c o n t r o l p l a t e was d e t e r m i n e d i m m e d i a t e l y f o l l o w i n g i r r a d i a t i o n and a f t e r 18 h o u r s i n c u b a t i o n i n t h e d a r k . Changes i n t u r b i d i t y were a v e r a g e d t o g i v e a s i n g l e r e p r e s e n t a t i v e change i n OD. The r e s u l t i n g OD was a d i r e c t r e f l e c t i o n of t h e i n i t i a l c e l l u l a r i n a c t i v a t i o n a n d / o r i n d i c a t i o n o f c e l l u l a r damage i n c u r r e d d u r i n g i r r a d i a t i o n . The r a t i o between OD's o f i r r a d i a t e d and n o n - i r r a d i a t e d p l a t e s y i e l d e d a measure o f t h e r e l a t i v e e f f e c t i v e n e s s of t h e dose ( s e n s i t i z e r + i r r a d i a t i o n ) . D i v i d i n g t h i s v a l u e by t h e number of i n c i d e n t p h o t o n s g i v e s t h e r e l a t i v e p h o t o n e f f i c i e n c y a t e a c h w a v e l e n g t h . T h i s v a l u e was p l o t t e d a g a i n s t w a v e l e n g t h f o r t h e a c t i o n s p e c t r u m . The d a t a p r e s e n t e d a r e a v e r a g e s of a t l e a s t t h r e e e x p e r i m e n t s c o n d u c t e d on d i f f e r e n t d a y s and a r e p l o t t e d w i t h s t a n d a r d d e v i a t i o n . 57 Rec Mutant A s s a y I s o g e n i c s t r a i n s o f E. c o l i K-12 r e c o m b i n a t i o n d e f i c i e n t ( r e c ) m u t a n t s were o b t a i n e d from D r . A. J . C l a r k e , U n i v e r s i t y of C a l i f o r n i a , B e r k e l e y . The s t r a i n s and t h e i r r e l e v a n t c h a r a c t e r i s t i c s a r e l i s t e d i n T a b l e 9. A l l s t r a i n s were grown i n n u t r i e n t b r o t h a t 37°C w i t h s h a k i n g a t 250 rpm. O v e r n i g h t c u l t u r e s were d i l u t e d 1 i n 100 and 0.1 ml was s p r e a d o n t o n u t r i e n t a g a r p l a t e s . S i x p l a t e s were p r e p a r e d f o r e a c h s t r a i n . S t e r i l e Whatman #1 f i l t e r p a p e r d i s c s w h i c h had been p r e v i o u s l y l o a d e d w i t h a l p h a - T (10 u g / d i s c ) , BBT (10 u g / d i s c ) , b l e o m y c i n (15 u g / d i s c ) and 8-MOP (20 u g / d i s c ) were p l a c e d o n t o t h e a g a r p l a t e s w h i c h were t h e n i n c u b a t e d f o r 1 hour a t 37°C. T h r e e r e p l i c a t e p l a t e s were kept d a r k f o r c o n t r o l s w h i l e t h e o t h e r t h r e e r e p l i c a t e p l a t e s were i r r a d i a t e d by f o u r S y l v a n i a F20T12/BLB lamps f o r 1 h o u r . AIT p l a t e s were t h e n i n c u b a t e d i n t h e d a r k f o r 48 h o u r s a t 37°C. I n h i b i t o r y z o n e s (edge t o edge) were measured i n m i l l i m e t e r s . The e x p e r i m e n t was r e p e a t e d t h r e e t i m e s on s e p a r a t e d a y s . R e p r e s e n t a t i v e d a t a from one e x p e r i m e n t i s p r e s e n t e d w i t h s t a n d a r d d e v i a t i o n s . S c a v e n g e r S t u d i e s A l l p r o c e d u r e s r e g a r d i n g p r e p a r a t i o n Of E. c o l i B c u l t u r e s , i r r a d i a t i o n and p l a t i n g f o r v i a b l e c e l l c o u n t s were d e s c r i b e d p r e v i o u s l y ( s e e A s s a y P r o c e d u r e ) . S t o c k s o l u t i o n s o f 1M s o d i u m a z i d e and 50 mM b u t y l a t e d h y d r o x y t o l u e n e (BHT) were p r e p a r e d f r e s h f o r a l l e x p e r i m e n t s by d i s s o l v i n g t h e 58 T a b l e 9. S t r a i n s of E. c o l i u s e d and t h e i r r e l e v a n t c h a r a c t e r i s t i c ! CBachmann and Low, 1980). S t r a i n P a r e n t P h e n o t y p i c T r a i t ' AB 1 157 AB 1133(Kl2) w i l d t y p e JC 2926 AB 1 157 r e c A" JC 3881 AB 1 157 r e c A,B,C" JC 5519 AB 1 1 57 r e c B,C" JC 5547 AB 1 1 57 r e c B,C,F" JC 9239 AB 1 1 57 r e c F" a p p r o p r i a t e c h e m i c a l i n d i s t i l l e d water and 95% e t h a n o l , r e s p e c t i v e l y . The a z i d e s o l u t i o n was' f i l t e r s t e r i l i z e d ( M i l l i p o r e , Type HA) w h i l e t h e BHT s o l u t i o n was u s e d w i t h o u t f u r t h e r s t e r i l i z a t i o n . A l i q u o t s of e a c h s t o c k s o l u t i o n were added a s e p t i c a l l y t o c u l t u r e s y i e l d i n g f i n a l c o n c e n t r a t i o n s of 20 mM sodium a z i d e and 50 uM BHT. The s c a v e n g e r s were added t o b a c t e r i a l s u s p e n s i o n s 15 m i n u t e s a f t e r a l p h a - T a d d i t i o n and f o l l o w e d by 15 m i n u t e s i n c u b a t i o n i n t h e d a r k b e f o r e i r r a d i a t i o n . A l l e x p e r i m e n t s were r e p e a t e d a t l e a s t t h r e e t i m e s . The d a t a p r e s e n t e d a r e r e p r e s e n t a t i v e . T e m p e r a t u r e S t u d i e s A l l p r o c e d u r e s r e g a r d i n g p r e p a r a t i o n o f E . c o l i B c u l t u r e s , i r r a d i a t i o n and s u r v i v a l a s s a y were d e s c r i b e d p r e v i o u s l y ( s e e A s s a y P r o c e d u r e ) . C e l l s were i n o c u l a t e d i n t o t h e r e a c t i o n f l a s k c o n t a i n i n g a i r s a t u r a t e d BM2 g r o w t h medium a t 37°C and i n c u b a t e d f o r 30 m i n u t e s . A l p h a - T (1 ug/ml) i n 95% 59 e t h a n o l was t h e n added and t h e c e l l s i n c u b a t e d i n t h e d a r k f o r a f u r t h e r 30 m i n u t e s . The t e m p e r a t u r e o f t h e c u l t u r e was t h e n a d j u s t e d t o - t h e d e s i r e d i r r a d i a t i o n t e m p e r a t u r e and e q u i l i b r a t e d f o r 15 m i n u t e s p r i o r t o i r r a d i a t i o n f o r 30 m i n u t e s . The e f f e c t of i n c u b a t i o n t e m p e r a t u r e on t h e p h o t o s e n s i t i z e d i n a c t i v a t i o n o f E . c o l i by a l p h a - T was i n v e s t i g a t e d by l o w e r i n g t h e c u l t u r e t e m p e r a t u r e t o 5°C f o r 15 m i n u t e s b e f o r e t h e a d d i t i o n o f a l p h a - T . F o l l o w i n g t h i s p r e c h i l l and 30 m i n u t e s i n c u b a t i o n w i t h a l p h a - T a t 5°C i n t h e d a r k , t h e c u l t u r e was i r r a d i a t e d a t 5°C and c e l l s u r v i v a l d e t e r m i n e d . A l l g r a p h s r e p r e s e n t a t l e a s t t h r e e s e p a r a t e e x p e r i m e n t s . C r o s s l i n k i n q o f E. c o l i P r o t e i n s C u l t u r e s of E. c o l i B were grown o v e r n i g h t t o s t a t i o n a r