UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Polarographic, potentiometric and kinetic studies of NAD model compounds Norris, Donald James 1975

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1975_A1 N67_7.pdf [ 9.82MB ]
Metadata
JSON: 831-1.0061082.json
JSON-LD: 831-1.0061082-ld.json
RDF/XML (Pretty): 831-1.0061082-rdf.xml
RDF/JSON: 831-1.0061082-rdf.json
Turtle: 831-1.0061082-turtle.txt
N-Triples: 831-1.0061082-rdf-ntriples.txt
Original Record: 831-1.0061082-source.json
Full Text
831-1.0061082-fulltext.txt
Citation
831-1.0061082.ris

Full Text

P O L A RO GRAPHIC, POTENTIOMETRIC AND - KINETIC STUDIES OF NAD MODEL COMPOUNDS by DONALD JAMES NORRIS B. Eng., M c G i l l U n i v e r s i t y , 1970 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department of CHEMISTRY 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 A p r i l , 1975 In presenting th i s thes is in pa r t i a l fu l f i lment of the requirements for an advanced degree at the Univers i ty of B r i t i s h Columbia, I agree that the L ibrary shal l make it f ree ly ava i lab le for reference and study. I further agree that permission for extensive copying of th i s thes is for scho lar ly purposes may be granted by the Head of my Department or by his representat ives. It i s understood that copying or pub l i ca t i on of this thesis for f i nanc ia l gain shal l not be allowed without my written permission. Department of C / e m ; s Ye*- cj The Univers i ty of B r i t i s h Columbia Vancouver 8, Canada Date J / ^ ^ / r /<£ /j? ABSTRACT S u p e r v i s o r : P r o f e s s o r Ross S t e w a r t The e f f e c t s o f v a r y i n g t h e 1- and 3 - s u b s t i t u e n t s o f NAD + (I) model compounds on reaction's''. (1) t h r o u g h (4) have i i R R been i n v e s t i g a t e d . The o n e - e l e c t r o n r e d u c t i o n i n r e a c t i o n (1) was observed p o l a r o g r a p h i c a l l y . A r e a c t i o n c o n s t a n t p* of +3.7 was found f o r the e f f e c t of 1 - s u b s t i t u e n t s and a r e a c t i o n constant p of +11 to +12 was found f o r the e f f e c t of 3 - s u b s t i t u e n t s . The p o t e n t i a l of the two-electron r e d u c t i o n of p y r i d i n i u m ions i n r e a c t i o n (2) was measured p o t e n t i o -m e t r i c a l l y . Reaction c o n s t a n t s of +2.8 and +9 to +11 were found f o r the e f f e c t s of 1- and 3 - s u b s t i t u e n t s , r e s p e c t i v e l y . Reaction constants of -1.9 and -6 were found for the e f f e c t s of 1- and 3 - s u b s t i t u e n t s on the r a t e s of o x i d a t i o n of i i i D m 1 , 4 - d i h y d r o p y r i d i n e s by f l a v i n s , r e a c t i o n (3) . A s i r a i l a r r e a c t i o n c o n s t a n t o f -2.0 t o -2.6 was f o u n d f o r t h e e f f e c t of 1 - s u b s t i t u e n t s on the a c i d d e c o m p o s i t i o n o f 1 , 4 - d i h y d r o -v r i d i n e s , r e a c t i o n ( 4 ) . These r e s u l t s s u g g e s t t h a t t h e e c h a n i s m o f o x i d a t i o n o f 1 , 4 - d i h y d r o p y r i d i n e s by f l a v i n s may be more complex t h a n the commonly a c c e p t e d mechanism o f h y d r i d e t r a n s f e r . R e a c t i o n s (1) t h r o u g h (4) were a l s o i n v e s t i g a t e d u s i n g HAD +, n i c o t i n a m i d e m o n o n u c l e o t i d e (NliN) and two 1,1'-a l k y l e n e b i s ( 3 - c a r b a m o y l p y r i d i n i u m ) compounds ( I I ) . The ( I D o x i d a t i o n - r e d u c t i o n p r o p e r t i e s o f t h e s e f o u r compounds be e x p l a i n e d by c o n s i d e r i n g t h e i n d u c t i v e e f f e c t s o f t 1 - s u b s t i t u e n t s . i v TABLE OF CONTENTS Page 1. INTRODUCTION 1 1.1 A c i d Decomposition of 1 , 4 - D i h y d r o p y r i d i n e s . . . 