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UBC Theses and Dissertations

Affinity chromatography : variations on a heme Hamilton, Andrew D. 1976

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AFFINITY CHROMATOGRAPHY: VARIATIONS ON A HEME by ANDREW D. HAMILTON B . S c , U n i v e r s i t y o f E x e t e r , 1974 A THESIS SUBMITTED IN THE REQUIREMENTS MASTER PARTIAL'FULFILMENT OF FOR THE DEGREE OF OF SCIENCE i n t h e Department o f C h e m i s t r y We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA Sept ember, 19 7 6 © A n d r e w D a v i d H a m i l t o n , 1976 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r ee t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r ag ree 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 pu rpo se s may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f CfjczP^/^l /2.V The U n i v e r s i t y o f B r i t i s h Co lumb ia 2075 Wesbrook P l a c e Vancouver, Canada V6T 1W5 Date i ABSTRACT The d e v e l o p m e n t o f a new a f f i n i t y c h r o m a t o g r a p h y s y s t e m f o r p o r p h y r i n and heme b i n d i n g p r o t e i n s i s r e p o r t e d . The l i g a n d was b a s e d o n a p r o t o p o r p h y r i n I X d e r i v a t i v e s u b s t i t u t e d o n e i t h e r t h e 2- o r 4 - v i n y l g r o u p . I : R 1 = R 2 = V i n y l , R ^ H I I : R 1 = C H O , R 2 = V i n y I , R 3 = t B u I I I : R 1 = V i n y l f R 2=CHO,R 3=tBu IV: R = - C=C-COOH,R 2=Vinyl,R 3=tBu V: R ^ V i n y l , R 2=-C=C-COOH, R 3 = t B u V I : R =-C=C-CONH ( C H 2 ) 3 N H 2 , R 2 = V i n y l , R 3 = H V I I : R = V i n y l , R 2=-C=C-CONH ( C H 2 ^ NH^R^K VIII:R 1=-C=C-CONH(CH 2 ) gNH 2, R 2=Viny 1, R3=H IX : R x = V i n y l ; R 2=-C=C-CONH (CH2> 6NH 2,R 3=H The l i g a n d was s y n t h e s i s e d f r o m p r o t o p o r p h y r i n I X ( I ) by o x i d a t i o n t o t h e m o n o f o r m y l m o n o v i n y l d e r i v a t i v e s ( I I and I I I ) f o l l o w e d b y a K n o e v e n a g e l c o n d e n s a t i o n w i t h m a l o n i c a c i d t o t h e m o n o a c r y l i c a c i d d e r i v a t i v e s ( I V a n d V ) . The a l i p h a t i c d i a m i n e was a t t a c h e d t o t h e a c r y l i c a c i d p o r p h y r i n t h r o u g h a n a c i d c h l o r i d e m e d i a t e d a m i d e l i n k a g e . The l i g a n d s V I , V I I , V I I I a n d I X w e r e p r e p a r e d b y u s i n g p r o p a n e 1,3— d i a m i n e o r h e x a n e 1 , 3 - d i a m i n e r e s p e c t i v e l y . The s e p a r a t i o n o f t h e 2- and 4- i s o m e r i c p o r p h y r i n s was c a r r i e d o u t by i i e m p l o y i n g t h e i n t e r m e d i a c y o f p h o t o p r o t o p o r p h y r i n I X i n t h e f o r m a t i o n o f m o n o f o r m y l m o n o v i n y l d e u t e r o p o r p h y r i n I X ( I I and I I I ) . T h i s g a v e r i s e t o t h e m o n o f o r m y l p o r p h y r i n i s o m e r s ( I I o r I I I ) on a p r e p a r a t i v e s c a l e a n d a l t h o u g h n o t u s e d i n t h e l a t e r s t a g e s o f t h e a f f i n i t y l i g a n d s y n t h e s i s , t h e e s t a b l i s h m e n t o f a r e l i a b l e s e p a r a t i o n t e c h n i q u e e n h a n c e d t h e p o t e n t i a l s e l e c t i v i t y a n d h e n c e e x t e n d e d t h e g e n e r a l a p p l i c a b i l i t y o f t h e a f f i n i t y l i g a n d . The p o r p h y r i n a f f i n i t y l i g a n d was c o u p l e d t o c y a n o g e n b r o m i d e a c t i v a t e d s e p h a r o s e t o g i v e a g e l w i t h a l i g a n d 3 c o n c e n t r a t i o n t h a t c o u l d b e v a r i e d f r o m 4.7 y m o l e s / c m t o 3 0.1 y m o l e s / c m b y v a r y i n g t h e a c t i v a t i o n p r o c e d u r e . I n c o r p o r a t i o n o f i r o n i n t o t h e p o r p h y r i n a f f i n i t y l i g a n d g a v e r i s e t o a heme b a s e d a f f i n i t y l i g a n d . T h i s i n t u r n was c o u p l e d t o c y a n o g e n b r o m i d e a c t i v a t e d s e p h a r o s e f o r f u t u r e u s e w i t h t h e heme b i n d i n g enzyme h e m o p e x i n . A t t e m p t s w e r e made t o f o r m a p o r p h y r i n o g e n a f f i n i t y l i g a n d by r e d u c t i o n o f t h e p o r p h y r i n w i t h s o d i u m b o r o h y d r i d e b u t w e r e u n s u c c e s s f u l . I n i t i a l a f f i n i t y c h r o m a t o g r a p h y r e s u l t s w i t h t h e p o r p h y r i n b i n d i n g enzyme f e r r o c h e l a t a s e a r e p r o m i s i n g a s b i n d i n g o n t o t h e c o l u m n h a s b e e n d e f i n i t e l y shown t o o c c u r . The p o t e n t i a l s y n t h e t i c u s e f u l n e s s o f t h e i n t e r m e d i a t e m o n o v i n y l m o . n o a c r y l i c a c i d d e u t e r o p o r p h y r i n i s a l s o d i s c u s s e d . i i i T ABLE OF CONTENTS ABSTRACT TABLE OF CONTENTS ACKNOWLEDGEMENTS V ABBREVIATIONS v i i 1. INTRODUCTION 1 1.1. A B r i e f O v e r v i e w o f t h e P o r p h y r i n M a c r o c y l e .... 1 1.11. The N a t u r e o f t h e M a c r o c y c l e 2 1.12. N o m e n c l a t u r e 4 1.13. E l e c t r o n i c S p e c t r a 5 1.14. B i o l o g i c a l O c c u r r e n c e 6 1.15. B i o s y n t h e s i s 9 1.2. The N a t u r e o f t h e P r o b l e m 13 PAGE l i i i 1.3. The Enzymes 18 i.31« F e r r o c h e l a t a s e 18 1.32. P r o t o p o r p h y r i n o g e n O x i d a s e 20 1.33. Hemopexin 2 2 2. DESIGN OF THE A F F I N I T Y LIGAND 26 2.1. S y n t h e t i c R e q u i r e m e n t s 26 2.2. A p p r o a c h e s 2 9 2.3. The S y n t h e t i c B a se 3 0 2.4. I n t r o d u c t i o n o f t h e C h a i n 33 2.41. The V i l s m e i e r A p p r o a c h 3 3 2.42. The V i n y l G r o u p A p p r o a c h 4 3 2.43. M o n o f o r m y l M o n o v i n y l D e u t e r o p o r -p h y r i n IX DTBE '. 45 2.44. M o n o a c r y l i c A c i d M o n o v i n y l D e u t e r o -p o r p h y r i n IX DTBE 51 i v PAGE 2.5. E x t e n s i o n o f t h e C h a i n - 54 2.51. The D i c y c l o h e x y l c a r b o d i i m i d e Method ... 56 2.52. The A c i d C h l o r i d e A p p r o a c h 60 2.6. Removal o f t h e T e r t i a r y B u t y l G r o u p s 6 6 2.7. A P o s s i b l e A l t e r n a t i v e L i g a n d S y n t h e s i s 68 3. A F F I N I T Y CHROMATOGRAPHY 71 3.1. I n t r o d u c t i o n 71 3.2. P r e p a r a t i o n o f t h e Column 75 3.3. A l t e r n a t i v e L i g a n d s « 79 3.31. P r o t o p o r p h y r i n o g e n IX D e r i v a t i v e s ..... 7 9 3.32. Heme D e r i v a t i v e s 81 3.4. E p i l o g u e 84 4. MONOACRYLIC ACID MOMOVINYL DEUTEROPORPHYRIN IX DTBE ... 85 5. EXPERIMENTAL 94 6. REFERENCES 121 7. SPECTRAL APPENDIX 130 P r o t o p o r p h y r i n IX 131 P r o t o p o r p h y r i n IX DTBE 132 P i m e l i c A c i d D i b e n z y l E s t e r 133 P i m e l i c A c i d M o n o b e n z y l E s t e r 133 P i m e l i c A c i d M o n o b e n z y l E s t e r . M o n o N - M e t h y l a n i l i d e 134 Z i n c ( I I ) P r o t o p o r p h y r i n IX DTBE 134 C o b a l t ( I I ) P r o t o p o r p h y r i n IX DTBE 134 2 - P h o t o p r o t o p o r p h y r i n IX DTBE 135 4 - P h o t o p r o t o p o r p h y r i n IX DTBE 13 6 V 2 - F o r m y l 4 - V i n y l D e u t e r o p o r p h y r i n I X DTBE 137 4 - F o r m y l 2 - V i n y l D e u t e r o p o r p h y r i n I X DTBE 138 2 (and 4 ) - M o n o a c r y l i c A c i d 4 (and 2 ) - M o n o v i n y l D u e t e r o p o r p h y r i n I X DTBE 139 2 (and 4 ) - M o n o m e t h y l A c r y l a t e 4 (and 2 ) -M o n o v i n y l D e u t e r o p o r p h y r i n I X DTBE 14 0 2 (and 4)-Mono 3-Amino 1-Amido P r o p a n e A c r y l a t e 4 (and 2 ) - M o n o v i n y l D e u t e r o -p o r p h y r i n I X DTBE 141 2 ( a n d 4)-Mono 6-Amino 1-Amido Hexane ; A c r y l a t e 4 (and 2 ) - M o n o v i n y l D e u t e r o -p o r p h y r i n I X DTBE 142 2 (and 4)-Mono 3-Amino 1-Amido P r o p a n e A c r y l a t e 4 (and 2 ) - M o n o v i n y l D e u t e r o -p o r p h y r i n I X „ 14 3 S e p h a r o s e B ound A f f i n i t y L i g a n d 143 P h o t o p r o t o p o r p h y r i n D e r i v a t i v e o f 2 (and 4 ) -Mono 3-Amino 1-Amido P r o p a n e A c r y l a t e 4 (and 2 ) - M o n o v i n y l D e u t e r o p o r p h y r i n I X DTBE „ 144 P y r i d i n e Hemochrome o f I r o n ( I I ) 2 (and 4 ) -Mono 3-Amino 1-Amido P r o p a n e A c r y l a t e 4 (and 2 ) - M o n o v i n y l D e u t e r o p o r p h y r i n I X DTBE . 14 4 P h o t o p r o t o p o r p h y r i n D e r i v a t i v e o f 2 (and 4 ) -M o n o a c r y l i c A c i d 4 (and 2 ) - M o n o v i n y l D e u t e r o p o r p h y r i n I X DTBE 145 2 ( a n d 4 ) - M o n o f o r m y l 4 (and 2 ) - M o n o a c r y l i c A c i d D e u t e r o p o r p h y r i n I X DTBE 14 5 v i ABBREVIATIONS DTBE D i t e r t i a r y b u t y l e s t e r DMF D i m e t h y l f o r m a m i d e HMPA He x a m e t h y l p h o s p h o r a m i d e OEP O c t a e t h y l p o r p h y r i n PBG P o r p h o b i l i n o g e n S S i n g l e t m M u l t i p l e t d D o u b l e t t T r i p l e t dd D o u b l e t o f d o u b l e t s v i i ACKNOWLEDGEMENTS Thanks a r e due t o a number o f p e o p l e whose h e l p , e n c o u r a g e m e n t , c a j o l i n g and g e n e r a l t o l e r a n c e o f my more a n t i s o c i a l e x c e s s e s c o n t r i b u t e d t o t h i s work. P r i m a r i l y , I am g r a t e f u l t o D a v i d D o l p h i n f o r h i s s u p e r v i s i o n and q u i e t s t i m u l a t i o n o v e r t h e s e two y e a r s . A l s o my t h a n k s go t o t h e D o l p h i n r e s e a r c h g r o u p who have had t o s u f f e r me a t t h e c l o s e s t r a n g e o f a l l . P a r t i c u l a r l y Bob D i N e l l o f o r a l l h i s h e l p and Gene J o h n s o n f o r h i s p r o o f r e a d i n g , b u t a l s o C a r l A l l e y n e and R i c h a r d Sawka f o r a l l t h e i r e q u i p m e n t and t o l e r a n c e . I w o u l d a l s o l i k e t o t h a n k Don C l a r k and B r i a n M c C a r r y f o r t h e i r h e l p w i t h t h e a f f i n i t y c h r o m a t o g r a p h y a s p e c t o f t h i s work and Rozanne P o u l s o n , D e n y s e Simpson and C h r i s Chang f o r t h e i r h e l p f u l d i s c u s s i o n . F i n a l l y I am most g r a t e f u l t o Luanna L a r u s s o n whose h a r d work, w h i r l w i n d t y p i n g and t r e m e n d o u s o r g a n i s a t i o n were a m a j o r f a c t o r i n t h e w r i t i n g o f t h i s t h e s i s . 1 INTRODUCTION 1.1. A BRIEF OVERVIEW OF THE PORPHYRIN MACROCYCLE The g r o w t h o f c h e m i s t r y o v e r t h e l a s t q u a r t e r c e n t u r y has been a l m o s t b a c t e r i a l i n b o t h i t s v o r a c i t y and i t s d i v i s i v e n e s s . F e d on t h e most u n p a l a t a b l e d i e t o f a c a d e m i c i n t r o v e r s i o n i t has d e v e l o p e d i n t o a c o l o n y o f r a r e l y c o i r a n u n i c a t i n g - o r i n t e r -a c t i n g d i s c i p l i n e s . W i t h i n s u c h a c u l t u r e t h e p o r p h y r i n m a cro-c y c l e s t a n d s o u t above a l l o t h e r c h e m i c a l s y s t e m s a s a u n i f y i n g f a c t o r ; i t o c c u p i e s , i f n o t a c e n t r a l , an i m p o r t a n t p l a c e i n a l l a r e a s o f t h e s u b j e c t . T h i s r e m a r k a b l y s i m p l e t e t r a p y r r o l i c r i n g s y s t e m e x h i b i t s a c o m p l e x a r r a y o f p r o p e r t i e s t h a t a r e s t u d i e d f r o m t h e s p e c t r a l e x t r e m e s o f c h e m i c a l p h y s i c s t o b i o l o g i c a l c h e m i s t r y t o g e o c h e m i s t r y . The s o l i d s t a t e c h e m i s t r y o f p o r p h y r i n s h a s r e c e n t l y r e -v e a l e d t h e e n o r m o u s l y p r o m i s i n g p r o p e r t y o f s u p e r c o n d u c t i v i t y " ' " . M e t a l l o p o r p h y r i n s a r e f o u n d i n c r u d e o i l and c a u s e many p r o b l e m s 2 by b e i n g e x a s p e r a t i n g l y d i f f i c u l t t o remove . The v a r i e d v i s i b l e s p e c t r a o f p o r p h y r i n s t e s t t h e p r e d i c t a b i l i t y o f m o l e c u l a r o r b i t a l c a l c u l a t i o n s t o t h e i r l i m i t . T h e i r i n o r g a n i c c h e m i s t r y i s a l l e n c o m p a s s i n g as t h e y a r e a b l e t o c h e l a t e e v e r y m e t a l i n t h e p e r i o d i c t a b l e , a n d ev e n some n o n m e t a l s , a n d , i n 3 d o i n g s o , o f t e n s t a b i l i s e v e r y u n u s u a l o x i d a t i o n s t a t e s . T h e i r o r g a n i c c h e m i s t r y i s e x t e n s i v e and u n i q u e r e f l e c t i n g b o t h t h e r e a c t i v i t y o f a l a r g e a r o m a t i c m a c r o c y c l e a n d t h e s u b t l e v a r i a t i o n s w r o u g h t on t h e s y s t e m by c h a n g e s i n t h e p e r i p h e r a l s u b s t i t u e n t s o r c e n t r a l m e t a l . T h e i r r o l e i n l i f e p r o c e s s e s 2 i s i n d e e d fundamental s i n c e i t i s t h o u g h t t h a t a s i m p l e p r e -b i o t i c p h o t o s y n t h e s i s a p p a r a t u s based on a m e t a l l o p o r p h y r i n i n t h e ' p r i m o r d i a l soup 1 was r e s p o n s i b l e f o r f o r m i n g t h e oxygen r i c h atmosphere c r u c i a l f o r t h e l a t e r development o f complex organisms w h i c h i n t u r n i n c o r p o r a t e d t h e m e t a l l o p o r p h y r i n s , i n s i m i l a r r o l e s , as t h e a d m i n i s t r a t o r s o f energy change. 1.11. The N a t u r e o f t h e M a c r o c y c l e A l l o f t h e s e p r o p e r t i e s c e n t e r on two fundamental a s p e c t s o f t h e t e t r a p y r r o l i c m a c r o c y c l e . The f i r s t i s t h e h i g h s t a b i l i t y endowed by t h e p r e s e n c e o f e l e v e n d o u b l e bonds i n t h e r i n g . N i ne o f t h e s e a r e i n d i r e c t c o n j u g a t i o n (1) g i v i n g an 18 e l e c -t r o n , a r o m a t i c a l l y d e l o c a l i s e d , TT system. The r e m a i n i n g two double bonds a r e c r o s s - c o n j u g a t e d and can be r e d u c e d , as i n t h e c h l o r i n s (2) o r b a c t e r i o c h l o r i n s ( 3 ) , w i t h o u t l o s s o f a r o m a t i c i t y . 3 The a r o m a t i c c h a r a c t e r i s e x e m p l i f i e d by t h e t h e r m a l s t a b i l i t y o f t h e m a c r o c y c l e , i t s s u s c e p t i b i l i t y t o e l e c t r o p h i l i c a t t a c k and t h e a r o m a t i c r i n g c u r r e n t s h i e l d i n g o f t h e i n t e r n a l NH (<5 % -2 ->• -4) p r o t o n s and d e s h i e l d i n g o f t h e e x t e r n a l p r o t o n s (6 % 1 0 ) . The second major f e a t u r e i s t h e a b i l i t y o f p o r p h y r i n s t o c h e l a t e m e t a l i o n s i n t o t h e c e n t r a l c a v i t y . The major bond-i n g i n t e r a c t i o n i s t h e a d o n a t i o n o f f o u r e l e c t r o n s from t h e n i t r o g e n s t o t h e c e n t r a l m e t a l i o n . T h i s c auses a major change i n t h e e l e c t r o n i c p r o p e r t i e s o f t h e c e n t r a l m e t a l i o n , o f t e n w i t h t h e s t a b i l i s a t i o n o f u n u s u a l o x i d a t i o n s t a t e s ; f o r 3 example b o t h Co (I) and P e ( I V ) can be formed i n m e t a l l o p o r p h y n n s . I n f a c t t h e p o r p h y r i n m a c r o c y c l e has been l i k e n e d t o t h e bed o f P r o c r u s t e s ^ , a s i n g u l a r l y u n p l e a s a n t r o b b e r o f Greek myth-o l o g y , who f o r c e d h i s v i c t i m s t o l i e on h i s bed and t h e n e i t h e r 7 s t r e t c h e d them o r c u t o f f t h e i r l i m b s t o make them f i t . However the e f f e c t i s s y n e r g i c and t h e e l e c t r o n e g a t i v i t y o f t h e m e t a l w i l l e x e r t an i n f l u e n c e on t h e e l e c t r o n d e n s i t y o f t h e p o r p h y r i n TT system and hence on t h e r e a c t i v i t y o f t h e r i n g p e r i p h e r y (see C h a p t e r 2 ) . The c o m b i n a t i o n o f t h e s e two major f a c t o r s r e s u l t s i n t h e most i m p o r t a n t r e d u c t i o n / o x i d a t i o n p r o p e r t i e s o f m e t a l l o p o r p h y r i n s where changes i n t h e c e n t r a l m e t a l o r e x t e r n a l s u b s t i t u e n t s cause changes i n t h e o x i d a t i o n and r e d u c t i o n p o t e n t i a l s o f t h e r i n g g i v i n g r i s e t o t h e i r use i n b i o l o g i c a l and n o n - b i o l o g i c a l systems as e l e c t r o n t r a n s f e r a g e n t s . 4 1.12. N o m e n c l a t u r e An I.U.P.A.C. n o m e n c l a t u r e b a s e d on a c o m p l e t e n u m e r a t i o n o f t h e p a r e n t p o r p h y r i n r i n g has been d e v e l o p e d , b u t , as i s o f t e n t h e c a s e , t h e c o m p l e x i t y o f n a t u r a l p r o d u c t s r e s u l t s i n a c l u m s y naming scheme arid so i s l a r g e l y i g n o r e d . C o n s e q u e n t l y t r i v i a l names s t i l l abound o f w h i c h t h e most common a r e l a b l e d b elow: P o r p h y r i n 1 2 3 4 5 6 7 8 m e s o P r o t o p o r p h y r i n I X Me V Me V Me H P H P Me H M e s o p o r p h y r i n I X Me E t Me E t Me H P K P Me H E t i o p o r p h y r i n I Me E t Me E t Me E t Me E t H D e u t e r o p o r p h y r i n I X Me H Me H Me H P H P Me H C o p r o p o r p h y r i n I I I Me H P Me H P Me H P H P Me H U r o p o r p h y r i n I A H H P Me H P Me H P Me H P H P h y l l o p o r p h y r i n X V Me E t Me E t Me H Me H P H P y r r o p o r p h y r i n X V Me E t Me E t H P H H P Me H A b b r e v . : M e = M e t h y l , E t = e t h y l ; V = v i n y l , p H = - C H 2 C H 2 C O H ; A H = - C H 2 C 0 2 H . The n u m b e r i n g s y s t e m , a g a i n i n c o n f l i c t w i t h I.U.P.A.C. i s q b a s e d on t h a t u s e d by Hans F i s c h e r . I t i s t h e most f r e q u e n t l y u s e d b e c a u s e i t e n a b l e s t h e p e r i p h e r a l 3 p y r r o l e and b r i d g i n g meso p o s i t i o n s t o be r e a d i l y d i s t i n g u i s h e d / 5 1.13. E l e c t r o n i c S p e c t r a The v a r i e t y o f c o l o u r s shown by p o r p h y r i n t y p e compounds i s r e f l e c t e d i n t h e v a r i e t y o f t h e i r v i s i b l e and u l t r a v i o l e t s p e c t r a . T h i s i s a u s e f u l t o o l f o r m o n i t o r i n g t h e p r o g r e s s o f p o r p h y r i n r e a c t i o n s . A l l t e t r a p y r r o l e s w i t h a c o n j u g a t e d n u c l e u s have a v e r y i n t e n s e band ( e % 10 a r o u n d 4 00 nm known as t h e S o r e t band, and a number o f s m a l l e r bands i n t h e v i s i b l e , a l l a r i s i n g f r o m v a r i o u s TT-^TT* t r a n s i t i o n s " ^ . The v i s i b l e b ands a r e e m p i r i c a l l y t h e more u s e f u l s i n c e t h e y a r e more s e n s i t i v e 10 t o c h a n g e s b o t h on t h e p e r i p h e r y and i n t h e c e n t r e o f t h e r i n g I n most p o r p h y r i n s t h e r e a r e f o u r bands i n t h i s v i s i b l e r e g i o n w h i c h a p p e a r t o be two p a i r s o f bands t h a t , i f t h e p o r p h y r i n was p e r f e c t l y s q u a r e and s y m m e t r i c a l l y s u b s t i t u t e d , w o u l d be superirnposable"'"^. I n m e t a l l o p o r p h y r i n s o f d i v a l e n t c a t i o n s , a s w e l l a s p r o t o n a t e d and d e p r o t o n a t e d p o r p h y r i n s e a c h p a i r c o l l a p s e s i n t o one band g i v i n g r i s e t o a c h a r a c t e r i s t i c two banded spectrum"'"'*". The r e l a t i v e i n t e n s i t i e s o f t h e f o u r v i s i b l e p e a k s a r e f a i r l y s e n s i t i v e t o t h e n a t u r e and c h a n g e s o f t h e p e r i p h e r a l g r o u p s , g i v i n g r i s e t o f o u r m a i n s p e c t r a l c l a s s i f i c a t i o n s . I f t h e p o r p h y r i n i s s u b s t i t u t e d by a l k y l o r e v e n v e r y w e a k l y e l e c t r o n w i t h d r a w i n g g r o u p s , e . g . v i n y l , on t h e 3 p o s i t i o n s t h e common e t i o - t y p e s p e c t r u m o c c u r s . One s t r o n g l y e l e c t r o n w i t h d r a w i n g g r o u p , e . g . f o r m y l , c a r b o x y l i c a c i d e t c . w A on a 3 p o s i t i o n r e s u l t s i n a r h o d o t y p e s p e c t r u m w h e r e a s two e l e c t r o n w i t h d r a w i n g g r o u p s on a d j a c e n t p y r r o l e r i n g s m o d i f y e a c h o t h e r ' s e f f e c t t o g i v e an e t i o s p e c t r u m . When two e l e c t r o -n e g a t i v e g r o u p s a r e on d i a g o n a l l y o p p o s i t e p y r r o l e r i n g s t h e n t h e o x o - r h o d o s p e c t r u m i s o b s e r v e d . F i n a l l y t h e p h y l l o t y p e "spectrum i s p a r t i c u l a r l y r e p r e s e n t a t i v e o f mono meso a l k y l s u b -s t i t u t e d p o r p h y r i n s " ^ . More comp l e x p o r p h y r i n s n a t u r a l l y g i v e r i s e t o more complex s p e c t r a . / 1.14. B i o l o g i c a l O c c u r r e n c e Much o f t h e r e s e a r c h i n t e r e s t i n p o r p h y r i n s ' i s s t i m u l a t e d by a d e s i r e t o u n d e r s t a n d t h e i r v i t a l y e t complex r o l e s as oxygen c a r r i e r s i n hemoglobin and m y o g l o b i n , e l e c t r o n t r a n s f e r c e n t r e s i n t h e cytochromes and hydrogen p e r o x i d e s c a v e n g e r s i n t h e c a t a l a s e s and p e r o x i d a s e s . The p r o s t h e t i c group f o r most o f t h e s e h e m e p r o t e i n s i s f e r r o p r o t o p o r p h y r i n IX ( 4 ) . A l t h o u g h t h i s i s t h e most commonly o c c u r r i n g n a t u r a l p o r p h y r i n , o t h e r s a r e found s e r v i n g s i m i l a r f u n c t i o n s i n o t h e r systems, e.g. heme a (5) i n t h e c y t o c h r o m e s - a . (4) (5) In an e x t r e m e l y s i m p l i s t i c way t h e b i o l o g i c a l p r o p e r t i e s can be seen as d e r i v i n g from a s y m b i o t i c i n t e r a c t i o n between t h e m e t a l and t h e p o r p h y r i n i n w h i c h each i s dependent on t h e o t h e r f o r t h e g e n e r a t i o n o f t h e f u n c t i o n a l l y c o r r e c t e l e c t r o n i c s t a t e . In hemoglobin t h e l i g a n d f i e l d o f t h e p o r p h y r i n n i t r o -gens and p r o t e i n h i s t i d i n e s c r e a t e s t h e u n u s u a l y e t e n t a t i c a l l y s t a b i l i s e d e q u i l i b r i u m between h i g h and low s p i n F e ( I I ) which 8 a l l o w s f o r t h e r a p i d b i n d i n g a n d r e l e a s e o f oxygen"1"'". i n t h e c y t o c h r o m e s t h e low e l e c t r o n e g a t i v i t y o f F e ( I I ) i s t r a n s m i t t e d t h r o u g h t h e i r o n - n i t r o g e n bonds c a u s i n g an i n c r e a s e i n e l e c t r o n d e n s i t y on t h e r i n g , a l o w e r i n g o f t h e o x i d a t i o n p o t e n t i a l o f t h e c o m p l e x and t h e f a c i l e l o s s o f an e l e c t r o n a t a p o t e n t i a l d i c t a t e d by t h e p o s i t i o n o f t h e heme p r o t e i n i n t h e e l e c t r o n 4 - 1-13 t r a n s p o r t c h a i n A l t h o u g h t h e s e g r o s s p r o p e r t i e s a r e p r e s e n t i n t h e s i m p l e heme c o m p l e x e s , t h e i m p o r t a n t s u b t l e e f f e c t s a r e i m p a r t e d by t h e e l e c t r o n i c and c o n f o r m a t i o n a l c o n t r o l o f a p r o t e i n . Some o f t h e s e e f f e c t s come f r o m t h e a r r a n g e m e n t o f amino a c i d s i n s p a c e a r o u n d t h e p o r p h y r i n and o t h e r f r o m t h e d i r e c t l i n k a g e o f t h e p r o t e i n t o t h e m e t a l l o p o r p h y r i n s . S i n c e d i f -f e r e n t e l e c t r o n i c s t a t e s a r e r e q u i r e d i n d i f f e r e n t h e m e p r o t e i n s y s t e m s t h e r e a r e a number o f ways i n w h i c h t h i s b i n d i n g c a n t a k e p l a c e . A b r i e f r e v i e w o f t h e w e l l - k n o w n h e m e p r o t e i n s i s w o r t h w h i l e h e r e s i n c e t h e n a t u r e and s e n s i t i v i t y o f p o r p h y r i n p r o t e i n i n t e r a c t i o n i s o f c o n s i d e r a b l e i m p o r t a n c e i n t h e p r o b l e m a t hand. I n a l l h e m e p r o t e i n s t h e c e n t r a l i r o n atom i s t h e f o c a l p o i n t o f a t t a c h m e n t w i t h b o n d i n g t a k i n g p l a c e t o n u c l e o p h i l i c p r o t e i n r e s i d u e s . Two h i s t i d i n e i m i d a z o l e s i d e c h a i n s a r e r e s p o n s i b l e , i n h e m o g l o b i n , m y o g l o b i n and c y t o c h r o m e b5, f o r t h i s c o m p l e x a t i o n w h e r e a s i n c y t o c h r o m e - c one o f t h e a x i a l p o s i t i o n s i s t a k e n by t h e s u l p h u r o f a m e t h i o n i n e r e s i d u e . I n 9 o t h e r h e m e p r o t e i n s t h e s i t u a t i o n i s a l i t t l e l e s s c e r t a i n . P e r o x i d a s e i s t h o u g h t t o have a t l e a s t one h i s t i d i n e a x i a l 14 . . . l i g a n d and t h e . a v a i l a b l e e v i d e n c e p o i n t s t o a c y s t e i n e t h i o l p a r t i c i p a t i n g i n t h e b i n d i n g o f t h e h e p a t i c monooxygenase, 15 c y t o c h r o m e P-450" . T h i s m e t a l - p r o t e i n b i n d i n g i s i n v a r i a b l y a c c o m p a n i e d by o t h e r i n t e r a c t i o n s between t h e p r o t e i n and t h e p e r i p h e r y o f t h e p o r p h y r i n r i n g . The most common o f t h e s e i s t h e h y d r o g e n b o n d i n g o f two f r e e p r o p i o n i c a c i d g r o u p s w i t h b a s i c amino a c i d s , o c c u r i n g i n a l m o s t a l l h e m e p r o t e i n s . O t h e r t y p e s o f b o n d i n g o c c u r more s p e c i f i c a l l y . C y t o c h r o m e - c , i n a d d i t i o n t o t h e m e t a l - p r o t e i n bond, c o n t a i n s two t h i o - e t h e r l i n k a g e s b e tween two c y s t e i n e r e s i d u e s and t h e v i n y l g r o u p s o f p r o t o h e m e . I n c y t o c h r o m e - a t h e p r o s t h e t i c g r o u p c o n t a i n s a f o r m y l g r o u p and a l o n g h y d r o p h o b i c s i d e c h a i n ; s i n c e no c o v a l e n t bond has be e n d e m o n s t r a t e d i t a p p e a r s t h a t h y d r o g e n b o n d i n g and Van d e r Waals f o r c e s must p l a y a r o l e . As y e t t h e p r o s t h e t i c g r o u p s • o f l a c t o p e r o x i d a s e and m y l o p e r o x i d a s e h a ve n o t been i d e n t i f i e d , however heme a p p e a r s t o be l i n k e d t o t h e p r o t e i n by a c o v a l e n t bond. A t h i o e t h e r l i n k t o t h e v i n y l g r o u p s was e l i m i n a t e d and an amide o r e s t e r l i n k a g e t o t h e p r o p i o n i c a c i d g r o u p s 17 has r e c e n t l y been i n v o k e d C l e a r l y t h e i r o n f r e e p o r p h y r i n s and p o r p h y r i n o g e n s e n c o u n t e r e d i n t h e t e r m i n a l s t e p s o f p o r p h y r i n b i o s y n t h e s i s > c a n n o t b i n d t o t h e i r f u n c t i o n a i i s i n g enzymes t h r o u g h a m e t a l ; 10 c o n s e q u e n t l y t h e t o t a l b i n d i n g s t r e n g t h comes f r o m v a r i o u s i n t e r a c t i o n s o f t h e p r o t e i n w i t h t h e p e r i p h e r a l g r o u p s , o f t h e s e , t h e p r o p i o n i c a c i d h y d r o g e n bonds a p p e a r t o be most d o m i n a n t . 