y phase a t 37°C w i t h s h a k i n g i n BM2 c o n t a i n i n g 0.4% g l u c o s e . Two, 10 ml p o r t i o n s were s u b c u l t u r e d i n 990 ml o f f r e s h BM2 s e p a r a t e l y and grown t o ah o p t i c a l d e n s i t y o f 0.6 a t 520 nm. The c e l l s were h a r v e s t e d by c e n t r i f u g a t i o n a t 8000 rpm f o r 10 m i n u t e s and r e s u s p e n d e d i n 100 ml o f 10 mM p h o s p h a t e b u f f e r pH 7.0. The r e s u l t i n g s u s p e n s i o n ( a p p r o x i m a t e l y 3 x 10 9 c e l l s / m l ) was i n c u b a t e d w i t h slow s h a k i n g f o r 15 m i n u t e s a t 37°C p r i o r t o th e a d d i t i o n of 1 ug/ml or of a l p h a - T i n 95% e t h a n o l . The s u s p e n s i o n was t h e n i n c u b a t e d f o r 30 m i n u t e s and s u b s e q u e n t l y i r r a d i a t e d by two h o r i z o n t a l S y l v a n i a F15T8/BL lamps s u s p e n d e d 20 cm above t h e s u s p e n s i o n s . Ten ml a l i q u o t s were removed p r i o r t o a l p h a - T a d d i t i o n , a f t e r 30 m i n u t e s i n c u b a t i o n w i t h a l p h a - T 60 and f o l l o w i n g 15 and 30 m i n u t e s UV-A i r r a d i a t i o n . V i a b l e c e l l c o u n t s were d e t e r m i n e d f rom e a c h of t h e sa m p l e s w h i c h were k e p t d a r k a n d on i c e d u r i n g t h e c o u r s e o f t h e e x p e r i m e n t . C e l l s were b r o k e n i n a F r e n c h p r e s s a t 14,000 p s i a f t e r t h e a d d i t i o n o f p a n c r e a t i c DNase I (20 ug/ml; S i g m a ) . The r e s u l t i n g b r o k e n c e l l s u s p e n s i o n s were c e n t r i f u g e d a t 10,000 x g f o r 1 5 m i n u t e s t o remove t h e r e m a i n i n g i n t a c t c e l l s . The s u s p e n s i o n was t h e n c e n t r i f u g e d a t 180,000 x g f o r 1 hour i n a Beckman 50 T i r o t o r . The p e l l e t c o n t a i n i n g membranes and r i b o s o m e s was r e s u s p e n d e d i n 0.5 ml d i s t i l l e d w a t e r . The s u p e r n a t a n t c o n t a i n i n g s o l u b l e o r c y t o p l a s m i c p r o t e i n s was e t h a n o l p r e c i p i t a t e d w i t h 0.5% N a C l and t w i c e t h e volume o f e t h a n o l (18 m l ) . The s o l u b l e p r o t e i n s were c o l l e c t e d a f t e r o v e r n i g h t p r e c i p i t a t i o n a t -20°C by c e n t r i f u g a t i o n i n an E p p e n d o r f Model 54-12 c e n t r i f u g e . S D S - P o l y a c r y l a m i d e G e l E l e c t r o p h o r e s i s S l a b g e l e l e c t r o p h o r e s i s was u s e d t o s e p a r a t e t h e s o l u b l e and p e l l e t a b l e p r o t e i n s of E. c o l i f o l l o w i n g i r r a d i a t i o n i n t h e p r e s e n c e of a l p h a - T . The b a s i c method was p r e v i o u s l y d e s c r i b e d by L u g t e n b e r g e t a l . ( 1 9 7 5 ) . S e p a r a t i n g o r l o w e r g e l s o f 7% ( v o l / v o l ) a c r y l a m i d e were p r e p a r e d from a s t o c k a c r y l a m i d e s o l u t i o n c o n t a i n i n g 30% ( w t / v o l ) a c r y l a m i d e and 0.8% ( w t / v o l ) N , N ' - m e t h y l e n e b i s a c r y l a m i d e . The g e l s were p o l y m e r i z e d by t h e a d d i t i o n ( p e r 12.5 ml o f g e l mix) o f 20 u l o f TEMED (N,N,N',N'-t e t r a m e t h y l e t h y l e n e d i a m i n e ) and 0.165 ml of 1% ( w t / v o l ) ammonium p e r s u l f a t e . The s t a c k i n g or upper g e l s were a l s o 61 p r e p a r e d f r o m t h e 30:0.8 a c r y l a m i d e s t o c k and was p o l y m e r i z e d by t h e a d d i t i o n ( p e r 5 ml of g e l mix) of 10 u l TEMED and 0.12 ml o f 1% ammonium p e r s u l f a t e . P r o t e i n s amples were a s s a y e d by t h e method of Sandermann and S t r o m i n g e r ( 1 9 7 2 ) . S o l u b i l i z a t i o n o f p r o t e i n s was a c c o m p l i s h e d by d i l u t i o n o f t h e sample i n an e q u a l volume of s o l u b i l i z a t i o n r e d u c t i o n mix c o n t a i n i n g 4% ( w t / v o l ) SDS, 10% ( v o l / v o l ) 2 - m e r c a p t o e t h a n o l , t w o f o l d - c o n c e n t r a t e d u p p e r g e l b u f f e r and 20% ( v o l / v o l ) g l y c e r o l f o l l o w e d by h e a t i n g a t 88°C f o r 10 m i n u t e s i n a Temp-Block module h e a t e r (Lab L i n e I n s t r u m e n t s I n c . , M e l r o s e P a r k , 111.) f i l l e d w i t h g l y e r o l . A p p r o x i m a t e l y 20 ug o f p r o t e i n was added p e r w e l l . The g e l s were run f o r 3 t o 3-1/2 h o u r s a t a c o n s t a n t v o l t a g e o f 150 v o l t s . The s e p a r a t e d p r o t e i n s were s t a i n e d u s i n g C o o m a s s i e B r i l l i a n t B l u e (Hancock and C a r e y , 1979). RESULTS AND DISCUSSION S u r v i v a l C u r v e s The s y n e r g i s t i c a c t i o n o f a l p h a - T and UV-A i r r a d i a t i o n o r BBT and UV-A on t h e s u r v i v a l of E. c o l i i s d e m o n s t r a t e d i n F i g u r e s 15 and 16. The d i f f e r e n c e s i n c e l l u l a r i n a c t i v a t i o n c a u s e d by t h e s e b i o s y n t h e t i c a l l y r e l a t e d t h i o p h e n e s s u g g e s t t h a t t h e compounds may have d i f f e r e n t mechanisms o f a c t i o n . F i g u r e 15 shows t h e s u r v i v a l o f c e l l s e x p o s e d t o v a r y i n g d o s e s of a l p h a - T and UV-A. A l l o f t h e c u r v e s had a p r o n o u n c e d l a g o r s h o u l d e r a f t e r t h e s t a r t of i r r a d i a t i o n w h i c h was f o l l o w e d by Figure 15. Survival curves of g. c o l i B-23<+> in resp various doses of alpha-T and UV-A i r r a d i a t i o n . 63 an e x p o n e n t i a l d e c l i n e i n c e l l v i a b i l i t y . T h i s t y p e o f s u r v i v a l i s s i m i l a r t o t h e a c t i o n o f many p h o t o s e n s i t i z e r s on m i c r o o r g a n i s m s ( O g i n s k y e t a_l. , 1959; S m i t h and H a n a w a l t , 1969; G r o s s w e i n e r and S m i t h , 1981; G r o v e r e_t a l . , 1981) and has been i n t e r p r e t e d t o mean t h a t c e l l s were e i t h e r c a p a b l e of a c c u m u l a t i n g c o n s i d e r a b l e damage b e f o r e d e a t h o r t h a t t h e y c o u l d r e p a i r i n c u r r e d c e l l u l a r damage b e f o r e a c r i t i c a l p o i n t when e x p o n e n t i a l k i l l i n g was a l l o w e d t o p r o c e e d (Zimmer, 1961; E l k i n d and Whitmore, 1967; C l a y t o n , 1971). I t was o b s e r v e d d u r i n g t h e s e and o t h e r e x p e r i m e n t s w i t h a l p h a - T and E. c o l i t h a t c o l o n i e s r e s u l t i n g f r o m t r e a t e d c e l l s d i s p l a y e d c o n s i d e r a b l e s i z e v a r i a t i o n . T h i s v a r i a t i o n may be a r e f l e c t i o n of t h e e x t e n t of damage i n c u r r e d by c e l l s d u r i n g i r r a d i a t i o n . I n a c t i v a t i o n o f c e l l s by BBT and UV-A ( F i g u r e 16) was f o u n d t o be q u i t e d i f f e r e n t from t h a t c a u s e d by a l p h a - T p l u s i r r a d i a t i o n . S u r v i v a l c u r v e s showed immediate e x p o n e n t i a l d e c l i n e i n c e l l v i a b i l i t y w h i c h may i n d i c a t e t h a t t h e c e l l u l a r t a r g e t a n d / o r mechanism o f p h o t o s e n s i t i z e d k i l l i n g i s d i s t i n c t f rom t h a t o f a l p h a - T . BBT, u n l i k e a l p h a - T , c o n t a i n s an u n s a t u r a t e d , f o u r c a r b o n s i d e c h a i n w h i c h r e s e m b l e s t h e s t r u c t u r e of a p o l y a c e t y l e n e from w h i c h t h e t h i o p h e n e s i n n a t u r e a r e u l t i m a t e l y d e r i v e d (Bohlmann e_t a l . , 1973). The p o l y a c e t y l e n e s a r e b e l i e v e d t o r e p r e s e n t a new c l a s s o f n a t u r a l p h o t o s e n s i t i z e r s (Towers, 1980). P h e n y l h e p t a t r i y n e (PHT), w h i c h has been s t u d i e d i n t h e most d e t a i l , does not show e n h a n c e d p h o t o a c t i v i t y t o E. c o l i i n oxygen ( A r n a s o n e t a l . , 1980) and doe s not form i n t e r s t r a n d Figure 16. Survival curves of E. c o l i B-23(+) in response various doses of BBT and UV-A i r r a d i a t i o n . 65 l i n k a g e s w i t h DNA (Wat e t a l . , 1977) l i k e t h e f u r o c o u m a r i n s . B a s e d on t h e s e and o t h e r d a t a t o be p r e s e n t e d , BBT a p p e a r s t o d i s p l a y an i n t e r m e d i a t e r e a c t i o n mechanism between t h a t o f a l p h a - T and t h a t of PHT. A c t i o n S p e c t r u m The a c t i o n s p e c t r u m f o r E. c o l i s u r v i v a l f o l l o w i n g t r e a t m e n t w i t h a l p h a - T and UV-A v e r s u s t h e a b s o r p t i o n s p e c t r u m f o r a l p h a - T i s shown i n F i g u r e 17. A c l o s e a g r e e m e n t between a c t i v i t y and l i g h t a b s o r p t i o n was o b s e r v e d . I n b o t h c u r v e s , t h e maximum was a t 350 nm w i t h d e c l i n i n g ' e f f e c t i v e n e s s a t s h o r t e r and l o n g e r w a v e l e n g t h s . T h e s e c u r v e s p r o v i d e good e v i d e n c e t h a t a l p h a - T , r a t h e r t h a n a p h o t o p r o d u c t formed d u r i n g i r r a d i a t i o n , was t h e p r i m a r y a b s o r b i n g m o l e c u l e r e s p o n s i b l e f o r t h e p h o t o s e n s i t i z e d r e a c t i o n . Had a p h o t o p r o d u c t been formed a d i f f e r e n t a c t i o n s p e c t r u m w o u l d have r e s u l t e d w h i c h would have r e s e m b l e d t h e a b s o r p t i o n s p e c t r u m o f t h e p h o t o p r o d u c t . T h i s r e s u l t a g r e e s w i t h t h e a c t i o n s p e c t r u m o f a l p h a - T on m o s q u i t o l a r v a e ( A r n a s o n e t a_l. , 1980). The maximum w a v e l e n g t h f o u n d i n t h a t s t u d y was 360 nm. The 10 nm d i f f e r e n c e c o u l d be due t o l i g h t s c a t t e r i n g amongst l a r v a e . T h i s w o u l d be g r e a t l y r e d u c e d by u s i n g v e r y d i l u t e s u s p e n s i o n s of E . c o l i c e l l s . 66 Wavelength Figure 17. The photoinduced b a c t e r i c i d a l action (o o> on E. coJi. B compared with the absorption <• •) 0 f alpha-T at di f f e r e n t wavelengths between 320nm and 400nm. 67 S t u d i e s on R e p a i r Def i c i e n t M u t a n t s of E. c o l i The i n a b i l i t y of v a r i o u s m u t a n t s of-. E. c o l i t o p e r f o r m g e n e t i c r e c o m b i n a t i o n o r p o s t - r e p l i c a t i o n r e p a i r was u s e d t o i n d i c a t e whether DNA damage c o u l d be c a u s e d by t h e a c t i o n of a l p h a - T o r BBT. R e c o m b i n a t i o n d e f i c i e n t ( r e c ) m u t a n t s a r e u n a b l e t o i n c o r p o r a t e e x t r a c h r o m o s o m a l DNA i n t o t h e i r genome or t o r e p a i r damaged s i t e s on t h e DNA ( S m i t h , 1977). T h e s e damaged s i t e s c o u l d i n c l u d e p y r i m i d i n e d i m e r s , monoadducts o r c r o s s l i n k a g e s i n t h e DNA s t r a n d o r n i c k s i n t h e DNA. I t was t h o u g h t t h a t r e c o m b i n a t i o n m u t a n t s would show g r e a t e r s e n s i t i v i t y t o compounds w h i c h damage DNA t h a n w i l d t y p e c e l l s w h i c h were c a p a b l e of r e p a i r . T h i s was shown t o be t h e c a s e i n th e e x p e r i m e n t s r e p o r t e d h e r e w i t h b l e o m y c i n and 8-MOP ( T a b l e 10), two compounds wh i c h have been shown t o r e a c t w i t h DNA. B l e o m y c i n , w h i c h c a u s e s t h e d e g r a d a t i o n o f i n t r a c e l l u l a r DNA ( O n i s h i e t a l . , 1973), i n h i b i t s DNA s y n t h e s i s ( S u z u k i e_t a l . , 1968), and i n d u c e s t h e s y n t h e s i s o f r e c A p r o t e i n i n E. c o l i (Gudas and P a r d e e , 1976), was u s e d as a n o n - p h o t o a c t i v a t e d c o n t r o l . G r e a t e r z o n e s o f i n h i b i t i o n were e x h i b i t e d by a l l of th e r e c ~ s t r a i n s a s compared w i t h w i l d t y p e (AB 1157). T h i s r e s u l t a g r e e s w i t h a r e c e n t s t u d y w h i c h showed t h a t g r o w i n g c u l t u r e s o f r e p a i r d e f i c i e n t s t r a i n s o f E. c o l i were more s e n s i t i v e t o t h e e f f e c t o f b l e o m y c i n (Yamamoto and H u t c h i n s o n , 1979) t h a n w i l d t y p e . 8-MOP, on t h e o t h e r hand, w h i c h k i l l s m i c r o o r g a n i s m s v i a p h o t o i n d u c e d monoadduct f o r m a t i o n and i n t e r s t r a n d c r o s s l i n k a g e s w i t h DNA (Song and T a p l e y , 1979), was s e l e c t e d a s a p h o t o a c t i v a t i b l e c o n t r o l . T h i s DNA i n t e r c a l a t o r 68 T a b l e 10. Response of v a r i o u s E . c o l i r e c mut a n t s t o a l p h a - T , BBT, b l e o m y c i n , and 8-MOP i n UV-A and d a r k . I n h i b i t o r y z o n e s (mm) a r e f o l l o w e d by s t a n d a r d d e v i a t i o n s i n p a r e n t h e s e s . S t r a i n A l p h a - T UV-A BBT UV-A UV-A Bleomyc i n Dark 8-MOP UV-A AB 1 1 57 15. 