1.15. B i o s y n t h e s i s The somewhat s e l f - a b u s i v e d i s c o v e r y o f Shemin, i n 1945, t h a t g l y c i n e was t h e p r i m a r y p o r p h y r i n p r e c u r s o r has h e r a l d e d t h r e e d e c a d e s o f i n t e n s i v e s t u d y i n t o t h e pathway o f p o r p h y r i n b i o s y n t h e s i s i n w h i c h a l l t h e m a j o r i n t e r m e d i a t e s have b e e n i s o l a t e d and c h a r a c t e r i s e d . The 'coming o f age' o f t h e b i o -s y n t h e t i c r o u t e c a n be s e e n by t h e i n c r e a s e d e m p h a s i s i n r e c e n t y e a r s , on t h e d e t a i l e d mechanisms o f t h e t r a n s f o r m a t i o n s and on t h e a c t u a l enzymes t h a t e x e c u t e them. When v i e w e d as a w h o l e t h e p o r p h y r i n b i o s y n t h e t i c pathway p r o v i d e s a w o n d e r f u l example o f t h e k e e n s i m p l i c i t y o f b i o -c h e m i c a l s y s t e m s a s d e t e r m i n e d by t h e t h e r m o d y n a m i c a l l y 19 f a v o u r a b l e r e d u c t i o n i n t h e a c t i v a t i o n e n t r o p y o f e a c h s t e p O n l y a t t h e v e r y f i r s t s t e p o f t h e b i o s y n t h e s i s do two u n l i k e m o l e c u l e s come t o g e t h e r , g l y c i n e (6) and s u c c i n i c a c i d ( 7 ) , 20 t o f o r m 5 - a m i n o l e c u l i n i c a c i d (8) . T h i s i s f o l l o w e d : by a d i m e r i s a t i o n , a t e t r a m e r i s a t i o n and t h e s e q u e n t i a l breakdown o f a l a r g e r s y s t e m t o t h e f i n a l p o r p h y r i n m e t a b o l i t e , a l l e n t r o p i c a l l y more f a v o u r a b l e p r o c e s s e s . Thus by a j u d i c i a l \ The heme biosynthetic pathway 12 c h o i c e of p r e c u r s o r s and a few of the "gimmicks" r e f e r r e d t o 21 by O r g e l , nature has overcome some of the e n e r g e t i c r e s t r i c -t i o n s , i n h e r e n t i n the s y n t h e t i c p r o c e s s , t h a t h i n d e r the attempts of mere m o r t a l s . The d i m e r i s a t i o n s t e p i n v o l v e s the c o n d e n s a t i o n of two molecules o f 5 a m i n o l e c u l i n i c a c i d (8) t o p o r p h o b i l i n o g e n (9) 22 (P.B.G.), the p o r p h y r i n monomer . The 3 p o s i t i o n s o f the p y r r o l e c o n t a i n the impo r t a n t a c e t i c a c i d (A ) and p r o p i o n i c a c i d (P ) combinations t h a t form the b a s i s f o r the p o r p h y r i n p e r i p h e r a l s u b s t i t u e n t s . The combination o f f o u r PBG molecules a p p a r e n t l y o c c u r s i n a s e q u e n t i a l way by deamination of one PBG t o a methylene p y r r o l e n i n e (15) which then a t t a c k s the a f r e e p o s i t i o n o f a second PBG molecule t o form a pyrromethane (16). 13 The process continues w i t h a l l four PBG molecules t o form the m a c r o c y c l i c uroporphyrinogen. When c a r r i e d out i n the presence of the PBG deaminase enzyme the l o g i c a l l y p r e d i c t a b l e uropor-p h y r i n I (17) i s formed. However, when a second enzyme, uro-porphyrinogen I I I cosynthetase, i s present the product i s e x c l u s i v e l y the n a t u r a l l y o c c u r r i n g uroporphyrinogen I I I (10). Thus the enzymic system uses the i n t e r a c t i o n of two p r o t e i n components to e f f e c t a remarkable s w i v e l i n the D r i n g and forir what i s the c h e m i c a l l y unexpected type I I I isomer. The nature of the cooperative a c t i o n between deaminase and cosynthetase and the mechanism of the r i n g D rearrangement i s as yet u n c l e a r , but appears to proceed through a ' s p i r o ' (10) (18) 13 a The f i n a l s t e p s o f t h e pathway i n v o l v e t h e m o d i f i c a t i o n o f t h e s i d e c h a i n s . F i r s t l y t h e f o u r a c e t i c a c i d g r o u p s o f u r o p o r p h y r i n o g e n I I I (10) a r e e n z y m a t i c a l l y and s e q u e n t i a l l y d e c a r b o x y l a t e d t o f o u r m e t h y l g r o u p s , a t p o s i t i o n s 1, 3, 5 and 8 o f t h e t e t r a c a r b o x y l i c c o p r o p o r p h y r i n o g e n I I I ( 1 1 ) . T h e r e t h e n f o l l o w s an o x i d a t i v e d e c a r b o x y l a t i o n o f t h e p r o -p i o n i c a c i d g r o u p s i n p o s i t i o n s 2 and 4 t o t h e v i n y l g r o u p s o f p r o t o p o r p h y r i n o g e n IX ( 1 2 ) . The mechanism o f t h i s t r a n s f o r -m a t i o n i s s t i l l u n d e r s t u d y b u t t h r o u g h t h e i s o l a t i o n o f a 24 25 number o f i n t e r m e d i a t e s ' i t a p p e a r s t h a t t h e p r o p i o n i c ac a t p o s i t i o n 2 r e a c t s b e f o r e t h a t a t 4 and b o t h go t h r o u g h a 3-h y d r o x y i n t e r m e d i a t e . However l a r g e q u a n t i t i e s o f a 2 - a c r y l a t e 4 - p r o p i o n a t e i n t e r m e d i a t e have a l s o been i s o l a t e d and may 2 6 s u g g e s t a n ^ a l t e r n a t i v e m e c h a n i s m (see C h a p t e r 4 ) . The f i n a l s t e p s o f t h e pathway i n v o l v e t h e e n z y m i c aroma-t i s a t i o n o f p r o t o p o r p h y r i n o g e n IX (12) t o p r o t o p o r p h y r i n IX (13) and t h e n , a g a i n , e n z y m i c a l l y c a t a l y s e d i n c o r p o r a t i o n o f i r o n i n t o t h e m a c r o c y c l e f o r m i n g t h e heme p r o s t h e t i c g r o u p (14) S i n c e t h e s e two t r a n s f o r m a t i o n s a r e o f c e n t r a l i m p o r t a n c e i n t h i s p r o j e c t t h e y a r e c o n s i d e r e d i n more d e t a i l i n s e c t i o n 1.2. 1.2. The N a t u r e o f t h e P r o b l e m The m a i n a i m o f t h i s i n v e s t i g a t i o n was t h e s y n t h e s i s o f a p o r p h y r i n d e r i v a t i v e t h a t c o u l d be u s e d , p e t s s i b l y i n a m o d i f i e d f o r m , as t h e i m m o b i l i s e d l i g a n d o f an a f f i n i t y c h r o m a t o g r a p h y 14 c o l u m n , w i t h t h e o b j e c t o f i s o l a t i n g p o r p h y r i n c r heme b i n d i n g enzymes. A f f i n i t y c h r o m a t o g r a p h y i s d i s c u s s e d i n more d e t a i l i n C h a p t e r 3, b u t b r i e f l y , i n v o l v e s t h e i m m o b i l i s a t i o n , by means o f a l o n g f u n c t i o n a l i s e d s i d e c h a i n , o f an a r t i f i c i a l subst3:ate o n t o a s o l i d , s u p p o r t . The enzyme u n d e r s t u d y i s p a s s e d down t h e column and b i n d s t o t h e s u b s t r a t e i s o l a t i n g i t s e l f f r o m t h e o r i g i n a l m i x t u r e . By a l t e r i n g t h e s o l v e n t s y s t e m i t c a n t h e n be e l u t e d , i n a much p u r i f i e d f o r m , f r o m t h e c o l u m n . T h i s v e r y p o w e r f u l t e c h n i q u e h a s been i n g e n e r a l u s e f o r almpst. a d e c a d e and has been s u c c e s s f u l l y a p p l i e d t o a l a r g e number o f enzyme and p r o t e i n s y s t e m s r e s u l t i n g i n p r e p -a r a t i o n s o f a p u r i t y t h a t was p r e v i o u s l y u n a t t a i n a b l e . N o t a b l y a b s e n t i n t h i s s u c c e s s s t o r y , however, a r e t h e p o r p h y r i n and heme b i n d i n g enzymes. A t t e m p t s h a v e , o f c o u r s e , been made, p a r t i c u l a r l y w i t h t h e heme b i n d i n g p r o t e i n hemopoxin, b u t t h e s e 27 2 8 ' f a i l e d t o e f f e c t any p u r i f i c a t i o n ' . The r e a s o n f o r t h e f a i l u r e was u n c l e a r b u t was p r o b a b l y due t o t h e f a c t t h a t b o t h i n v e s t i g a t o r s u s e d t h e p r o p i o n i c a c i d g r o u p s o f p r o t o p o r p h y r i n IX, o r a d e r i v a t i v e , a s t h e means o f a t t a c h m e n t t o t h e s o l i d s u p p o r t . I t i s most l i k e l y t h a t t h e s e g r o u p s f o r m i m p o r t a n t b i n d i n g i n t e r a c t i o n s i n most heme p r o t e i n s and so t h e i r r e -m o v a l w o u l d i n t e r f e r e w i t h enzyme-heme b i n d i n g . T h e r e was t h o u g h t t o be a good c h a n c e t h a t t h e s e d i f f i -c u l t i e s m i g h t be overcome by m o v i n g t h e p o i n t o f a t t a c h m e n t t o a n o t h e r p a r t o f t h e p o r p h y r i n p e r i p h e r y t h a t w o u l d n o t have s u c h a d a m a g i n g i n f l u e n c e o n t h e b i n d i n g t o t h e enzyme. T h e r e -f o r e t h e m a i n t h r u s t o f t h i s s t u d y became t h e f u n c t i o n a l i s a t i o n o f a p o s i t i o n o n t h e p o r p h y r i n r i n g , t h e e x t e n s i o n o f t h i s i n i t i a l c e n t r e t o a l o n g s i d e c h a i n a n d t h e n t h e a t t a c h m e n t o f t h i s s i d e c h a i n t o a s u i t a b l e s o l i d s u p p o r t . C l e a r l y t h e c e n t r a l o b j e c t i v e was t h e i s o l a t i o n and p u r i -f i c a t i o n o f a n enzyme; h o w e v e r a t t h e e a r l y s t a g e s a ' c a t c h 22' d i l e m m a e x i s t e d . F o r e f f e c t i v e . d e s i g n o f t h e a f f i n i t y l i g a n d a k n o w l e d g e o f c e r t a i n a c t i v e s i t e c h a r a c t e r i s t i c s , s u c h a s t h e s p a t i a l r e l a t i o n s h i p o f t h e p o r p h y r i n t o t h e enzyme, was n e e d e d b u t t h e v e r y f a c t t h a t t h e enzyme ha d t o u n d e r g o a f f i n i t y c h r o m a t o g r a p h y f o r p u r i f i c a t i o n means t h a t j t h i s i n f o r m a t i o n , a s w e l l a s many o t h e r i m p o r t a n t p h y s i c a l p r o p e r t i e s o f t h e (19) 16 enzyme, was n o t a l w a y s a v a i l a b l e . T h i s c r e a t e d a c e r t a i n b l i n d n e s s i n t h e s y n t h e t i c r a t i o n a l e s i n c e i t c o u l d n o t be known u n t i l t h e l i g a n d had b e e n s y n t h e s i s e d w h e t h e r b i n d i n g w o u l d t a k e p l a c e . The s t r e n g t h o f b i n d i n g o f t h e e n z y m e - l i g a n d c o m p l e x , as e x p r e s s e d by t h e d i s s o c i a t i o n c o n s t a n t I<D where K D = [ L i g ] [Enz] . , . • . . . ,. , , . . . . r . _ =- , was i m p o r t a n t s i n c e i t must f a l l w i t h i n a c e r t a i n [ L i g - Enz] ^ -2 -5 w o r k i n g r a n g e ( u s u a l l y c o n s i d e r e d as 10 ->- 10 M) . I f i t . was t o o h i g h t h e n no m e a n i n g f u l r e t a r d a t i o n , and t h u s p u r i f i -c a t i o n , would o c c u r as t h e enzyme p a s s e d down t h e a f f i n i t y c o l u mn; i f t o o low t h e n t h e s e v e r i t y o f t h e e l u a n t n e e d ed t o remove t h e enzyme f r o m t h e l i g a n d m i g h t be l i k e l y t o c a u s e s e r i o u s d e n a t u r a t i o n . T h i s l a t t e r p r o b l e m c o u l d o c c u r w i t h t h e heme p r o t e i n s t h a t u t i l i s e t h e heme as a p r o s t h e t i c g r o u p s i n c e b o t h t h e i r s e l e c t i v i t y and d i s s o c i a t i o n c o n s t a n t s a r e o f t e n f a i r l y low. However, w i t h b i o s y n t h e t i c enzymes t h a t e x e c u t e a t r a n s f o r m a t i o n on t h e p o r p h y r i n , i t i s t h e d i s s o c -i a t i o n c o n s t a n t o f t h e e n z y m e - p r o d u c t complex t h a t d i c t a t e s t h e e a s e o f e l u t i o n ; t h e s e a r e u s u a l l y h i g h , s t i m u l a t i n g an e f f i c i e n t t u r n o v e r o f t h e enzyme. I t a p p e a r e d t h a t t h e u n c e r t a i n t i e s i n h e r e n t i n t h e l a t e r s t a g e s o f t h e s t u d y c o u l d a t l e a s t be m i n i m i s e d by a v o i d i n g o t h e r p r o b l e m s e a r l y on and b u i l d i n g i n t o t h e s y n t h e t i c scheme t h e p o s s i b i l i t y o f a l t e r a t i o n . 17 To t h i s end t h e n a t u r a l p o r p h y r i n , p r o t o p o r p h y r i n I X , was chosen as t h e l i g a n d (19). T h i s i s t h e most commonly o c c u r r i n g n a t u r a l p o r p h y r i n and u s u a l l y has t h e l o w e s t b i n d i n g c o n s t a n t o f any p o r p h y r i n f o r a g i v e n p o r p h y r i n o r heme b i n d i n g enzyme. I f t h e d i s s o c i a t i o n c o n s t a n t i s t o o low t h e n a l t e r a -t i o n s c o u l d be made t o d i s t o r t t h e m o l e c u l e and h i n d e r b i n d i n g ; i f t o o h i g h t h e n i t was hoped t h a t t h e s y n t h e t i c scheme would be such t h a t a s l i g h t change i n d i r e c t i o n would g e n e r a t e a s l i g h t l y m o d i f i e d l i g a n d w i t h , p o s s i b l y , a l o w e r b i n d i n g c o n s t a n t . T h i s f l e x i b i l i t y was p a r t i c u l a r l y i m p o r t a n t s i n c e , i f c o m p l e t e l y s u c c e s s f u l , t h i s n o v e l a f f i n i t y chromatograph system may f i n d widespread- use w i t h heme p r o t e i n s and enzymes a l l o w i n g t h e i r s t u d y a t a new and more m e a n i n g f u l l e v e l . I n i t i a l l y i t was hoped' t o a p p l y t h i s method, i n c o l l a b o r a -t i o n w i t h Rozanne P o u l s o n and Denyse Simpson o f t h e U.B.C. B i o c h e m i s t r y Department, t o t h r e e p r o t e i n s w i t h d i f f e r e n t 29 s u b s t r a t e s p e c i f i c i t i e s ; f e r r o c h e l a t a s e ( p r o t o p o r p h y r i n ) , p r o t o p o r p h i n o g e n o x i d a s e " ^ ( p r o t o p o r p h y r i n o g e n ) and hemopexin"^ (heme). > 18 1.3. THE ENZYMES 1.31. F e r r o c h e l a t a s e The f i n a l s t e p o f heme b i o s y n t h e s i s i n v o l v e s t h e i n c o r -p o r a t i o n o f i r o n i n t o p r o t o p o r p h y r i n IX; t h i s t r a n s f o r m a t i o n 32 c a n be a c h i e v e d r e l a t i v e l y e a s i l y i n o r g a n i c s o l v e n t s o r , 33 w i t h c a r e f u l l y c o n t r o l l e d c o n d i t i o n s , i n aqu e o u s s o l v e n t D e s p i t e t h e e a r l y v i e w t h a t i n c o r p o r a t i o n was s t r i c t l y c h e m i -c a l , a number o f enzyme p r e p a r a t i o n s t h a t c a t a l y s e t h e m e t a l l o -p o r p h y r i n f o r m a t i o n h a v e b e e n i s o l a t e d f o r m a v a r i e t y o f b i o -. 29 l o g i c a l s y s t e m s i n c l u d i n g mammalian l i v e r m i t o c h r o n d r i a , o v i a n e r y t h r o c y t e s 3 ^ b a c t e r i a 3 " 3 and c h l o r o p l a s t s 3 ^ ; p r e s u m a b l y , i n t h e l a s t two s y s t e m s , i n c o r p o r a t i n g i r o n i n t o t h e c y t o c h r o m e s . 19 C e r t a i n f e a t u r e s o f t h e e n z y m i c a n d a q u e o u s n o n - e n z y m i c p r e p a r a -33 t i o n s a r e v e r y s i m i l a r ( e . g . pH o p t i m u m 8.0 and a s p e c i -f i c i t y f o r f r e e c a r b o x y l a t e p o r p h y r i n s ) and t h i s c a s t d o u b t on t h e v a l i d i t y o f t h e enzyme p r e p e r a t i o n s . However t h e o b s e r v a t i o n s t h a t on b o i l i n g t h e p r e p a r a t i o n s l o s t t h e i r c a t a -l y t i c a c t i v i t y , a n d t h a t a m u t a n t w h i c h r e q u i r e d p r o t o h e m e 37 f o r g r o w t h l a c k e d a l l f e r r o c h e l e t a s e a c t i v i t y e s s e n t i a l l y c o n f i r m t h e e x i s t e n c e o f s u c h a n enzyme. The enzyme a p p e a r e d t o be membrane b o u n d b e i n g a s s o c i a t e d , p a r t i c u l a r l y , w i t h t h e m i t o c h o n d r i a o f e u k a r i o t s a n d t h e c y t o -35 p l a s m i c membrane o f b a c t e r i a . E x t e n s i v e p u r i f i c a t i o n h a s b e e n h i n d e r e d b e c a u s e o f t h e p a r t i c u l a t e n a t u r e o f t h e p r e p -e r a t i o n . N e v e r t h e l e s s , w o r k done on t h e s p e c i f i c i t y o f t h e enzyme p o i n t e d t o p o s s i b l e a l t e r a t i o n s o f t h e a f f i n i t y l i g a n d 3 8 f o r m o s t e f f i c i e n t b i n d i n g The enzyme, f r o m l i v e r m i t o c h o n d r i a , was f a i r l y s p e c i f i c f o r F e ( I I ) , i n c o r p o r a t i n g Z n ( I I ) a n d C o ( I I ) t o a s m a l l 39 e x t e n t , b u t f o r t h i s o x i d a t i o n s t a t e t o be m a i n t a i n e d , t h e c o n d i t i o n s m u s t be a n a e r o b i c . E s t e r i f i c a t i o n o f t h e p o r p h y r i n 4 0 d i c a r b o x y l i c a c i d g r o u p s d e s t r o y e d a c t i v i t y c o n f i r m i n g t h e n e c e s s i t y f o r t h e f r e e c a r b o x y l i c a c i d g r o u p s i n t h e a f f i n i t y l i g a n d . The v i n y l g r o u p s ^ w e r e n o t , h o w e v e r , e s s e n t i a l f o r b i n d i n g s i n c e many 2 , 4 - d e u t e r o p o r p h y r i n d e r i v a t i v e s h a v e b e e n m e t a l l a t e d • u s i n g t h e enzyme p r e p a r a t i o n s ; t h e r a t e o f i n c o r p o r a -t i o n d e p e n d s , a s m i g h t be e x p e c t e d , s o l e l y o n t h e b a s i c i t y 20 of the porphyrin n i t r o g e n s . In t h i s respect protoporphyrin was one of the l e s s e f f i c i e n t s u b s t r a t e s s i n c e the v i n y l groups have a weakly e l e c t r o n withdrawing e f f e c t . This e l e c -t r o n i c e f f e c t was considered u n l i k e l y to hinder enzyme b i n d i n g and the success of the a f f i n i t y l i g a n d ; i t seemed reasonable t h a t the u n a l t e r e d protoporphyrin IX l i g a n d i t s e l f would be s u f f i c i e n t l y e f f e c t i v e . 1.32. Protoporphyrinogen Oxidase The a r o m a t i s a t i o n of protoporphyrinogen IX (12) t o p r o t o -porphyrin IX (13) i s a f a c i l e r e a c t i o n , o c c u r i n g r e a d i l y i n l i g h t or the presence of oxygen to generate the s t a b l e . c o n -jugated macrocycle. I t was, t h e r e f o r e , thought f o r many years t h a t the o x i d a t i o n was spontaneous and non-enzymic i n b i o l o g i c a l systems. E a r l y b i o s y n t h e t i c experiments, which i n v a r i a b l y i n v o l v e d an exposure to a i r or l i g h t d i d not, or course, r e f u t e t h i s . The e x i s t e n c e of an enzyme was f i r s t suggested by N i s h i d a 41 on very l i t t l e evidence . Much l a t e r t r i t i u m l a b e l i n g s t u d i e s showed th a t h i s p r e d i c t i o n was probably c o r r e c t . Doubly l a b e l l e d coproporphyrinogen I I I , t r i t i a t e d at the meso p o s i -t i o n s , was prepared by treatment of "^C l a b e l l e d magnesium 42 coproporphyrin w i t h deuterium oxide . The meso t r i t i a t e d compound was incubated w i t h e r y t h r o c y t e s and. a measurement of t h e H/ C l a b e l r a t i o showed t h a t 50% o f t h e t r i t i u m had been l o s t . When l a b e l l e d c o p r o p o r p h y r i n o g e n I I I was a u t o o x i d i s e d v i r t u a l l y no t r i t i u m was l o s t b e c a u s e o f i t s i s o t o p e e f f e c t . S t e r e o s p e c i f i c r e m o v a l o f t h e p r o t o n s was t a k i n g p l a c e i n t h e n a t u r a l s y s t e m s t r o n g l y s u g g e s t i n g t h e p r e s e n c e o f an enzyme. R e s u l t s o b t a i n e d by B a t t e r s b y u s i n g d o u b l y l a b e l l e d p o r p h o -43 b i l i n o g e n l e d t o a s i m i l a r c o n c l u s i o n . 30 F i n a l c o n f i r m a t i o n came when P o u l s o n and P o l g l a s e i s o l a t e d a c a t a l y t i c a l l y a c t i v e f r a c t i o n f r o m y e a s t m i t o c h o n -d r i a . They p a r t i a l l y p u r i f i e d t h e enzyme on s e p h a d e x and showed i t t o have a m o l e c u l a r w e i g h t o f a r o u n d 180,000. Compared t o f e r r o c h e l e t a s e t h e enzyme was much more s p e c i f i c f o r i t s -s u b s t r a t e , p r o t o p o r p h y r i n o g e n IX, w i t h a M i c h a e l i s c o n s t a n t — 6 3 0 o f a r o u n d 4.8x10 M . The r a t e o f o x i d a t i o n was r e d u c e d by 8 0% when m e s o p o r p h y r i n o g e n IX v/as u s e d as t h e s u b s t r a t e , s u g g e s t i n g an i n v o l v e m e n t o f t h e v i n y l g r o u p s i n enzyme b i n d -i n g . However more r e c e n t work i n d i c a t e s t h a t o n l y t h e v i n y l g r o u p i n p o s i t i o n 4 i s v i t a l ^ . I n d e s i g n i n g o u r a f f i n i t y l i g a n d t h i s f a c t w o u l d have t o be t a k e n i n t o a c c o u n t , p a r t i c u l a r l y when r e d u c i n g t h e p r o t o -p o r p h y r i n IX l i g a n d b a s e t o t h e s u b s t r a t e p o r p h y r i n o g e n . The 3 0 o b s e r v a t i o n t h a t heme r e v e r s i b l y b i n d s and i n h i b i t s p r o t o -p o r p h y r i n o g e n o x i d a s e i n d i c a t e d a n o t h e r p o s s i b l e d i r e c t i o n f o r t h e a f f i n i t y c h r o m a t o g r a p h y o f t h e enzyme w i t h a heme as t h e - l i g a n d . 1.33. Hemopexin Hemopexin i s a serum g l y c o p r o t e i n w i t h an e x c e e d i n g l y 4 5 h i g h a f f i n i t y f o r heme . I t s m e t a b o l i c f u n c t i o n i s , as y e t , u n c l e a r b u t i t a p p e a r s t h a t i t may p l a y a r o l e a s a c a r r i e r 31 d u r i n g t h e d i s p o s a l o f heme . The c u r r e n t v i e w i s t h a t t h e m a j o r i t y o f h e m o g l o b i n f r o m e r y t h r o c y t e s , a f t e r e x p u l s i o n , i s bound by h a p t o g l o b i n and t h e t w o - p r o t e i n c o m p l e x removed 47 t o t h e l i v e r where breakdown o f t h e heme t o b i l i r u b i n o c c u r s Unbound p l a s m a h e m o g l o b i n t h a t e x c e e d s t h e h a p t o g l o b i n b i n d -i n g c a p a c i t y , d i s s o c i a t e s i n t o i t s component g l o b i n and heme and t h e l a t e r i s o x i d i s e d t o t h e F e ( I I I ) f o r m . The heme t h e n c o m p l e x e s w i t h t h e two serum p r o t e i n s , a l b u m i n and he m o p e x i n . A l b u m i n has a low a f f i n i t y f o r heme c o n c e i v a b l y r e l e a s i n g i t l a t e r f o r f e e d b a c k and i n d u c t i o n c o n t r o l o f heme m e t a b o l i s m . Hemopexin h a s a s t r o n g e r a f f i n i t y f o r t h e heme and a p p e a r s t o c a r r y i t s t r a i g h t t o t h e l i v e r where d e g r a d a t i o n t o b i l e p i g m e n t s 48 46 t a k e s p l a c e . The i n a b i l i t y o f h e m o p e x i n t o b i n d b i l i r u b i n s u g g e s t s t h a t o x i d a t i o n o f t h e heme may t a k e p l a c e w h i l e i t i s s t i l l c o m p l e x e d t o t h e h e m o p e x i n , f o l l o w e d by e a s y r e l e a s e o f t h e b i l i r u b i n and r e u t i l i s a t i o n o f hemopexin; however t h e e x a c t n a t u r e o f b o t h t h e o x i d i s i n g s p e c i e s and heme p r o t e i n 49 complex i n t h i s t r a n s f o r m a t i o n has n o t b e e n e l u c i d a t e d , and so t h e r o l e o f hemopexin r e m a i n s u n c l e a r . 23 The o b s e r v a t i o n t h a t h e m o p e x i n c a n remove t h e heme g r o u p f r o m h e m o g l o b i n 5 0 , b a c t e r i a l c y t o c h r o m e P - 4 5 0 5 1 , a d e n a t u r e d 52 53 f o r m o f mammalian P-4 50 and methemealbumin t e n t a t i v e l y p o i n t s t o a n o t h e r r o l e f o r hemopexin as t h e r e m o v e r o f heme d i r e c t l y f r o m h e m e p r o t e i n s . T h i s p r o p e r t y may a l s o be o f some s y n t h e t i c u s e i n p r e p a r i n g apoenzymes o f h e m e p r o t e i n s w i t h a v e r y low d i s s o c i a t i o n c o n s t a n t . An example o f s u c h a heme-54 p r o t e i n i s c h l o r o p e r o x i d a s e ; r e m o v a l o f t h e s t r o n g l y bound heme a l s o r e s u l t s i n d e n a t u r a t i o n o f t h e enzyme. A p r e p a r a t i o n of. a pohemopexin, w i t h i t s h i g h a f f i n i t y f o r heme, may be a b l e t o e f f e c t t h i s r e m o v a l w i t h o u t d amaging t h e enzyme l e a v i n g i t a v a i l a b l e f o r r e c o n s t i t u t i o n s t u d i e s w i t h d i f f e r e n t m e t a l l o -p o r p h y r i n s . I n o r d e r f o r s t u d i e s on b o t h heme e x c h a n g e w i t h h e m o p e x i n and t h e m e t a b o l i c r o l e o f hemopexin t o a d v a n c e , a more p u r i -f i e d p r e p a r a t i o n o f t h e enzyme i s n e e d e d . T h i s may be p r o -v i d e d by an e f f i c i e n t a f f i n i t y c h r o m a t o g r a p h y s y s t e m . The v e r y n a t u r e o f h e m o p e x i n may, however, c r e a t e some . p r o b l e m s w i t h t h i s . The v e r y low d i s s o c i a t i o n c o n s t a n t (Kd << 10 8 M ) 4 6 f o r t h e hemopexin-heme i n t e r e a c t i o n s u g g e s t e d t h a t i f a heme b a s e d a f f i n i t y l i g a n d was u s e d , e l u t i o n f r o m t h e column w o u l d be i m p o s s i b l e w i t h o u t d e s t r o y i n g t h e enzyme. Hemopexin i s a v e r y n o n s p e c i f i c h e m e p r o t e i n b i n d i n g t o a l m o s t a n y t h i n g w i t h a t e t r a p y r r o l i c n u c l e u s , i n c l u d i n g m e t a l s u b -55 55 4 6 s t i t u t e d heme , p o r p h y r i n s and e v e n d i a q u o c o b x n a m i d e , 24 a l t h o u g h n o t v i t a m i n 5^.2^°" T h e m a ^ - n b i n d i n g i n t e r a c t i o n a p p e a r s t o be t h a t o f t h e c e n t r a l m e t a l t o t h e p r o t e i n g i v i n g - 7 4 6 d i s s o c i a t i o n c o n s t a n t s o f a p p r o x i m a t e l y 10 M f o r heme and 1 I I I I eve n t h e m e t a l s u b s t i t u t e d hemes ( e . g . Co ' and N i ) . C l e a r l y o t h e r p o i n t s o f a t t a c h m e n t t o t h e enzyme e x i s t s i n c e p r o t o -— 6 3 2 p o r p h y r i n IX (Kd ^  10 M) b i n d s so s t r o n g l y . Removal o f t h e v i n y l g r o u p s has no e f f e c t ( d e u t e r o p o r p h y r i n and 2,4-— 6 32 d i s u l p h o n i c a c i d d e u t e r o p o r p h y r i n ; Kd ^ 10 ) and s o , p r e s u m a b l y , a s t r o n g l i n k t o t h e p r o p i o n i c a c i d g r o u p s i s p r e s e n t . I n t e r m s o f t h e d e s i g n o f t h e a f f i n i t y l i g a n d t h e d i s -s o c i a t i o n c o n s t a n t (<< 10 ^M) f o r a heme b a s e d l i g a n d may be t o o low, d e p e n d i n g on t h e amount o f d i s t u r b a n c e c a u s e d by t h e l o n g s i d e c h a i n . A more r e a s o n a b l e a p p r o a c h w o u l d be t h e u s e o f t h e b a s i c p o r p h y r i n l i g a n d (19) w i t h i t s h i g h e r d i s s o c i a t i o n c o n s t a n t (Kd ^ 10 6 ) w h i c h s h o u l d f a c i l i t a t e e l u t i o n f r o m t h e col u m n . I f a g a i n t h e b i n d i n g i n t e r a c t i o n i s t o o s t r o n g , an a l t e r n a t i v e might be t o u s e a heme b a s e d l i g a n d , a l l o w t h e enzyme t o b i n d and t h e n t o c a r r y o u t a c o u p l e d o x i d a t i o n o f t h e heme w h i l e i t i s s t i l l bound t o t h e c o l u m n and t h e enzyme. T h i s c o u l d , c o n c e i v a b l y , be c a r r i e d o u t w i t h an a s c o r b i c a c i d -56 57 oxyg e n o r a h y d r a z i n e - o x y g e n s y s t e m o r t h e more d i r e c t h y d r o g e n p e r o x i d e method. A f t e r h y d r o l y s i s t h e p r o d u c t , s t i l l bound t o t h e colu m n , w o u l d be b i l i v e r d i n ; t h i s h as a low a f f i n i t y f o r h e m o p e x i n and t h u s w o u l d a l l o w t h e enzyme t o be e l u t e d ) 25 e a s i l y f r o m t h e c o l u m n . I n t h i s p r o c e d u r e t h e heme l i g a n d w o u l d , c l e a r l y , be d e s t r o y e d f o r f u r t h e r u s e b u t t h i s may be t h e o n l y a p p r o a c h f o r t h e p u r i f i c a t i o n o f t h i s s t r o n g l y b i n d i n g and v e r y i n t e r e s t i n g p r o t e i n . 