5(0 .9) 20. 7(0 .9) 20. 7(1 .2) 21 .0(0 .8) 10. 0(0 .8) JC 2926 17. 3(1 .2) 26. 3(0 .9). 31 . 7(0 .5) 28 .7(0 .9) 19. 0(0 .8) JC 3881 16. 3(0 .5) 25. 0(0 .0) 32. 7(0 .5) 27 .3(2 .5) 20. 0(0 .0) JC 5519 16. 0(0 .8) 25. 0(0 .0) 28. 7(1 .2) 26 .7(1 .2) 16. 0(0 .8) JC 5547 16. 3(0 .9) 24. 7(1 .9) 32. 7(2 .1) 24 .7(2 .6) 15. 3(0 .5) JC 9239 14. 7(0 .5) 22. 7(0 .9) 24. 2(0 .5) 21 .7(0 .5) 15. 3(0 .5) a l s o i n h i b i t e d mutant s t r a i n s more t h a n s t r a i n AB 1157, t h e w i l d t y p e . T h i s f i n d i n g a g r e e s w i t h t h e r e s u l t s of G r o s s w e i n e r and S m i t h (1981) who r e c e n t l y showed t h a t r e p a i r d e f i c i e n t s t r a i n s of E. c o l i were more s e n s i t i v e t o 8-MOP and UV-A t h a n w i l d t y p e c e l l s . The d a t a f u r t h e r show t h a t t h e r e c ~ s t r a i n s and t h e p a r e n t a l w i l d t y p e (AB 1157) were e q u a l l y s e n s i t i v e t o a l p h a - T . Had t h e - m u t a n t s been more s e n s i t i v e t h a n AB 1157 t h e n t h i s would have been good e v i d e n c e f o r a l p h a - T damage t o DNA. T h i s a g r e e s w i t h one o t h e r p u b l i s h e d s t u d y by MacRae e_t a_l. (1980a) who showed t h a t a l p h a - T and UV-A do n o t i n c r e a s e t h e f r e q u e n c y of chromosomal a b e r a t i o n s i n c u l t u r e d S y r i a n h amster c e l l s . In a n o t h e r s t u d y , however, Kagan's g r o u p (Kagan e_t a l . , 1980) fo u n d t h e o p p o s i t e r e s u l t , t h a t a l p h a - T d i d r e a c t w i t h c a l f thymus DNA i n v i t r o and C a n d i d a u t i l i s DNA i n v i v o . T h i s 69 f i n d i n g may be e x p l a i n e d by t h e h i g h c o n c e n t r a t i o n s of a l p h a - T w h i c h were u s e d (40 ug/ml as compared w i t h 1 ug/ml u s e d i n t h e s e s t u d i e s ) a l o n g w i t h t h e l o n g i r r a d i a t i o n t i m e s (2 t o 4 h o u r s ) . I t can be a r g u e d t h a t a l p h a - T may r e a c t w i t h DNA g i v e n a s u f f i c i e n t c o n c e n t r a t i o n and i r r a d i a t i o n t i m e , however e v i d e n c e f o r r e a c t i o n o f a l p h a - T w i t h DNA was n o t f o u n d i n t h i s i n v i v o s t u d y u s i n g b a c t e r i c i d a l c o n c e n t r a t i o n s . BBT, a l t h o u g h b i o s y n t h e t i c a l l y r e l a t e d t o a l p h a - T , e l i c i t e d a v e r y d i f f e r e n t r e s p o n s e from t h e E . c o l i m u t a n t s . A l l r e c ~ s t r a i n s e x c e p t JC 9239 ( r e c F) were more s e n s i t i v e t o BBT and UV-A th a n t h e w i l d t y p e (AB 1157). T h i s may i n d i c a t e t h a t t h e mechanism of a c t i o n o f BBT c o u l d i n v o l v e DNA damage i n c e l l u l a r k i l l i n g . No i n h i b i t o r y z o n e s were formed i n r e s p o n s e t o any o f t h e p h o t o a c t i v a t e d compounds i n t h e d a r k . I n t e r c a l a t i o n o f 8-MOP i n t o DNA i n t h e d a r k may c a u s e t h e i n h i b i t i o n o f DNA s y n t h e s i s (Song and T a p l e y , 1979), b u t i n h i b i t o r y z o n e s were not f o u n d w i t h t h e c o n c e n t r a t i o n of 8-MOP u s e d i n t h i s s t u d y . A e r o b i c - A n a e r o b i c S t u d i e s The p h o t o i n d u c e d a n t i b i o t i c a c t i v i t y o f a l p h a - T was f o u n d t o o c c u r o n l y i n t h e p r e s e n c e of a i r o r oxygen and was i n d e p e n d e n t of t h e a e r o b i c or a n a e r o b i c s t a t u s o f c e l l s j u s t p r i o r t o i r r a d i a t i o n . C u l t u r e s o f E . c o l i grown and i r r a d i a t e d a e r o b i c a l l y i n e i t h e r a i r o r He/02 (4:1) i n t h e p r e s e n c e o f a l p h a - T showed a marked d e c r e a s e i n c e l l v i a b i l i t y ( F i g u r e 1 8 ) . 70 The t r e a t m e n t o f a n a e r o b i c a l l y grown c e l l s i r r a d i a t e d u nder He w i t h UV-A and a l p h a - T c a u s e d no d e c r e a s e i n c e l l s u r v i v a l ( F i g u r e 19). C e l l s grown i n He and t r a n s f e r r e d t o an a i r a t m o s p h e r e d u r i n g i r r a d i a t i o n were k i l l e d i n t h e p r e s e n c e o f a l p h a - T . T h e r e was a b r i e f d e l a y i n t h e s t a r t o f k i l l i n g , however, w h i c h was p r o b a b l y r e l a t e d t o t h e d i f f u s i o n r a t e of o x y g e n i n t h e s u s p e n s i o n . I n c o n t r a s t , c e l l s r e s p i r i n g a e r o b i c a l l y were p r o t e c t e d f r o m t h e e f f e c t s of a l p h a - T by t h e i n t r o d u c t i o n o f He p r i o r t o i r r a d i a t i o n . S i m i l a r r e s u l t s were o b t a i n e d w i t h Pseudomonas a e r u g i n o s a ( F i g u r e 2 0 ) , a l t h o u g h h i g h e r d o s e s o f a l p h a - T (2.5 ug/ml) were n e c e s s a r y t o y i e l d e q u i v a l e n t d e c r e a s e s i n c e l l v i a b i l i t y . C o n t r o l c o n d i t i o n s w i t h a l p h a - T i n t h e d a r k or UV-A i r r a d i a t i o n o f c e l l s i n t h e a b s e n c e of a l p h a - T may have had a s m a l l e f f e c t on t h e s u r v i v a l o f E. c o l i and P. a e r u g i n o s a . J a g g e r (1976) has d e m o n s t r a t e d t h a t e x p o s u r e of E. c o l i t o UV-A i n d u c e s growth d e l a y w h i c h may have o c c u r r e d i n t h e s e s t u d i e s , however t h i s r e s p o n s e was n o t i n v e s t i g a t e d . The r e q u i r e m e n t of oxygen f o r p h o t o t o x i c i t y of a l p h a - T i s c o n s i s t e n t w i t h a Type I I r e a c t i o n mechanism ( K r i n s k y , 1977; S m i t h , 1977). The s e n s i t i v i t y o f E . c o l i a n d P. a e r u g i n o s a t o t r e a t m e n t w i t h a l p h a - T and UV-A was not f o u n d t o be a f u n c t i o n o f t h e t y p e of c e l l u l a r r e s p i r a t i o n i n d i f f e r e n t c u l t u r e s as r e c e n t l y s u g g e s t e d ( A r n a s o n e t a l . , 1981). I n s t e a d , c e l l u l a r i n a c t i v a t i o n was o n l y d e p e n d e n t on t h e a v a i l i b i l i t y of oxygen d u r i n g i r r a d i a t i o n . Wat e t a l . (1980) s t u d i e d t h e e f f e c t ' o f a l p h a - T and UV-A 71 Figure 18. The survival of aerobically grown E. c o l i B ir r a d i a t e d with UV-A in the presence of alpha-T under aerobic conditions. Alpha-T was added to cultures at 0 minutes and i r r a d i a t i o n was started at 30 minutes. 72 Anaerobic Conditions aT(l ug/ml) UV-A Air -Jr 1 - J - 1 1 1 I I l_ O 20 40 60 BO Time (minutes) Figure 19. The survival of anaerobically grown E. c o l i B ir r a d i a t e d with UV-A in the presence of alpha-T under aerobic and anaerobic conditions. Alpha-T was added to cultures at 0 minutes and i r r a d i a t i o n was started at 30 minutes. C e l l s i n i t i a l l y in He < o — — o) were transferred to a i r at 60 minutes. C e l l s i n i t i a l l y in a i r ( a a) were switched to He at 30 minutes. 73 a7(2.5^/ro/) UV-A P. aeruginosa o o CD '5 "4 -5 He \ \ \ \ ^ Air 20 40 Time (minutes) 60 Figure 20. The survival of Pseudomonas aeruoinosa in the presence of alpha-T and UV-A i r r a d i a t i o n under aerobic and anaerobic conditions. Alpha-T was added to cultures at 0 minutes and i r r a d i a t i o n was started at 30 minutes. 74 i r r a d i a t i o n on human e r y t h r o c y t e s . T h e i r r e s u l t s c l e a r l y showed t h a t b o t h h e m o l y s i s and RBC a c e t y l c h o l i n e s t e r a s e i n a c t i v a t i o n were oxygen d e p e n d e n t . A r n a s o n e_t a l . (1981) a l s o d e m o n s t r a t e d t h a t t h e s u r v i v a l o f s t a r v e d , s t a t i o n a r y phase c e l l s i r r a d i a t e d i n t h e p r e s e n c e of a l p h a - T was oxygen d e p e n d e n t . ' K a g a n e_t a l . ( 1 9 8 0 ) , on t h e o t h e r hand, c o n c l u d e d t h a t a l p h a - T was a non-p h o t o d y n a m i c p h o t o t o x i c compound o r a Type I p h o t o s e n s i t i z e r . T h i s r e s u l t c a n n o t be e x p l a i n e d i n view of t h e f i n d i n g s p r e s e n t e d h e r e . The a c t i o n of a l p h a - T was u n e q u i v o c a l l y d e p e n d e n t on oxygen under t h e c o n d i t i o n s o f t h e e x p e r i m e n t s r e p o r t e d . E f f e c t of S c a v e n g e r s on A l p h a - T P h o t o t o x i c i t y The i n a c t i v a t i o n r a t e o f E. c o l i c e l l s e x p o s e d t o a l p h a - T (1 ug/ml) and UV-A i r r a d i a t i o n i n t h e p r e s e n c e o f 20 mM a z i d e ( F i g u r e 21) was l e s s t h a n t h e k i l l i n g r a t e o f c e l l s i n t h e a b s e n c e o f a z i d e ( c o n t r o l ) . The most o b v i o u s d i f f e r e n c e i n v o l v e d t h e s u r v i v a l o f a z i d e t r e a t e d c e l l s . C o n t r o l c u l t u r e s showed more t h a n two o r d e r s of m a g n i t u d e g r e a t e r i n a c t i v a t i o n f o l l o w i n g 10 m i n u t e s i r r a d i a t i o n t h a n c e l l s i n t h e p r e s e n c e o f a z i d e . A z i d e , an e f f e c t i v e q u e n c h e r of s i n g l e t oxygen ir\ v i t r o ( H a s t y e t a l . , 1972) and jjn v i v o ( I t o , 1978), was n o t f o u n d t o a f f e c t c e l l v i a b i l i t y d u r i n g t h e 15 m i n u t e s d a r k p e r i o d p r i o r t o UV-A e x p o s u r e . C e l l u l a r p r o t e c t i o n i n d i c a t e s t h a t s i n g l e t oxygen may be i n v o l v e d i n t h e p h o t o t o x i c mechanism of a l p h a - T ( I t o , 1978). A r n a s o n e t a_l. (1981) a l s o d e m o n s t r a t e d p r o t e c t i o n o f s t a r v e d , s t a t i o n a r y p h a s e E. c o l i c e l l s f r o m a l p h a - T and UV-75 <y(lMs /m/) UV-A E. coli B •J 1 1 1 i i i _ 0 20 40 60 Time (minutes) Figure 21. The effect of 20 mM sodium azide on the surv i v a l of E. c o l i B in the presence of alpha-T and UV-A i r r a d i a t i o n . Alpha-T was added to cultures at 0 minutes* azide at 15 minutes/ and i r r a d i a t i o n was started at 30 minutes. 76 A by 23 mM a z i d e . Kagan e t a l . ( 1 9 8 0 ) , on t h e o t h e r hand, f o u n d no p r o t e c t i o n of C a n d i d a u t i l i s by 0.153 mM a z i d e and c o n c l u d e d t h a t c e l l u l a r k i l l i n g by a l p h a - T d i d n o t i n v o l v e s i n g l e t o x y g e n . They s t u d i e d c e l l u l a r g r o w t h r a t e s r a t h e r t h a n i n a c t i v a t i o n r a t e s , w h i c h made i t n e c e s s a r y f o r them t o use a s u b - i n h i b i t o r y c o n c e n t r a t i o n o f a z i d e (0.153 mM). T h i s c o n c e n t r a t i o n was a p p r o x i m a t e l y 100 t i m e s l o w e r t h a n t h a t recommended by I t o (1978) t o d e m o n s t r a t e _in v i v o p r o t e c t i o n f r o m s i n g l e t o x ygen. Kagan's g r o u p was u n a b l e t o use h i g h e r c o n c e n t r a t i o n s of t h i s c y t o t o x i c compound b e c a u s e o f i t s i n h i b i t o r y e f f e c t on r e s p i r a t i o n ( i . e . g r o w t h ) . I n a c t i v a t i o n r a t e s r a t h e r t h a n g r o w t h r a t e s were measured i n t h e e x p e r i m e n t s r e p o r t e d h e r e b e c a u s e o f t h i s e f f e c t . BHT, a f r e e r a d i c a l s c a v e n g e r ( F o o t e , 1976), was a l s o a d d e d t o E. c o l i s u s p e n s i o n s t o d e t e r m i n e i t s e f f e c t on t h e p h o t o s e n s i t i z e d r e a c t i o n ( F i g u r e 2 2 ) . The e f f e c t of BHT on c e l l s u r v i v a l was l e s s d r a m a t i c t h a n w i t h a z i d e , a l t h o u g h a s i g n i f i c a n t r e d u c t i o n i n t h e r a t e of k i l l i n g o v e r t h e i r r a d i a t i o n p e r i o d was o b s e r v e d . T h i s i n d i c a t e s t h e i n v o l v e m e n t of a Type I or r a d i c a l component i n t h e p h o t o t o x i c mechanism of a l p h a - T and UV-A. The e x i s t e n c e of a r a d i c a l mechanism was o r i g i n a l l y i n d i c a t e d by t h e work o f A r n a s o n e t a_l. ( 1 9 8 1 ) , who showed t h a t E. c o l i , i n t h e p r e s e n c e of a l p h a - T and UV-A was p r o t e c t e d by t h e exogenous a d d i t i o n of s u p e r o x i d e d i s m u t a s e . T h i s a l t e r n a t e pathway was f u r t h e r i n d i c a t e d by t h e p r o t e c t i o n of BHT p r e s e n t e d h e r e . As w i t h a z i d e , no b a c t e r i c i d a l e f f e c t s were o b s e r v e d w i t h 50 uM BHT and a l p h a - T i n t h e d a r k p e r i o d 77 Figure 22. The su r v i v a l of E. c o l i B in the presence of alpha-T. UV-A i r r a d i a t i o n and 50 uM BHT. Alpha-T w a s added t o cultures at 0 minutes, BHT at 15 minutes, and i r r a d i a t i o n w a s started at 30 minutes. 