2. DESIGN OF THE AFFINITY LIGAND 2.1. Synthetic Requirements The ligand for an a f f i n i t y chromatography column i s , i n essence, a 'molecular t o o l ' , and as such the requirements of i t s synthetic approach are conceptually d i f f e r e n t from those usually encountered i n that f e l i c i t o u s world of organic synthe-5 8 s i s . "Elegance" as Eschenmoser pointed out "must be l e f t to t a i l o r s and cobblers" and other more mundane, but nonetheless v i t a l , goals must dominate. The a f f i n i t y ligand acts as an a r t i f i c i a l substrate causing the enzyme to bind and so slowing i t s progress down the column. To e f f e c t t h i s at the active s i t e of the enzyme the ligand must, i n general, bear as close a resemblance as p o s s i b l e to the natural substrate and so maintain a s u f f i c i a n t l y low d i s s o c i a t i o n constant to gain a worthwhile p u r i f i c a t i o n . A major aim of the synthesis was, thus, to prepare an o a f f i n i t y ligand from a natural porphyrin and i n doing so to preserve the basic s i m i l a r i t y to the substrate despite the i n s t a b i l i t y of the natural porphyrins. The ligand and natural substrate structures must, c l e a r l y , d i f f e r . since the former i s attached to a s o l i d support. This attachment takes place through a long a l i p h a t i c chain which i s introduced onto the porphyrin periphery i n place of one of the natural e substituents. This can a l t e r both the e l e c t r o n i c and 27 c o n f o r m a t i o n a l p r o p e r t i e s o f 'the n a t u r a l - p o r p h y r i n a n d c o n -s e q u e n t l y t h e p c r p h y r i n - e a z y - m e i n t e r a c t i o n . The . e x t e n t o f t h e a l t e r a t i o n a n d s e n s i t i v i t y o f t h e enzyme t o i t d e p e n d o n a number o f f a c t o r s . S u b s t i t u t i o n d i r e c t l y onto t h e meso p o s i t i o n t e n d s t o c a u s e a b u c k l i n g i n t h e p o r p h y r i n r i n g a n d , i n t u r n , t h i s d e v i a t i o n f r o m p l a n a r i t y d i s t u r b s t h e a r o m a t i c s t a b i l i t y o f 5Q t h e m a c r o c y c l e " . A l t e r a t i o n of a 8 s u b s t i t u e n t wil 1 -have l e s . s o f an e f f e c t on t h e s h a p e o f t h e r i n g b u t w i l 1 c h a n g e b o t h t h e s p a t i a l a n d , i f s u b s t i t u t i o n o c c u r s a t t h e v i n y l o r p r o -p i o n i c a c i d g r o u p s , t h e e l e c t r o n i c p r o f i l e o f t h e p e r i p h e r y . The s e n s i t i v i t y , a n d r e s u l t a n t i n c r e a s e i n d i s s o c i a t i o n c o n s t a n t , o f t h e enzyme t o a n y d e v i a t i o n f r o m t h e n o r m a l s u b -s t r a t e s t r u c t u r e w i l l d e p e n d on t h e way i n w h i c h t h e enzyme b i n d s t h e p o r p h y r i n , b o t h i n t h e d i r e c t p o r p h y r i n - e n z y m e b o n d and t h e more d i s t a n t d i s t r i b u t i o n o f t h e p r o t e i n a r o u n d t h e p o r p h y r i n . I f t h e enzyme i s p r e v e n t e d f r o m b i n d i n g t o t h e p o r p h y r i n a t any o f i t s u s u a l a t t a c h m e n t s i t e s ( e . g . c e n t r a l m e t a l , v i n y l g r o u p s o r p r o p i o n i c a c i d g r o u p s ) b y t h e p r e s e n c e o f a l o n g c h a i n s u b s t i t u e n t t h e n c o m b i n a t i o n w i l l n o t t a k e p l a c e . I t i s a l s o p r o b a b l e t h a t , on c e r t a i n p o s i t i o n s a r o u n d t h e r i n g , t h e l o n g c h a i n w i l l s t e r i c a l l y i n t e r a c t w i t h some p a r t s o f t h e p r o t e i n , i n t e r f e r i n g w i t h t h e f o l d i n g o f t h e enzyme, and p r e v e n t i n g t h e s u b s t r a t e - i n d u c e d g e n e r a t i o n o f t h e c o r r e c t a c t i v e s i t e g e o m e t r y . T h i s w i l l , h o w e v e r , d e p e n d o n t h e ) 28 p o s i t i o n on t h e r i n g t h a t i s s u b s t i t u t e d and t h e l o c a t i o n o f t h e a c t i v e s i t e i n t h e enzyme t e r t i a r y s t r u c t u r e . The more t h e p o r p h y r i n s u b s t r a t e i s e x p o s e d above t h e s u r f a c e o f t h e enzyme t h e l e s s i m p o r t a n t t h e s t e r i c p r o b l e m becomes. F o r -t u n a t e l y t h e a c t i v e s i t e s o f most p o r p h y r i n o r heme b i n d i n g enzymes a r e c l o s e t o t h e s u r f a c e . A c o m p l e t e l y b u r i e d a c t i v e s i t e w o u l d s e v e r e l y r e d u c e t h e l i k e l i h o o d t h a t an a f f i n i t y c h r o m a t o g r a p h y p u r i f i c a t i o n w o u l d be s u c c e s s f u l . Many o f t h e s e f a c t o r s h a v e n o t b e e n c o m p l e t e l y e l u c i d a t e d f o r w e l l - k n o w n s y s t e m s and so w i t h i n c o m p l e t e l y c h a r a c t e r i s e d enzymes a c o n s i d e r a b l e u n c e r t a i n t y i s i n t r o d u c e d i n t o any a f f i n i t y c h r o m a t o g r a p h y i n v e s t i g a t i o n . T h i s u n d e r l i n e s a n e e d f o r f l e x i b i l i t y i n t h e l i g a n d s y n t h e s i s . I d e a l l y i t s h o u l d be p o s s i b l e t o a l t e r s l i g h t l y t h e s y n t h e t i c scheme and g e n e r a t e a d i f f e r e n t l i g a n d w h i c h w o u l d accommodate t h e a c t i v e s i t e c h a r a c t e r i s t i c s o f d i f f e r e n t enzymes. A l t e r n a t i v e l y t h e a p p r o a c h s h o u l d p r o v i d e a number o f e a s i l y i s o l a t e d and c h a r a c t e r i s e d i s o m e r s w h i c h c a n t h e n be t e s t e d on t h e a f f i n i t y c o l u m n f o r e f f e c t i v e n e s s . L a s t l y t h e s y n t h e s i s s h o u l d be r e l a t i v e l y s i m p l e . T h e r e a r e many enzyme s y s t e m s i n many l a b o r a t o r i e s t h a t m i g h t b e n e -f i t f r o m a p o r p h y r i n a f f i n i t y c o l u m n . • I f t h e method i s t o f i n d g e n e r a l u s e i t i s i m p o r t a n t t h a t t h e l i g a n d s h o u l d n o t be made i n a c c e s s i b l e by a c o m p l e x and t e c h n i c a l l y d i f f i c u l t s y n t h e s i s , . 29 2.2. A p p r o a c h e s The m a j o r i t y o f p o r p h y r i n s y n t h e s e s f o u n d i n t h e l i t e r a t u r e ^ " * " , 6 2 i n c l u d i n g t h o s e w i t h l o n g s i d e c h a i n s , h a v e i n v o l v e d c o n -s t r u c t i o n o f t h e f o u r p y r r o l i c u n i t s and t h e n a b u i l d up o f t h e m a c r o c y c l e t h r o u g h a '2+2' J o r a l i n e a r t e t r a p y r r o l i c s e q u e n c e ^ . T h i s h a s t h e a d v a n t a g e o f l a r g e s c a l e p o t e n t i a l and an u n e q u i v o c a l p r o d u c t . However any f l e x i b i l i t y i n t h e n a t u r e o f t h e p r o d u c t i s l o s t s i n c e a d i f f e r e n t , o r i s o m e r i c , l i g a n d c a n o n l y be f o r m e d by a t o t a l l y new s y n t h e s i s . The s t e p w i s e method i s a l s o t i m e c o n s u m i n g , c o s t l y , c o m p l i c a t e d and t h e r e f o r e u n s u i t a b l e f o r an a f f i n i t y l i g a n d s y n t h e s i s . As t h e p r o s p e c t i v e l i g a n d was t o be a m o d i f i e d n a t u r a l p o r p h y r i n i t seemed more r e a s o n a b l e t o s t a r t f r o m one o f t h e n a t u r a l l y o c c u r r i n g p o r p h y r i n s . The a d v a n t a g e s o f u s i n g a p e r -f o r m e d p o r p h y r i n a r e numerous. The c o m p l e x c o n s t r u c t i o n o f an u n s y m m e t r i c a l t e t r a p y r r o l e , w h i c h r e q u i r e s a c o n s i d e r a b l e e f f o r t i n t h e s t e p w i s e s y n t h e s i s , i s a c h i e v e d w i t h consummate e a s e i n n a t u r e l e a v i n g t h e s y n t h e s i s o f t h e a f f i n i t y l i g a n d s i m p l y a m a t t e r o f t h e m o d i f i c a t i o n o f p e r i p h e r a l g r o u p s . The v a r i e t y o f t h e p o r p h y r i n s s i d e c h a i n s f a c i l i t a t e s v a r i a t i o n i n t h e n a t u r e o f t h e l i g a n d p r o d u c t . To i t s d i s a d v a n t a g e , however, t h e f u n c t i o n a l g r o u p s a r e n o t u n i q u e and any g i v e n r e a c t i o n may t a k e p l a c e a t a number o f p o s i t i o n s . T h i s i n t r o -d u c e s t h e p r o b l e m o f s e p a r a t i n g a number o f v e r y s i m i l a r i s o m e r s , a d i f f i c u l t t a s k g i v e n t h e i n h e r e n t i n s t a b i l i t y o f n a t u r a l p o r p h y r i n s . 30 2.3. The S y n t h e t i c Base The two n a t u r a l l y o c c u r r i n g t e t r a p y r r o l i c compounds t h a t a r e most r e a d i l y a v a i l a b l e f o r p r e p a r a t i v e s c a l e work a r e c h l o r o p h y l l (20) and h e m a t o p o r p h y r i n ( 2 1 ) . E i t h e r c o u l d be u s e d a s t h e s t a r t i n g p o i n t o f a p o t e n t i a l s y n t h e t i c scheme. However, b e f o r e any m o d i f i c a t i o n o f t h e c h l o r o p h y l l m o l e c u l e c o u l d o c c u r i t w o u l d have t o be d e g r a d e d t o t h e more u s e f u l 64 v i n y l p y r r o p o r p h y r i n ( 2 2 ) ; a f a r f r o m t r i v i a l p r o c e d u r e The u s e o f c h l o r o p h y l l a i s f u r t h e r c o m p l i c a t e d by i t s un-a v o i d a b l e c o n t a m i n a t i o n w i t h 2 0% o f c h l o r o p h y l l b. S e p a r a t i o n 65 i s p o s s i b l e , b u t d i f f i c u l t , and t h e p u r e compounds a r e p r o h i b i t i v e l y e x p e n s i v e t o buy c o m m e r c i a l l y . On t h e o t h e r hand h e m a t o p o r p h y r i n (21) i s r e a d i l y a v a i l -a b l e f r o m b l o o d by t r e a t m e n t w i t h c o n c e n t r a t e d s u l p h u r i c a c i d . C02Phytyl (20) (21) (22) 31 T h i s t r e a t m e n t b o t h removes t h e c e n t r a l i r o n atom and h y d r a t e s t h e v i n y l g r o u p s o f t h e p a r e n t heme. C o m m e r c i a l l y t h e p i g m e n t i s a v a i l a b l e f r o m a number o f s o u r c e s ( e . g . I.C.N, and Sigma) and a l l i n v a r i a b l y c o n t a i n s m a l l amounts o f t h e monohydroxy e t h y l m o n o v i n y l (21a) and d i v i n y l d e r i v a t i v e s (23) as a r e -s u l t o f t h e i n s t a b i l i t y o f t h e p o r p h y r i n i c a l c o h o l . T h e s e im-p u r i t i e s were u n i m p o r t a n t i n t h i s work s i n c e t h e f i r s t s t e p o f t h e s y n t h e s i s was t o u t i l i s e t h e v e r y i n s t a b i l i t y t h a t i n i t i a l l y f o r m e d them as i m p u r i t i e s t o g e n e r a t e p r o t o p o r p h y r i n IX (23) . T h i s t r a n s f o r m a t i o n h as been c a r r i e d o u t w i t h an o-d i c h l o r o b e n z e n e / p - t o l u e n e s u l p h o n y l c h l o r i d e t r e a t m e n t ^ ^ b u t was more s i m p l y a c h i e v e d h e r e by t h e a d d i t i o n o f hemato-p o r p h y i n IX (21) t o b o i l i n g d i m e t h y l f o r m a m i d e f o r 30 s e c o n d s . D e v e l o p e d i n t h i s g r o u p by R.K. D i N e l l o , t h i s method gave a v e r y p u r e p r o t o p o r p h y r i n IX p r o d u c t w i t h y i e l d s t h a t were c o n s i s t e n t l y above 90%. In t h e n a t u r a l d i c a r b o x y l i c a c i d f o r m , p r o t o p o r p h y r i n IX (23) i s s o l u b l e o n l y i n s t r o n g b a s e o r t h e o r g a n i c ' s u p e r s o l v e n t s ' s u c h a s p y r i d i n e , DMF, DMSO and HMPA. C o n s e q u e n t l y i t i s i m p r a c t i c a l t o work w i t h and i m p o s s i b l e t o p u r i f y by any l a r g e s c a l e c h r o m a t o g r a p h i c t e c h n i q u e . T h e s e p r o b l e m s a r e u s u a l l y o vercome by f o r m i n g t h e d i m e t h y l e s t e r (24a) d e r i v a -t i v e w i t h e i t h e r d i a z o m e t h a n e o r a m e t h a n o l - m i n e r a l a c i d 6 8 m i x t u r e . T h i s , i n t u r n , c o u l d c r e a t e a p r o b l e m a t t h e end o f t h e s y n t h e t i c scheme when t h e n a t u r a l d i a c i d s t a t e o f t h e p o r p h y r i n must be r e g e n e r a t e d . A c i d o r b a s e h y d r o l y s i s o f t h e d i m e t h y l e s t e r (24a) w o u l d , a t t h a t s t a g e , u n d o u b t e d l y c a u s e 6 8 h y d r a t i o n o f t h e s e n s i t i v e v i n y l g r o u p s . A more e x p e d i e n t c h o i c e f o r t h e e s t e r f u n c t i o n w o u l d be t e r t i a r y b u t y l e s t e r g r o u p s w h i c h , b e i n g s u s c e p t i b l e t o a c i d c a t a l y s e d a l k y l o x y g e n 69 f i s s i o n , c a n r e a d i l y be removed by d r y h y d r o g e n c h l o r i d e i n m e t h y l e n e c h l o r i d e o r t o l u e n e s u l p h o n y l c h l o r i d e i n r e f l u x i n g b e n z ene; u n d e r w h i c h c o n d i t i o n s t h e v i n y l g r o u p s o f p r o t o -69 p o r p h y r i n IX s h o u l d be s t a b l e . ' 33 The d i t e r t i a r y b u t y l e s t e r o f p r o t o p o r p h y i n IX (24b) was made v i a t h e d i a c i d c h l o r i d e by t r e a t m e n t o f t h e d i c a r -b o x y l i c a c i d w i t h o x a l y l c h l o r i d e f o l l o w e d by d r y t e r t i a r y b u t y l a l c o h o l . E s t e r i f i c a t i o n c a u s e d t h e p o r p h y r i n t o be e x t r e m e l y s o l u b l e i n m e t h y l e n e c h l o r i d e and c h l o r o f o r m . I n t h e s e s o l v e n t s i t e x h i b i t e d t h e e x p e c t e d e t i o - t y p e v i s i b l e s p e c t r u m o f p r o t o p o r p h y r i n IX; i n t r o d u c t i o n and v a r i a t i o n o f t h e e s t e r g r o u p s h a s v i r t u a l l y no e f f e c t on t h e s p e c t r u m 1 0 . However c a r e had t o be t a k e n , p a r t i c u l a r l y when t h e e s t e r was i n s o l u t i o n , t o a v o i d e x p o s i n g i t t o l i g h t a nd h e n c e a v o i d the. f a c i l e 1,4 a d d i t i o n o f s i n g l e t o x y g e n t o t h e v i n y l g r o u p s g i v i n g t h e g r e e n p h o t o p r o t o p o r p h y r i n d e r i v a t i v e (see s e c t i o n 2 . 42). 2.4. I n t r o d u c t i o n o f t h e C h a i n The p r o b l e m o f i n t r o d u c i n g a l o n g c h a i n c e n t r e d on t h e u t i l i s a t i o n o r g e n e r a t i o n o f some f u n c t i o n a l i s e d c e n t r e i n t h e m o l e c u l e t o w h i c h a f u r t h e r , l o n g c h a i n , s u b s t i t u e n t c o u l d be a t t a c h e d . 2.41. The V i l s m e i e r A p p r o a c h M o l e c u l a r o r b i t a l c a l c u l a t i o n s ^ 0 and e x p e r i m e n t a l r e s u l t s ^ 1 b o t h i n d i c a t e t h a t t h e m e t h i n e b r i d g e s o f t h e p o r p h y r i n m a cro-c y c l e a r e more r e a c t i v e t h a n t h e a and \i p o s i t i o n s o f t h e f o u r 34 r r o l e r i n g s to a l l types of r e a c t i o n ; t h i s i s seen p a r t i c u -i r l y i n e l e c t r o p h i l i c but a l s o n u c l e o p h i l i c and r a d i c a l 71 :actions . The most important e l e c t r o p h i l i c r e a c t i o n s c a r r i e d it on the periphery of the p o r p h y r i n have been V i l s m e i e r 72 73 74 75 rmylation , bromination , n i t r a t i o n and d e u t e r a t i o n a l l cases the r e a c t i v i t y and meso s e l e c t i v i t y of these e c t r o p h i l i c a t t a c k s were g r e a t l y enhanced by the presence of i e l e c t r o n r i c h metal i n the centre of the porphyrin r i n g . This f a s c i n a t i n g aspect of the i n f l u e n c e of a metal ion the organic r e a c t i v i t y o f the l i g a n d has been the subject 71 76 much study ' . As might be expected i t was found that t r o d u c t i o n of a metal i o n of low e l e c t r o n e g a t i v i t y (e.g. Z n 1 1 , C u 1 1 , N i 1 1 , C o 1 1 ) i n t o the porphyrin enhanced the s e e p t i b i l i t y to e l e c t r o p h i l i c a t t a c k . T h i s i s due to the creased e l e c t r o n d e n s i t y on the r i n g , donated from the metal, id hence a lowered o x i d a t i o n p o t e n t i a l and increased nucleo-t i l i c i t y . At the other extreme h i g h l y e l e c t r o n e g a t i v e ions :.g. S n I V) d e a c t i v a t e the r i n g towards e l e c t r o p h i l i c attack 71 id , i n f a c t , promote n u c l e o p h i l i c a t t a c k The reason f o r the i n f l u e n c e of the metal on the r i n g i s irder to d e l i n e a t e . Although an a t t r a c t i v e r a t i o n a l i s a t i o n , -pn bonding does not appear to be present i n most me t a l l o ->rphyrins, because the energy of the antibonding o r b i t a l s of ie macrocycle i s p r o h i b i t i v e l y high f o r o v e r l a p with the metal o r b i t a l s to take place. Of more importance are the metal-35 n i t r o g e n a bonds and t h e i r a b i l i t y t o t r a n s m i t c h a n g e s i n 71 e l e c t r o n e g a t i v i t y o f t h e m e t a l t o t h e p o r p h y r i n The r e a c t i v i t y p a r a m e t e r s o f t h e V i l s m e i e r r e a c t i o n , w i t h r e g a r d t o t h e c e n t r a l m e t a l e f f e c t , f o l l o w t h e g e n e r a l . p a t t e r n . However s i n c e t h e r e a c t i o n i s c a r r i e d o a t u n d e r t h e a c i d i c c o n d i t i o n s o f p h o s p h o r u s o x y c h l o r i d e and DMF t h e a c i d l a b i l e m e t a l l o p o r p h y r i n s , d e s p i t e b e i n g p o t e n t i a l l y more r e a c t i v e due t o t h e i r v e r y low e l e c t r o n e g a t i v i t y , a r e d e -"76 7 6 I I I I m e t a l l a t e d . J o h n s o n showed t h a t Cu and N i p o r p h y r i n s r e a d i l y gave t h e mono m e s o - f o r m y l p r o d u c t (25) whereas C o 1 1 i o n s so a c t i v a t e t h e p e r i p h e r y t h a t t h e u s u a l d e a c t i v a t i o n o f one f o r m y l g r o u p c o u l d be o v e r c o m e t o g i v e t h e d i f o r m y l d e r i v a t i v e . (25) P r e d i c t a b l y t h e Zn1"*" s p e c i e s was d e m e t a l l a t e d and, u n d e r t h i s c o n d i t i o n , o f a d i l u t e d r e a c t i o n m i x t u r e , t h e more e l e c t r o -n e g a t i v e t r i v a l e n t F e 1 1 1 and M n 1 1 1 s u b s t i t u t e d p o r p h y r i n s d i d n o t r e a c t . 36 The e a s e and s e l e c t i v i t y o f t h e V i l s m e i e r f o r m y l a t i o n i s a l s o i n f l u e n c e d by t h e s t e r i c i n t e r f e r e n c e between t h e p o r -p h y r i n s i d e c h a i n s and a t t a c k i n g V i l s m e i e r c o m p l e x . I t i s t h i s i n t e r a c t i o n t h a t p r e s u m a b l y p r e v e n t s F r i e d e l - C r a f t s 71 a c y l a t i o n , wxth i t s l a r g e a c t i v e i n t e r m e d i a t e , on s u b s t i t u t e d p o r p h y r i n s w h i l s t a l l o w i n g a c c e s s t o t h e s m a l l e r V i l s m e i e r c o mplex. 72 I n h o f f e n showed t h a t f o r m y l a t i o n o f c o p p e r ( I I ) d e u t e r o -p o r p h y r i n (2 6) gave a number o f mono and d i f o r m y l a t e d p r o d u c t s w h i c h . c o u l d be i s o l a t e d on a l u m i n a ; t h e s e i n c l u d e d m o n o f o r m y l p o r p h y r i n s s u b s t i t u t e d i n t h e 2 and 4 3 p o s i t i o n s and t h e a and 8 meso p o s i t i o n s ( 2 7 ) . T h e s e a r e t h e l e a s t h i n d e r e d p o s i t i o n s o f t h e p o r p h y r i n . S i m i l a r d i r e c t i o n a l e f f e c t s were 77 f o u n d when n a t u r a l i r o n ( I I I ) p o r p h y r i n s were f o r m y l a t e d 37 The use o f t h e V i l s m e i e r f o r m y l a t i o n i n an a f f i n i t y l i g a n d s y n t h e s i s grew o u t o f t h e hope t h a t , by u s i n g a s u i t a b l e c e n t r a l m e t a l , i t would be p o s s i b l e t o i n t r o d u c e a l o n g c h a i n s u b s t i t u e n t i n t o t h e l e a s t h i n d e r e d meso p o s i t i o n o f p r o t o -p o r p h y r i n IX d i t e r t i a r y b u t y l e s t e r (DTBE). I n t h e o r y t h e l e a s t h i n d e r e d y s u b s t i t u t i o n s h o u l d p r e d o m i n a t e w i t h l e s s e r amounts of. t h e a and 8 form. These s h o u l d a l l be i s o l a b l e 72 and i d e n t i f i a b l e g i v i n g r i s e t o t h r e e i s o m e r i c a f f i n i t y l i g a n d s . 72,76,7 The p o r p h y r i n V i l s m e i e r r e a c t i o n s r e p o r t e d up t o now have a l l i n v o l v e d f o r m y l a t i o n s . A t t e m p t s t o f u r t h e r r e a c t t h e 7 8 f o r m y l group and e x t e n d t h e s i d e c h a i n (e.g. W i t t i g land 7 6 Knoevenagel r e a c t i o n s ) had a l l p r e v i o u s l y f a i l e d . T h i s was due t o t h e h i g h c o n t r i b u t i o n o f t h e e n o l f o r m t o t h e c a r b o n y l s t r u c t u r e w h i c h i n t u r n was caused by t h e v i n y l o g o u s amide e f f e c t o f t h e p o r p h y r i n . F o r t h e a f f i n i t y l i g a n d more hope l a y i n t h e a p p l i c a t i o n o f t h e V i l s m e i e r a c y l a t i o n . A l t h o u g h never, a p p l i e d t o p o r p h y r i n s t h i s r e a c t i o n has been e x t e n s i v e l y 79 used i n p y r r o l e c h e m i s t r y t o i n t r o d u c e l o n g c h a i n a c y l groups o n t o p y r r o l e a p o s i t i o n s . I t was a r e a s o n a b l e a s s u m p t i o n t h a t p o r p h y r i n meso p o s i t i o n s would a l s o undergo t h i s s u b s t i t u t i o n . C H f ^ N H  3 H P0C13 (CH3)iNCOR R B2H6 R P i m e l i c a c i d m o n o b e n z y l e s t e r mono N - m e t h y l a n i l i d e (30) v;a c h o s e n a s t h e d i s u b s t i t u t e d a m i d e s i n c e i t w o u l d g i v e a s u f -f i c i e n t s p a c e r d i s t a n c e f r o m t h e s o l i d s u p p o r t a n d s i n c e t h e b e n z y l e s t e r c o u l d be r e m o v e d s p e c i f i c a l l y p r i o r t o b i n d i n g t o t h e c o l u m n . I t was p r e p a r e d f r o m p i m e l i c a c i d (28) by d i -b e n z y l a t i o n t h r o u g h t h e a c i d c h l o r i d e f o l l o w e d by c o n t r o l l e d h y d r o g e n o l y s i s o f one b e n z y l g r o u p o v e r p a l l a d i s e d c h a r c o a l . The N - m e t h y l N - p h e n y l a m i d e (30) was p r e p a r e d by r e a c t i o n o f N - m e t h y l a n i l i n e w i t h t h e a c i d c h l o r i d e o f ( 2 9 ) . iSO-Cfe / ; H0 2 C(CH 2 ) 5 C0 2 H - ^ c H z O H ^ P "CH 20 2C fCH 2) 5C0 2CH 2-p (28) H 2 RVC ^OCfCH 2 ) , -CO 2CHr0 ^ 2 _ C J ^ H n r / r M . r r r , r> ClH3 2 o 2 2.CH3-.^H. ^U 2 C(CH 2 ) 5 CU 2uh 2-p (3.0) 0 " (29) The o v e r a l l p l a n was t o i n t r o d u c e t h e l o n g c h a i n a c y l g r o u p i n t o t h r e e p o s i t i o n s a r o u n d t h e r i n g , s e p a r a t e t h e i s o m e r s and t h e n r e d u c e t h e k e t o n e w i t h d i b o r a n e t o g i v e , a f t e r r e -m o v a l o f t h e b e n z y l g r o u p b y c a t a l y t i c h y d r o g e n a t i o n a n d d e -m e t a l l a t i o n , t h r e e i s o m e r i c a f f i n i t y l i g a n d s ( 3 3 ) ; e a c h one u s e f u l f o r a d i f f e r e n t a c t i v e s i t e g e o m e t r y . a , M = Zn b, M = Co * a l t e r n a t i v e a t t a c h m e n t s i t e I n s u c h a scheme t h e c e n t r a l m e t a l i s o f p a r a m o u n t i m p o r t a n c e s i n c e n o t o n l y m u s t i t a c t i v a t e t h e meso p o s i t i o n s t o e l e c t r o p h i l i c a t t a c k b u t i t m u s t a l s o be r e m o v e d e a s i l y l a t e r o n i n t h e s y n t h e s i s w i t h o u t a f f e c t i n g t h e a c i d l a b i l e v i n y l a n d d i t e r t i a r y b u t y l e s t e r g r o u p s . The m o s t w i d e l y u s e d I I I I m e t a l s , Cu a n d N i _ , w e r e i n a p p r o p r i a t e s i n c e t h e i r r e s p e c -t i v e m e t a l l o p o r p h y r i n s a r e e x t r e m e l y s t a b l e r e q u i r i n g t h e f o r c i n g c o n d i t i o n s o f c o n c e n t r a t e d s u l p h u r i c a c i d t o r e m o v e 72 t h e m 4 0 Z i n c (I.I) , w i t h i t s low e l e c t r o n e g a t i v i t y and d"1"^ c o n f i g u r a t i o n , o f f e r e d a compromise b e i n g h i g h l y a c t i v a t i n g y e t e a s i l y removed. U s i n g a g e n e r a l l y a p p l i c a b l e method o f p o r p h y r i n m e t a l l a t i o n t h e z i n c , a s i t s a c e t a t e s a l t , was i n t r o -d u c e d i n t o p r o t o p o r p h y r i n IX DTBE i n b o i l i n g DMF. A l m o s t i n s t a n t a n e o u s l y t h e p o r p h y r i n f o u r l i n e v i s i b l e s p e c t r u m c h a n g e d t o t h e c h a r a c t e r i s t i c two l i n e s o f t h e m e t a l l o p o r -p h y r i n (3.1a) . N o r m a l l y V i l s m e i e r r e a c t i o n s a r e r u n i n DMF and p h o s -p h o r u s o x y c h l o r i d e , h o w e v e r , J o h n s o n showed t h a t z i n c p o r -p h y r i n s were r a p i d l y d e m e t a l l a t e d u n d e r t h e s e a c i d i c c o n d i t i o n s . I t a p p e a r e d t h a t t h i s p r o b l e m m i g h t be overcome by u s i n g a 8 0 m o d i f i e d p r o c e d u r e , i n t r o d u c e d by C l e z y f o r p y r r o l e s w i t h a c i d l a b i l e s i d e c h a i n s . He s u b s t i t u t e d b e n z o y l c h l o r i d e f o r p h o s p h o r u s o x y c h l o r i d e a n d o b t a i n e d h i g h y i e l d s o f t h e f o r -m y l a t e d p r o d u c t . 7 6" The method o f J o h n s o n was a d o p t e d f o r u s e w i t h mono-b e n z y l e s t e r mono N- m e t h y l a n i l i d e , b e n z o y l c h l o r i d e and z i n c p r o t o p o r p h y r i n IX DTBE ( 3 1 a ) . The e x t e n d e d amide and b e n z o y l c h l o r i d e f o r m e d a b r i g h t r e d V i l s m e i e r c o m p l e x b u t t h e e x p e c t e d c o l o u r change i n t h e p o r p h y r i n , f r o m r e d t o g r e e n , on meso a c y l a t i o n d i d n o t o c c u r . D u r i n g t h e e i g h t h o u r r e a c t i o n some d e m e t a l l a t i o n d i d o c c u r b u t , on work up and c o m p l e t e d e m e t a l l a -t i o n , no change i n t h e p o r p h y r i n was s e e n . The e x p e c t e d p h y l l o t y p e s p e c t r u m o f a meso s u b s t i t u t e d p o r p h y r i n was n o t 4 0a p r o d u c e d , i n s t e a d t h e e t i o - t y p e s p e c t r u m o f p r o t o p o r p h y r i n IX was r e g e n e r a t e d . A s s u m i n g t h e p r o b l e m l a y w i t h e i t h e r e x t e n s i v e d e m e t a l l a -t i o n c r d e c r e a s e d a c t i v a t i o n u n d e r t h e r e a c t i o n c o n d i t i o n s o t h e r m e t a l i o n s were i n v e s t i g a t e d . U s i n g o c t a e t h y l p o r p h y r i n 81 (OEP) as a m odel s y s t e m i t was f o u n d t h a t c o b a l t ( I I ) OEP c o u l d be r e a d i l y d e m e t a l l a t e d i n m e t h y l e n e c h l o r i d e (CI^Cl.-,) by d r y HC1. T h i s d i s c o v e r y was p a r t i c u l a r l y a t t r a c t i v e s i n c e 16 I I J o h n s o n had shown Co p o r p h y r i n s t o be s t r o n g l y V i l s m e i e r a c t i v e and a l s o s i n c e t h e f a c i l e d e m e t a l l a t i o n w o u l d n o t a f f e c t ' t h e d i t e r t i a r y b u t y l g r o u p s w h i c h r e q u i r e e x t e n d e d t r e a t m e n t w i t h d r y H C 1 6 9 . C o b a l t ( I I ) p r o t o p o r p h y r i n IX DTBE (31b) was p r e p a r e d by 8 2 t h e method o f F a l k i n w h i c h a b o i l i n g s a t u r a t e d s o l u t i o n o f c o b a l t ( I I ) a c e t a t e was a d d e d t o a c h l o r o f o r m s o l u t i o n o f t h e p o r p h y r i n . A g a i n t h e two band m e t a l l o p o r p h y r i n s p e c t r u m 8 2 was f o r m e d , a l t h o u g h o f a c h a r a c t e r i s t i c a l l y d i f f e r e n t band shape t o t h e Zn ( I I ) p o r p h y r i n . The V i l s m e i e r r e a c t i o n was c a r r i e d o u t w i t h t h e d i s u b s t i t u t e d amide (30) and b e n z o y l 77 c h l o r i d e . The e x p e c t e d h y p s o c h r o m i c s h i f t on meso a c y l a t i o n , o f t h e Co"1"1 p o r p h y r i n d i d n o t o c c u r i n s t e a d t h e s h o u l d e r o f t h e I I Co p o r p h y r i n s p e c t r u m c h a n g e d t o a m a j o r peak s u g g e s t i n g t h a t T J J o x i d a t i o n t o Cc " had o c c u r r e d . O x i d a t i o n s h o u l d n o t h a v e I I a f f e c t e d t h e a c t i v a t i o n o f t h e m e t a l s i n c e , a l t h o u g h Co i s 8 3 one o f t h e most a c t i v e m e t a l s , D o l p h i n h a s shown i n d e u t e r a t i o n 41 e x p e r i m e n t s , t h a t Co I I I a l s o s t r o n g l y a c t i v a t e d t h e meso p o s i -t i o n t o e l e c t r o p h i l i c a t t a c k . C o m p l e t e d e m e t a l l a t i o n w i t h d r y HCI c o n f i r m e d t h a t no r e a c t i o n h a d t a k e n p l a c e . R e p e a t e d a t t e m p t s a t t h i s V i l s m e i e r a c y l a t i o n a l l y i e l d e d s i m i l a r n e g a -t i v e r e s u l t s . The c h o i c e o f c o b a l t ( I I ) a s t h e c e n t r a l m e t a l was a g o o d one s i n c e no d e m e t a l l a t i o n o c c u r r e d d u r i n g t h e r e a c t i o n . The p r o b l e m p r e s u m a b l y l a y more w i t h t h e V i l s m e i e r c o m p l e x u s e d . 16 I n t h e s t a n d a r d r e a c t i o n DMF a n d p h o s p h o r u s o x y c h l o r i d e p r o v i d e a v e r y s m a l l a n d h i g h l y a c t i v a t e d i n t e r m e d i a t e , u s u a l l y r e p r e s e n t e d a s t h e i o n p a i r h y b r i d (34) , w h i c h c a n a p p r o a c h a nd r e a c t w i t h t h e p o r p h y r i n meso p o s i t i o n . I n t h i s m o d i f i e d m e thod t h e i n t e r m e d i a t e was c o n s i d e r a b l y d i f f e r e n t ( 3 5 ) . F i r s t l y i t was much l a r g e r a n d t h u s h a d more o f a p o s s i b i l i t y o f s t e r i c h i n d r a n c e , w i t h t h e p o r p h y r i n 8 - s u b s t i t u e n t s . S e c o n d l y t h e r e a c t i v i t y o f t h e c e n t r a l c a r b o n atom o f (35) was (34) 0=0 (35) d i m i n i s h e d due t o t h e i n c r e a s e d e l e c t r o n d o n a t i o n o f t h e a l k y l c h a i n as compared t o t h e h y d r o g e n o f DMF and t h e d e c r e a s e d e l e c t r o n w i t h d r a w i n g e f f e c t o f t h e b e n z o y l c h l o r i d e o v e r t h e h i g h l y p o l a r i s e d p h o s p h o r u s o x y c h l o r i d e . I n t h e r a t h e r f o r l o r n hope o f r e d u c i n g t h e i n h i b i t o r y s t e r i c and e l e c t r o n i c e f f e c t s b e n z o y l c h l o r i d e was r e s u b -s t i t u t e d f o r p h o s p h o r u s o x y c h l o r i d e i n t h e Co ( I I ) p r o t o p o r -p h y r i n IX DTBE r e a c t i o n . As was a n t i c i p a t e d t h e l o n g r e a c t i o n t i m e c a u s e d b o t h d e m e t a l l a t i o n and d e e s t e r i f i c a t i o n o f t h e p o r p h y r i n w i t h no meso s u b s t i t u t i o n . T h i s l a s t a t t e m p t u n d e r l i n e d t h e i n h e r e n t l i m i t a t i o n s and d i f f i c u l t i e s t h a t had been i m p o s e d on t h e V i l s m e i e r method by c o n s i d e r a t i o n s l a t e r i n t h e s y n t h e t i c s e q u e n c e ; n a m e l y t h e n e c e s s i t y o f an a c t i v a t i n g y e t e a s i l y removed m e t a l , o f e a s i l y removed e s t e r f u n c t i o n s and t h e i n a b i l i t y o f f u r t h e r r e a c t i o n s on a meso f o r m y l g r o u p . W i t h t h e s e u n a v o i d a b l e c o n d i t i o n s and i n s u r m o u n t a b l e p r a c t i c a l c o n s t r i c t i o n s i n mind, t h e V i l s m e i e r method was abandoned i n f a v o u r o f a l e s s d i r e c t a p p r o a c h . 