78 preceding i r r a d i a t i o n . The p o s s i b i l i t y of enhanced c e l l u l a r protection by the simultaneous addition of 20 mM azide and 50 uM BHT was also investigated (Figure 23). Slight protection was observed, but differences in k i l l i n g rates occurred only during the f i r s t f i v e minutes of i r r a d i a t i o n . Protection above that observed with either scavenger alone was not found. The simultaneous presence of the three compounds (azide, BHT and alpha-T) may be toxic to the c e l l s , which could explain the lack of enhanced protection. This aspect, however, was not studied further. The p a r t i c i p a t i o n of singlet oxygen in the reaction mechanism of alpha-T has also been demonstrated in. v i t r o . Bakker e_t a l . (1979) showed that glucose-6-phosphate dehydrogenase inactivation by alpha-T was protected by various quenchers of singlet oxygen including azide, h i s t i d i n e , tryptophan, and methionine. They also reported enhanced enzymatic inactivation in D20 where the l i f e t i m e of singlet oxygen i s increased ten fold (Merkel et a l . , 1972). Temperature Studies The ef f e c t of i r r a d i a t i o n temperature on the photodynamic inac t i v a t i o n of E. c o l i by alpha-T was investigated (Figure 24). C e l l s grown and incubated with the s e n s i t i z e r at 37°C were irr a d i a t e d at various temperatures between 5°C and 42°C. In general, c e l l u l a r inactivation showed a temperature e f f e c t . Survival during i r r a d i a t i o n at 42°C showed s i n g l e - h i t kinetics and high rates of k i l l i n g . As temperatures were lowered the lag 79 -1 0) 0 o o it -3 c co E. coli B BHT+NaN, ~~ Control w 20 40 Time (minutes) 60 F i g u r e 23. The s u r v i v a l of E. c o l i B i n the presence o f a l p h a - T , UV-A i r r a d i a t i o n , 20 mM a z i d e and 50 uM B H T . A l p h a - T was added t o c u l t u r e s a t 0 minutes, a z i d e and B H T a t 15 minutes and i r r a d i a t i o n was s t a r t e d a t 30 minutes. 80 F i g u r e 24. The s u r v i v a l of E. c o l i B i n the presence of a l p h a - T and UV-A i r r a d i a t i o n a t d i f f e r e n t i r r a d i a t i o n t e m p e r a t u r e s between 5 and 42 C. A l l c u l t u r e s were i n c u b a t e d w i t h a l p h a - T a t 37 C f o r 30 minutes. The c u l t u r e temperatures were then a d j u s t e d t o the i r r a d i a t i o n t emperatures and a l l o w e d to e q u i l i b r a t e f o r 15 minutes p r i o r t o i r r a d i a t i o n (45 m i n u t e s ) . 81 or s h o u l d e r r e g i o n s o f t h e g r a p h s became more p r o n o u n c e d and were f o l l o w e d by s l o w e r k i l l i n g r a t e s . The most p r o t e c t i o n , o r g r e a t e s t r e s i s t a n c e t o i n a c t i v a t i o n , was a t 5°C. R e c e n t s t u d i e s by v a r i o u s w o r k e r s have shown in v i v o p h o t o t o x i c i t y r e s p o n s e s w h i c h a r e a f f e c t e d by t e m p e r a t u r e . Reduced i n a c t i v a t i o n r a t e s o f S a c c h a r o m y c e s c e r e v i s i a e were d e m o n s t r a t e d a t l o w e r t e m p e r a t u r e s i n r e s p o n s e t o p h o t o s e n s i t i z a t i o n by b o t h h e m a t o p o r p h y r i n (HP) and t o l u i d i n e b l u e (TB) ( S t e n s t r o m e t a l . , 1980; I t o , 1981). The same t r e n d was a l s o s e e n i n an E . c o l i - T B s y s t e m (Wakayama e t a l . , 1980) and i n a human e r y t h r o c y t e - p r o t o p o r p h y r i n (PP) s y s t e m (Dubbelman e t a l . , 1980). F i g u r e 25 g i v e s t h e A r r h e n i u s r e l a t i o n s h i p between r a t e o f i n a c t i v a t i o n d u r i n g t h e l o g a r i t h m i c phase ( l o g o f i n a c t i v a t i o n r a t e , k) and t e m p e r a t u r e (1/°K) from t h e d a t a o f F i g u r e 24. A l i n e a r r e s p o n s e was o b t a i n e d f o r i n a c t i v a t i o n between 5°C and 37°C w i t h an a p p a r e n t a c t i v a t i o n e n e r g y o f 6.63 K c a l / m o l e . The r a t e of c e l l u l a r k i l l i n g a t 42°C d i d not show t h e l i n e a r n a t u r e of t h e o t h e r r a t e s , w h i c h s u g g e s t s t h e p o s s i b i l i t y o f t h e i n v o l v e m e n t of a d d i t i o n a l f a c t o r s p a r t i c i p a t i n g i n c e l l u l a r i n a c t i v a t i o n . T h e s e f a c t o r s may be r e l a t e d t o e f f e c t s of t e m p e r a t u r e on DNA, p r o t e i n s , o r membrane components. I t has been s u g g e s t e d t h a t , t e m p e r a t u r e e f f e c t s on p h o t o d y n a m i c k i l l i n g r e s u l t f r o m c h a n g e s i n t h e f l u i d i t y o f c y t o p l a s m i c membranes. I t o (1981) s t u d i e d t h e e f f e c t s o f t e m p e r a t u r e on p h o t o d y n a m i c k i l l i n g of S. c e r e v i s i a e by TB. An A r r h e n i u s p l o t o f t h e d a t a showed a s l i g h t d i s c o n t i n u i t y a t 21-82 O ~~* 33 3A 3J5 3 6 * do') Figure 25. The Arrhenius relationship of the data presented in Figure 24 (Ea =6.6 kcal/mole). 83 22°C; I t o (1981) s u g g e s t e d t h a t t h i s was a r e f l e c t i o n o f t h e c y t o p l a s m i c membrane phase t r a n s i t i o n . T h i s i s t h e t e m p e r a t u r e where t h e l i p i d m a t r i x c h a n g e s f r o m a l i q u i d c r y s t a l l i n e s t a t e t o a g e l o r o r d e r e d s t a t e . Cohn and T s e n g (1977) r e p o r t e d a s u b s t a n t i a l i n c r e a s e i n t h e p h o t o d y n a m i c k i l l i n g e f f i c i e n c y of S. c e r e v i s i a e by e o s i n Y a t 30°C w h i c h was n o t seen a t 27°C. They c o n c l u d e d t h a t t h e marked dependence of i n a c t i v a t i o n r a t e on t e m p e r a t u r e m i g h t have been c a u s e d by a t e m p e r a t u r e d e p e n d e n t i n c r e a s e i n membrane f l u i d i t y w h i c h e i t h e r a l l o w e d s i n g l e t oxygen t o m i g r a t e more r e a d i l y o r c a u s e d a t e m p e r a t u r e i n d u c e d change i n l i p i d - p r o t e i n i n t e r a c t i o n s o r i n membrane p r o t e i n c o n f o r m a t i o n w h i c h r e s u l t e d i n i n c r e a s e d s e n s i t i v i t y . E. c o l i c y t o p l a s m i c membranes show a v e r y b r o a d phase t r a n s i t i o n b e g i n n i n g a t a p p r o x i m a t e l y 10°C below t h e g r o w t h t e m p