84 However, a v e r y r e c e n t r e p o r t s u g g e s t s a p o s s i b l e f u t u r e u s e o f t h i s a p p r o a c h . U s i n g t h e u n u s u a l t i t a n i u m t e t r a -8 5 c h l o r i d e as a c t i v a t o r f o r K n o e v e n a g e l c o n d e n s a t i o n , d i e t h y l m a l o n a t e and mono meso f o r m y l OEP (2 5) were c o n d e n s e d t o g i v e a 70% y i e l d o f t h e meso e t h y l a c r y l a t e p o r p h y r i n ( 3 6 ) . (35) (26) Extension of t h i s r e a c t i o n to the previously unreacted meso formyl d e r i v a t i v e s of natural porphyrins could lead to a meso a c r y l i c acid a f f i n i t y ligand. 2.42. The V i n y l Group Approach Having been unable to make use of the i n t r i n s i c r e a c t i -v i t y of the porphyrin meso positions towards Vilsmeier attack, the next l o g i c a l step was to create a r e a c t i v e f u n c t i o n a l i t y on one of the 6 substituents and then to use t h i s as the basis f o r lengthening the side chain. The v i n y l groups of protoporphyrin IX were c l e a r l y the best choice f o r consideration since the propionic acid groups are usually important i n enzyme binding and the methyl groups are unreactive. The problem of interference with the enzyme may also occur with the v i n y l groups which are often used i n enzyme binding (see i n t r o d u c t i o n ) . This d i f f i c u l t y could be minimised by d i r e c t s u b s t i t u t i o n onto the v i n y l groups without removing the unsaturation and by f u n c t i o n a l i s i n g j u s t one of the two v i n y l grups. This introduced the problem of isomer separation but once overcome, two isomeric a f f i n i t y ligands could be prepared. 44 The v i n y l groups o f p r o t o p o r p h y i n IX DTBE (24b) a r e i n d e e d q u i t e r e a c t i v e . They r e a d i l y undergo M a r k o w n i k o f f h y d r a t i o n i n aqueous a c i d t o g i v e t h e c o n j u g a t e d a l c o h o l w h i c h w i l l r e a d i l y 8 2 d e h y d r a t e back t o t h e v i n y l group ". Of more s y n t h e t i c i n t e r e s t i s t h e a n t i - M a r k o w n i k o f f a t t a c k a c h i e v e d by h y d r o -b o r a t i o n o r t h a l l i u m ( I I I ) n i t r a t e o x i d a t i o n ^ . P r o t o p o r p h y r i n IX can be o x i d i s e d t o t h e s t a b l e t e r m i n a l a c e t a l (37) w h i c h a f t e r f u r t h e r o x i d a t i o n g i v e s t h e a l d e h y d e (3 8 ) . T h i s r e p r e -s e n t s a p o s s i b l e c e n t r e f o r f i a r t h e r r e a c t i o n t o a l o n g s i d e 24a c h a i n . However, i n t h e p r o c e s s th e u n s a t u r a t e d c e n t r e would be l o s t c a u s i n g p o t e n t i a l d i f f i c u l t i e s a t t h e enzyme b i n d i n g s t a g e ; t h u s t h i s scheme was r e j e c t e d . A more d e s i r a b l e r o u t e was s u g g e s t e d by t h e o b s e r v a t i o n t h a t t h e v i n y l , groups can be r e a d i l y o x i d i s e d t o f o r m y l groups 8 7 7 8 and then r e a c t e d f u r t h e r under Knoevenagel o r W i t t i g c o n d i t i o n s t o a c r y l i c a c i d d e r i v a t i v e s . A s y n t h e t i c p l a n i n v o l v i n g o x i d a t i o n o f one v i n y l group t o t h e p o r p h y r i n a l d e -hyde (40) and then c o n d e n s a t i o n o f t h i s t o the a c r y l i c a c i d d e r i v a t i v e (41) t h u s p r e s e n t e d i t s e l f , c o n f i r m i n g t h e t h e o r y 45 o f P e r u t z t h a t i f y o u p u r s u e them l o n g enough, s y n t h e s e s , as w e l l as s t r u c t u r e s " l i e down f o r y o u t o c a t c h them, t h e y 8 8 become tame" . • 2.43. M o n o f o r m y l M o n o v i n y l D e u t e r o p o r p h y r i n IX DTBE The f i r s t o x i d a t i o n o f p r o t o p o r p h y i n IX t o i t s f o r m y l 8 7 d e r i v a t i v e was a c h i e v e d by S p a r a t o r e when he f o r m e d d i f o r m y l d e u t e r o p o r p h y r i n IX by o x i d a t i o n o f t h e v i n y l g r o u p s t o t h e b i s 1,2 g l y c o l w i t h osmium t e t r o x i d e and t h e n c l e a v a g e o f t h e d i o l w i t h s o d i u m p e r i o d a t e . A more a p p e a l i n g and s p e c i f i c 8 9 method was d e v e l o p e d by Caughey i n w h i c h an a c e t o n e s o l u t i o n o f p r o t o p o r p h y r i n IX d i m e t h y l e s t e r was o x i d i s e d by an e q u i -m o l a r amount o f p o t a s s i u m p e r m a n g a n a t e f o r a c o n t r o l l e d l e n g t h o f t i m e . He p r e p a r e d t h e m o n o f o r m y l and d i f o r m y l d e r i v a t i v e s i n r a t h e r low y i e l d s ( 1 5 % and 2 0% r e s p e c t i v e l y ) b u t c o u l d 46 s e p a r a t e them w i t h r e l a t i v e e a s e on an a l u m i n a column. F o r t h i s r e a s o n t h i s method was i n i t i a l l y c h o s e n f o r g e n e r a t i o n o f t h e m o n o f o r m y l p r e c u r s o r o f t h e a f f i n i t y l i g a n d . The r e a c t i o n was c a r r i e d o u t w i t h p o t a s s i u m p e r m a n g a n a t e on p r o t o p o r p h y r i n IX d i t e r t i a r y b u t y l e s t e r f o r 45 m i n u t e s . As• m i g h t be e x p e c t e d t h e s t r o n g s o l u b i l i s i n g e f f e c t o f t h e t e r t i a r y b u t y l e s t e r s , a s compared w i t h t h e m e t h y l e s t e r s , i n c r e a s e d t h e i r s o l u b i l i t y i n c h l o r o f o r m and made t h e s e p a r a t i o n o f t h e m o n o f o r m y l (40) and d i f o r m y l (42) d e r i v a t i v e s more d i f f i c u l t . I n f a c t s i l i c a g e l c o l u m n c h r o m a t o g r a p h y was c a r r i e d o u t t w i c e t o e f f e c t s e p a r a t i o n and t h e f i n a l y i e l d s were c o n s e q u e n t l y l o w e r t h a n t h o s e o f Caughey (monoformyl 14%, d i f o r m y l 1 1 % ) . The two compounds were r e a d i l y d i s t i n g u i s h e d by t h e i r v i s i b l e (24b) KMn04 (40 ) s p e c t r a . The m o n o f o r m y l d e r i v a t i v e ( 4 0 ) , w i t h one e l e c t r o n w i t h d r a w i n g g r o u p , g a v e a r h o d o t y p e s p e c t r u m w h e r e a s t h e d i f o r m y l ( 4 2 ) , w i t h e l e c t r o n w i t h d r a w i n g g r o u p s on two a d j a c e n t p y r r o l e r i n g s , e x h i b i t e d an e t i o - t y p e s p e c t r u m . 47 A l t h o u g h t h e p u r i t y o f t h e m o n f o r m y l compound was s u f f i c i e n t f o r f u r t h e r e x p l o r a t o r y work i t was i n f a c t a m i x t u r e o f t h e 2 - f o r m y l 4 - v i n y l (40) and 2 - v i n y l 4 - f o r m y l (43) i s o m e r s . I f t h e e f f i c i e n c y o f t h e a f f i n i t y l i g a n d was 9 0 t o be m a x i m i s e d t h e s e had t o be s e p a r a t e d . B o t h Sano and 91 O 'Keefe had e f f e c t e d a s e p a r a t i o n by p r e p a r a t i v e l a y e r chrom-a t o g r p h y b u t t h e p r o c e d u r e s were e x t r e m e l y t e d i o u s and l i m i t e d t o l e s s t h a n 10 mgs., an i m p r a c t i c a l l y s m a l l s c a l e . I t was q u i t e c o n c e i v a b l e t h a t s e p a r a t i o n o f t h e two i s o m e r s w o u l d be e a s i e r a t a l a t e r s t a g e i n t h e s y n t h e s i s when t h e s i d e c h a i n l e n g t h had been i n c r e a s e d b u t t h i s was by no means c e r t a i n , p a r t i c u l a r l y s i n c e a l l o ther.known 2 and 4 p o r p h y r i n i s o m e r s have been shown, w i t h o u t e x c e p t i o n , t o be d i f f i c u l t t o s e p a r a t e and a l s o s i n c e t h e t e r t i a r y b u t y l e s t e r s w o u l d t e n d t o com-p l i c a t e any s e p a r a t i o n . 92 T h i s p r o b l e m was e l e g a n t l y o v e rcome by I n h o f f e n i n h i s s y n t h e s i s o f s p i r o g r a p h i s p o r p h y r i n ( 2 - f o r m y l 4 - v i n y l d e u t e r o -p o r p h y r i n IX 4 0 ) . He made u s e o f t h e f a c i l e 1,4 a d d i t i o n o f s i n g l e t o x y g e n t o t h e d i e n e c e n t r e f o r m e d by a v i n y l g r o u p and one o f t h e two e x o c y c l i c d o u b l e bonds i n p r o t o p o r p h y r i n IX. The b r i g h t g r e e n p r o d u c t , l y r i c a l l y named p h o t o p r o t o p o r p h y r i n , (44a and 45a) c o n t a i n e d an a c r o l e i n g r o u p i n c o n j u n c t i o n w i t h t h e IT s y s t e m ; t h i s d e a c t i v a t e s t h e r i n g t o a t t a c k by a s e c o n d o x y g e n m o l e c u l e , l e a v i n g one v i n y l g r o u p u n t o u c h e d . 4 8 The d i s t o r t i o n i n t h e m a c r o c y c l e b r o u g h t a b o u t by t h e a c r o l e i n g r o u p f a c i l i t a t e s c h r o m a t o g r a p h i c s e p a r a t i o n o f t h e two i s o m e r s (44a) and (45) w h i c h c a n be a c h i e v e d on a p r e -p a r a t i v e s c a l e . (44a) (45a) (43) (40) (47a) a, R = C H 3 b, R = t B u A f t e r s e p a r a t i o n , I n h o f f e n f o r m e d t h e m o n o f o r m y l d e r i v a t i v e s by r e d u c t i o n o f t h e t e r m i n a l a l d e h y d e (44a and 45a) t o an a l l y l i c a l c o h o l f o l l o w e d by a c i d c a t a l y s e d r e a r r a n g e m e n t t o t h e p o r p h y r i n d i o l (4 6a, 47a) and f i n a l l y Sodium p e r i o d a t e 49 c l e a v a g e o f t h e 1,2 g l y c o l . H i s r e p o r t e d y i e l d s were v e r y h i g h (35% f o r e a c h p h o t o p o r t o p o r p h y r i n i s o m e r and 80% f o r 93 s p i r o g r a p h i s p o r p h y r i n ) a s , p e r h a p s , were h i s h o p e s . C l e z y r e p e a t e d t h e r e a c t i o n on p r o t o p o r p h y r i n I I w i t h o n l y a 25% 91 y i e l d i n t h e f i n a l s t e p s t o t h e a l d e h y d e and O ' Keefe was u n a b l e t o r e p r o d u c e t h e r e a c t i o n on p r o t o p o r p h y r i n IX d i m e t h y l e s t e r . D e s p i t e t h e s e u n c e r t a i n t i e s , t h e a d v a n t a g e s o v e r t h e 8 9 method o f Caughey o f e a s y i s o m e r s e p a r a t i o n and p o s s i b l y h i g h y i e l d s were a t t r a c t i v e and t h e method was a p p l i e d t o t h e a f f i n i t y l i g a n d s y n t h e s i s . When p h o t o l y s e d i n a m e t h y l e n e c h l o r i d e / p y r i d i n e s o l u t i o n f o r 8 h o u r s i n t h e p r e s e n c e o f o x y g e n p r o t o p o r p h y r i n IX DTBE gave 70% o f t h e m i x e d p h o t o p r o t o p o r p h y r i n IX i s o m e r s (4 4b, 4 5 b ) . T h e s e c o u l d be s e p a r a t e d on a s i l i c a g e l column ( g r a d e IV) w i t h m e t h y l e n e c h l o r i d e : e t h e r (20:1) a s t h e e l u e n t , d e s p i t e t h e s o l u b i l i t y e f f e c t o f t h e t e r t i a r y b u t y l e s t e r g r o u p s , w i t h a y i e l d o f 2 0% o f e a c h i s o m e r and 2 0% o f t h e m i x e d i s o m e r s . S u r p r i s i n g l y , s u b s t i t u t i o n o f c h l o r o f o r m f o r m e t h y l e n e c h l o r i d e , w h i c h u s u a l l y has l i t t l e e f f e c t , r e n d e r e d s e p a r a t i o n much more d i f f i c u l t . Any t r a c e s o f t h e o t h e r i s o m e r i n e i t h e r o f t h e f i n a l p u r e compounds c o u l d be removed by c r y s t a l l i s a t i o n . f r o m a c h l o r o f o r m / m e t h a n o l s o l u t i o n . 50 By c o m p a r i s o n t o t h e known s p i r o g r a p h i s p r o p h y r i n , 92 I n h o f f e n i d e n t i f i e d t h e f a s t e s t m o v i n g i s o m e r a s 4 p h o t o -p r o t o p o r p h y r i n d i m e t h y l e s t e r (44a) and t h e s l o w e r a s t h e 2 p h o t o p r o t o p o r p h y r i n d i m e t h y l e s t e r ( 4 5 a ) . D e s i g n a t i o n o f t h e d i t e r t i a r y b u t y l e s t e r i s o m e r s (44b a n d 45b) w e r e made on t h i s b a s i s . A s e r i e s o f t e s t r e a c t i o n s was r u n and t h e h i g h e s t y i e l d o f m o n o f o r m y l m o n o v i n y l d e u t e r o p o r p h y r i n I X DTBE (40 a n d 43) 92 was f o u n d when a p r o c e d u r e m o d x f i e d f r o m I n h o f f e n ' s was u s e d . T h i s i n v o l v e d t r e a t m e n t o f a m e t h y l e n e c h l o r i d e s o l u -t i o n o f p h o t o p r o t o p o r p h y r i n I X DTBE (44b a n d 45b) w i t h s o d i u m b o r o h y d r i d e , i n m e t h a n o l , f o r one h o u r f o l l o w e d b y p e r i o d a t e o x i d a t i o n i n an a c i d i f i e d 1,4 d i o x a n e s o l u t i o n f o r 5 h o u r s . 92 93 Compared t o t h e r a p i d m e t h o d s o f I n h o f f e n " a n d C l e z y a l e w a c i d c o n c e n t r a t i o n a n d l o n g r e a c t i o n t i m e w e r e f o u n d t o be most e f f e c t i v e i n t h e p e r i o d a t e o x i d a t i o n s t e p w i t h t h e l e a s t amount o f d e - e s t e r i f i c a t i o n . Y i e l d s o f t h e m o n o f o r m y l d e r i v a t i v e w e r e c o n s i s t e n t l y a r o u n d 60% w i t h t h e m a j o r i m p u r i t y ( o f t e n a s much a s 2 0%) b e i n g t h e m o n o d e - e s t e r i f i c a t i o n f o r m y l compound, s e p a r a t i o n o f t h e s e compounds was f a c i l e on s i l i c a g e l . D e s p i t e t h e e x t e n d e d p r o c e d u r e s t h e m o d i f i e d I n h o f f e n m e t h o d o f o x i d i z i n g p r o t o p o r p h y r i n I X was shown t o be p r e f e r a b l e 8 9 t o t h e C a u g h e y m e t h o d . The c o n s i s t e n t l y h i g h y i e l d s a n d f a c i l e s e p a r a t i o n o f t h e m o n o f o r m y l i s o m e r s c o n f i r m i t s u s e f u l n e s s when e i t h e r t h e p u r e compounds o r m i x e d i s o m e r s a r e r e q u i r e d . 2.44. M o n o a c r y l i c a c i d M o n o v i n y l D e u t e r o p o r p h y r i n I X DTBE The m o s t u s e f u l m e t h o d s f o r e x t e n s i o n o f t h e c h a i n , y e t w i t h r e t e n t i o n o f v i n y l g r o u p s i n t h e 2 and 4 p o s i t i o n s , i n v o l v e d e i t h e r t h e W i t t i g or. ' K n o e v e n a g e l r e a c t i o n s t o f o r m an a c r y l i c a c i d g r o u p . The W i t t i g r e a c t i o n was c a r r i e d o u t o n a p o r p h y r i n g 78 I I f o r m y l g r o u p b y C a l l o t when he f o r m e d mono a c r y l i c a c i d N i I I TTP f r o m m o n o f o r m y l N i ' TPP. The y i e l d was f a i r l y h i g h (71%) a l t h o u g h a m i x t u r e o f t h e c i s and t r a n s a c r y l a t e s was f o r m e d (3 0% c i s ; 4 1 % t r a n s ) . A l t h o u g h t h i s m i x t u r e w o u l d be u n -d e s i r a b l e i n t h e p r e s e n t l i g a n d s y n t h e s i s i t i s u n l i k e l y t h a t , i n t h e p r e s e n c e o f s t e r i c i n t e r f e r e n c e f r o m o t h e r 8 s u b s t i t u e n t s , a n y o f t h e more s t e r i c a l l y h i n d e r e d c i s i s o m e r w o u l d be f o r m e d . A s i m p l e r a l t e r n a t i v e , o v e r t h e r a t h e r c o m p l e x W i t t i g r e a c t i o n , was t h e K n o e v e n a g e l c o n d e n s a t i o n o f m a l o n i c a c i d , i n t h e p r e s e n c e o f a s t r o n g b a s e , w i t h a c a r b o n y l compound t o g i v e , a f t e r d e c a r b o x y l a t i o n , t h e a c r y l i c a c i d d e r i v a t i v e . T h i s r e a c t i o n h a s t w i c e ^ f ^ b e e n a p p l i e d t o p o r p h y r i n c h e m i s t r y , w i t h d i f o r m y l d e u t e r o p o r p h y r i n a s t h e c a r b o n y l com-pound on b o t h o c c a s i o n s . S t r a n g e l y n e i t h e r r e p o r t m e n t i o n e d V y i e l d s a l t h o u g h t h e s e a r e g e n e r a l l y h i g h and s t e r e o c h e m i c a l l y p u r e . C o n d e n s a t i o n s o f t h e ' a l d o l 1 t y p e u s u a l l y f o r m o l e f i n s o f t h e more s t a b l e t r a n s c o n f i g u r a t i o n ; when t h i s t h e r m o d y n a m i c 52 c o n t r o l was c o u p l e d w i t h t h e s t e r i c i n f l u e n c e s o f t h e p o r p h y r i n t h e t r a n s a c r y l i c a c i d d e r i v a t i v e s were e x c l u s i v e l y p r o d u c e d . U s i n g t h e common K n o e v e n a g e l c o n d i t i o n s o f p y r i d i n e and p i p e r i d i n e , t h e r e a c t i o n was c a r r i e d o u t on m o n o f o r m y l m o n o v i n y l d e u t e r o p o r p h y r i n IX DTBE (4 0 and 43) t o g i v e an 80% y i e l d o f mono a c r y l i c a c i d m o n o v i n y l d e u t e r o p o r p h y r i n IX DTBE ( 4 8 ) . (43) (48) A l t h o u g h r e f l u x was m a i n t a i n e d f o r f o u r h o u r s i t was f o u n d t h a t a s l o w r a i s i n g o f t h e t e m p e r a t u r e up t o r e f l u x was i m p o r t a n t due t o s o l u b i l i t y p r o b l e m s w i t h t h e m a l o n i c a c i d and i t s e a s y d e c a r b o x y l a t i o n . C h r o m a t o g r a p h i c p u r i f i c a t i o n o f t h e p r e -d i c t a b l y s l o w m o v i n g a c r y l i c a c i d p o r p h y r i n was a c h i e v e d on s i l i c a g e l p r e p a r a t i v e l a y e r p l a t e s ( C H C l ^ : m e t h a n o l 2 0 : 1 ) . When t h e m i x t u r e o f m o n o f o r m y l i s o m e r s (40 and 43) was u s e d i n t h e r e a c t i o n t h e a c r y l i c a c i d p o r p h y r i n band showed a d i s t i n c t , a l b e i t s l i g h t , r e s o l u t i o n i n t o t h e two i s o m e r s . A l t h o u g h n o t a t t e m p t e d , r e p e a t e d d e v e l o p m e n t o f t h e p l a t e s s h o u l d have 9 0 a f f e c t e d c o m p l e t e s e p a r a t i o n The s o l u b i l i t y o f t h e a c r y l i c a c i d p o r p h y r i n (48) i n c h l o r o f o r m , r e l a t i v e t o t h a t o f p r o t o p o r p h y r i n IX DTBE, was much r e d u c e d . N e v e r t h e l e s s i t was s u f f i c i e n t f o r a F o u r i e r T r a n s f o r m nmr s p e c t r u m t o be t a k e n . T h i s showed a c o u p l i n g c o n s t a n t o f 17 Hz f o r t h e v i n y l p r o t o n s o f t h e a c r y l i c a c i d g r o u p , c o n f i r m i n g t h e trans c o n f i g u r a t i o n o f t h e a c r y l i c a c i d 1 0 ^ . The p r e s e n c e o f t h e a c r y l i c a c i d g r o u p c o n j u g a t e d t o t h e p o r p h y r i n TT s y s t e m was n i c e l y d e m o n s t r a t e d by t h e v i s i b l e s p e c t r u m . I n c h l o r o f o r m t h e e l e c t r o n ' w i t h d r a w i n g c a r b o x y l i c a c i d p r o d u c e d a r h o d o - t y p e s p e c t r u m , however, on a d d i n g p y r i d i n e t h e c a r b o x y l i c a c i d g r o u p i s i o n i z e d , t h e e l e c t r o n w i t h d r a w i n g e f f e c t removed and t h e s p e c t r u m c h a n g e d t o t h e e t i o f o r m . The a c r y l i c a c i d p o r p h y r i n (4 8) d i d n o t g i v e a mass s p e c -t r u m d e s p i t e r a p i d h e a t i n g t o 4 5 0 ° C . The p o r p h y r i n r i n g i t s e l f 95 t e n d s t o r e q u i r e a h i g h p r o b e t e m p e r a t u r e so when compounded w i t h a f r e e c a r b o x y l i c a c i d c o m p l e t e i n v o l a t i l i t y r e s u l t s . A c o n f i r m a t o r y mass s p e c t r u m was, however, o b t a i n e d w i t h t h e m e t h y l e s t e r o f m o n o a c r y l i c a c i d d e u t e r o p o r p h y r i n IX DTBE .This was p r e p a r e d by t r e a t m e n t o f t h e a c r y l i c a c i d s w i t h d i a z o m e t h a n e i n m e t h y l e n e c h l o r i d e . I t g a v e t h e e x p e c t e d m o l e c u l a r i o n a t m/e = 7 32. However t h e c h a n g e i n t h e v i s i b l e 54 V~C02H CH2N2 (48) (49) s p e c t r u m , f r o m r h o d o t o e t i o t y p e , o n a d d i t i o n o f p y r i d i n e d i d n o t , o f c o u r s e , o c c u r w i t h t h e m e t h y l e s t e r . S i n c e a l a r g e amount o f m o n o a c r y l i c a c i d m o n o v i n y l d e u t e r o -p o r p h y r i n I X DTBE was n e e d e d f o r e x p l o r a t o r y r e a c t i o n s ; a. r a p i d s y n t h e s i s was d e v e l o p e d . P r o t o p o r p h y r i n I X (1.5 g) was c o n v e r t e d i n t o t h e m o n o a c r y l i c a c i d d e r i v a t i v e i n h i g h y i e l d s (48 ;0.4 5g, 3 0%) w i t h o u t s e p a r a t i o n o f t h e i s o m e r s a n d w i t h , s i m p l y , p r e p a r a t i v e l a y e r p u r i f i c a t i o n o f e a c h i n t e r m e d i a t e . 2.5. E x t e n s i o n o f t h e C h a i n The n e x t s t a g e i n t h e s y n t h e s i s o f t h e a f f i n i t y l i g a n d was t h e e x t e n s i o n o f t h e s i d e c h a i n o u t w a r d f r o m t h e a c r y l i c a c i d g r o u p . I d e a l l y t h e p r o c e s s s h o u l d be f l e x i b l e so t h a t t h e l e n g t h o f t h e s p a c e r arm b e t w e e n t h e p o r p h y r i n a n d t h e s o l i d s u p p o r t c o u l d be v a r i e d t o f i n d t h e most e f f e c t i v e l i g a n d f o r any g i v e n enzyme ( s e e C h a p t e r 3 ) . The s i m p l e s t , and t h u s t h e c h o s e n , r o u t e i n v o l v e d t h e f o r m a t i o n o f an a m i d e bond w i t h one a m i n e a>roup f r o m an a l i -p h a t i c d i a m i n e , l e a v i n g t h e o t h e r a m i n e f r e e f o r b o n d i n g t o t h e s o l i d s u p p o r t . The l e n g t h o f t h e a l i p h a t i c d i a m i n e c o u l d be v a r i e d between t h e two r e a s o n a b l e e x t r e m e s o f p r o p a n e 1,3 d i a m i n e and hexane 1,6 d i a m i n e . The f o r m a t i o n o f an amide bond i n v o l v e s , w i t h v e r y few 96 e x c e p t i o n s , t h e a m i n o l y s i s o f an a c t i v a t e d c a r b o x y l i c a c i d d e r i v a t i v e . The most common methods o f a c t i v a t i o n , p a r t i c u l a r A - R C02H > R-^P-X ) R-CONHR R NH 2 + HX 97 when r a c e m i s a t i o n o f t h e amide i s no p r o b l e m , a r e t h e a c i d c h l o r i d e and t h e c a r b o d i i m i d e methods. 56 2,51. The D i c y c l o h e x y l c a r b o c l i i m i d e Method The c a r b o d i i m i d e method was i n i t i a l l y c h o s e n f o r t h i s t r a n s f o r m a t i o n s i n c e i t i s a v e r y m i l d method and c a n u s u a l l y be r e l i e d upon t o g i v e h i g h y i e l d s o f t h e amide. The a c t i -v a t e d i n t e r m e d i a t e i s t h e o - a c y l i s o u r e a (51) f o r m e d by a t t a c k o f t h e c a r b o x y l a t e on t h e d i i m i d e ; t h e c a r b o n y l i s t h u s a c t i -v a t e d t o w a r d s n u c l e o p h i l i c a t t a c k by t h e amine t o p r o d u c e t h e amide (pathway a ) . However i f t h e a t t a c k i n g n u c l e o p h i l e i s s l u g g i s h , a c o m p e t i n g i n t r a m o l e c u l a r a t t a c k t o g i v e t h e N-a c y l u r e a (52) c a n o c c u r (pathway b ) . R-N=<£NhR R-&2^ ( 5 D RC02 ^ H 2 R 'b lr (52) The d i c y c l o h e x y l c a r b o d i i m i d e (R = c y c l o h e x y l ) i s u s u a l l y c h o s e n s i n c e i t i s s o l u b l e i n c h l o r o c a r b o n s o l v e n t s w h e r e a s t h e f i n a l d i c y c l o h e x y l u r e a (53 R = c y c l o h e x y l ) i s n o t . O t h e r c a r b o -9 9 d x i m i d e s , i n c l u d i n g w a t e r s o l u b l e o n e s , a r e a l s o a v a i l a b l e 98 57 The c o u p l i n g was i n i t i a t e d a t 0°C b y m i x i n g m o n o a c r y l i c a c i d m o n o v i n y l d e u t e r o p o r p h y r i n IX DTBE, d i c y c l o h e x y l c a r b o -d i i m i d e (DCC) a n d p r o p a n e d i a m i n e i n m e t h y l e n e c h l o r i d e . A f t e r an h o u r t h e r e a c t i o n was a l l o w e d t o warm up t o room t e m p e r a t u r e p r i o r t o w o r k up a n d c h r o m a t o g r a p h i c s e p a r a t i o n . A p a r t f r o m a s m a l l amount o f u n r e a c t e d a c r y l i c a c i d p o r p h y r i n , t h e m a j o r p r o d u c t was a r e d compound t h a t moved e x t r e m e l y f a s t on s i l i c a g e l t . l . c . r e l a t i v e t o t h e a c r y l i c a c i d ( C H C l ^ : m e t h a n o l 2 0 : 1 ) . The v i s i b l e s p e c t r u m c o n f i r m e d t h a t t h e c a r b o x y l i c a c i d g r o u p h a d r e a c t e d s i n c e no c h a n g e f r o m t h e r h o d o - t y p e was o b s e r v e d o n a d d i t i o n o f p y r i d i n e . The n.m.r. s p e c t r u m showed an a c r y l a t e p o r p h y r i n w i t h a l a r g e number o f u p f i e l d p r o t o n s , s e e m i n g l y s u g g e s t i n g t h a t t h e N - a c y l a r e a h a d b e e n f o r m e d . T h i s c o u l d n o t b e c o n f i r m e d by mass s p e c -t r o s c o p y b e c a u s e t h e compound was i n v o l a t i l e o n t h e probe.: The r e a c t i o n was r e p e a t e d w i t h i n c r e a s e d a m o u n t s o f t h e d i a -m i n e and DCC b u t e a c h t i m e t h e u n w a n t e d s i d e p r o d u c t was f o r m e d . I n r e t r o s p e c t t h e f o r m a t i o n o f t h e N - a c y l a r e a c o u l d be r a t i o n a l i z e d a s t h e s l u g g i s h a t t a c k o f t h e d i a m i n e on a c a r -b o x y l a t e t h a t i s d e a c t i v a t e d by b e i n g p a r t o f a v i n y l o g o u s a m i de s y s t e m . T h i s w o u l d s i g n i f i c a n t l y r e d u c e t h e e l e c t r o -p h i l i c i t y o f t h e c a r b o n y l ( 5 4 ) . C o n c e i v a b l y f u r t h e r h i n d r a n c e t o i n t e r m o l e c u l a r a t t a c k c o u l d come f r o m t h e s t e r i c c r o w d -i n g o f t h e O - a c y l a c t i v e i n t e r m e d i a t e ( 5 5 ) . The c y c l o h e x y l 58 r •NH (54) (55) g r o u p s w o u l d t a k e up a p r e f e r r e d p o s i t i o n a b o v e and b e l o w t h e p l a n e i n t e r f e r i n g w i t h t h e a p p r o a c h o f t h e n u c l e o p h i l e . I n t h e p r e s e n c e o f s u c h a d i l a t o r y n u c l e o p h i l e i t i s v e r y l i k e l y t h a t i n t r a m o l e c u l a r a t t a c k w o u l d p r e d o m i n a t e . In t h e s y n t h e s i s o f more n o r m a l amides t h i s p r o b l e m c a n g e n e r a l l y be overcome by t h e u s e o f s t r o n g l y n u c l e o p h i l i c N - h y d r o x y compounds, u s u a l l y N - h y d r o x y s u c c i n i m i d e o r N-h y d r o x y b e n z o t r i a z o l e . T h e s e a r e s u f f i c i e n t l y n u c l e o p h i l i c t o r e a c t w i t h a d e a c t i v a t e d O - a c y l u r e a t o g i v e an a c t i v e e s t e r i n t e r m e d i a t e w h i c h w i l l r a p i d l y r e a c t w i t h t h e amino component t o f o r m t h e p r e v i o u s l y u n a v a i l a b l e amide. HNC6H| N - h y d r o x y s u c c i n i m i d e ft > R-C-O-N •9 , R-C-N-R H 59 T h i s t e c h n i q u e was a p p l i e d t o t h e a c r y l i c a c i d p o r p h y r i n i n t h e hope o f o v e r c o m i n g t h e i n h i b i t i n g i n f l u e n c e s . Mono a c r y l i c a c i d m o n o v i n y l d e u t e r o p o r p h y r i n IX DTBE was d i s s o l v e d i n c h l o r o f o r m a t 0°C and d i c y c l o h e x y l c a r b o d i i m i d e , 1 - h y d r o x y b e n z t r i a z o l e and p r o p a n e 1,3 d i a m i n e were add e d . A f t e r one hour a t 0°C t h e s o l u t i o n was a l l o w e d t o warm up t o room tem-p e r a t u r e p r i o r t o work up a n d c h r o m a t o g r a p h i c a n a l y s i s . A s m a l l p e r c e n t a g e o f t h e p r o d u c t was u n r e a c t e d a c r y l i c a c i d p o r p h y r i n . I n s t e a d o f t h e e x p e c t e d r e d p o r p h y r i n w i t h a., s l i g h t l y h i g h e r Rf t h a n t h e f r e e a c r y l i c a c i d p o r p h y r i n , t h e b u l k o f t h e p r o d u c t was f o u n d t o be a g r e e n compound w i t h a v e r y h i g h Rf i n c h l o r o f o r m - m e t h a n o l ( 2 0 : 1 ) . The r e a c t i o n c o n d i t i o n s were v a r i e d a s were t h e r e l a t i v e amounts o f t h e d i f f e r e n t r e a c t a n t s b u t e a c h t i m e t h e p r e d o m i n a n t p r o d u c t was t h e f a s t m o v i n g g r e e n compound . When a l a r g e e x c e s s o f t h e d i a m i n e was u s e d a s m a l l amount o f t h e amino p o r p h y r i n a p p e a r e d t o be f o r m e d , b u t t h e y i e l d s were t o o s m a l l ( 5%) t o be u s e f u l . A s p e c t r a l a n a l y s i s o f t h e g r e e n compound gave v e r y i n t e r e s t i n g r e s u l t s b u t d i d n o t p e r m i t i d e n t i f i c a t i o n . The v i s i b l e s p e c t r u m e x h i b i t e d an o x o - r h o d o t y p e s p e c t r u m , w h i c h on t h e a d d i t i o n o f b a s e (NaOH/methanol) was c o n v e r t e d t o a r h o d o t y p e , t h e ch a n g e was r e v e r s e d on t h e a d d i t i o n o f a c e t i c a c i d . The f o r m a t i o n o f an o x o - r h o d o t y p e i s u n e x p e c t e d s i n c e 60 t h i s i s u s u a l l y a s s o c i a t e d w i t h p o r p h y r i n s w i t h e l e c t r o n w i t h d r a w i n g groups on d i a g o n a l l y o p p o s i t e p y r r o l e r i n g s ' ^ , an u n l i k e l y r e s u l t under t h e s e c o n d i t i o n s . However some 107 p o r p h y r i n s w i t h i s o c y c l i c r i n g s have been r e p o r t e d t o g i v e t h i s type, o f spectrum p o s s i b l y s u g g e s t i n g a c y c l i s e d p r o d u c t . The n.m.r. spectrum was complex b u t showed t h a t t h e a methine p r o t o n o f t h e a3 u n s a t u r a t e d c a r b o x y l a t e s y s -tem was ab s e n t . The mass spectrum gave a peak a t m/e = 732; t h i s , was h a r d t o . r a t i o n a l i s e i n terms o f t h e o t h e r e v i -dence e x c e p t , p o s s i b l y , as t h e m e t h y l e s t e r o f m o n o a c r y l i c ac mo n o v i n y l d e u t e r o p o r p h y r i n IX DTBE (48) formed by r e a c t i o n o f t h e g r e e n compound w i t h methanol i n t h e e l u t i n g m i x t u r e . The h i g h y i e l d and ease o f f o r m a t i o n o f t h e unknown green compound from t h e p o r p h y r i n a c r y l i c a c i d a c t i v e e s t e r s y n t h e s i s r e v e a l e d an i n h e r e n t and p r e d o m i n a t i n g s i d e r e a c -t i o n and u n d e r l i n e d t h e i n a p p l i c a b i l i t y o f t h e t e c h n i q u e t o t h i s p a r t i c u l a r p o r p h y r i n a 3 u n s a t u r a t e d c a r b o x y l i c a c i d . 