e r a t u r e (Nakayama e_t §_1. , 1980). No c r i t i c a l p o i n t was f o u n d n e a r t h i s t e m p e r a t u r e as c a n be seen i n F i g u r e 25 and t h e r e f o r e no d i r e c t r e l a t i o n s h i p between p h o t o d y n a m i c k i l l i n g of E . c o l i by a l p h a - T and membrane phase t r a n s i t i o n c o u l d be drawn i n t h e p r e s e n t s t u d y . A r e c e n t s t u d y a l s o i n d i c a t e s t h e p a r t i c i p a t i o n o f membranes i n t h e p h o t o d y n a m i c a c t i o n o f a l p h a - T . MacRae e t a l . (1980b) d e m o n s t r a t e d l e s i o n s i n t h e c y t o p l a s m i c membranes of human e r y t h r o c y t e s i r r a d i a t e d i n t h e p r e s e n c e of a l p h a - T . T h i s i n d i c a t i o n of membrane damage m i g h t r e f l e c t i n v o l v e m e n t of e i t h e r t h e l i p i d o r p r o t e i n components of membranes o r an i n t e r a c t i o n between t h e two. The e f f e c t of i n c u b a t i o n t e m p e r a t u r e 'on c e l l u l a r 84 i n a c t i v a t i o n by a l p h a - T was a l s o s t u d i e d . C u l t u r e s i n c u b a t e d w i t h a l p h a - T a t 5°C and 37°C f o r 30 m i n u t e s and s u b s e q u e n t l y i r r a d i a t e d a t 5°C gave n e a r l y i d e n t i c a l s u r v i v a l c u r v e s ( F i g u r e 2 6 ) . T h i s r e s u l t shows t h a t i n c u b a t i o n t e m p e r a t u r e i s a p p a r e n t l y n o t a c r i t i c a l f a c t o r f o r t h e p h o t o d y n a m i c a c t i o n o f a l p h a - T on E. c o l i . A l t h o u g h E. c o l i c y t o p l a s m i c membranes a t 5°C r e t a i n some f l u i d l i p i d d omains ( J a c k s o n and C r o n a n , 1978), t h e s i g n i f i c a n t . f l u i d i t y d i f f e r e n c e s between 5°C and 37°C p r e i n c u b a t e d membranes was n o t f o u n d t o i n f l u e n c e t h e s u b s e q u e n t a c t i v i t y of a l p h a - T . T h i s means t h a t t h e t e m p e r a t u r e e f f e c t s o b s e r v e d i n F i g u r e 24 do n o t r e s u l t from an i n h i b i t i o n of a l p h a - T t r a n s p o r t i n t o t h e c e l l a t t h e l o w e r t e m p e r a t u r e ( o t h e r w i s e t h e 37°C p r e i n c u b a t e d c e l l s i n F i g u r e 26 would more c l o s e l y r e s e m b l e t h e 37°C t r e a t e d c e l l s i n F i g u r e 2 4 ) . T h i s i s s u g g e s t i v e e v i d e n c e t h a t a l p h a - T need n o t n e c e s s a r i l y e n t e r t h e c e l l t o c a u s e c e l l d e a t h s u b s e q u e n t t o i r r a d i a t i o n . E f f e c t s of A l p h a - T on C e l l u l a r P r o t e i n s F i g u r e 27 shows an e l e c t r o p h e r o g r a m o f t h e s o l u b l e (A-C) and membrane a s s o c i a t e d (E-H) p r o t e i n s f r o m E. c o l i B b e f o r e and a f t e r t r e a t m e n t w i t h 1 ug/ml a l p h a - T and UV-A. The s o l u b l e p r o t e i n s b e f o r e a d d i t i o n (A) and a f t e r 30 m i n u t e s d a r k i n c u b a t i o n w i t h a l p h a - T (B) showed no o b v i o u s d i f f e r e n c e s . P r o t e i n s f r o m i r r a d i a t e d c e l l s ( C ) , however, showed r e d u c e d d e f i n i t i o n w h i c h was a c c o m p a n i e d w i t h g e n e r a l b l u r r i n g o f p r o t e i n b a n d s . M e m b r a n e - a s s o c i a t e d p r o t e i n s showed t h e r e s u l t s of p h o t o s e n s i t i z a t i o n even more d r a m a t i c a l l y . No v i s i b l e 85 Figure 26. The effect of incubation temperature on the survival of E. c o l i B irradiated in the presence of alpha-T at 5 C. P r e c h i l l treated cultures (• • ) were f i r s t cooled to 5 C prior to adding alpha-T and then incubated at 5 C for 30 minutes while p o s t c h i l l treated cultures ( •• •) were incubated with alpha-T for 30 minutes and then cooled to 5 C for 15 minutes. Irradiation of a l l cultures was started at 45 minutes. 86 tri mp B C D E F G H Figure 27. SDS-gel e1ectropherogram of the soluble or cytoplasmic proteins (A-C) and the membrane-associated proteins (E-H) of E_. c o l i B. Standards (D) included BSA (67,000), ovalbumin (45,000), pepsin (34,700), carbonic anhydrase (30,000), trypsinogen (25,000), trypsin i n h i b i t o r (20,000) and lysozyme (14,300). A) soluble proteins before treatment; B) after 30 minutes dark incubation with lug/ml alpha-T; C) aft e r 30 minutes UV-A; E) membrane-associated proteins before treatment; F) aft e r 30 minutes dark incubation with lug/ml; G) after 15 minutes UV-A and H> after 30 minutes UV-A. mp- 'matrix protein'* t r i - trimer of 'matrix protein'. Note the aggregation of protein at the tops of columns G and H and the non-specific blurring of proteins in C, G and H above 'matrix protein'. 87 d i f f e r e n c e s i n " p r o t e i n s b e f o r e a d d i t i o n and f o l l o w i n g 30 m i n u t e s d a r k i n c u b a t i o n w i t h 1 ug/ml a l p h a - T were e v i d e n t i n c o l u m n s E and F. The p r o t e i n s i n c o l u m n s G and H, however, show t h e e f f e c t s f o l l o w i n g 15 and 30 m i n u t e s i r r a d i a t i o n , r e s p e c t i v e l y . The h i g h e r m o l e c u l a r w e i g h t p r o t e i n s were t h e most a f f e c t e d . G e n e r a l b l u r r i n g of bands was f o u n d i n a d d i t i o n t o p r o t e i n a g g r e g a t i o n a t t h e t o p of g e l s . P h o t o d y n a m i c m o d i f i c a t i o n of membrane p r o t e i n f r o m human e r y t h r o c y t e s s e n s i t i z e d by p r o t o p o r p h y r i n has a l s o been r e p o r t e d ( D e G o e i j e t a l . , 1975 and 1976; G i r o t t i , 1976; Lamola and D o l e i d e n , 1980). SDS g e l e l e c t r o p h o r e s i s o f p r o t e i n from t r e a t e d c e l l s r e v e a l e d t h e b l u r r i n g of p r o t e i n bands and t h e a p p e a r a n c e o f p r o t e i n a g g r e g a t e s a t t h e t o p of g e l s ( D e G o e i j e_t a l . , 1976). T h i s e f f e c t was p o s t u l a t e d t o r e s u l t f r o m c r o s s l i n k i n g of membrane p r o t e i n s f o l l o w i n g p h o t o o x i d a t i o n o f amino a c i d r e s i d u e s by s i n g l e t oxygen (Dubbelman e t a_l. , 1980), but m i g h t a l s o r e s u l t f r o m p r o t e i n d e g r a d a t i o n . The e f f e c t on e r y t h r o c y t e s c l o s e l y m a t c h e s t h e p h o t o d y n a m i c e f f e c t of a l p h a - T on E. c o l i p