2.52. The A c i d C h l o r i d e Approach W i t h t h e f a i l u r e o f the DCC method t o form t h e l o n g c h a i n amide a t t e n t i o n was t u r n e d t o t h e a c i d c h l o r i d e approac T h i s had i n i t i a l l y been p a s s e d o v e r , i n f a v o u r o f t h e m i l d e r DCC method, f o r f e a r t h a t t h e a c i d c o n d i t i o n s would i r r e v e r -s i b l y a t t a c k t h e a c i d s e n s i t i v e v i n y l and d i t e r t i a r y b u t y l e s t e r groups of t h e a c r y l i c a c i d p o r p h y r i n ( 4 8 ) . 61 The f i r s t a t t e m p t s o f c o u p l i n g by t h e a c i d c h l o r i d e method i n v o l v e d a d d i t i o n o f o x a l y l c h l o r i d e , a s t h e c h l o r i n a t i n g a g e n t , t o a b o i l i n g m e t h y l e n e c h l o r i d e s o l u t i o n o f t h e mono-v i n y l m o n o a c r y l i c a c i d d e u t e r o p o r p h y r i n IX d i t e r t i a r y b u t y l e s t e r (48) f o l l o w e d by r e a c t i o n o f t h e a c i d c h l o r i d e w i t h , p r o p a n e 1,3 d i a m i n e . The d e s i r e d p r o d u c t was n o t , however, f o r t h c o m i n g . I t was a n t i c i p a t e d t h a t t h e amino p o r p h y r i n (50a) w ould r u n f a s t e r t h a n t h e a c r y l i c a c i d p o r p h y r i n (48) on s i l i c a g e l , b e c a u s e t h e c a r b o x y l a t e g r o u p had b e en removed, b u t s l o w e r t h a n t h e m e t h y l e s t e r ( 4 9 ) , b e c a u s e o f t h e p r e s e n c e o f a t e r m i n a l amine i n t h e m o l e c u l e . However t h e p r o d u c t r a n s l o w e r t h a n t h e a c r y l i c a c i d p o r p h y r i n and s o p r e s u m a b l y some a c i d d e c o m p o s i t i o n o f t h e v i n y l g r o u p s had o c c u r r e d . The r e a c t i o n was r e p e a t e d i n t h e p r e s e n c e o f p y r i d i n e , t o a v o i d any a c i d a t t a c k , b u t t h e r e s u l t s were e v e n l e s s e n c o u r a g i n g . The p r o d u c t d i d n o t move f r o m t h e o r i g i n o f s i l i c a g e l t . l . c . and was u n i d e n t i f i a b l e . I t i s p o s s i b l e t h a t t h e p r e s e n c e o f p y r i d i n e i n t h e r e f l u x i n g s o l u t i o n had c o n -v e r t e d t h e a c i d c h l o r i d e t o a k e t e n e - l i k e m a t e r i a l w h i c h w o u l d t h e n r e a c t f u r t h e r t o i n t r a c t a b l e p r o d u c t s . T h i s r e a c t i o n i s known t o o c c u r w i t h a c i d c h l o r i d e s a t h i g h e r t e m p e r a t u r e s 1 0 0 . The r e a c t i o n was r e p e a t e d w i t h d i f f e r e n t b a s e s , e a c h t i m e y i e l d i n g t h e same r e s u l t . The v a l i d i t y o f t h e a c i d c h l o r i d e a p p r o a c h was t e s t e d by r e p e a t i n g t h e r e a c t i o n a t room t e m p e r a t u r e , w i t h o u t p y r i -d i n e , w i t h m e t h a n o l as t h e a t t a c k i n g n u c l e o p h i l e . The mono-m e t h y l a c r y l a t e m o n o v i n y l p o r p h y r i n (4 9) was p r e p a r e d i n a h i g h y i e l d and i d e n t i f i e d by c o m p a r i s o n w i t h t h e compound for m e d f r o m d i a z o m e t h a n e t r e a t m e n t . From t h i s i t was c o n -c l u d e d t h a t t h e method i t s e l f was s a t i s f a c t o r y b u t t h e d i f -f i c u l t y l a y i n t h e u s e o f h i g h t e m p e r a t u r e . S u c c e s s was f i n a l l y a c h i e v e d by t r e a t i n g a m e t h y l e n e c h l o r i d e s o l u t i o n o f t h e a c r y l i c a c i d p o r p h y r i n (48) w i t h o x a l y l c h l o r i d e f o r a s h o r t t i m e , a t room t e m p e r a t u r e , t o m i n i m i s e a.ny a c i d d e c o m p o s i t i o n , b e f o r e pumping t o d r y n e s s . A m e t h y l e n e c h l o r i d e s o l u t i o n o f p r o p a n e 1,3 d i a m i n e was added and a l l o w e d t o r e a c t a t room t e m p e r a t u r e f o r two h o u r s . An e x c e s s o f d i a m i n e was u s e d t o p r e v e n t f o r m a t i o n o f a p o r -6 2 p h y r i n d i m e r and a l s o t o a c t as a ' p r o t o n s p o n g e ' f o r t h e s e c o n d s t e p o f t h e , r e a c t i o n . (48) (56) (50a) As e x p e c t e d t h e m o n o v i n y l mono 3-amino 1-amide p r o p a n e a c r y l a t e d e u t e r o p o r p h y r i n IX DTBE (50a) h a d a g r e a t e r R f t h a n t h e a c r y l i c a c i d p o r p h y r i n (4 8) on s i l i c a g e l w i t h some s t r e a k i n g o f t h e s p o t , due t o t h e f r e e amino g r o u p . The s t r e a k i n g c o u l d be e l i m i n a t e d by a d d i n g a s m a l l amount o f t r i e t h y l a m i n e t o t h e e l u e n t . The y i e l d , o f t h e amino p o r p h y r i n (50a) was d i s a p p o i n t i n g l y low ( a r o u n d 20%) and t . l . c . showed t h e m a i n i m p u r i t i e s t o be u n r e a c t e d a c r y l i c a c i d and a com-pound t h a t d i d n o t move from- t h e o r i g i n . The p r e s e n c e o f u n r e a c t e d a c r y l i c a c i d was due, p r e s u m a b l y , t o h y d r o l y s i s o f t h e i n t e r m e d i a t e a c i d c h l o r i d e by t r a c e s o f w a t e r i n t h e d i a -mine. When t h e r e a c t i o n was r e p e a t e d u n d e r s c r u p u l o u s l y d r y c o n d i t i o n s t h e y i e l d was r a i s e d t o 30%. The p r o d u c t w i t h a z e r o Rf was f i r s t i d e n t i f i e d a s a m o n o d e - e s t e r i f i e d p o r p h y r i n as a r e s u l t o f a c i d c l e a v a g e o f t h e e s t e r g r o u p , b u t i t s a b s e n c e i n t h e a c i d c h l o r i d e m e d i a t e d m e t h y l e s t e r (49) s y n t h e s i s s u g g e s t e d t h a t , e v e n a t room t e m p e r a t u r e , a d d i t i o n o f t h e n u c l e o p h i l i c d i a m i n e c a u s e d some d e c o m p o s i t i o n o f t h e a c i d c h l o r i d e t o k e t e n e d e r i v e d p r o d u c t s . The s t r u c t u r e c f t h e p o r p h y r i n amino amide (50a) was c o n f i r m e d f r o m i t s s p e c t r a l p r o p e r t i e s . The v i s i b l e s p e c t r u m was i n t e r m e d i a t e between an e t i o and r h o d o t y p e r e f l e c t i n g t h e w e a k l y e l e c t r o n w i t h d r a w i n g e f f e c t o f t h e amide bond. T h i s , c o n s e q u e n t l y , had a weakened ' r h o d o f y i n g ' e f f e c t on t h e s p e c t r u m , compared t o t h o s e o f t h e m o n o f o r m y l and 64 m o n o a c r y l i c a c i d compounds, r e s u l t i n g i n t h e e q u a l i n t e n s i t y o f b a n d s , I I I and IV, o f t h e s p e c t r u m . The weak e l e c t r o n w i t h d r a w i n g e f f e c t o f t h e amide, due t o a m a j o r c o n t r i b u t i o n t o t h e s t r u c t u r e f r o m t h e ' e n o l 1 f o r m , was a l s o d e m o n s t r a t e d by t h e e a s e i n w h i c h t h e p o r p h y r i n amino amide (50a) u n d e r w e n t 1,4 a d d i t i o n o f s i n g l e t o x y g e n t o g i v e t h e p h o t o p r o t o p o r p h y r i n d e r i v a t i v e ( 5 7 ) . The r a t e o f t h i s r e a c t i o n , on s t a n d i n g i n (50a) (57) s o l u t i o n u n d e r v i s i b l e l i g h t , was c o m p a r a b l e , i f s l i g h t l y s l o w e r , t o t h a t o f p r o t o p o r p h y r i n IX DTBE (24b) and much f a s t e r t h a n t h e m o n o f o r m y l (40) and m o n o a c r y l i c a c i d (48) com-pounds w h i c h a r e h i g h l y d e a c t i v a t e d by t h e i r c a r b o n y l s u b s t i t u e n t s . A l t h o u g h t h e n.m.r. s p e c t r u m was c o m p l e x t h e p r o t o n s f r o m t h e a l i p h a t i c s i d e c h a i n were v i s i b l e i n t h e 6•= 3 -*• 4 r e g i o n . F i n a l c o n f i r m a t i o n o f t h e s t r u c t u r e came f r o m t h e mass s p e c t r u m . In f a c t t h e p o r p h y r i n amino amide (50a) was i n v o l a t i l e on t h e p r o b e d e s p i t e r a p i d h e a t i n g t o 4 5 0 ° C . However when t h e p h o t o p r o t o p o r p h y r i n d e r i v a t i v e (57) was f o r m e d a peak a t m/e = 806, 32 u n i t s h i g h e r t h a n t h e p o r -p h y r i n amine amide (m/e = 7 7 4 ) , was o b t a i n e d . I t a p p e a r e d t h a t f o r m a t i o n o f t h e c h l o r i n had a s u f f i c i e n t l y d i s t o r t i n g 95 e f f e c t on t h e m a c r o c y c l e t o i n c r e a s e i t s v o l a t i l i t y The a c i d c h l o r i d e r e a c t i o n was r e p e a t e d u s i n g h exane 1,6 d i a m i n e as t h e a t t a c k i n g n u c l e o p h i l e i n o r d e r t o g e n e r a t e an a f f i n i t y l i g a n d w i t h a l a r g e r s p a c e r arm. A b e n z e n e s o l u -t i o n o f t h e s o l i d d i a m i n e was a d d e d t o t h e p o r p h y r i n a c i d c h l o r i d e (56) w h i c h had b e e n f o r m e d as b e f o r e . A g a i n y i e l d s were low ( a r o u n d 20%) w i t h f r e e a c r y l i c a c i d p o r p h y r i n and t h e z e r o Rf p r o d u c t as t h e m a j o r i m p u r i t i e s . As e x p e c t e d t h e m o n o v i n y l mono 6-amino l - a m i d o hexane a c r y l a t e d e u t e r o -p o r p h y r i n IX DTBE (50b) had a h i g h e r Rf t h a n t h e a m i n o p r o p a n e compound (50a) b u t i t was a c c o m p a n i e d by c h r o n i c s t r e a k i n g o f t h e band. A g a i n , t h i s c o u l d be e l i m i n a t e d by t h e a d d i t i o n o f t r i e t h y l a m i n e t o t h e e l u e n t and i n f a c t i s o l a t i o n was b e s t a c h i e v e d by u s i n g a c h l o r o f o r m - t r i e t h y l a m i n e m i x t u r e ( 1 0 0 : 1 ) . 66 The v i s i b l e s p e c t r u m o f t h e a m i n o h e x a n e p o r p h y r i n (50b) was a n a l o g o u s t o t h a t o f t h e a m ino p r o p a n e compound (50a) a l t h o u g h t h e n.m.r., a s e x p e c t e d , was more c o m p l e x . The a l i p h a t i c h e x a n e p r o t o n s c o u l d be d i s t i n g u i s h e d i n b o t h t h e 6 = 1.5 and 6 = 3 . 5 r e g i o n s b u r t h e r e was a c o n s i d e r a b l e o v e r l a p w i t h t h e n u c l e a r p o r p h y r i n p r o t o n s . N e v e r t h e l e s s t h e i n t e g r a t i o n g a v e a g o o d c o r r e l a t i o n w i t h t h e p r o p a n e s t r u c t u r e ( 5 0 b ) . The compound was, o n c e a g a i n , i n v o l a t i l e i n t h e 'mass s p e c t r o m e t e r a n d a l t h o u g h t h e p h o t o p r o t o p o r p h y r i n d e r i v a t i v e was e a s i l y p r e p a r e d i t d i d n o t s u f f i c i e n t l y e n h a n c e t h e v o l a t i l i t y o f t h e a m i n o h e x a n e compound t o o b t a i n a mass s p e c t r u m . 2.6. R e m o v a l o f t h e T e r t i a r y B u t y l G r o u p s The l a s t s t e p i n t h e s y n t h e s i s o f t h e a f f i n i t y l i g a n d i n v o l v e d r e m o v a l o f t h e t - b u t y l e s t e r g r o u p s and r e g e n e r a t i o n c f t h e n a t u r a l d i c a r b o x y l i c a c i d s t a t e . A l t h o u g h r e s i s t a n t t o h y d r o g e n o l y s i s and b a s e c a t a l y s e d 101 !'  h y d r o l y s i s t - b u t y l e s t e r g r o u p s a r e s u s c e p t i b l e t o v e r y '< m i l d a c i d c a t a l y s e d a l k y l - o x y g e n f i s s i o n w i t h l o s s o f i s o b u t y l e n e ^ ^ ' . The m o s t common m e t h o d s o f e f f e c t i n g t h i s 69 a r e t r e a t m e n t w i t h t r i f l u o r o a c e t i c a c i d a t room t e m p e r a t u r e o r d r y KC1 i n m e t h y l e n e c h l o r i d e a t 0°C. The l a t t e r p r o -cedure, was c h o s e n s i n c e i t h a d been shown i n t h e s e l a b o r a t o r i e s t h a t no a d d i t i o n t o t h e r e a c t i v e v i n y l g r o u p s t a k e s p l a c e u n d e r t h e s e c o n d i t i o n s . M o n o v i n y l mono 3-amino l - a m i d o p r o p a n e a e r y l a t e d e u t e r o -p o r p h y r i n IX DTBE (50a) was d i s s o l v e d i n m e t h y l e n e c h l o r i d e HCI/CH2CI COpH C 0 2 H (58) and s a t u r a t e d with, d r y HCI. The r e a c t i o n was shown by t . l . c . t o be c o m p l e t e a f t e r s e v e n h o u r s . W i t h a b e n z e n e m e t h a n o l e l u e n t (10:1) t h e d i o r m o n o e s t e r i f i e d compound moved on s i l i c w h i l e movement o f t h e d i a c i d r e q u i r e d t h e p r e s e n c e o f f o r m i c a c i d i n t h e e l u e n t . The m o n o v i n y l mono 3-amino l - a m i d o p r o p a n e a c r y l a t e d e u t e r o p o r p h y r i n IX (58) was s o l u b l e i n v e r y m i l d b a s e ( e . g . d i l u t e s o d i u m c a r b o n a t e s o l u t i o n ) due t o t h e s o l u b i l i s i n g e f f e c t o f t h e s i d e c h a i n amine. P r o t o -p o r p h y r i n IX n o r m a l l y r e q u i r e s a t l e a s t 0.1 M s o d i u m h y d r o x i d e s o l u t i o n 1 0 . The v i s i b l e s p e c t r u m o f t h e d i a c i d amino p o r p h y r i n (58) was t a k e n i n d i l u t e b a s e and shown t o be i n between an e t i o and r h o d o t y p e . The r e s o l u t i o n and r e l a t i v e i n t e n s i t i e s o f t h e p e a k s were s l i g h t l y a l t e r e d due t o t h e s o l v e n t e f f e c t o f t h e aqueous s o l u t i o n . However, t h e t w o ^ v i n y l g r o u p s c o u l d 68 be s e e n t o be i n t a c t s i n c e t h e r h o d o e f f e c t was s t i l l e v i d e n t , p r o o f o f an a 3 u n s a t u r a t e d c a r b o n y l , and i f l e f t i n t h e l i g h t a peak a t 690 nm emerged due t o 1,4 a d d i t i o n o f s i n g l e t o x y g e n t o t h e f r e e v i n y l g r o u p . 2.61. A P o s s i b l e A l t e r n a t i v e L i g a n d S y n t h e s i s A m a j o r u n c e r t a i n t y i n t h e u s e o f a v i n y l s u b s t i t u t e d a f f i n i t y l i g a n d i s t h a t an a l k y l s u b s t i t u t e n t on t h e v i n y l g r o u p s m i g h t s t e r i c a l l y i n t e r a c t w i t h t h e enzyme a t a c l o s e r a n g e and so i n h i b i t i t s b i n d i n g t o t h e v i n y l g r o u p s . T h i s i s a p o t e n t i a l p r o b l e m s i n c e many heme and p o r p h y r i n b i n d i n g enzymes a r e known t o f o r m l i n k a g e s w i t h t h e p o r p h y r i n v i n y l g r o u p s . T h i s p r o b l e m o f c l o s e r a n g e i n t e r f e r e n c e m i g h t be c i r c u m -v e n t e d by a t t a c h i n g t h e l o n g s i d e c h a i n t o one o f t h e p o r p h y r i n m e t h y l g r o u p s , w h i c h a r e n o t u t i l i z e d i n enzyme b i n d i n g , and t h e r e b y l e a v i n g t h e enzyme a l e s s h i n d e r e d a c c e s s t o t h e v i n y l g r o u p s . T h e s e m e t h y l s u b s t i t u e n t s a r e c o m p l e t e l y u n r e a c t i v e t o any k i n d o f e l e c t r o p h i l i c o r n u c l e o p h i l i c a t t a c k . However, l i k e o t h e r m e t h y l g r o u p s , t h e y c o u l d be s u s c e p t i b l e t o r a d i c a l 102 h y d r o g e n a b s t r a c t i o n . I f c a r r i e d o u t w i t h t h e u s u a l non-s e l e c t i v i t y o f an i n t e r m o l e c u l a r r a d i c a l r e a c t i o n h y d r o g e n a b s t r a c t i o n w o u l d be o f no s y n t h e t i c u s e , b u t r e g i o s p e c i f i t y 69 c o u l d be i n t r o d u c e d i f t h e r e a c t i o n was s u b j e c t e d t o t h e s t e r e o -c h e m i c a l c o n s t r a i n t s o f a n i n t r a m o l e c u l a r r e a c t i o n . S u c h a c o n d i t i o n e x i s t s i n t h e B a r t o n r e a c t i o n " * " ^ ' 1. T h i s i n v o l v e s t h e h o m o l y t i c f i s s i o n o f a n i t r i t e e s t e r t o an o x y g e n r a d i c a l + NO H?C HO ... 21 - NO H?C HO and f r e e n i t r i c o x i d e . The o x y g e n r a d i c a l a b s t r a c t s a p r o t o n f r o m a n e a r b y m e t h y l g r o u p and t h e m e t h y l e n e r a d i c a l t h u s f o r m e d s u b s e q u e n t l y r e a c t s w i t h f r e e n i t r i c o x i d e t o g i v e t h e n i t r o s o d e r i v a t i v e o f t h e p r e v i o u s l y u n a c t i v a t e d m e t h y l g r o u p . The r e g i o s e l e c t i v i t y i s i m p o s e d b e c a u s e t h e u n a c t i v a t e d m e t h y l g r o u p must be i n c l o s e p r o x i m i t y t o t h e o x y g e n r a d i c a l . T h i s c o r r e c t a l i g n m e n t i s o n l y p r e s e n t i n a s i x membered r i n g a nd t h u s t h e r i g i d r e q u i r e m e n t o f t h e B a r t o n r e a c t i o n becomes t h e f o r m a t i o n o f a s i x membered c y c l i c t r a n s i t i o n s t a t e " * " ^ ' . I t was o b s e r v e d t h a t t h i s v e r y s i t u a t i o n e x i s t s i n mono-v i n y l m o n o h y d r o x y e t h y l d e u t e r o p o r p h y r i n I X ( 5 9 ) , an i s o l a b l e i n t e r m e d i a t e i n t h e d e h y d r a t i o n o f h e m a t o p o r p h y r i n I X t o p r o t o -p o r p h y r i n I X . A l t h o u g h c o m p l i c a t e d by t h e f a c i l e d e h y d r a t i o n o f t h e c o n j u g a t e d a l c o h o l , t h e f o r m a t i o n o f e t h e r s on t h e h y d r o x y e t h y l g r o u p i s w e l l known"*" I t s h o u l d t h e r e f o r e be p o s s i b l e t o f o r m t h e n i t r i t e e s t e r o f t h e h y d r o x y e t h y l p o r p h y r i n (59) by t r e a t m e n t w i t h n i t r o s y l c h l o r i d e a t 0°C 104 P h o t o l y s i s 6 9a 7 0 o f t h e n i t r i t e e s t e r (60) s h o u l d g i v e an o x y g e n r a d i c a l (61) t h a t e x i s t s i n t h e r e q u i r e d s i x membered c y c l i c t r a n s i t i o n s t a t e w i t h an a d j a c e n t m e t h y l g r o u p . A b s t r a c t i o n o f a m e t h y l p r o t o n f o l l o w e d by r e a c t i o n o f t h e m e t h y l e n e r a d i c a l (62) w i t h n i t r i c o x i d e w o u l d f o r m a m e t h y l n i t r o s o p o r p h y r i n (63) w h i c h s h o u l d r a p i d l y t a u t o m e r i s e t o t h e more s t a b l e c x i m e ( 6 4 ) . H y d r o l y s i s o f t h e oxime w i t h s t r o n g a c i d s h o u l d g i v e t h e m o n o f o r m y l monohydroxy e t h y l p o r p h y r i n (65) w h i c h s h o u l d r e a d i l y d e h y d r a t e t o 2 , 4 - d i v i n y l 3 - f o r m y l d e u t e r o p o r p h y r i n IX (66) a s s u m i n g s t a r t i n g p o r p h y r i n was t h e 2 - v i n y l 4 - h y d r o x y ~ e t h y l i s o m e r . The f o r m y l g r o u p i s t h e n a v a i l a b l e f o r u s e as an a c t i v e c e n t r e on t h e m a c r o c y c l e o n t o w h i c h a l o n g s i d e c h a i n c o u l d be a t t a c h e d i n t h e manner d i s c u s s e d i n t h i s c h a p t e r . S u c h an a f f i n i t y l i g a n d w o u l d h a v e b o t h v i n y l g r o u p s f r e e f o r b i n d i n g t o t h e enzyme. The p r o b l e m s o f r a d i c a l a t t a c k o n t o t h e r i n g a t t h e a d j a c e n t meso p o s i t i o n w o u l d p r o b a b l y be o v e r c o m e by i n c o r p o r a -t i o n o f a m e t a l w i t h a h i g h f i r s t o x i d a t i o n p o t e n t i a l , s u c h I I I I I I as Co o r Fe , i n t o t h e c e n t r e o f t h e m o n o v i n y l mono-h y d r o x y e t h y l p o r p h y r i n . 71 3. A F F I N I T Y CHROMATOGRAPHY 3.1. I n t r o d u c t i o n A f f i n i t y c h r o m a t o g r a p h y i s , p e r h a p s , t h e m o s t r e c e n t e x p r e s s i o n o f t h a t p r i m a r y b i o c h e m i c a l a x i o m t h a t ' b i o c h e m i s t s s h o u l d n o t w a s t e c l e a n t h o u g h t s on d i r t y enzymes 1 . I t h a s an a d v a n t a g e o v e r o t h e r common enzyme i s o l a t i o n t e c h n i q u e s s i n c e i t u t i l i s e s t h e n a t u r a l f u n c t i o n a l s e l e c t i v i t y o f t h e enzyme t o e f f e c t s e p a r a t i o n r a t h e r t h a n r e l y i n g o n g e n e r a l 113 p h y s i c o - c h e m i c a l d i f f e r e n c e s I n e s s e n c e i t i n v o l v e s t h e i m m o b i l i s a t i o n o f a s u b s t r a t e o n t o a s o l i d s u p p o r t a n d t h e p a c k i n g o f t h e s u p p o r t i n t o a c o l u m n . When a c r u d e enzyme p r e p a r a t i o n i s e l u t e d down t h e c o l u m n o n l y t h o s e p r o t e i n s o r enzymes t h a t h a v e a n a p p r e c i a b l e a f f i n i t y f o r t h e m o d i f i e d s u b s t r a t e w i l l be r e t a i n e d b y t h e c o l u m n ; a l l o t h e r s w i l l p a s s t h r o u g h . The s p e c i f i c a l l y b o u n d p r o t e i n , i s t h e n r e m o v e d f r o m t h e c o l u m n b y c h a n g i n g t h e e l u e n t t o one t h a t w i l l d i s s o c i a t e t h e e n z y m e - s u b s t r a t e c o m p l e x . 'A o O o ° 4 A •o 72 The t e c h n i q u e o f a f f i n i t y c h r o m a t o g r a p h y , o r , more c o r r e c t l y , b i o s p e c i f i c a d s o r p t i o n i s an o l d one. E a r l y u s e s t e n d e d t o be o f low s p e c i f i c i t y o f t e n u t i l i s i n g t h e s o l i d 116 s u p p o r t x t s e l f a s t h e a r t i f i c i a l s u b s t r a t e " . The m a j o r t h r u s t i n i t s a p p l i c a t i o n t o a m u l t i t u d e o f enzyme s y s t e m s came w i t h t h e d e v e l o p m e n t , by C u a t r e c a s a s , o f a f a c i l e method f o r a c t i v a t i o n and a t t a c h m e n t o f l i g a n d s t o an i n s o l u b l e 117 p o l y s a c c h a r i d e s u p p o r t . He t r e a t e d t h e p o l y d i s a c c h a r i d e a g a r o s e (67) w i t h c y a n o g e n b r o m i d e t o f o r m t h e a c t i v a t e d i m i d o c a r b o n a t e (68) w h i c h t h e n r e a d i l y r e a c t e d w i t h a l i g a n d c o n t a i n i n g a p r i m a r y amino g r o u p t o b i n d t h e l i g a n d t o t h e .120 s u p p o r t H20H — 0 i — 0 — (67.) HI ^—-OH CNfr A -OH -0 -a :C=NH RNH2 -OH •NH-R H OH -O-C-NH-R (69) (71) (68) (70) X 3 8 The mechanism o f a t t a c h m e n t i s u n c l e a r b u t a p p e a r s t o i n v o l v e a c o m b i n a t i o n o f t h e i s o u r e a (69) N - s u b s t i t u t e d i m i d o c a r b o n a t e (70) and N - s u b s t i t u t e d c a r b a m a t e (71) d e r i v a t i v e s , S i n c e t h i s d i s c o v e r y o f C u a t r e c a s e s many new s o l i d s u p p o r t s have been i n t r o d u c e d i n t o a t e c h n i q u e w h i c h h a s b e en a p p l i e d t o a l m o s t e v e r y a r e a o f b i o c h e m i s t r y . The l i g a n d s t h a t have b e e n bound t o a s o l i d s u p p o r t h a v e r a n g e d f r o m s m a l l amino-a c i d r e s i d u e s o r c o f a c t o r s t o c o m p l e t e enzymes o r v i r u s e s and by t h i s method a v a s t a r r a y o f enzyme s y s t e m s has b e e n 119 p u r i f x e d . I n many ways t h e t e c h n i q u e h a s r e a c h e d i t s z e n i t h and i s u n d e r g o i n g a p e r i o d o f r e a p p r a i s a l . " As t h e f i e l d o f e n z y m o l o g y has e x p a n d e d so has t h e c o m p l e x i t y and s p e c i f i c i t y o f t h e a f f i n i t y l i g a n d s n e e d e d t o e f f e c t enzyme p u r i f i c a t i o n „ T h u s , i n some c a s e s , a l o n g and c o m p l e x s y n t h e -s i s o f t h e l i g a n d has t o be u n d e r t a k e n b e f o r e a f f i n i t y c hroma-t o g r a p h y c a n be u s e d . S u c h a p r o c e d u r e i s o f t e n b e y o n d t h e f a c i l i t i e s o f a b i o c h e m i s t r y l a b o r a t o r y and t h e a f f i n i t y c h r o m a t o g r a p h y t e c h n i q u e l o s e s some o f i t s i m m e d i a t e u s e f u l n e s s . I n a f f i n i t y c h r o m a t o g r a p h y t h e most i m p o r t a n t f a c t o r i s t h e s t r e n g t h and s e l e c t i v i t y o f t h e p r o t e i n - l i g a n d i n t e r -a c t i o n . The s t r e n g t h o f t h i s i n t e r a c t i o n i s m e a s u r e d i n t e r m s o f t h e d i s s o c i a t i o n c o n s t a n t o f t h e enzyme s u b s t r a t e complex"*""^ , where K = [ E ] [ S ] D [ES] and E = enzyme; S = s u b s t r a t e and ES = e n z y m e - s u b s t r a t e complex. The e f f e c t i v e n e s s o f an a f f i n i t y c h r o m a t o g r a p h y c o l u m n i s 74 dependent on the d i s s o c i a t i o n constant f a l l i n g within a given range. The upper l i m i t of the range i s approximately _3 K D = 10 M above which no meaningful retardation, and hence p u r i f i c a t i o n of the enzyme w i l l occur on the column. The -5 lower l i m i t i s usually considerd as K D = 10 M below which binding i s so strong that the enzyme cannot be eluted from the column without a l t e r a t i o n of the s e n s i t i v e t e r t i a r y structure of the protein and denaturation. The i d e a l value for K D i s -4 around 10 M as t h i s expresses a s i t u a t i o n where binding i s s u f f i c i e n t l y strong to e f f e c t i s o l a t i o n but can be disrupted by s u i t a b l y mild reagents. Fortunately these l i m i t s are not absolute since i n the p r a c t i c a l i s o l a t i o n of enzymes d i s s o c i a t i o n constants out -5 -3 side the 10 -*• 10 M range are often encountered. The enzyme-substrate i n t e r a c t i o n i s an equilibrium process and as such the rate at which the enzyme moves down the column can be altered by varying the concentration of the substrate on the column. I f the a f f i n i t y of the enzyme f o r the ligand i s too low ( i . e . K D too high) then retardation w i l l be increased by r a i s i n g the concentration of the substrate on the support 13 or, a l t e r n a t i v e l y , increasing the length of the column . If the' d i s s o c i a t i o n constant i s too low then the movement of the enzyme down the column can be accelerated by decreasing the concentration of substrate on the column or shortening the column length. The substrate concentration i s a fundamental parameter i n the enzyme-substrate interaction"*""*"^ but the influence 75 o f column g e o m e t r y i s l e s s c l e a r and a p p e a r s t o e x e r t t h e g r e a t e s t e f f e c t w i t h enzyme o f low a f f i n i t y o r where h i g h 113 s u b s t r a t e c o n c e n t r a t i o n i s u n a t t a i n a b l e 3.2. P r e p a r a t i o n o f t h e Column In t h e i n i t i a l p r e p a r a t i o n o f an a f f i n i t y c h r o m a t o g r a p h y column f o r a s p e c i f i c enzyme s y s t e m t h e two most i m p o r t a n t f a c t o r s t o be c o n s i d e r e d a r e t h e l i g a n d and t h e s o l i d s u p p o r t . The r a t i o n a l e b e h i n d t h e c h o i c e and d e s i g n o f t h e p o r -p h y r i n l i g a n d was d i s c u s s e d i n C h a p e r 2. The f i n a l s t e p o f t h e s y n t h e s i s i n v o l v e d t h e c o u p l i n g o f a l o n g c h a i n d i a m i n e t o t h e a c r y l i c a c i d p o r p h y r i n ( 4 8 ) . The l e n g t h o f t h e d i a m i n e , o t h e r v / i s e known a s t h e s p a c e r arm, i s an i m p o r -119 t a n t p a r a m e t e r i n a f f i n i t y c h r o m a t o g r a p h y . Once a g a i n i t must f a l l w i t h i n a c e r t a i n r a n g e (72) f o r e f f i c i e n t s e p a r a t i o n t o o c c u r . The s p a c e r arm must be l o n g enough (73) t o a l l o w t h e enzyme f r e e a c c e s s t o t h e l i g a n d w i t h o u t a d e t r i m e n t a l i n t e r a c t i o n w i t h t h e s o l i d s u p p o r t . However i f t o o l o n g an arm i s u s e d (74) t h e p o s s i b i l i t y o f n o n - b i o s p e c i f i c a d s o r p t i o n o f c o n t a m i n a n t p r o t e i n s o n t o t h e h y d r o p h o b i c s i d e c h a i n i n c r e a s e s , r e d u c i n g c o n s i d e r a b l y t h e e f f i c i e n c y o f t h e p u r i f i c a t i o n . The p r o b l e m o f t h i s n o n - s p e c i f i c h y d r o p h o b i c i n t e r a c t i o n has b e e n overcome by t h e u s e o f h y d r o p h i l i c s p a c e r 121 arms and h a s , i n f a c t , r e c e n t l y a l s o been e x p a n d e d f o r u s e J 7 2 as a n o n - s p e c i f i c p r o t e i n p u r i f i c a t i o n method " ' . (72) (73) (74) I n t h e p o r p h y r i n a f f i n i t y c h r o m a t o g r a p h y s y s t e m t h e c h o i c e o f t h e s p a c e r arm was b e s e t by t h e same u n c e r t a i n t i e s as t h e d e s i g n o f t h e l i g a n d i t s e l f . An i n f o r m e d d e c i s i o n , c o u l d n o t be made w i t h o u t a k n o w l e d g e o f t h e enzyme t o p o g r a p h y , and c o n s e q u e n t l y t h e c h o i c e was d i c t a t e d more by e x p e d i e n c y . The 3-amino l - a m i d o p r o p a n e a c r y l a t e p o r p h y r i n (50a) was p r e p a r e d more e a s i l y i n l a r g e r q u a n t i t i e s and so i n i t i a l s t u d i e s were b a s e d on t h i s l i g a n d . A s y n t h e s i s o f an a l t e r n a t i v e l i g a n d , 6-amino l - a m i d o h e x a n e a c r y l a t e p o r p h y r i n (50b) was a l s o c a r r i e d o u t , b u t t h i s l i g a n d was n o t u s e d i n t h e s e i n i t i a l e x p e r i m e n t s . The c h o i c e o f s o l i d s u p p o r t i s an i m p o r t a n t one i n a f f i n i t y c h r o m a t o g r a p h y . The m a t r i x must p l a y an i n e r t r o l e i n t h e p a s s a g e o f t h e enzyme down t h e column t o r e d u c e n o n -b i o s p e c i f i c i n t e r a c t i o n s , y e t i t must h a v e an a b u n d a n t s u p p l y o f c h e m i c a l g r o u p s on t h e s u r f a c e t h a t c a n be r e a d i l y a c t i v a t e d t o f o r m c o v a l e n t b o n d s w i t h t h e a f f i n i t y l i g a n d . 77 I d e a l l y t h e m a t r i x s h o u l d a l s o be o f a. s u f f i c i e n t l y h i g h p o r o s i t y t o a l l o w a f r e e p a s s a g e t o l a r g e m o l e c u l e s and a l s o , o f c o u r s e , be s t a b l e u n d e r t h e v a r i o u s c o n d i t i o n s t o w h i c h i t i s s u b j e c t e d . 113 T h e r e a r e a number o f c o m m e r c i a l l y a v a i l a b l e m a t r i c e s and t h e c h o i c e between them i s l a r g e l y an e m p i r i c a l one. 123 A g a r o s e ( S e p h a r o s e 2B. P h a r m a c i a F i n e Chem. ) was c h o s e n f o r t h i s p o r p h y r i n a f f i n i t y c h r o m a t o g r a p h y s y s t e m . I t has a v e r y h i g h p o r o s i t y and i s e a s i l y a c t i v a t e d by c y a n o g e n b r o m i d e . The method u s e d t o a c t i v a t e a g a r o s e w i t h c y a n o g e n b r o m i d e 124 was t h a t o f C u a t r e c a s a s . T h i s i s a s i m p l i f i e d method and d o e s n o t r e q u i r e t h e t e d i o u s t i t r a t i o n p r o c e d u r e s p r e v i o u s l y 120 e m p l o y e d . The g e n e r a l a c t i v a t i o n p r o c e d u r e i n v o l v e d t r e a t m e n t o f a s o d i u m c a r b o n a t e s o l u t i o n (2.0 M) o f t h e a g a r o s e s l u r r y w i t h an a c e t o n i t r i l e s o l u t i o n o f c y a n o g e n b r o m i d e f o r two m i n u t e s f o l l o w e d by e x t e n s i v e w a s h i n g and s t o r a g e a t 4°C i n an aqueous s o l u t i o n (pH 7 ) . The a c t i v a t e d i m i d o c a r b o n a t e compound (68) i s s l o w l y h y d r o l y s e d by b a s i c s o l u t i o n s and so must be s t o r e d a t pH 7 r a t h e r t h a n t h e c o u p l i n g c o n d i t i o n s o f pH 9.5. A l s o c y a n o g e n b r o m i d e i s a f a i r l y h a r s h r e a g e n t and e x p o s u r e o f t h e s e n s i t i v e s e p h a r o s e m a t r i x t o e x t e n d e d t r e a t -ment must be k e p t t o a minimum. The e x t e n t o f a c t i v a t i o n o f t h e s e p h a r o s e c a n r e a d i l y be v a r i e d by a l t e r i n g t h e c o n d i t i o n s o f a c t i v a t i o n . C u a t r e c a s e s 1 " ^ f o u n d t h a t when a c t i v a t i o n was c a r r i e d o u t a t 4°C t h e f i n a l 78 l i g a n d c o u p l i n g was i n c r e a s e d by 3 5-4 0% r e l a t i v e t o t h a t a c t i -v a t e d a t room t e m p e r a t u r e . I n t h i s s t u d y i t was f o u n d t h a t t h e c o n c e n t r a t i o n o f t h e f i n a l p o r p h y r i n l i g a n d on t h e 3 3 s e p h a r o s e c o u l d be v a r i e d f r o m 0.1 uM/cm t o 4.7 yM/cm by a s i m p l e v a r i a t i o n i n t h e amount o f t h e a c e t o n i t r i l e s o l u -t i o n o f c y a n o g e n b r o m i d e u s e d i n t h e a c t i v a t i o n s t e p . T h i s r e p r e s e n t s a u s e f u l r a n g e o f l i g a n d c o n c e n t r a t i o n s f o r g e n e r a l a p p l i c a t i o n o f t h e p o r p h y r i n a f f i n i t y c h r o m a t o g r a p h y s y s t e m . C o u p l i n g o f t h e l i g a n d t o t h e a c t i v a t e d s e p h a r o s e t o o k p l a c e i n s o d i u m b i c a r b o n a t e s o l u t i o n (0.2 M, pH 9.5) a t 4°C f o r , on a v e r a g e , 24 h o u r s . The c o u p l i n g r e a c t i o n i s , i n f a c t , o v e r i n two h o u r s b u t t h e e x t e n d e d t i m e p e r i o d i s u s e d t o e n s u r e a l l r e a c t i v e i m i d o c a r b o n a t e g r o u p s a r e h y d r o l y s e d and u n a b l e t o i r r e v e r s i b l y i n t e r a c t w i t h t h e enzyme. A t t h e end o f t h e c o u p l i n g s t e p t h e p o r p h y r i n - b o u n d s e p h a r o s e was p a c k e d i n t o a s m a l l c o l u m n , washed t h o r o u g h l y and s t o r e d , i n an aqueous s o l u t i o n (pH = 7 . 0 ) , a t 4°C i n a l i g h t f r e e c o n t a i n e r . S h i e l d i n g f r o m l i g h t was a n e c e s s a r y p r e c a u t i o n s i n c e t h e l i g a n d s t i l l p o s s e s s e d t h e r e a c t i v e v i n y l g r o u p s w h i c h , e v e n on t h e c o l u m n , c o u l d u n d e r g o a ' p h o t o p r o t o ' r e a c t i o n . A f u r t h e r p r e c a u t i o n , p a r t i c u l a r l y when t h e column was t o be l e f t f o r l o n g p e r i o d s o f t i m e , was t h e a d d i t i o n o f a s o d i u m a z i d e s o l u t i o n (0.02%) t o a c t as a 123 b a c t e r i o s t a t i c a g e n t 79 Measurement of the l i g a n d c o n c e n t r a t i o n on t h e column was c a r r i e d o u t by m e asuring t h e d i f f e r e n c e i n t h e v i s i b l e a b s o r p -t i o n o f t h e p o r p h y r i n s o l u t i o n s . 3.3. A l t e r n a t i v e L i g a n d s 3.31. P r o t o p o r p h y r i n o g e n IX D e r i v a t i v e The p u r i f i c a t i o n o f p r o t o p o r p h y r i n o g e n o x i d a s e r e q u i r e d a p r o t o p o r p h y r i n o g e n a f f i n i t y l i g a n d w i t h a f r e e v i n y l group i n p o s i t i o n 4 (see C h a p t e r I ) . T h i s c o u l d c l e a r l y be p r o v i d e d by a r e d u c t i o n o f the 3-amino, l-amido d e u t e r o p o r p h y r i n IX l i g a n d , p r e f e r a b l y w h i l e i t was bound t o t h e sepharose s i n c e p o r p h y r i n o g e n s a r e r e a c t i v e s p e c i e s and r e a d i l y a u t o x i d i s e t o t h e s t a b l e p o r p h y r i n s ; a f f i n i t y p u r i f i c a t i o n o f t h e enzyme s h o u l d r a p i d l y f o l l o w t h e r e d u c t i o n s t e p . A s epharose bound r e d u c t i o n o f t h e p o r p h y r i n would a l s o a l l o w t h e column t o be r e c y c l e d by f a c i l e r e g e n e r a t i o n o f t h e p o r p h y r i n o g e n from t h e e n z y m i c a l l y formed p o r p h y r i n . The most commonly used r e d u c i n g agents f o r t h i s t r a n s f o r -m a t i o n a r e sodium amalgam and sodium b o r o h y d r i d e . Sodium amalgam was o f l i t t l e use i n t h i s c a s e s i n c e i t i s known t o 125 cause e x t e n s i v e r e d u c t i o n o f p o r p h y r i n v i n y l groups and would a l s o d r a s t i c a l l y i n t e r f e r e w i t h t h e enzyme i f any t r a c e s were l e f t i n t h e g e l a t t h e p u r i f i c a t i o n s t a g e . Sodium 80 b o r o h y d r i d e a p p e a r e d t o be more a p p l i c a b l e . G r a n i c k J " " " J h a s shown t h a t 2 , 4 - d i a c r y l i c d e u t e r o p o r p h y r i n was r e d u c e d w i t h a 70% e f f i c i e n c y and f r e e v i n y l g r o u p s were l e s s s u s c e p t i b l e t o r e d u c t i o n u n d e r t h e s e c o n d i t i o n s . I n t h i s r e s p e c t a 70% y i e l d r e a c t i o n w o u l d s t i l l g i v e r i s e t o a p o r p h y r i n o g e n a f f i n i t y column o f s u f f i c i e n t p u r i t y t o e f f e c t p u r i f i c a t i o n o f p r o t o -p o r p h y r i n o g e n o x i d a s e . The r e d u c t i o n was a t t e m p t e d by p a s s i n g a s o l u t i o n o f s o d i u m 3 b o r o h y d r i d e (90mg) i n a t r i s b u f f e r (pH 8.0, 10cm ) s l o w l y t h r o u g h t h e p o r p h y r i n bound s e p h a r o s e o v e r a p e r i o d o f 2 h o u r s . The d a r k r e d c o l o u r o f t h e g e l d i s a p p e a r e d t o g i v e r i s e t o a w h i t e u n t i n t e d g e l , a s i t u a t i o n t h a t w o u l d be e x -p e c t e d f o r t h e f o r m a t i o n o f t h e c o l o u r l e s s p o r p h y r i n o g e n . However on p a s s i n g o x y g e n a t e d b u f f e r t h r o u g h t h e c o l u m n and a f t e r e x t e n d e d p h o t o l y s i s , c o n d i t i o n s known t o c a u s e a r a p i d r e g e n e r a t i o n o f t h e p o r p h y r i n , o n l y a s l i g h t c o l o u r c h a n g e bac k t o t h e r e d p o r p h y r i n was n o t e d . C l e a r l y some o f t h e s e p h a r o s e bound p o r p h y r i n had e i t h e r b e en d e g r a d e d o r d e t a c h e d f r o m t h e column. I n r e t r o s p e c t e x t e n s i v e d e g r a d a t i o n was u n l i k e l y s i n c e p o r p h y r i n o g e n s h a ve been shown t o be s t a b l e t o w a r d s h i g h c o n -125 c e n t r a t i o n s o f s o d i u m b o r o h y d r i d e . I t seems more l i k e l y t h a t t h e b o r o h y d r i d e i s a s u f f i c i e n t l y s t r o n g r e d u c i n g a g e n t t o e f f e c t r e d u c t i o n o f t h e a c t i v a t e d s e p h a r o s e p o r p h y r i n bond. 81 An i s o u r e a d e r i v a t i v e i s one o f t h e p r o p o s e d b o n d i n g s i t u a t i o n s i n t h e c o u p l i n g r e a c t i o n and i t i s c o n c e i v a b l e t h a t t h i s i s s u s c e p t i b l e t o r e d u c t i o n by b o r o h y d r i d e t o t h e p r i m a r y amine w h i c h w o u l d t h e n decompose w i t h d e t a c h m e n t o f t h e l i g a n d . A H ~ J H -0-O-N—PORP ^NH NaBH4 H | _ § > _ N - P OlH A 1 — o C - N - P NH C l e a r l y more work i s n e e d e d t o d i s c o v e r t h e a p p r o p r i a t e c o n d i t i o n s and c o n c e n t r a t i o n s o f b o r o h y d r i d e r e q u i r e d t o e f f e c t t h i s r a t h e r s e n s i t i v e t r a n s f o r m a t i o n . 3.32. Heme D e r i v a t i v e A m a j o r aim o f t h i s s t u d y was t h e p r e p a r a t i o n o f an a f f i n i -l i g a n d b a s e d on t h e heme p r o s t h e t i c g r o u p f o r p o s s i b l e u s e w i t h t h e many heme enzymes and h e m e p r o t e i n s f o u n d i n b i o -c h e m i s t r y . A s o l u t i o n t o t h i s a p p e a r e d t o be p r o v i d e d by t h e s i m p l e m e t a l l a t i o n o f mono 3-amino l - a m i d o p r o p a n e a c r y l a t e 82 m o n o v i n y l d e u t e r o p o r p h y r i n IX (75) t o i t s i r o n ( I I I ) d e r i v a t i v e . S i n c e t h e l i g a n d was t o be u s e d t o i s o l a t e an enzyme t h e f i r s t a t t e m p t a t m e t a l l a t i o n i n v o l v e d , l o g i c a l l y enough, t h e t r e a t m e n t o f t h e ' s e p h a r o s e bound p o r p h y r i n w i t h f e r r o c h e l a t a s e i n t h e a p p r o p r i a t e a s s a y c o n d i t i o n s o f f e r r o u s s u l p h a t e (0.1 mM) t r i s - H C L b u f f e r (50mM, pK 9.5) and c y s t e i n e (lOmM). A f t e r i n c u b a t i o n a t 3 7°C, t h e optimum t e m p e r a t u r e f o r t h e 44 enzyme , f o r 2 h o u r s t h e s e p h a r o s e bound l i g a n d s t x l l showed t h e f o u r l i n e r h o d o / o x o r h o d o s p e c t r u m and t h u s no m e t a l l o -p o r p h y r i n had b e e n f o r m e d . The e x p e r i m e n t was r e p e a t e d w i t h a l o n g e r i n c u b a t i o n p e r i o d b u t a g a i n no r e a c t i o n t o o k p l a c e . A more r e l i a b l e o r g a n i c s y n t h e s i s was a t t e m p t e d and p r o v e d s u c c e s s f u l . The unbound mono 3-amino 1-amido p r o p a n e a c r y l a t e m o n o v i n y l d e u t e r o p o r p h y r i n IX was m e t a l l a t e d by a s l i g h t m o d i f i c a t i o n o f t h e method o f Falk"*"^. The p o r p h y r i n was d i s s o l v e d i n p y r i d i n e a c e t i c a c i d m i x t u r e and a f e r r o u s s u l -p h a t e s o l u t i o n was a d d e d . Under an a t m o s p h e r e o f n i t r o g e n t h e s o l u t i o n was h e a t e d a t 80° f o r 10 m i n u t e s . A f t e r t h e a d d i t i o n o f h y d r o c h l o r i c a c i d t h e i r o n ( I I I ) p o r p h y r i n s a r e n o r m a l l y e x t r a c t e d i n e t h e r s o l u t i o n . However t h e p r e s e n c o f a t e r m i n a l amino s i d e c h a i n on t h e a f f i n i t y l i g a n d r e d u c e d i t s s o l u b i l i t y i s e t h e r and so p r e c i p i t a t i o n w i t h s o d i u m b i c a r b o n a t e s o l u t i o n was e f f e c t e d . 3 The i r o n ( I I I ) 3-amino l-amido propane a c r y l a t e deutero-porphyrin (75) was c o l l e c t e d and found to be s o l u b l e i n d i l u t e base. The v i s i b l e spectrum, i n d i l u t e base, showed a broad unresolved s e r i e s of peaks, as i s o f t e n the case w i t h 3 porphyrin complexes of t r i v a l e n t metal ions . The p y r i d i n e hemochrome spectrum was much more d i s t i n c t i v e . The charac-t e r i s t i c a l l y strong a and (3 bands were found at 569 and 526 nm r e s p e c t i v e l y . These values f e l l i n between the l i t e r a t u r e 68 values f o r F e ( I I ) p r o t o p o r p h y r i n IX (557 nm and 526 nm) and Fe(II) monoformyl monovinyl. dueteroporphyrin IX (58 3 nm and 54 5) r e f l e c t i n g the intermediate e l e c t r o n withdrawing e f f e c t of the amide group of (7 5) r e l a t i v e to the v i n y l group of protoporphyrin and the formyl group of monoformyl monovinyl deuteroporphyrin. Coupling of the i r o n ( I I I ) a f f i n i t y l i g a n d complex to cyanogen bromide a c t i v a t e d sepharose was c a r r i e d out i n an e x a c t l y s i m i l a r way to t h a t of the metal f r e e l i g a n d . Again 8 4 t h e amount o f h e m i n bound t o t h e g e l was e s t i m a t e d by t h e d i f f e r e n c e i n t h e v i s i b l e s p e c t r a o f t h e h e m i n s o l u t i o n b e f o r e a n d a f t e r c o u p l i n g ; t h i s was f o u n d t o be a p p r o x i m a t e l y 1.0 pM/cm 3. The g r e e n / b r o w n c o l o u r e d g e l was s h i e l d e d f r o m l i g h t a n d s t o r e d a t 4°C f o r f u t u r e u s e w i t h h e m o p e x i n . 3.4. E p i l o g u e The v e r y e a r l i e s t r e s u l t s f r o m t h e B i o c h e m i s t r y D e p a r t -ment a t U . B . C , show c o n s i d e r a b l e p r o m i s e f o r t h e e f f e c t i v e n e s s o f t h e p o r p h y r i n a f f i n i t y l i g a n d . U s i n g a c o l u m n c o n t a i n i n g 3 a p p r o x i m a t e l y 0.1 ymole/cm o f l i g a n d a p r e p a r a t i o n o f f e r r o -c h e l a t a s e f r o m r a t l i v e r h o m o g e n a t e was c h r o m a t o g r a p h e d . A t 2 0°C no b i n d i n g o c c u r r e d and t h e enzyme p a s s e d t h r o u g h t h e c o l u m n . However when t h e t e m p e r a t u r e was r a i s e d t o t h e o p -t i m u m t e m p e r a t u r e o f 37°C 4 0 % o f t h e enzyme i n t h e c r u d e p r e p a r a t i o n b ound t o t h e c o l u m n . T h i s t e m p e r a t u r e v a r i a n c e s u g g e s t s t h a t t h e b i n d i n g was n o t a r t i f a c t u a l a nd t h a t on a c o l u m n w i t h a g r e a t e r l i g a n d c o n c e n t r a t i o n a g r e a t e r d e g r e e o f enzyme b i n d i n g w i l l o c c u r . T h e s e i n i t i a l r e s u l t s a r e , o f c o u r s e , s i m p l y t h e h o p e f u l b e g i n n i n g o f t h e n e x t m a j o r p h a s e i n t h e l o n g t e r m s t u d y o f p o r p h y r i n b i o c h e m i s t r y w h i c h w i l l i n v o l v e t h e e l u c i d a t i o n o f t h e c o r r e c t c o n d i t i o n s f o r e f f i c i e n t a f f i n i t y p u r i f i c a t i o n o f a number o f heme and p o r -p h y r i n b i n d i n g e nzymes. 85 4. MONOACRYLIC ACID MONOVINYL DEUTEROPORPHYRIN I X DITERTIARY BUTYL ESTER. The m a j o r i n t e r m e d i a t e s i n t h e a f f i n i t y l i g a n d s y n t h e s i s w ere t h e i s o m e r i c a c r y l i c a c i d p o r p h y r i n s ( 4 8 ) . T h e s e com-pounds a l s o p r e s e n t t h e m s e l v e s a s p o t e n t i a l l y u s e f u l i n t e r -(48) m e d i a t e s i n t h e s y n t h e s i s o f a number o f b i o s y n t h e t i c a l l y i n t e r e s t i n g 2- and 4- d i f f e r e n t i a l l y s u b s t i t u t e d d e u t e r o p o r -20 p h y r i n s , whxch a t p r e s e n t a r e o n l y a v a i l a b l e b y e x t e n d e d s t e p w i s e s y n t h e s e s . I n a n y n a t u r a l p o r p h y r i n d e r i v e d s y n t h e s i s t w o m a j o r p r o b l e m s a r e o f t e n e n c o u n t e r e d . The f i r s t i s t h e s e p a r a t i o n and i d e n t i f i c a t i o n o f t h e 2- a n d 4- i s o m e r s a n d t h e s e c o n d i s t h e c h e m i c a l d i f f e r e n t i a t i o n o f t h e 2- a n d , t h e -4- s u b s t i t u e n t s 86 f o r f u r t h e r m o d i f i c a t i o n s t o be c a r r i e d o u t . I n t h e case o f m o n o a c r y l i c m o n o v i n y l d e u t e r o p o r p h y r i n IX bo t h o f t h e s e p r o -blems have been overcome. The s e p a r a t i o n and i d e n t i f i c a t i o n o f t h e 2- and 4- f u n c t i o n a l i s e d i s o m e r s were c a r r i e d o u t a t t h e p h o t o p r o t o p o r p h y r i n IX s t a g e and t h e d i f f e r e n t c h e m i c a l r e a c t i v i t i e s o f t h e v i n y l and a c r y l a t e groups made s e l e c t i v e m o d i f i c a t i o n p o s s i b l e . The most u s e f u l s e l e c t i v e r e a c t i o n o f t h e v i n y l group was, s i m p l y , a r e p e t i t i o n o f t h e sequence c a r r i e d o u t on t h e d i v i n y l compound. The mixed mono a c r y l i c a c i d m o n o v i n y l p o r p h y r i n i s o m e r s ( 4 8 ) , i n c h l o r o f o r m , were i r r a d i a t e d w i t h a desk lamp i n t h e p r e s e n c e o f oxygen. The 1 , 4 - a d d i t i o n o f s i n g l e t oxygen t o t h e d i e n e formed by t h e v i n y l group and c r o s s c o n j u g a t e d d o u b l e bond t o g i v e t h e p h o t o p r o t o p o r p h y r i n d e r i v a t i v e (76a) was e x c e e d i n g l y slow (1 week) due t o t h e de~ 108 a c t i v a t i n g e f f e c t o f t h e c o n j u g a t e d carboxylate" 1" . The green p r o d u c t was p u r i f i e d by s i l i c a g e l chromatography (CHCl^: MeOK 20:1) and had a s m a l l e r Rf than t h e p a r e n t p o r p h y r i n . S u r p r i s i n g l y t h e green compound showed, on t . l . c , a d i s t i n c -t i v e s e p a r a t i o n i n t o t h e 2- and 4- i s o m e r s . T h i s was a much more marked s e p a r a t i o n t h a n was seen w i t h p h o t o p r o t o p o r p h y r i n IX DTBE i t s e l f (44a, 45a) and was presumably due t o t h e predominant e l e c t r o s t a t i c e f f e c t o f t h e c a r b o x y l a t e i n t h e a c r y l i c a c i d p o r p h y r i n . A l t h o u g h n o t e x p l o i t e d here t h i s o b s e r v a t i o n o f f e r s an a l t e r n a t i v e and, p e r h a p s , easier-* r o u t e by w h i c h t h e l a b o r i o u s s e p a r a t i o n o f the 2~ and 4- i s o m e r i c d e u t e r o p o r p h y r i n s 8 7 c o u l d be e f f e c t e d . OHC-CH V ^ 1 C O O R (•48) O2 hv COgtBu C02tBu (76a) + t h e o t h e r isomer b a, R = H b, R = Me The v i s i b l e spectrum o f t h e p h o t o l y s e d d e r i v a t i v e showed th e c h a r a c t e r i s t i c s t r o n g c h l o r i n peak a t 68 7 nm, bypso-c h r o m i c a l l y s h i f t e d r e l a t i v e t o p r o t o p o r p h y r i n IX (671 nm) as a r e s u l t o f t h e e l e c t r o n w i t h d r a w i n g a c r y l a t e f u n c t i o n i n th e m o l e c u l e . However, on a d d i t i o n o f base t h e e l e c t r o n w i t h -d r a wing e f f e c t was d i m i n i s h e d and t h e peak s h i f t e d t o 677 nm. T h i s b a t h o c h r o m i c s h i f t c o n f i r m e d t h e p r e s e n c e i n t h e m o l e c u l e o f an a3 u n s a t u r a t e d c a r b o x y l a t e and t h a t a d d i t i o n o f s i n g l e t oxygen had t a k e n p l a c e a t t h e f r e e v i n y l group. T h i s would be e x p e c t e d s i n c e , a l t h o u g h t h e t r u e n a t u r e o f t h e ' p h o t o p r o t o ' r e a c t i o n i s unknown, i t appears t o f o l l o w t h e c o n s t r a i n t s o f a D i e l s - A l d e r r e a c t i o n and t h u s would be l a r g e l y d e a c t i v a t e d by a c a r b o x y l a t e on t h e d i e n e component. 88 The a c r y l i c a c i d p h o t o p r o t o p o r p h y r i n IX DTBE (76a) was l e s s s o l u b l e t h a n t h e p a r e n t p o r p h y r i n i n CDCl^ and gave a p o o r l y r e s o l v e d n.m.r. I t was a l s o i n v o l a t i l e i n t h e mass s p e c t r o m e t e r . F u r t h e r e v i d e n c e f o r t h i s s t r u c t u r a l Cqesignationyvzas o b t a i n e d when t h e p h o t o l y s i s was c a r r i e d o u t on monomethyl a c r y l a t e m o n o v i n y l d e u t e r o p o r p h y r i n DTBE (49 ) . T h i s r e a c t i o n was c o r r e s p o n d i n g l y s l o w and t h e 2- and 4- i s o m e r s showed no s e p a r a t i o n on t . l . c . However, t h e m e t h y l a c r y l a t e p h o t o p r o t o p o r p h y r i n compound (76b) was s u f f i c i e n t l y v o l a t i l e t o g i v e a mass spectrum and showed the e x p e c t e d peak a t m/3 = 7 64 due t o t h e a d d i t i o n o f one oxygen m o l e c u l e (m.wt. = 32) t o monomethyl a c r y l a t e d e u t e r o p o r p h y r i n (m.wt. = 732). The n e x t s t a g e i n t h e f u n c t i o n a l i s a t i o n o f t h e second v i n y l group s h o u l d i n v o l v e an analogous t r a n s f o r m a t i o n t o 9? t h a t c a r r i e d out on p h o t o p o r o t p o r p h y n n IX DTBE " (see Chapter 2 ) , namely r e d u c t i o n w i t h sodium b o r o h y d r i d e f o l l o w e d by sodium p e r i o d a t e o x i d a t i o n o f t h e g l y c o l formed by a c i d rearrangement t o , f i n a l l y , y i e l d t h e f o r m y l d e r i v a t i v e . T h i s r e a c t i o n was a t t e m p t e d on t h e a c r y l i c a c i d p h o t o p r o t o -p o r p h y r i n (76]) and showed p r o m i s i n g , a l t h o u g h not c o n c l u s i v e , r e s u l t s . The brown p r o d u c t was p u r i f i e d by s i l i c a g e l chromatography and showed a s i n g l e s p o t . The compound was o n l y s l i g h t l y s o l u b l e i n c h l o r o f o r m , as would be e x p e c t e d f o r a p o r p h y r i n w i t h b o t h a f o r m y l and a c r y l i c a c i d group on t h e r i n g . The v i s i b l e spectrum showed, i n i t i a l l y , an e t i o t y p e 39 o f s p e c t r u m w h i c h on a d d i t i o n o f b a s e c o l l a p s e d i n t o a r h o d o / o x o r h o d o t y p e . However t h e p r e s e n c e o f s h o u l d e r s i n t h e s p e c t r u m s u g g e s t e d some i m p u r i t y a n d made a b s o l u t e c h a r -a c t e r i s a t i o n d i f f i c u l t . The e x p e c t e d p r o d u c t f o r t h e r e a c t i o n was 2- (and 4-) f o r m y l 4- ( a n d 2-) a c r y l i c a c i d d e u t e r o p o r p h y r i n I X DTBE ( 7 7 ) . T h i s compound, w i t h e l e c t r o n w i t h d r a w i n g g r o u p s a t a d j a c e n t (77) p y r r o l e r i n g s , w o u l d be e x p e c t e d t o g i v e a n e t i o - t y p e s p e c -t r u m , w h i c h , on a d d i t i o n o f b a s e a n d r e m o v a l o f t h e e l e c t r o n w i t h d r a w i n g e f f e c t o f t h e c a r b o x y l i c a c i d , s h o u l d c h a n g e t o a r h o d o t y p e . T h i s b e h a v i o r i s i n d i c a t e d b y t h e v i s i b l e s p e c t r a b u t t h e p o s s i b l e i n t e r f e r e n c e f r o m i m p u r i t i e s made u n a m b i g u o u s a n a l y s i s o f t h e r e s u l t d i f f i c u l t . The m o n o f o r m y l m o n o a c r y l i c a c i d p o r p h y r i n (77) i s a p o t e n t i a l l y v e r y u s e f u l p r e c u r s o r f o r t h e S y n t h e s i s o f some o f t h e p o r p h y r i n o g e n s t h a t h a v e b e e n p r o p o s e d t o s u c c e e d 90 c o p r o p o r p h y r i n o g e n (11) i n t h e b i o s y n t h e t i c p r o d u c t i o n o f 20 p r o t o p o r p h y r i n IX ( C h a p t e r 1 ) . The p r e s e n c e o f t h e f o r m y l and a c r y l i c a c i d g r o u p s a t p o s i t i o n s 2- and 4- f a c i l i t a t e t h e c h e m i c a l d i f f e r e n t i a t i o n and s e l e c t i v e i s o t o p i c l a b e l l i n g o f t h e s e p o s i t i o n s i n t h e f i n a l c o p r o p o r p h y r i n o g e n d e r i v a t i v e s . Some o f t h e s e d e r i v a t i v e s ha.ve b e e n s y n t h e s i s e d , b u t o n l y v i a 4- • 4 4-U ,,109,110 a t e d i o u s s t e p w i s e method The n a t u r e o f t h e i n t e r m e d i a t e s between c o p r o p o r p h y r i n o -gen I I I and p r o t o p o r p h y r i n o g e n IX i n t h e b i o s y n t h e t i c pathway i s , as y e t , u n c l e a r . An o x i d a t i v e d e c a r b o x y l a t i o n o f two p r o p i o n i c a c i d g r o u p s t o g i v e two v i n y l g r o u p s i s i n v o l v e d and t h e 3 - h y d r o x y c a r b o x y l a t e s (81) h a v e b e e n p o s t u l a t e d as 20 i n t e r m e d i a t e s . I t a p p e a r s t h a t t h e 2 - p r o p i o n a t e i s d e -c a r b o x y l a t e d b e f o r e t h e 4 - p r o p i o n a t e 1 1 1 b u t a g r e a t d e a l more work i s n e e ded i n t h i s a r e a t o e l u c i d a t e t h e s e q u e n c e and n a t u r e o f t h e t r a n s f o r m a t i o n . By s i m p l y a r e p e t i t i o n o f t h e K n o e v e n a g e l r e a c t i o n s e q u e n c e m o n o f o r m y l m o n o a c r y l i c a c i d d e u t e r o p o r p h y r i n (77) o f f e r s a r o u t e t o t h e c o p r o p o r p h y r i n I I I d e r i v a t i v e s , i n w h i c h , by an a p p r o p r i a t e c h o i c e o f i s o t o p i c a l l y l a b e l l e d r e a g e n t s , t h e 2- and 4- f u n c t i o n a l i t i e s c a n be d i f f e r e n t i a t e d . S u c h s p e c i f i c a l l y l a b e l l e d d e r i v a t i v e s may be u s e f u l i n b i o -s y n t h e t i c i n c o r p o r a t i o n e x p e r i m e n t s . C o p r o p o r p h y r i n I I I (80) i t s e l f i s a v a i l a b l e f r o m m o n o f o r m y l m o n o a c r y l i c a c i d d e u t e r o p o r p h y r i n (77) by c a t a l y t i c h y d r o g e n a t i o n o f t h e a c r y l a t e v i n y l group f o l l o w e d by Knoevenagel c o n d e n s a t i o n o f m a l o n i c a c i d t o the a c r y l i c a c i d d e r i v a t i v e (79) and c a t a l y t i c h y d r o g e n a t i o n o f t h e second a c r y l a t e f u n c t i o n , (77) Hp/Pd CO,H Hyp •C02H (78) H„/Pd M a l o n i c a c i d P y r i d i n e , p i p e r i d i n e CO^H (79! • cPaH ( 8 0 ) Use o f l a b e l l e d m a l o n i c a c i d o r d e u t e r i u m gas i n a g i v e n s y n -t h e t i c s t e p would g i v e r i s e t o a v a r i e t y o f l a b e l l e d p r o d u c t s . The mono g hydroxy p r o p i o n a t e m o n o p r o p i o n i c a c i d p o r p h y r i n s (81) has been proposed as t h e major i n t e r m e d i a t e i n t h e enzymatic d e c a r b o x y l a t i o n and i s , i n f a c t , an i n t e r m e d i a t e i n t h e Knoevenagel c o n d e n s a t i o n . I t would be more r e a d i l y -a v a i l a b l e from a h y d r a t i o n o f t h e a p p r o p r i a t e m o n o a c r y l i c monopropionate isomer ( 7 9 ) . H y d r a t i o n o f t h e d i a c r y l i c a c i d compound has been a c h i e v e d by t r e a t m e n t w i t h H B r - a c e t i c a c i d f o l l o w e d by aqueous h y d r o l y s i s t o g i v e e x c l u s i v e l y t h e 3-hydroxy c a r b o x y l a t e porphyrin 1"'""'" and t h i s s h o u l d be a p p l i c a b l e t o t h e m o n o a c r y l i c a c i d m o n o p r o p i o n i c a c i d p o r p h y r i n s . 