r o t e i n s . The c r o s s l i n k i n g or d e g r a d a t i o n o f membrane p r o t e i n s a p p e a r s n e a r l y i d e n t i c a l . The p h o t o s e n s i t i z e d e f f e c t on s o l u b l e p r o t e i n ,however, i s a n o t e w o r t h y d i f f e r e n c e . The c y t o p l a s m i c or s o l u b l e p r o t e i n s of E. c o l i a l s o were f o u n d t o be s u s c e p t i b l e t o p h o t o o x i d a t i o n and s u b s e q u e n t c r o s s l i n k i n g i n t o a g g r e g a t e s . The s o l u b l e p r o t e i n s ( c y t o p l a s m i c enzymes) o f r e d b l o o d c e l l s have been shown t o r e s p o n d v a r i a b l y t o d i f f e r e n t p h o t o d y n a m i c s e n s i t i z e r s . A l p h a - T c a u s e d o n l y s l i g h t e f f e c t s on e n z y m a t i c a c t i v i t y p r i o r t o c e l l l y s i s w h i l e 88 methylene blue was responsible for considerable inactivation of cytoplasmic enzymes (Yamamoto et a l . , 1979). These differences probably, r e f l e c t the d i f f e r i n g a b i l i t i e s of these s e n s i t i z e r s to penetrate the c e l l membrane. Lysis of E. c o l i was not found to result from photosensitization by alpha-T despite substantial crosslinking of the proteins. Intact c e l l s could s t i l l be observed microscopically following 30 minutes i r r a d i a t i o n in the presence of alpha-T, although most c e l l movement had ceased. This suggests that the peptidoglycan layer or c e l l wall remained intact. Observations at the electron microscopic l e v e l were not made to determine whether alpha-T induced membrane lesions in outer membranes as observed in red blood c e l l s . CONCLUSION The in vivo mechanism of action of alpha-T and BBT was studied. Alpha-T was found to require oxygen for the expression of photoinduced b a c t e r i c i d a l action. C e l l u l a r k i l l i n g of E. c o l i resembled multiple-hit kinetics and was not dependent on the p r i o r respiratory status of c e l l s before i r r a d i a t i o n in the presence of alpha-T. This Type II s e n s i t i z e r was shown to e l i c i t b a c t e r i c i d a l a c t i v i t y at d i f f e r e n t wavelengths between 320 nm and c400 nm which resembled the absorption of alpha-T in the same wavelength range. This suggested that alpha-T, rather than a photoproduct, was the s e n s i t i z e r molecule. Protection of E. c o l i c e l l s by sodium azide and BHT indicated the p a r t i c i p a t i o n of both singlet oxygen and free radicals in the 89 i n a c t i v a t i o n o f c e l l s . S t u d i e s w i t h r e c o m b i n a t i o n d e f i c i e n t m u t a n t s o f E. c o l i d i d n o t s u g g e s t t h a t damage t o c e l l u l a r DNA r e s u l t e d f r o m a t t a c k by t h e p h o t o s e n s i t i z e r . C e l l u l a r p r o t e i n s , h o w e v e r , were d r a m a t i c a l l y a f f e c t e d by UV-A i r r a d i a t i o n i n t h e p r e s e n c e o f a l p h a - T . SDS g e l e l e c t r o p h o r e s i s r e v e a l e d t h a t b o t h c y t o p l a s m i c a n d membrane p r o t e i n s o f E. c o l i m i g h t be c r o s s l i n k e d by t h e a c t i o n o f a l p h a - T i n t h e p r e s e n c e o f UV-A i r r a d i a t i o n . BBT was a l s o s t u d i e d b u t t o a l e s s e r e x t e n t . A d i f f e r e n t m e c h a n i s m o f a c t i o n f r o m a l p h a - T was i n d i c a t e d . C e l l u l a r k i l l i n g o f E. c o l i f o l l o w e d d i f f e r e n t i n a c t i v a t i o n k i n e t i c s t h a n k i l l i n g c a u s e d by a l p h a - T a n d s t u d i e s w i t h r e c o m b i n a t i o n d e f i c i e n t m u t a n t s s u g g e s t e d t h e p o s s i b i l i t y o f damage t o DNA i n t h e m e c h a n i s m o f c e l l u l a r i n a c t i v a t i o n . 90 APPENDIX I The f o l l o w i n g i s a l i s t i n g o f t h e s p e c t r a l d a t a o b t a i n e d f r o m t h e r e f e r e n c e compounds i s o l a t e d f r o m T. p a t u l a o r s y n t h e s i z e d c h e m i c a l l y . UV, IR, MS and f l u o r e s c e n c e s p e c t r a f o r most compounds a r e g i v e n . UV a b s o r p t i o n maxima a r e f o l l o w e d by p u b l i s h e d e x t i n c t i o n c o e f f i c i e n t s i n p a r e n t h e s e s . Mass s p e c t r a s h o wing r e l a t i v e i n t e n s i t i e s o f . m/e p e a k s a r e a l s o g i v e n . F l u o r e s c e n c e d a t a s h o w i n g e x c i t a t i o n maxima a r e f o l l o w e d by e m i s s i o n maxima ( f l u o r e s c e n c e ) i n p a r e n t h e s e s (nm). V e r i f i c a t i o n of t h e s t r u c t u r e s o f V I I , V I I I , IX and X were b a s e d on c o m p a r i s o n of s p e c t r a l d a t a w i t h p r e v i o u s l y p u b l i s h e d r e s u l t s . The s t r u c t u r e o f a l p h a - T was a l s o c o n f i r m e d by c h e m i c a l s y n t h e s i s . . 2 , 2 ' : 5 ' , 2 " - t e r t h i e n y l ( a l p h a - T ) MW 2 4 8 ; UV(nm) 3 5 0 ( 2 2 4 0 0 ) , 2 5 2 ( 9 1 0 0 ) ; I R ( l / c m ) 8 3 0 , 8 0 0 , 6 9 0 ; MS (m/e>l0%) 6 9 ( 1 8 ) , 1 2 4 ( 1 0 ) , 1 2 7 ( 1 9 ) , 1 7 1 ( 1 4 ) , 2 0 3 ( 1 8 ) , 2 4 8 ( 1 0 0 ) , 2 4 9 ( 1 8 ) , 2 5 0 ( 1 5 ) ; f l u o r e s c e n c e ( n m ) 3 5 0 ( 4 2 5 ) . 5 - ( b u t e n - 1 - y n y l ) - 2 , 2 ' - b i t h i e n y l (BBT) MW 2 1 6 ; UV(nm) 3 4 6 ( 2 7 8 0 0 ) 2 5 4 ( 9 7 5 0 ) ; I R ( l / c m ) 3 0 7 0 , 2 2 0 0 , 1 6 0 0 , 9 7 5 , 9 2 0 , 8 5 0 ; MS(m/e>l0%) 9 5 ( 1 3 ) , 1 7 1 ( 1 8 ) , 2 1 5 ( 1 1 ) , 2 1 6 ( 1 0 0 ) , 2 1 7 ( 1 8 ) ; f l u o r e s c e n c e ( n m ) 3 4 6 ( 4 1 0 ) . 5 - ( 4 - a c e t o x y - 1 - b u t e n y l ) - 2 , 2 ' - b i t h i e n y l (BBT-OAc) MW 2 7 9 ; UV(nm) 3 3 3 ( 2 1 8 0 0 ) , ( 3 2 5 ) ( 2 1 5 0 0 ) , 2 4 6 ( 5 5 4 0 ) ; MS (m/e>l0%) 4 3 ( 4 7 ) , 9 5 ( 1 6 ) , 1 0 8 ( 1 4 ) , 1 4 9 ( 1 1 ) , 1 7 1 ( 1 4 ) , 2 0 3 ( 1 8 ) , 2 1 6 ( 1 0 0 ) , 2 1 7 ( 1 8 ) , 2 1 8 ( 2 2 ) , 2 7 6 ( 1 9 ) ; f l u o r e s c e n c e ( n m ) 3 3 3 ( 3 9 0 ) . 5 - ( 4 - h y d r o x y - 1 - b u t e n y l ) - 2 , 2 ' - b i t h i e n y l (BBT-OH) MW 2 3 4 ; UV(nm) 3 3 3 ( 2 2 7 0 0 ) , ( 3 2 7 ) ( 2 2 5 0 0 ) , 2 4 0 ( 6 6 0 0 ) ; I R ( l / c m ) 3 6 5 0 , 3600, 3400, 3060, 1050, 845; f l u o r e s c e n c e ( n m ) 333(387). 92 LITERATURE CITED A r n a s o n , T.; Swain, T.; Wat, C.-K.; Graham, E.A.; P a r t i n g t o n , S.; Towers, G.H.N, and Lam, J . 1980. 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