92 C02H C02H CO^H HBr/HAc H CC^H (79) (31) The p o r p h y r i n i s o l a t e d f r om t h e H a r d e r i a n g l a n d o f 51 . . . . . . r a t s and named, w i t h a p p r o p r i a t e s c i e n t i f i c i m a g i n a t i o n , h a r d e r o p o r p h y r i n (82) c o n t a i n s v i n y l and p r o p i o n i c a c i d groups i n t h e 2- and 4- p o s i t i o n s . The p o r p h y r i n o g e n o f (82) p r o t o p o r p h y r i n IX and i s c l e a r l y s t r u c t u r a l l y s i m i l a r t o m o n o a c r y l i c a c i d m o n o v i n y l d e u t e r o p o r p h y r i n IX DTBE ( 4 8 ) . I t s h o u l d t h e o r e t i c a l l y , be formed by s e l e c t i v e r e d u c t i o n o f t h e c a r b o x y l a t e s u b s t i t u t e d v i n y l g r o u p , however, t h i s w o u l d be d i f f i c u l t t o e f f e c t due t o t h e r e a c t i v i t y o f t h e unsub-s t i t u t e d v i n y l g r o u p . A more r e a s o n a b l e a p p r o a c h w o u l d be t o p r o t e c t t h e v i n y l group by a t e r m i n a l o x i d a t i o n w i t h 112 t h a l l i u m ( I I I ) n i t r a t e , r e d u c e t h e a c r y l i c a c i d and t h e n r e g e n e r a t e t h e v i n y l group by c o n v e r s i o n o f t h e a l c o h o l t o t h e c h l o r o e t h y l s u b s t i t u e n t f o l l o w e d by base c a t a l y s e d e l i m i n a t i o n o f i t s z i n c ( I I ) complex. A l t e r n a t i v e l y , t h e r e d u c t i o n o f t h e a c r y l a t e group on the m o n o a c r y l a t e monoformyl p o r p h y r i n (77) would y i e l d t h e monoformyl m o n o p r o p i o n i c a c i d p o r p h y r i n (83) w h i c h s h o u l d has been p r o p o s e d 111 as an i n t e r m e d i a t e i n t h e f o r m a t i o n o f OH (82) f o rm h a r d e r o p o r p h y r i n (82) by W i t t i g r e a c t i o n w i t h t h e a p p r o p r i a t e y l i d . (7 3) The m o n o a c r y l i c a c i d m o n o p r o p i o n i c a c i d d e u t e r o p o r p h y r i n IX ( 7 9 ) , i n v o k e d as an i n t e r m e d i a t e i n some o f t h e s e t r a n s -f o r m a t i o n s i s i t s e l f a n a t u r a l p o r p h y r i n . I t has been i s o l a t e d from meconium (the f i r s t f a e c e s o f a baby) i n 52 r e l a t i v e l y l a r g e q u a n t i t i e s and named ' p o r p h y r i n S411' The i n i t i a l s t r u c t u r a l d e s i g n a t i o n was a s t h e 2 - c i s a c r y l i c a c i d compound but subsequent model,, s t u d i e s and a s t e p -w i s e s y n t h e s i s show t h i s t o be i n c o r r e c t and t h e t r a n s 93a c o n f i g u r a t i o n has been c o n f i r m e d *\ The r o l e t h a t t h e p o r -p h y r i n o g e n p l a y s i n t h e b i o s y n t h e t i c pathway i s an e n i g m a t i c ] 09 one s i n c e , by l a b e l l i n g e x p e r i m e n t s , J a c k s o n , has shown i t n o t t o be an i n t e r m e d i a t e i n t h e f o r m a t i o n o f p r o t o -p o r p h y r i n IX. However t h e l a r g e q u a n t i t i e s o f t h e p o r p h y r i n i n meconium s u g g e s t , p o s s i b l y , an a l t e r n a t i v e pathway. In summary mono a c r y l i c a c i d m o n o v i n y l d e u t e r o p o r p h y r i n IX (48) and, more p a r t i c u l a r l y , i t s m o n o f o r m y l m o n o a c r y l i c a c i d d e r i v a t i v e (77) p r o v i d e a p o t e n t i a l l y r a p i d s y n t h e t i c r o u t e t o a number o f p r o p o s e d m e t a b o l i c i n t e r m e d i a t e s i n t h e c o n v e r s i o n o f c o p r o p o r p h y r i n o g e n I I I t o p r o t o p o r p h y r i n o g e n IX. The f a c i l i t y o f s p e c i f i c i s o t o p i c l a b e l l i n g i n t h e s e , a d m i t t e d l y s p e c u l a t i v e , s y n t h e t i c s e q u e n c e s may sh e d l i g h t , t h r o u g h i n c o r p o r a t i o n e x p e r i m e n t s , on a p a r t i c u l a r l y murky a r e a o f t h e b i o s y n t h e t i c pathway. 94 5. EXPERIMENTAL S p e c t r o s c o p i c A l l i n f r a r e d s p e c t r a were r e c o r d e d on a P e r k i n Elmer 457 s p e c t r o p h o t o m e t e r . They were t a k e n i n c h l o r o f o r m s o l u t i o n and c a l i b r a t e d r e l a t i v e t o t h e 1601 cm 1 band o f p o l y s t y r e n e . U n l e s s o t h e r w i s e s t a t e d a i l n.m.r. s p e c t r a were t a k e n i n d e u t e r i o c h l o r o f o r m on e i t h e r a V a r i a n model T-60 o r XL100. T e t r a m e t h y l s i l a n e was used as an i n t e r n a l s t a n d a r d and a l l c h e m i c a l s h i f t s a r e r e p o r t e d on t h e 6 s c a l e . The UV-Vis s p e c t r a were r e c o r d e d on a Cary 17 r e c o r d i n g s p e c t r o p h o t o m e t e r . The e x t i n c t i o n c o e f f i c i e n t s f o r a l l new chromophoric systems were, where q u a n t i t y p e r m i t t e d , measured. For o t h e r known chromophores t h e r a t i o s o f t h e v i s i b l e a b s o r -bances are g i v e n i n p a r e n t h e s e s . The s p e c t r a were g e n e r a l l y run i n CHCl^ and a b s o r p t i o n s a r e g i v e n i n nm. The mass s p e c t r a were r e c o r d e d on an A t l a s CH-4b. The i m p o r t a n t masses a r e r e p o r t e d and t h e r e l a t i v e i n t e n s i t i e s measured r e l a t i v e t o t h e m o l e c u l a r i o n . Other S i l i c a g e l was o b t a i n e d from ICN P h a r m a c e u t i c a l s I n c . , the c o r r e c t a c t i v i t y was p r e p a r e d as per i n s t r u c t i o n s . T h i n l a y e r chromatography was c a r r i e d o u t w i t h p r e c o a t e d (250 m i c r o n s ) s i l i c a g e l GF p l a t e s from A n a l T e c h I n c . The p r e p a r a t i v e l a y e r p l a t e s were from the same s o u r c e and were s i l i c a g e l GF (2000 m i c r o n s ) . 94a The a g a r o s e s u p p o r t was o b t a i n e d f r o m P h a r m a c i a F i n e C h e m i c a l s a s S e p h a r o s e 2B. The s o l v e n t s e m p l o y e d w e r e e i t h e r r e a g e n t g r a d e , p a r t i c u l a r l y f o r t h e l a r g e s c a l e w o r k , o r c e r t i f i e d o r ' s p e c t r o ' g r a d e f o r t h e s m a l l s c a l e w o r k . H e m a t o p o r p h y r i n I X was o b t a i n e d f r o m N u t r i t i o n a l B i o c h e m i c a l s . O r; P r o t o p o r p h y r i n IX (23) Under an a t m o s p h e r e o f n i t r o g e n , d i m e t h y l f o r m a m i d e 3 (2000 cm', r e a g e n t g r a d e ) was b r o u g h t t o b o i l i n g i n an e r l e n m e y e r f l a s k on a h o t p l a t e . H e m a t o p o r p h y r i n IX d i h y d r o -c h l o r i d e (25 g; 0.042 m o l e s ) , i n a s m a l l b e a k e r , was d r o p p e d i n t o t h e s o l v e n t and s t i r r e d v i g o r o u s l y f o r 60 s e c o n d s . The d a r k r e d s o l u t i o n was i m m e d i a t e l y c o o l e d i n an i c e b a t h and t h e n , a f t e r t r a n s f e r t o a r o u n d b o t t o m e d f l a s k , e v a p o r a t e d t o d r y n e s s on a m e c h a n i c a l l y pumped r o t a r y e v a p o r a t o r . The r e d s o l i d was d i s s o l v e d i n a minimum o f f o r m i c a c i d and p r e -c i p i t a t e d w i t h e t h e r . The p r e c i p i t a t e was f i l t e r e d u n d e r vacuum and washed w i t h e t h e r and w a t e r . A f t e r a i r d r y i n g f o r two h o u r s t h e s o l i d was d e s i c c a t e d u n d e r vacuum o v e r n i g h t ( y i e l d : 23.5 g; 95%) . T . l . c . on s i l i c a g e l w i t h b e n z e n e - m e t h a n o l - f o r m i c a c i d (11:1.7:0.2) showed a s i n g l e c l e a n p r o d u c t . The p u r i t y o f e a c h c o m m e r c i a l sample o f h e m a t o p o r p h y r i n IX was c h e c k e d by t . l . c . i n b e n z e n e : m e t h a n o l : f o r m i c a c i d ( 1 1 : 3 : 0 . 1 ) . N.M.R.: ( i n p y r i d i n e d 5 ) ; 3.36 (m, 16H), 4.40 ( t . 4H), 6.04 (dd, 4H), 8.14 (m. 2H), 10.02 (n, 4 H ) . S p e c t r a - page 131 P r o t o p o r p h y r i n IX D i t e r t b u t y l E s t e r (24b) P r o t o p o r p h y r i n IX (1.5 g; 2.5 mmoles) was s u s p e n d e d i n 3 ' d r y m e t h y l e n e c h l o r x d e (100cm ) and h e a t e d t o r e f l u x u n d e r 3 a r g o n on a s t i r r i n g h o t p l a t e . O x a l y l c h l o r i d e (15cm ) was V. 96 c a r e f u l l y added t o t h e s o l u t i o n w i t h s t i r r i n g . The r e a c t i o n m i x t u r e was a l l o w e d t o r e f l u x f o r 10 m i n u t e s and t h e n e v a p o r -a t e d t o d r y n e s s i n a fume hood. F u r t h e r m e t h y l e n e c h l o r i d e 3 (50cm ) was a d d e d and t h e n e v a p o r a t e d t o d r y n e s s . The r e s u l t -3 i n g , g r e e n s o l i d was r e d i s s o l v e d i n d r y CI-J^ CI-p (100cm ), b r o u g h t t o r e f l u x and d r y t e r t i a r y b u t a n o l (10cm J) was a d d e d . The m i x t u r e was a l l o w e d t o r e f l u x f o r 45 m i n u t e s . A t t h e 3 end o f t h i s t i m e more t - b u t a n o l (10cm ) was added and r e f l u x c o n t i n u e d f o r a f u r t h e r 4 5 m i n u t e s . The work up o f t h e d a r k r e d s o l i d i n v o l v e d w a s h i n g w i t h s o d i u m b i c a r b o n a t e s o l u t i o n ( 5 % ) , u n t i l t h e a c i d was n e u t r a i i s f o l l o w e d by e x t r a c t i o n w i t h w a t e r . The o r g a n i c p h a s e was d r i e d w i t h s o d i u m s u l p h a t e and t a k e n t o d r y n e s s . P u r i f i c a t i o n was c a r r i e d o u t on a s i l i c a g e l (250 g; g r a d e IV) c o l u m n u s i n g C H C l 3 : E t 2 0 (100:1) as t h e e l u e n t and r e c r y s t a l l i s a t i o n f r o m b e nzene y i e l d e d 1.15 g (76.7% b a s e d on t h e i n i t i a l amount o f p r o t o p o r p h y r i n IX) o f p r o t o p o r p h y r i n IX d i t e r t i a r y b u t y l e s t e r . v i s : 504, 539, 576, 631 ( R a t i o s ; 2.7:2.2:1.3:1) E t i o - t y p e . I.R.: 3320 (N-H), 1720 ( e s t e r c a r b o n y l ) , 990 ( v i n y l ) . N.M.R.:. 1.35 ( s , 18H, tBu) , 3.14 ( t , 4H,i3 p r o p i o n y 1-CH 2) 3.60 (d, 12H,-CH 3), 4.32 ( t , 4H,a p r o p i o n y l C H 2 ) , 6.20 (dd, 4H, v i n y l CH ) , 8.24 (dd, 2H, v i n y l CH), 10.04 (m, 4H, m e t h i n e 97 P i m e l i c A c i d D.ibenzyl E s t e r (28a) 3 P i m e l i c a c i d (5-3 g) was d i s s o l v e d i n CE^CJ^ (100cm ) 3 and t h i o n y l c h l o r i d e (45cm ) was added s l o w l y . The mixture was r e f l u x e d on an o i l bath f o r 2 hours and then evaporated 3 to dryness. More CH 2C1 2 (50cm~) was added and a f t e r evapora-3 t i o n the o l i v e green o i l was d i s s o l v e d i n CH„C1 (100cm ) ^* z 3 and benzyl a l c o h o l (50cm ) was added; the mixture was r e f l u x e d f o r 5 hours. At the end of t h i s time the s o l u t i o n was wash-ed w i t h water, NaHC0 3 s o l u t i o n (5%) and water and d r i e d over magnesium sulphate. Rotary evaporation of the methylene c h l o r i d e l e f t a bright-orange s o l u t i o n which v;as d i s t i l l e d under vacuum using a short path condenser. The main f r a c t i o n was taken at 208-213°C (pressure = 1.0 mmHg). Y i e l d = 8.02 g; 71.2% based on the p i m e l i c a c i d . N.M.R.: 1.50 (m, 6H), 2.25 ( t , 4H), 5.01 (s, 4H), '7.30 (s, 10H) . Spectrum - page 133 P i m e l i c Acid Monobenzyl E s t e r (29) P a l l a d i s e d carbon (0.3 g) v/as t r a n s f e r r e d to a 3 necked 3 3 500cm :round bottomed f l a s k , ethanol (150cm ) was c a r e f u l l y add-ed and the f l a s k was attached to a hydrogenation apparatus. The system was flushed w i t h hydrogen and d i b e n z y l pimelate (3.97 g:0.0117 moles) was introduced v i a a septum. A f t e r hydro-gen (262. l c i n J ,0. 0117 moles) had been taken up over a p e r i o d of 5 hours the p a l l a d i s e d carbon was f i l t e r e d o f f and the solution. e v a p o r a t e d t o d r y n e s s . The r e s u l t a n t y e l l o w o i l was d i s -3 s o l v e d i n NaHCO^ s o l u t i o n (5%:50cm") and t h e c l o u d y s o l u t i o n washed w i t h e t h e r t o remove t h e u n r e a c t e d d i b e n z y l p i m e l a t e . The aqueous p h a s e was n e u t r a l i s e d w i t h h y d r o c h l o r i c a c i d (10%) and t h e n e x t r a c t e d t w i c e w i t h c h l o r o f o r m t o remove t h e mo n o b e n z y l p i m e l a t e f r o m p i m e l i c a c i d . The c h l o r o f o r m l a y e r was d r i e d w i t h MgSO^ and e v a p o r a t e d t o y i e l d p i m e l i c a c i d m o n o b e n z y l e s t e r (1.60 g ; 5 5 % ) . N.M.R.: 1.50 (m, 6H), 2.32 (m, 4H), 5.09 ( s , 2H), 7.35 ( s , 5H), 10.80 ( s , I H ) . S p e c t r u m - page 133 P i m e l i c a c i d m o n o b e n z y l e s t e r mono N - m e t h y l a n i l i d e (30) P i m e l i c a c i d m o n o b e n z y l e s t e r (2g) was d i s s o l v e d i n ' 3 t h i o n y l c h l o r i d e (5cm ) and h e a t e d f o r one h o u r a t 60°C. E x c e s s t h i o n y l c h l o r i d e was removed b y d i s t i l l a t i o n a nd t h e s u l t a n t o i l d i s s o l v e d i n e t h e r . A s o l u t i o n o f N - m e t h y l 3 3 a n i l i n e (10cm ) i n e t h e r (50cm ) was adde d and t h e m i x t u r e a l l o w e d t o s t i r a t room t e m p e r a t u r e o v e r n i g h t . The m i x t u r e was t h e n washed w i t h h y d r o c h l o r i c a c i d (10%) and w a t e r and d r i e d o v e r MgSO^. A f t e r f i l t r a t i o n t h e e t h e r was e v a p o r a t e d o f f t o y i e l d a p a l e s t r a w c o l o u r e d o i l ( y i e l d = 2.45 g; 81.4% b a s e d on p i m e l i c a c i d m o n o b e n z y l e s t e r ) . N.M.R.: 1.50 (m, 6H), 2.20 (m, 4H), 3.23 ( s , 3H), 5.08 (s , 2H), 7.36 ( s , 10H). 99 Z i n c ( I I ) P r o t o p o r p h y r i n IX d i t e r t i a r y b u t y l e s t e r (31a) P r o t o p o r p h y r i n IX DTBE (0.15 g; 0.23 nunoles) was d i s s o l v e d 3 i n DMF (25cm. ) and b r o u g h t t o r e f l u x u n d e r a r g o n f o r 30 m i n u t e s . Z i n c a c e t a t e (0.21 g; 5 e q u i v a l e n t s ) was add e d t o t h e s o l u -t i o n . M e t a l l a t i o n was f o l l o w e d by v i s i b l e s p e c t r o s c o p y and shown t o be a l m o s t i n s t a n t a n e o u s . A f t e r r e f l u x i n g f o r one m i n u t e t h e h e a t was removed and t h e s o l u t i o n a l l o w e d t o c o o l . 3 The r e a c t i o n m i x t u r e was t h e n d i l u t e d w i t h C H C l ^ (3 0cm ) and washed f o u r t i m e s w i t h w a t e r . A f t e r d r y i n g w i t h a n h y d r o u s magnesium s u l p h a t e and e v a p o r a t i o n t o d r y n e s s , t h e w i n e r e d s o l i d was r e c r y s t a l l i s e d f r o m c h l o r o f o r m - m e t h a n o l (1:1,-4cm 3) y i e l d i n g d a r k r e d c o l o u r e d c r y s t a l s ( y i e l d = 0.13 g : S 4 . 0 % ) . v i s : 584, 547 ( R a t i o ; 1 . 0 4 : 1 ) . A t t e m p t e d V i l s m e i e r f o r m y l a t i o n w i t h Zn ( I I )  p r o t o p o r p h y r i n IX DTBE P i m e l i c a c i d m o n o b e n z y l e s t e r monomethyl a n i l i d e (0.3g; 1.0 mmoles) was c o o l e d i n i c e and b e n z o y l c h l o r i d e (0.17 5g; 1.25 mmoles) was adde d d r o p w i s e . The m i x t u r e was a l l o w e d t o s t a n d a t room t e m p e r a t u r e f o r 30 m i n u t e s d u r i n g w h i c h t i m e t h e d a r k r e d c o l o u r o f t h e V i l s m e i e r c o m p l e x was f o r m e d . Z i n c ( I I ) p r o t o p o r p h y r i n IX DTBE (0.02g; 30 pinoles) i n C H 2 C 1 2 3 (15cm ) :was add e d d r o p w i s e o v e r a p e r i o d o f 15 m i n u t e s w i t h c o n s t a n t s t i r r i n g . The t e m p e r a t u r e was r a i s e d t o 50°C and m a i n t a i n e d t h e r e f o r 8 h o u r s . .100 A t t h e end o f t h i s t i m e a s a t u r a t e d s o l u t i o n o f s o d i u m 3 a c e t a t e was added (15cm ) "and t h e s o l u t i o n warmed, w i t h s t i r r i n g , f o r 6 h o u r s . The aqueous l a y e r was removed and 3 e x t r a c t e d t w i c e w i t h e t h e r (20cm ) w h i c h was added t o t h e o r g a n i c p h a s e . T h i s p h a s e was washed w i t h w a t e r , . d r i e d o v e r MgS0 4 and e v a p o r a t e d t o d r y n e s s . B o t h t h e v i s i b l e s p e c t r u m and t . l . c . showed t h a t no r e a c t i o n had o c c u r r e d . C o b a l t ( I I ) p r o t o p o r p h y r i n IX d i t e r t i a r y b u t y l e s t e r (31b) P r o t o p o r p h y r i n IX DTBE (0.2g; 0.31 mmoles) was d i s s o l v e d 3 i n C H C l ^ (12cm ) and b r o u g h t t o r e f l u x u n d e r a r g o n . W i t h v i g o r o u s s t i r r i n g a b o i l i n g s a t u r a t e d s o l u t i o n o f c o b a l t 3 a c e t a t e (1cm ) was added and a l l o w e d t o r e f l u x f o r one m i n u t e . 3 The r e a c t i o n m i x t u r e was d i l u t e d w i t h C H C l ^ (2 00cm ) and washed f o u r t i m e s w i t h w a t e r . The d a r k r e d / m a r o o n s o l u t i o n was d r i e d w i t h Na^SO^ and e v a p o r a t e d t o d r y n e s s . The s o l i d was d i s s o l v e d i n a minimum o f C H C l ^ and c h r o m a t o g r a p h e d on s i l i c a g e l w i t h c h l o r o f o r m as t h e e l u e n t ( y i e l d = 46 g, 7 3 % ) . v i s : 560, 526 ( R a t i o : 1 . 8 8 : 1 ) . S p e c t r u m - page 13 4 101' ? ( ° r 4) M o n o f o r m y l 4 ( o r 2) M o n o v i n y l D e u t e r o p r o p h y r i n I X  DTBE (40 and 43) by P o t a s s i u m P e r m a n g a n a t e O x i d a t i o n P r o t o p o r p h y r i n I X DTBE (0.2 g; 0.31 mmoles) was d i s s o l v e d 3 i n a c e t o n e (8 00cm ) and b r o u g h t t o r e f l u x . A s o l u t i o n o f 3 p o t a s s i u m p e r m a n g a n t e (0.2 g) i n a c e t o n e (50 cm') was a d d e d d r o p w i s e t o t h e b o i l i n g s o l u t i o n o v e r a p e r i o d o f 4 5 m i n u t e s . A t t h e end o f t h i s t i m e t h e s o l u t i o n was f i l t e r e d t o r e m o v e 3 t h e manganese d i o x i d e a nd d i l u t e d w i t h C H C l ^ (4 00cm ) . A f t e r w a s h i n g t h r e e t i m e s w i t h w a t e r , t h e w a s h i n g s w e r e e x t r a c t e d w i t h more C H C l ^ and t h e o r g a n i c e x t r a c t s w e r e c o m b i n e d . D r y i n g was e f f e c t e d by a n h y d r o u s s o d i u m s u l p h a t e a n d t h e s o l u t i o n was e v a p o r a t e d t o d r y n e s s . The r e s u l t i n g r e d / b r o w n s o l i d was d i s s o l v e d i n a minimum, o f c h l o r o f o r m and c h r o m a t o g r a p h e d o n s i l i c a g e l (2x.l00g) t o a c h i e v e a s a t i s f a c t o r y s e p a r a t i o n . R e c r y s t a l l i s a t i o n was c a r r i e d o u t f r o m c h l o r o f o r m -m e t h a n o l t o g i v e m aroon c r y s t a l s o f 2 ( o r 4 ) - m o n o f o r m y l 4 - ( o r 2) m o n o v i n y l d e u t e r o p o r p h y r i n I X DTBE ( y i e l d = 0.028 g; 14%) a n d t h e r e d / b r o w n c r y s t a l s o f 2,4 d i f o r m y l d e u t e r o p o r -p h y r i n I X DTBE ( y i e l d = 0.022 g; 1 1 % ) . 2 ( a n d 4 ) - m o n o f o r m y l ; 4 (and 2 ) - m o n o v i n y l d e u t e r o p o r p h y r i n I X DTBE. v i s : 640, 582, 559, 515 ( R a t i o s ; 1:4.1:4.9:4.8) r h o d o -t y p e . I . R . : 3410 (N-H), 1720 ( e s t e r ) , 1 6 6 3 > ( a l d e h y d e ) , 2,4 d i f o r m y l d e u t e r o p o r p h y r i n I X DTBE. v i s : 648, 592, 561, 525 ( R a t i o s ; 1:1.5:2.0:2.9), e t i o - t y p e . 102 2 (or 4) P h o t o p r o t o p o r p h y r i n IX D i t e r t i a r y b u t y l e s t e r (44b o r 45 b) P r o t o p o r p h y r i n IX DTBE (3 g; 0.4 5 mmoles) was d i s s o l v e d 3 3 xn d x c h l o r o m e t h a n e (2200cm ) and p y r i d i n e (200cm"), and o x y g e n b u b b l e d t h r o u g h t h e s o l u t i o n . P h o t o l y s i s o f t h e r e s u l t a n t s o l u t i o n was c a r r i e d o u t w i t h a 450 w a t t m e r c u r y lamp i n a w a t e r c o o l e d p y r e x p h o t o l y s i s a p p a r a t u s . The r e a c t i o n was c h e c k e d p e r i o d i c a l l y by t . l . c . (CHC1 0 :MeOI-1, 5 0 : 1 ) , A t t h e end o f 8 h o u r s t h e s o l u t i o n was c o n c e n t r a t e d , washed t h r e e t i m e s w i t h w a t e r t o remove p y r i d i n e and f i n a l l y e v a p o r a -t e d t o d r y n e s s on an m e c h a n i c a l l y pumped r o t a r y e v a p o r a t o r . The d a r k g r e e n s o l i d was d i s s o l v e d i n a minimum o f C H C l ^ and c h r o m a t o g r a p h e d on s i l i c a g e l ( g r a d e IV) w i t h d i c h l o r o -m e t h a n e - e t h e r (20:1) as t h e e l u e n t . I n i t i a l l y a r e d band o f u n r e a c t e d p r o t o p o r p h y r i n IX DTBE (0.51 g; 17%) was t a k e n o f f , f o l l o w e d by t h e main g r e e n p r o d u c t . T w e l v e f r a c t i o n s o f 3 500cm were t a k e n o f f and c h e c k e d by t . l . c . ( C H 2 C 1 2 : E t 2 0 , 1 0 : 1 ) . T h i s showed a smooth t r a n s i t i o n f r o m t h e 4 p h o t o p r o t o -p o r o h y r i n d e r i v a t i v e t o t h e 2 i s o m e r . The f i r s t f o u r f r a c -t i o n s were com b i n e d t o g i v e 4 p h o t o p r o t o p o r p h y r i n IX DTBE (0.60 g; 2 0 % ) , t h e l a s t f o u r g a v e p u r e 2 p h o t o p r o t o p o r p h y r i n DTBE (0.47 g; 16%) and t h e m i d d l e f o u r f r a c t i o n s were a m i x -t u r e o f t h e two i s o m e r s (0.44.g; 1 5 % ) . Any t r a c e o f t h e o p p o s i t e i s o m e r c o u l d be removed f r o m t h e p u r e compounds by c r y s t a l l i s a t i o n f r o m c h l o r o f o r m - m e t h a n o l . 103 4 - P h o t o p r o t o p o r p h y r i r i I X DTBE v i s : 672, 613, 575 ( R a t i o s ; 1 : 0 . 3 3 : 0 . 3 6 ) . N.M.R.: 1.22 ( s , 3H, a l i p h a t i c C H 3 ) , 1.38 ( s , .1811, t B u ) , 3.15 (m, 4H, 3 p r o p i o n y l C H 9 ) , 3.28, 2.32, 3.50 ( 3 s , 9H, a r o m a t i c C K 3 ) , 4.16 (m, 4 K ) , a p r o p i o n y l - C H 2 ) , 6.55 ( d , I H , o l e f i n i c C H ) , 6.03 (m, 2H, v i n y l - C H 2 ) , 7.48 (m, I H , v i n y l -CH) , 7.70, 8.42, 9.52, 9.74 ( 4 s , 4H, m e t h i n e ) , 9.98 (d , I H , a l d e h y d e ) . 2 - P h o t o p r o t o p o r p h y r i n I X DTBE v i s : 6 70, 6 1 1 , 573 ( R a t i o s ; .1: 0. 32 : 0 . 3 8 ) . N.M.R.: 1.19 ( s , 3H, a l i p h a t i c C H 3 ) , 1.39 ( s , 18H, t B u ) , 3.12 (m, 4H, 3 p r o p i o n y l - C H 2 ) , 2.76, 3.28, 3.32 ( 3 s , 9M, a r o m a t i c C H 3 ) , 4.14 (m, 4H,, a p r o p i o n y l - C H 2 ) , 6.12 (m, 3H, v i n y l C H 2 and o l e f i n C H ) , 7.90 (m, I H , v i n y l - C H ) , 7.71, 8.32, 9.54, 9.66 ( 4 s , 4H, m e t h i n e ) . 2 ( o r 4 ) - M o n o f o r m y l 4 ( o r 2 ) - M o n o v i n y l D e u t e r o p o r p h y r i n I X  D i t e r t i a r y B u t y l E s t e r (4 0 o r 43) P h o t o p r o t o p o r p h y r i n I X DTBE (0.25 g; 0.38 mmoles o f e i t h e r t h e 2 o r 4 i s o m e r o r t h e m i x e d i s o m e r s ) was d i s s o l v e d 3 m d r y m e t h y l e n e c h l o r i d e (125cm ) and s o d i u m b o r o h y d r i d e 3 (0.25 g) i n d r y m e t h a n o l (8cm ) was a d d e d . The s o l u t i o n was a l l o w e d t o s t i r a t room t e m p e r a t u r e . A l m o s t i m m e d i a t e l y a s h i f t i n t h e m a j o r p e a k f r o m 67 0 nm t o 6 62 t o o k p d a c e . A t t h e e n d o f one h o u r s u f f i c i e n t a c e t i c a c i d was a d d e d t o d e s t r o y any-e x c e s s s o d i u m b o r o h y d r i d e . 104 The r e a c t i o n m i x t u r e was p o u r e d i n t o w a t e r (600cm ) , e x t r a c t e d w i t h c h l o r o f o r m a n d e v a p o r a t e d t o d r y n e s s . The d a r k brown s o l i d was c o l l e c t e d , d i s s o l v e d i n 1 , 4 - d i o x a n e 3 3 (100cm ) a n d s o d i u m p e r i o d a t e (0.5 g) i n w a t e r (8cm ) was a d d e d , f o l l o w e d i m m e d i a t e l y by c o n c e n t r a t e d s u l p h u r i c a c i d ( 0 . 6 c m ^ ) . T h i s s o l u t i o n was t h e n s t i r r e d a t room t e m p e r a t u r e f o r 5 h o u r s . The r e s u l t a n t p u r p l e s o l u t i o n was p o u r e d i n t o a d i l u t e 3 s o d i u m c h l o r i d e s o l u t i o n (600cm ) a n d e x t r a c t e d w i t h m e t h y l e n e 3 c h l o r i d e (100cm ) . The o r g a n i c p h a s e was w a s h e d w i t h NaHCO^ s o l u t i o n (5%) a n d w a t e r a nd t h e n e v a p o r a t e d t o d r y n e s s . The r e d s o l i d was d i s s o l v e d i n a minimum o f c h l o r o f o r m a n d c h r o m a -t o g r a p h e d on s i l i c a g e l ( c h l o r o f o r m - m e t h a n o l , 2 0 : 1 ) . The f i r s t r e d f r a c t i o n t o be r e m o v e d was m o n o f o r m y l m o n o v i n y l d e u t e r o p o r p h y r i n I X DTBE (0.148 g; 5 9 % ) . T h i s was f o l l o w e d b y a s l o w e r m o v i n g band, w i t h an e x a c t l y s i m i l a r r h o d o - t y p e s p e c t r u m w h i c h was p r e s u m e d t o be t h e m o n o d e e s t e r i f i e d mono-f o r m y l m o n o v i n y l d e u t e r o p o r p h y r i n I X (0.024 g; 1 0 % ) . The m o n o f o r m y l m o n o v i n y l d e u t e r o p o r p h y r i n I X DTBE c o u l d be r e c r y s t a l l i s e d f r o m CHCl 3/MeOH t o g i v e m a r o o n c r y s t a l s . 4 - f o r m y l 2 - v i n y l d e u t e r o p o r p h y r i n I X DTBE v i s : 6 43, 584, 560, 519 ( R a t i o s ; 1 : 2 . 6 : 4 . 1 : 3 . 1 ) , r h o d o -t y p e . N.M.R.: 1.36 ( s , 18H, t B u ) , 3.08, 3.38, 3.44, 3.50, 3.54 (m, 16H, r i n g C H 3 and 3 p r o p i o n y l CE^) , 4.1*8 (m, 4H, a p r o p i o n y l C H 2 ) , 6.08 (m, 2H, v i n y l - C H 2 ) , 7.86 ( d d , I H , v i n y l - C H ) , 9.38 105 9.60, 9.73, 10.41 (4s, 4H, m e t h i n e ) , 11.09 ( s , H i , a l d e h y d e ) . S p e c t r a - page 138 2 - f o r m y l , 4 - v i n y l d e u t e r o p o r p h y r i n IX DTBE v i s : 641, 583, 559, 518 ( R a t i o s : 1:3.4:5.2:3.7), r h o d o - t y p e . N.M.R.: 1.37 (d, 18H, t B u ) , 3.08, 3.41 ( t , s, 16H, 3 p r o p i o n y l - C H 2 and r i n g - C H ^ ) , 4.23 (m, 4H, a p r o p i o n y l - C H 2 ) , 6.23 (m, 2H, v i n y l - C H 2 ) , 8.04 (dd, IH, v i n y l - C H ) , 9.30, 9.67, 9.71, 10.15 (4s, 4H, m e t h i n e ) , 10.99 ( s , IH, a l d e h y d e ) . S p e c t r a - page 137 2 (and 4 ) - M o n o a c r y l i c A c i d 4 (and 2 ) - M o n o v i n y l  D e u t e r o p o r p h y r i n IX D i t e r t i a r y B u t y l E s t e r (43) 2 (and 4) m o n o f o r m y l 4 (and 2) m o n o v i n y l d e u t e r o p o r p h y r i n 3 IX DTBE (0.13 g; 0.2 mmoles) was d i s s o l v e d i n p y r i d i n e (100cm") and m a l o n i c acid. (2.5 g) was added. The m i x t u r e was s l o w l y 3 h e a t e d t o 50°C, w i t h s t i r r i n g , and p i p e r i d m e (0.2cm ). added. The t e m p e r a t u r e o f t h e r e a c t i o n m i x t u r e was t h e n r a i s e d t o 80°C and m a i n t a i n e d t h e r e f o r f o u r h o u r s . A f t e r c o o l i n g t h e d a r k r e d s o l u t i o n was p o u r e d i n t o d i l u t e h y d r o c h l o r i c a c i d (200cm 3; 0.001M) and t h i s p r o d u c e d a d a r k r e d / b r o w n p r e c i p i t a t e . The s u s p e n s i o n was e x t r a c t e d w i t h c h l o r o f o r m and t h e o r g a n i c p h a s e was washed w i t h w a t e r and e v a p o -r a t e d t o d r y n e s s . The s o l i d was d i s s o l v e d i n c h l o r o f o r m and c h r o m a t o g r a p h e d on a s i l i c a g e l p r e p a r a t i v e l a y e r p l a t e (2000 m i c r o n t h i c k n e s s ) w i t h c h l o r o f o r m - m e t h a n o l ' (20 :1) as t h e e l u e n t . 106 The s l o w m o v i n g band was removed f r o m t h e s i l i c a g e l w i t h a c h l o r o f o r m - m e t h a n o l - a c e t i c a c i d s o l u t i o n (10:1:0.5) t o y i e l d m o n o a c r y l i c a c i d m o n o v i n y l d e u t e r o p o r p h y r i n IX DTBE (0.105 g; 7 8 % ) . A f t e r a d d i t i o n o f t h e a c e t i c a c i d s o l u t i o n t h e s i l i c a g e l was r a p i d l y f i l t e r e d o f f s i n c e i t was f o u n d t h a t i f t h e d e l a y was t o o g r e a t t h e a c r y l i c a c i d p o r p h y r i n was c o n v e r t e d q u a n t i t a t i v e l y i n t o i t s z i n c ( I I ) d e r i v a t i v e . v i s : ( C H C 1 3 ) : 637, 580, 552,.512, 419 ( r a t i o s ; 1:2.32: 3.67:3.19:32.24) R h o d o - t y p e . ( C H C l 3 + p y r i d i n e ) , 634, 580, 547, 510, 413 ( R a t i o s ; 1:1.57:2.56:2.67) E t i o - t y p e . I.R.: 3300 (N-H), 1720 ( e s t e r c a r b o n y l ) , 1665 ( c a r b o x y l a t e c a r b o n y l ) , 1615 ( a g v i n y l g r o u p ) , 1153 ( e s t e r ) . N.M.R.: 1.36 ( s , 18H, t B u ) , 3.35 (m, 16H, 6 p r o p i o n y l -C H 2 and r i n g - C H . ^ ) , 4.17 (m, 4H, a p r o p i o n y l - C H ^ ) , 6.20 (dd, 2H, v i n y l CH ) , 6.85 (d, IH, a a c r y l a t e - C H , J = 17 Hz) 8.0 (m, IH, v i n y l - C H ) , 8.99 (d, IH B a c r y l a t e - C H , J = 17 H z ) , 9.24-9.80 (m, 4H, m e t h i n e ) . S p e c t r a - page 139 A R a p i d S y n t h e s i s o f 2 (and 4 ) - M o n o a c r y l i c A c i d 4 (and 2 ) - M o n o v i n y l D e u t e r o p o r p h y r i n IX DTBE f r o m P r o t o p o r p h y r i n IX P r o t o p o r p h y r i n IX (1.5 g; 0.23 mmoles) was s u s p e n d e d i n 3 d r y m e t h y l e n e c h l o r i d e (10 0cm ) and b r o u g h t t o r e f l u x . O x a l y l 3 c h l o r i d e (15cm ) was added u n d e r an a t m o s p h e r e o f a r g o n and t h e s o l u t i o s t i r r e d f o r 10 m i n u t e s , and t h e n e v a p o r a t e d t o d r y n e s s . More 3 d r y CH-C1„ (50cm ) was added and e v a p o r a t e d ^ The s o l i d was 107 r e d , d i s s o l v e d i n d r y CH 2C1 (lOOcm" 3), b r o u g h t t o r e f l u x , 3 d r y t e r t i a r y b u t a n o l (15cm ) added and r e f l u x e d f o r 4 0 3 m i n u t e s . A f t e r w h i c h more t - b u t a n o l (15cm ) was added and t h e s o l u t i o n r e f l u x e d f o r a f u r t h e r 4 0 m i n u t e s . The s o l u t i o n was c o o l e d , washed w i t h w a t e r , NaHCO^ (5%) and w a t e r and e v a p o r a t e d t o d r y n e s s . No f u r t h e r p u r i f i c a t i o n was a t t e m p t e d . The c o m p l e t e y i e l d o f p r o t o p o r p h y r i n IX DTBE was d i s -3 3 s o l v e d i n CH C l ^ (1100cm ) a n d p y r i d i n e (100cm ) and p h o t o -l y s e d , u n d e r a s t r e a m o f o x y g e n , f o r 8 h o u r s i n a p y r e x w a t e r c o o l e d v e s s e l . T h i s s o l u t i o n was t h e n washed w i t h w a t e r and e v a p o r a t e d t o d r y n e s s on a r o t a r y e v a p o r a t o r . The g r e e n / brown s o l i d was c h r o m a t o g r a p h e d on two p r e p a r a t i v e l a y e r p l a t e s (2000 m i c r o n s t h i c k ) e l u t i n g f i r s t l y w i t h C H 2 C l 2 : E t 2 0 (100:1) and t h e n C H 2 C l 2 : E t 2 0 (20:1) t o e f f e c t s e p a r a t i o n o f t h e u n r e a c t e d p r o t o p o r p h y r i n IX DTBE and p h o t o p r o t o p o r p h y r i n IX DTBE. The p h o t o p r o t o p o r p h y r i n was removed f r o m t h e s i l i c a g e l w i t h a CH 2C1 :MeOH s o l u t i o n . The r e s u l t i n g t o t a l y i e l d o f p h o t o p r o t o p o r p h y r i n IX was 3 d i s s o l v e d i n d r y C H 2 C 1 2 (500cm ), s o d i u m b o r o h y d r i d e (1 g) 3 i n m e t h a n o l (2 0cm ) was added and t h e s o l u t i o n was s t i r r e d a t room t e m p e r a t u r e f o r one h o u r . A c e t i c a c i d was t h e n added t o d e s t r o y e x c e s s b o r o h y d r i d e . The s o l u t i o n was p o u r e d 3 i n t o w a t e r (6 00cm ), e x t r a c t e d w i t h C H 2 C 1 2 and e v a p o r a t e d t o 3 d r y n e s s . The s o l i d was d i s s o l v e d i n 1 , 4 - d i o x a n (400cm ) and 3 s o d i u m p e r i o d a t e (2 g ) , f o l l o w e d by s u l p h u r i c a c i d (2.4cm ; c o n c ) , .108 was a d d e d . The m i x t u r e was s t i r r e d a t room t e m p e r a t u r e f o r 5 h o u r s , a f t e r w h i c h t h e s o l u t i o n was p o u r e d i n t o d i l u t e b r i n e , e x t r a c t e d w i t h C H C l ^ , w a s h e d w i t h w a t e r and e v a p o r a t e d t o d r y n e s s . The maroon . r e d s o l i d was c h r o m a t o g r a p h e d on one p r e p a r a t i v e l a y e r p l a t e (CHCl^:MeOH;20:1). The m o n o f o r m y l m o n o v i n y l d e u t e r o p o r p h y r i n I X DTBE t h u s 3 r e c o v e r e d was d i s s o l v e d i n p y r i d i n e (300cm ) , a n d m a l o n i c a c i d (10 g) a d d e d . The s o l u t i o n was h e a t e d t o 50°C, p i p e r i d i n e 3 (0.8cm ) a d d e d and t h e t e m p e r a t u r e r a i s e d t o 8 0°C f o r one h o u r ; a f t e r w h i c h r e f l u x was m a i n t a i n e d , f o r f o u r h o u r s . The 3 s o l u t i o n was c o o l e d , a d d e d t o d i l u t e HCI (1000cm ;0.001M) a n d e x t r a c t e d w i t h c h l o r o f o r m . The e x t r a c t was w a s h e d w i t h w a t e r , e v a p o r a t e d t o d r y n e s s a n d c h r o m a t o g r a p h e d on one s i l i c a g e l p r e p a r a t i v e l a y e r p l a t e (CHCl^rMeOH; 2 0 : 1 ) . The s l o w m o v i n g r e d / b r o w n b a n d was r e m o v e d w i t h a c h l o r o f o r m - m e t h a n o l - a c e t i c a c i d m i x t u r e ( 1 0 : 1 : 0 . 5 ) . The o v e r a l l y i e l d o f m o n o a c r y l i c a c i d m o n o v i n y l d e u t e r o -p o r p h y r i n I X DTBE f r o m p r o t o p o r p h y r i n I X was 0.45 g ( 3 0 % ) . 2 (and 4 ) - M o n o m e t h y l A c r y l a t e 4 (and 2 ) - M o n o v i n y l  D e u t e r o p o r p h y r i n I X D i t e r t i a r y B u t y l E s t e r (48) f r o m D i a z o m e t h a n e M o n o a c r y l i c a c i d m o n o v i n y l d e u t e r o p o r p h y r i n I X DTBE 3 (0.01 g; 0.02 mmoles) was d i s s o l v e d i n d i c h l o r o m e t h a n e (10cm ) 3 and an e t h e r e a l s o l u t i o n o f d i a z o m e t h a n e (2cm ) was a d d e d . The 109 s o l u t i o n was e v a p o r a t e d t o d r y n e s s and c h r o m a t o g r a p h e d on s i l i c a g e l (CHC1 3--methanol, 2 0 : 1 ) . The f a s t m o v i n g r e d band was removed and was f o u n d t o be m o n o m e t h y l a c r y l a t e m o n o v i n y l d e u t e r o p o r p h y r i n IX DTBE (9 mgs; 90%) . v i s : ( i n CHC1 , e x l 0 ~ 3 ) , 638 ( 4 . 2 9 ) , 580 ( 8 . 0 3 ) , 552 (1 3 . 3 2 ) , 513 ( 1 1 . 8 0 ) , 420 ( 1 3 9 . 5 9 ) . R h o d o - t y p e . I.R.: 3321 (N-H), 1723 ( b r o a d ; p r o p i o n i c and a c r y l a t e C=0) , 1630 (a, |3 a c r y l a t e v i n y l ) , 1165 ( b r o a d , p r o p i o n i c and a c r y l a t e e s t e r s ) . N.M.R.: 1.36 ( s , 18H, t B u ) , 3.13 (m, 4H, 3 p r o p i o n y l -C H 2 ) , 3.53, 3.58, 3.63 (3s, 12H, r i n g C H 3 ) , 4.06 ( s , 3H, -OCH 3), 4.28 (m, 4H, a p r o p i o n y l - C H 2 ) , 6.25 (m, 2H, v i n y l -C H 2 ) , 7.01 (d, IH, a a c r y l a t e - C H , J = 17 H z ) , 8.21 (m, IH, v i n y l - C H ) , 9.22 (d, IH, 3 a c r y l a t e - C H , J = 17 H z ) , 9.93, 9.99, •10.05 (3s, 4H, m e t h i n e ) . Mass S p e c t r u m (Low R e s o l u t i o n ) : 734 ( 1 6 ) , 733 ( 5 6 ) , 732 (1 0 0 ) , 678 ( 3 ) , 677 ( 6 ) , 676 ( 1 6 ) , 622 ( 3 ) , 523 ( 9 ) , 624 (19) . S p e c t r a - page 140 2 (and 4)-Monomethyl A c r y l a t e 4 (and 2 ) - M o n o v i n y l D e u t e r o -p o r p h y r i n IX D i t e r t i a r y B u t y l E s t e r f r o m t h e A c i d C h l o r i d e M o n o a c r y l i c a c i d m o n o v i n y l d e u t e r o p o r p h y r i n IX DTBE (0.01 g; 20 pinoles) was d i s s o l v e d i n d r y m e t h y l e n e c h l o r i d e 3 3 (.10 cm ) and o x a l y l c h l o r i d e (2cm ) a d d e d . » The s o l u t i o n was 110 s t i r r e d at room temperature for 10 minutes and then evaporated to dryness. A green/red s o l i d was c o l l e c t e d and dissolved i n 3 3 more dry CH 2C1 2 (10cm ) and dry methanol (5cm ) was added. The s o l u t i o n was then washed with NaHC03 (5%) and water, evaporated to dryness and chromatographed on s i l i c a gel (CHCl^: MeOH, 10:1) to y i e l d monomethyl acrylate monovinyl deutero-porphyrin IX DTBE (9 mg:90%). The spectral data were found to be the same as that of the methyl acrylate porphyrin from diazomethane. 2 (and 4)-Monovinyl 4 (and 2)-Mono 3-amino 1-amido Propane  A c r y l a t e Deuteroporphyrin IX D i t e r t i a r y Butyl Ester (50a) Monoacrylic acid monovinyl deuteroporphyrin IX DTBE 3 (0.1 g; 0.15 mmoles) was dissolved i n dry CH 2C1 2 (25cm ) and 3 o x a l y l c h l o r i d e (2cm ) was added. The solution was s t i r r e d at room temperature for two minutes and then pumped to dryness on a water pump. The green s o l i d was dissolved i n dry CH 2C1 2 3 3 (25cm ) and propane 1,3 diamine (5cm ) was added. The colour reverted to porphyrin red with p r e c i p i t a t i o n of propane diamine hydrochloride. The mixture was s t i r r e d at room temperature for two hours and then washed three times with water followed by evaporation to dryness. The red s o l i d was dissolved i n a minimum of chloroform and chromatographed on s i l i c a g el (CHCl^:methanol, 20:1). The fast-moving band was removed with a chloroform-methanol solution I l l t o y i e l d t h e m o n o v i n y l mono 3-amino l - a m i d o p r o p a n e a c r y l a t e d e u t e r o p o r p h y r i n I X DTBE (31 mg:31%). v i s : ( i n C H 2 C 1 2 , e x l 0 ~ 3 ) : 640 ( 4 . 5 2 ) , 582 ( 7 . 8 3 ) , 552 ( 1 0 . 6 5 ) , 512 ( 1 0 . 9 7 ) , 405 ( 1 1 9 . 7 2 ) , r h o d o / e t . i o - t y p e . I . R . : 3320 (N-H), 1724 ( p r o p i o n y l e s t e r C = 0 ) , 1655 (amide C=0), 1614 (ct3 v i n y l ) , 1155 ( p r o p i o n y l e s t e r ) . N.M.R.: 1.37 ( s , t B u , and c e n t r a l a l k y l CH ) , 2.70 •* 3.70 ( b r o a d M, 3 p r o p i o n y l - C H ^ r r i n g CH^, t e r m i n a l a . l k y l - C H 2 ) , 4.20 (m, a p r o p i o n y l - N H 2 ) , 5.60 -> 6.20 (m, v i n y l C H 2 amide-NH) , 7.00 ( d , c t a c r y l a t e CH) , 7.64 (rn, v i n y l - C H ) , 8.34 ( d , a c r y l a t e - C H ) , 8.92 -> 9.41 (m, m e t h i n e ) . S p e c t r a - p age 141 P h o t o p r o t o p o r p h y r i n D e r i v a t i v e o f M o n o v i n y l Mono 3-Am.ino, 1-Am.ido P r o p a n e A c r l y a t e D e u t e r o p o r p h y r i n I X D i t e r t i a r y B u t y l E s t e r (57) 3-Amino l - a m i d o p r o p a n e a c r y l a t e d e u t e r o p o r p h y r i n I X DTBE (3 mg) was d i s s o l v e d i n c h l o r o f o r m a n d i r r a d i a t e d [ w i t h a d e s k lamp f o r 2 4 h o u r s . The r e a c t i o n was c h e c k e d by t . l . c . a nd t h e f o r m a t i o n o f t h e new g r e e n c h r o m o p h o r e was m o n i t o r e d by i t s v i s i b l e s p e c t r u m . The p r o d u c t s w e r e c h r o m a t o g r a p h e d on s i l i c a g e l ( C H C l ^ M e O K , 2 0 : 1 ) . The g r e e n p h o t o p r o t o -p o r p h y r i n d e r i v a t i v e r a n s l i g h t l y s l o w e r t h a n t h e p a r e n t a m i n o p o r p h y r i n a n d was r e m o v e d f r o m t h e s i l i c a g e l w i t h a c h l o r o -f o r m - m e t h a n o l m i x t u r e . 112 v i s : 680, 578, 553, 505 ( R a t i o s : 1:0.8:0.85:1.02). Mass S p e c t r u m (Low R e s . ) : 808 ( 2 5 ) , 807 (33) 806 ( 1 0 0 ) , 750 ( 3 3 ) , 749 ( 5 0 ) , 748 ( 1 0 0 ) , 747 ( 7 5 ) . S p e c t r u m - page 14 4 M o n o v i n y l Mono 6-Amino 1-Amido Hexane A c r y l a t e  D e u t e r o p o r p h y r i n IX D i t e r t i a r y B u t y l E s t e r (5 0b) M o n o v i n y l m o n o a c r y l i c a c i d d e u t e r o p o r p h y r i n IX DTBE (0.02 g; 0.03 mmoles) was d i s s o l v e d i n C ^ C l ^ and o x a l y l c h l o r i d e 3 (lcm ) added. The s o l u t i o n was s t i r r e d f o r two m i n u t e s and pumped t o d r y n e s s . A s o l u t i o n o f h e x a n e 1 , 6 - d i a m i n e (0.1 g) i n b e n z e n e (5cm 3) was added and a l l o w e d t o s t i r f o r 2 h o u r s . The d a r k r e d s o l u t i o n was washed w i t h w a t e r , pumped -t o d r y n e s s and t h e n p u r i f i e d by s i l i c a g e l p r e p a r a t i v e l a y e r c h r o m a t o g r a p h y w i t h C H C l ^ - m e t h a n o l - t r i e t h y l a m i n e (20:1:0.5)= The r e d / b r o w n band was removed f r o m t h e p l a t e and washed w i t h s o d i u m h y d r o x i d e s o l u t i o n (0.1%) t o y i e l d m o n o v i n y l mono 6-amino 1-amido hexane a c r y l a t e d e u t e r o -p o r p h y r i n IX DTBE (22 mg; 2 2 % ) . v i s : 640, 581, 551, 510 ( R a t i o s : 1:1.79:2.22:2.35), r h o d o / e t i o - t y p e . N.M.R.: 1.3 4 (m, t B u and 2,3,4,5 and 6 a l k y l C H 2 ) , 2.96 -> 3.84 (m, r i n g - C H 3 , 3 p r o p i o n y l ( , 1 a l k y l - C H 2 ) 4.24, (m, a p r o p i o n y l - C H 2 ) , 6.18 (m, v i n y l - C H 2 ) , 6.97 (d, a a c r y l a t e -CH), 7.78 (m, v i n y l - C H ) , 8.50 (m, 3 a c r y l a t e - C H ) , 8.85 + 9.75 (m, m e t h i n e ) . S p e c t r a 14 2 1.13 M o n o v i n y l Mono 3-Amino 1-Amido P r o p a n e A c r y l a t e  D e u t e r o p o r p h y r i n IX (58) M o n o v i n y l mono 3-amino l - a m i d o p r o p a n e a c r y l a . t e d e u t e r o -p o r p h y r i n IX DTBE was d i s s o l v e d i n m e t h y l e n e c h l o r i d e and d r y HCI was p a s s e d t h r o u g h t h e s o l u t i o n f o r s e v e n h o u r s . The s o l v e n t volume o f t h e r e a c t i o n was k e p t c o n s t a n t by a d d i t i o n o f C H 0 C 1 „ t o r e p l a c e e v a p o r a t i o n l o s s e s . The r e d s o l u t i o n r a p i d l y t u r n e d g r e e n , and s l o w l y t h e d e - e s t e r i f i e d p o r p h y r i n o i l e d o u t o f s o l u t i o n . A t t h e end o f 7 h o u r s n i t r o g e n was blown t h r o u g h t h e s o l u t i o n and t h e s o l v e n t e v a p o r a t e d t o d r y -n e s s . The p r o g r e s s o f t h e r e a c t i o n was c h e c k e d by t . l . c . ( b e n z e n e : m e t h a n o l , 1 0 : 1 ) . The mono and d i e s t e r s e l u t e d i n t h i s s y s t e m whereas t h e d i a c i d r e m a i n e d s t a t i o n a r y , u n t i l a s m a l l volume o f f o r m i c a c i d was add e d t o t h e s y s t e m . v i s : ( i n NaHC0 3 s o l u t i o n , 0.2 M), 640, 682, 560, 516 ( R a t i o s : 1:2.0:2.21:2.50), r h o d o - e t i o t y p e . S p e c t r u m - page 143 Cyanogen Bromide A c t i v a t i o n o f A g a r o s e 3 A s l u r r y o f a g a r o s e b e a d s ( s e p h a r o s e 2B; 30cm o f a 1:1 g e l and w a t e r m i x t u r e ) was t r a n s f e r r e d t o an E r l e n m e y e r f l a s k , s o d i u m 3 c a r b o n a t e s o l u t i o n was add e d (3 0cm , 2M) and s t i r r e d s l o w l y . The r a t e o f s t i r r i n g was i n c r e a s e d and c y a i r o g e n b r o m i d e (3 g) 114 3 i n a c e t o n i t r i l e (1.5cm ) a d d e d . A f t e r two m i n u t e s o f v i g o r o u s s t i r r i n g t h e s l u r r y was f i l t e r e d i n a c o a r s e s i n t e r e d g l a s s 3 f u n n e l , w a s h ed w i t h s o d i u m b i c a r b o n a t e s o l u t i o n (2 0 0cm , 0.1 M, 3 pH 9.5) and w a t e r (200cm ) and t r a n s f e r r e d , a s a n a q u e o u s s l u r r y (pH 7 . 0 ) , t o a p l a s t i c b o t t l e . S o d i u m a z i d e (0.02%) was a d d e d t o t h e g e l w h i c h was s t o r e d a t 4°C. T h i s a c t i v a t i o n p r o c e d u r e g a v e a s o l i d s u p p o r t w i t h a c o u p l i n g c a p a c i t y o f 0.1 3 yM/cm w i t h r e s p e c t t o t h e p o r p h y r i n a f f i n i t y l i g a n d . 3 The a c t i v a t i o n was r e p e a t e d o n s e p h a r o s e (30cm o f a 1:1 3 g e l t o w a t e r m i x t u r e ) . S o d i u m b i c a r b o n a t e (30cm , 2M) was a d d e d a n d t h e s o l u t i o n was s t i r r e d a t 0°C. C y a n o g e n b r o m i d e 3 . (20 g ) , i n a c e t o n i t r i l e (10cm ) was a d d e d a n d t h e v i g o r o u s l y s t i r r e d s o l u t i o n was a l l o w e d t o r e a c t , a t 4°C, f o r 5 m i n u t e s . The s o l u t i o n was f i l t e r e d , w a s h e d w i t h a c o o l e d s o d i u m b i c a r -3 3 b o n a t e d s o l u t i o n ( 0.1 M, 200cm ) and w a t e r (200cm ) . T h i s l o w t e m p e r a t u r e t r e a t m e n t o f t h e a g a r o s e w i t h a g r e a t e r q u a n t i t y o f c y a n o g e n b r o m i d e s o l u t i o n f o r a n e x t e n d e d t i m e i n -c r e a s e d t h e c o u p l i n g c a p a c i t y o f t h e CNBr a c t i v a t e d a g a r o s e 3 t o 4.7 yM/cm w i t h r e s p e c t tcf t h e p o r p h y r i n l i g a n d . The C o u p l i n g , o f Mono 3-Amino 1-Amido P r o p a n e A c r y l a t e  D e u t e r o p o r p h y r i n I X DTBE t o C y a n o g e n B r o m i d e A c t i v a t e d A g a r o s e 3 An a q u e o u s s l u r r y o f t h e a c t i v a t e d a g a r o s e (3.0cm" o f a 1:1 g e l w a t e r m i x t u r e ) was c e n t r i f u g e d a n d t h e a q u e o u s s o l u -3 t i o n was r e m o v e d . S o d i u m b i c a r b o n a t e s o l u t i o n (3.0cm , 0.2M 115 pH 9.5) V7as added, t h e s o l u t i o n was s h a k e n , r e c e n t r i f u g e d and t h e aqueous l a y e r was removed. A s o d i u m b i c a r b o n a t e s o l u t i o n 3 (3.0cm ,0.2M; pll 9.5) o f mono 3-amino l - a m i d o p r o p a n e a c r y l a t e m o n o v i n y l d e u t e r o p o r p h y r i n IX was a d d e d t o t h e a c t i -v a t e d s e p h a r o s e and t h e m i x t u r e was s h a k e n on a m e c h a n i c a l s h a k e r f o r 24 h o u r s a t 4°C. The c o u p l e d s e p h a r o s e was t h e n f i l t e r e d i n t o a p i p e t t e p a c k e d a t t h e end w i t h g l a s s w o o l , w h i c h was t o s e r v e as t h e c o l u m n . The r e d / b r o w n 3 c o l o u r e d g e l was washed w i t h w a t e r (30cm ) and s t o r e d a t 4 0 ° C and away f r o m t h e l i g h t . The c o n c e n t r a t i o n o f p o r p h y r i n bound t o t h e c o l u m n was m e a s u r e d by t h e d i f f e r e n c e i n t h e v i s i b l e a b s o r p t i o n o f t h e p o r p h y r i n s o l u t i o n s . A t t e m p t e d E n z y m i c P r e p a r a t i o n o f S e p h a r o s e Bound Hemin A sample o f t h e s e p h a r o s e bound p o r p h y r i n was m e a s u r e d o u t (1cm 3 o f a 1:1 g e l w a t e r m i x t u r e ) and i t s v i s i b l e s p e c t r u m 3 was t a k e n by v i g o r o u s l y s h a k i n g a s q u a r e c u v e t t e (0.5cm ) and u s i n g a f a s t c h a r t s p e e d ( l O n m / s e c ) . The g e l .was t r a n s f e r r e d t o an e r l e n m e y e r f l a s k and a f r e s h l y p r e p a r e d 3 s o l u t i o n (20cm ) c o n t a i n i n g T r i s - H C l (50cM, pH 7 . 5 ) , f e r r o u s s u l p h a t e (O.lmM) and c y s t e i n e (lOmM, pH 7.5) was a d d e d . The 3 f e r r o c h e l a t a s e e x t r a c t f r o m l i v e r h o mogenates (2.0cm ) was a d d -ed and t h e m i x t u r e was i n c u b a t e d u n d e r an a t m o s p h e r e o f a r g o n 116 a t 37°C; t h e a r g o n s t r e a m was n o t b u b b l e d t h r o u g h t h e r e a c -t i o n s o l u t i o n b e c a u s e t h i s t e n d e d t o c a u s e f r o t h i n g . A f t e r 24 h r s . t h e p o r p h y r i n b o u n d g e l was f i l t e r e d o f f i n t o a g l a s s w o o l b l o c k e d p i p e t t e a n d i t s v i s i b l e s p e c t r u m t a k e n . No c h a n g e f r o m t h e c h a r a c t e r i s t i c f o u r b a n d v i s i b l e s p e c t r u m was s e e n . V i s i b l e s p e c t r u m o f p o r p h y r i n b o u n d s e p h a r o s e : 64 0, 571 , 560, 521 ( R a t i o s ; 1:2.5:2.8:2.7) r h o d o / o x o r h o d o t y p e . S p e c t r u m - p a g e 14 3 I r o n ( I I I ) Mono 3-Amino 1-Amido P r o p a n e A c r y l a t e  M o n o v i n y l D e u t e r o p o r p h y r i n I X Mono 3-amino 1-amido p r o p a n e a c r y l a t e m o n o v i n y l d e u t e r o -p o r p h y r i n I X (4mg , 6.15 y m o l e s ) was d i s s o l v e d i n p y r i d i n e 3 (1.0cm ) a n d a c e t i c a c i d (25cm) a n d a s a t u r a t e d s o l u t i o n o f 3 f e r r o u s s u l p h a t e (2cm ) a d d e d . The s o l u t i o n was h e a t e d t o 80°C f o r 10 m i n u t e s u n d e r a s t r e a m o f n i t r o g e n . A c o l o u r c h a n g e f r o m r e d / b r o w n t o b r o w n was e v i d e n t . The s o l u t i o n was a l l o w e d t o c o o l i n t h e a i r w h i l e o x i d a t i o n t o t h e i r o n ( I I I ) p o r p h y r i n t o o k p l a c e . The p r o d u c t was d i l u t e d w i t h h y d r o -3 c h l o r i c a c i d (50cm , 25% w / v ) , and t h e h e m m was p r e c i p i t a t e d w i t h s a t u r a t e d s o d i u m b i c a r b o n a t e s o l u t i o n a n d t h e n f i l t e r e d u n d e r vacuum t o g i v e t h e d a r k b r o w n F e ( I I I ) mono 3-amino 1-amido p r o p a n e a c r y l a t e m o n o v i n y l d e u t e r o p o r p h y r i n I X (3mg, 7 0 % ) . 117 A p y r i d i n e hemochrome was t a k e n u s i n g t h e s t a n d a r d c o n -d i t i o n s o f p y r i d i n e ( 0 . 6 4 c m " ) , p o t a s s i u m h y d r o x i d e s o l u t i o n 3 3 (0.40cm , 1.0N), w a t e r ( 0 . 9 6 c m " ) , d i t h i o n i t e (2-3mg) a n d one d r o p o f t h e p o r p h y r i n s o l u t i o n . a Band - 5 69 nm, (5 band 527 nm. R a t i o : 1.7:1. S p e c t r u m - p a g e 144 S o d i u m B o r o h y d r i d e . R e d u c t i o n o f S e p h a r o s e B o u n d  Mono 3-Amino 1-Amido P r o p a n e A c r y l a t e M . o n o v i n y l D e u t e r o p o r p h y r i n I X A s a m p l e o f t h e r e d p o r p h y r i n b o u n d s e p h a r o s e was f i l t e r e d i n t o a g l a s s - w o o l p l u g g e d p i p e t t e and d e o x y g e n a t e d T r i s b u f f e r (pH 8, 0.5M) was washed, t h r o u g h . The c o l u m n was p r o t e c t e d f r o m l i g h t and s o d i u m b o r o h y d r i d e (100 mg) i n d e -o o x y g e n a t e d T r i s b u f f e r (pH 8, 0.5M, lOcm" 1) p a s s e d t h r o u g h . The f l o v ; was h a l t e d a n d t h e r e a c t i o n m i x t u r e was a l l o w e d t o s t a n d f o r two h o u r s ; t h e r e l e a s e o f h y d r o g e n f r o m t h e r e a c t i o n m i x t u r e was s u f f i c i e n t t o s t i r t h e s o l u t i o n . The w h i t e g e l was f i n a l l y w a s h e d w i t h d e o x y g e n a t e d T r i s b u f f e r (pH 8, 0.5M) and p r o t e c t e d f r o m l i g h t . 2 ( o r 4 ) - A c r y l i c A c i d P h o t o p r o t o p o r p h y r i n 2 (and 4 ) - A c r y l i c a c i d 4 (and 2 ) - v i n y l d e u t e r o p o r -p h y r i n I X DTBE (lOmg, 20 p i n o l e s ) was d i s s o l v e d i n c h l o r o f o r m 3 3 (10cm ) and p y r i d i n e (2cm ) a n d i r r a d i a t e d f o r one week w i t h a 60 w a t t desk. lamp. The r e s u l t i n g g r e e n s o l u t i o n was w a s h e d 118 w i t h w a t e r , c o n c e n t r a t e d a n d c h r o m a t o g r a p h e d on a s i l i c a g e l p r e p a r a t i v e l a y e r p l a t e (2000 m i c r o n s t h i c k ) , w i t h c h l o r o f o r m m e t h a n o l (20:1) as t h e s o l v e n t . The s l o w m o v i n g g r e e n p r o d u c t s e p a r a t e d i n t o t w o d i s t i n c t i v e b a n d s and was r e m o v e d f r o m t h e s i l i c a g e l w i t h a c h l o r o f o r m - m e t h a n o l -a c e t i c a c i d s o l u t i o n ( 1 0 : 1 : 0 . 1 ) . The s o l u t i o n was pumped t o d r y n e s s t o y i e l d t h e g r e e n 2 (and 4) a c r y l i c a c i d p h o t o -p r o t o p o r p h y r i n (8mg, 75% y i e l d ) . V i s : ( i n C H C 1 3 , e x l 0 ~ 3 ) 682 ( 1 1 . 1 5 ) , 635 ( 3 . 8 2 ) , 578 ( 5 . 3 0 ) , 506 ( 5 . 1 4 ) , 509 ( 6 . 0 5 ) , 416 ( 5 6 . 1 4 ) . ( C H C 1 3 + p y r i d i n e ) 678, 618, 566, 507 ( r a t i o s : 1:0.2: 0.37:0.36). S p e c t r a - p a g e 145 2 (and 4 ) - M o n o m e t h y l A c r y l a t e P h o t o p r o t o p o r p h y r i n I X DTBE 2 (and 4 ) - M o n o m e t h y l a c r y l a t e 4 (and 2 ) - m o n o v i n y l d e u t e r o -p o r p h y r i n I X DTBE (2mg, 4 m o l e s ) was d i s s o l v e d i n c h l o r o f o r m 3 3 (5cm ) and p y r i d i n e (1cm ) and i r r a d i a t e d f o r one week w i t h a 60 w a t t d e s k l amp. Work up was s i m i l a r t o t h a t o f t h e a c r y l i c d e r i v a t i v e e x c e p t t h a t t h e R f o n s i l i c a g e l was much g r e a t e r . V i s : 675, 619, 570, 501 ( R a t i o s ; 1 : 0 . 3 : 0 . 4 9 : 0 . 5 6 ) . Mass S p e c t r u m (Low R e s ) : 766 ( 1 3 ) , 765 ( 6 6 ) , 764 ( 1 0 0 ) , 750 ( 3 ) , 749 ( 6 ) , 748 ( 1 9 ) , 740 ( 2 ) , 739 ( 6 ) , 738 ( 1 9 ) , 723 ( 4 ) , 722 ( 1 0 ) , 720 ( 4 ) , 709 ( 1 0 ) , 708 J 3 2 ) , 680 ( 3 0 ) , 652 (91 ) , 636 ( 5 8 ) , 635 ( 5 8 ) , 634 ( 5 8 ) , 619 ( 7 1 ) , 609 ( 7 1 ) . 119 A t t e m p t e d P r e p a r a t i o n o f 2 (and 4 ) - F o r m y l 4 (and 2 ) - A c r y l i c A c i d D e u t e r o p o r p h y r i n I X D i t e r t i a r y B u t y l E s t e r 2 (and 4 ) - M o n o a c r y l i c a c i d p h o t o p r o t o p o r p h y r i n I X DTBE 3 (5mg) was d i s s o l v e d i n m e t h y l e n e c h l o r i d e (10cm ) and sodium-3 b o r o h y d r i d e (2 5mg) i n m e t h a n o l (1cm ) was a d d e d . The s o l u t i o n was s t i r r e d f o r one h o u r , a t t h i s t i m e t h e m a j o r p e a k o f t h e s p e c t r u m showed a h y p s o c h r o m i c s h i f t o f 4nm a n d a c h a n g e i n c o l o u r f r o m g r e e n t o y e l l o w / b r o w n o c c u r e d . The s o l u t i o n was w a s hed w i t h w a t e r a n d e v a p o r a t e d t o d r y n e s s . The r e s u l t a n t b r o w n s o l i d was d i s s o l v e d i n 1 , 4 - d i o x a n e a f t e r 3 w h i c h s o d i u m p e r i o d a t e (5 0mg) i n w a t e r (1cm ) and c o n c e n t r a t e d 3 s u l p h u r i c a c i d 0.05cm w e r e a d d e d . The r e a c t i o n m i x t u r e was a l l o w e d t o s t i r f o r 5 h o u r s . The r e s u l t i n g b l u e s o l u t i o n was p o u r e d i n t o d i l u t e b r i n e and e x t r a c t e d w i t h c h l o r o f o r m ; t h e o r g a n i c p h a s e was w a s h e d w i t h w a t e r and e v a p o r a t e d t o d r y n e s s t o y i e l d a b r o w n s o l i d . T h i s was d i s s o l v e d i n a m i n i -mum o f c h l o r o f o r m and c h r o m a t o g r a p h e d o n s i l i c a g e l . The m a i n brown f r a c t i o n h a d a l o w e r R f t h a n t h e p a r e n t a c r y l i c a c i d p o r p h y r i n (as e x p e c t e d f o r a f o r m y l s u b s t i t u t e d d e r i v a -t i v e ) a nd was r e m o v e d f r o m t h e s i l i c a g e l w i t h a c h l o r o f o r m -m e t h a n o l s o l u t i o n t o y i e l d a brown s o l i d (^2mg;40%). The p r e s e n c e o f a s h o u l d e r . i n t h e v i s i b l e s p e c t r u m showed t h e p r o d u c t t o be c o n t a m i n a t e d . 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'.'i o!. • | - I.: ft* ] <V-C0ftfcH2),NH COgtBu C02tBu (57) + A l t e r n a t i v e Isomer Pyridine Hemochrome of —pj~ Fe(II) Mono-3-Amino 1- ——k Amido Propane Acr y l a t e Monovinyl Deuteroporphyrin IX _:. :\ .! ..Li: 1 '•' I'-W ™ '-•\T ". i . r ... - - -" .1 i nm o 145 COgiBu C02tBu + Al t e r n a t i v e Isomer 

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