3 1.2 N u c l e o p h i l i c A d d i t i o n to P y r i d i n i u m S a l t s . . . 5 1.3 O x i d a t i o n of 1,4-Dihydropyridines by F l a v i n s . . 6 1.4 O x i d a t i o n - R e d u c t i o n P o t e n t i a l s of P y r i d i n i u m / D i h y d r o p y r i d i n e Systems 11 1.5 E l e c t r o c h e m i c a l Reduction of P y r i d i n i u m S a l t s . 18 1.6 S u b s t i t u e n t E f f e c t s i n P y r i d i n i u m / D i h y d r o -p y r i d i n e Systems 31 2. SCOPE OF THE INVESTIGATION 35 3. RESULTS 38 3.1 The A c i d - c a t a l y z e d Decomposition of Dihydropyr i d i n e s 38 3.2 B a s e - c a t a l y z e d Decomposition Reactions 52 3.3 L i m i t a t i o n s on pH i n the Study of NAD Model Compounds . 5 6 3.4 S u b s t i t u e n t Constants of the 3-carbamoyl-p y r i d i n i u m Group 61 3.5 Polarography of P y r i d i n i u m S a l t s 65 3.5.1 A n a l y s i s of Wave I 70 3.5.2 Prewave Formation 87 3.5.3 The E f f e c t s of 1-Substituents on P o l a r o g r a p h i c Reduction P o t e n t i a l s . . . 97 3.5.4 The E f f e c t s of 3-Substituents on the P o l a r o g r a p h i c Reduction P o t e n t i a l s . . .119 v Page 3.6 Reduction P o t e n t i a l s of P y r i d i n i u m -D i h y d r o p y r i d i n e H a l f - c e l l s 128 3.6.1 S u b s t i t u e n t E f f e c t s a t the 1 - P o s i t i o n . 144 3.6.2 S u b s t i t u e n t E f f e c t s at the 3 - P o s i t i o n . 147 3.7 The O x i d a t i o n of D i h y d r o p y r i d i n e s by F l a v i n s . 150 4. DISCUSSION. 163 4.1 P o l a r o g r a p h i c Reduction Mechanisms 163 4.2 The E f f e c t s of S u b s t i t u e n t s on the Reduction of P y r i d i n i u m Ions 170 4.3 The E f f e c t s of S u b s t i t u e n t s i n D i h y d r o p y r i d i n e s on the Rates of O x i d a t i o n by F l a v i n s 185 4.4 Comparisons of 1,1'-Alkylenebis(3-carbamoyl-pyridinium) Compounds with the S e r i e s I Compounds 193 4.5 Comparisons of NAD+ and NMN+ with the S e r i e s I Compounds 201 5. SUGGESTIONS FOR FURTHER RESEARCH 210 6. EXPERIMENTAL. . "; 214 6.1 B u f f e r s 214 6.2 Oxygen-free Work 216 6.3 Sy n t h e s i s of Quaternary P y r i d i n i u m S a l t s . . . 218 6.4 Sy n t h e s i s of 1, 4-Dihydropyr i d i n e s 228 6.5 K i n e t i c s 237 6.5.1 A c i d - c a t a l y z e d Decomposition of 1, 4-Dihydropyridines 237 6.5.2 Reactions Between F l a v i n s and 1, 4-Dihydropyr i d i n e s 239 v i Page 6.6 P o l a r o g r a p h y . 244 6.7 P o t e n t i o m e t r y 248 6.8 A b s o r b a n c e S p e c t r a 259 BIBLIOGRAPHY 263 v i i LIST OF TABLES Page I Redox p o t e n t i a l s of NAD models 15 II Half-wave p o t e n t i a l s of NAD models 26 I I I S u b s t i t u e n t s on the compounds st u d i e d 36 IV D i h y d r o p y r i d i n e acid-decomposition r a t e constants . . . . . . . . . . . 43 V A l i s t of a* values used i n t h i s work 49 VI S t a b i l i t y l i m i t s f o r p y r i d i n i u m s a l t s and d i h y d r o p y r i d i n e s 57 VII Slopes and i n t e r c e p t s of p l o t s of p o t e n t i a l versus l o g ( i d - i ) / ( i) 2 / 3 and log ( i d - i ) / ( i) 1 / 2 . . 77 VIII Slopes and half-wave p o t e n t i a l s obtained by p l o t t i n g a p p l i e d p o t e n t i a l versus l o g ( i d - i ) / ( i ) 82 IX Mean i n t e r c e p t s from p l o t s of a p p l i e d p o t e n t i a l versus l o g ( i d - i ) / ( i ) 2 / 3 and l o g ( i d - i ) / ( i ) 1 / 2 . . 84 X L i s t of compounds p l o t t e d i n F i g u r e 12 89 XI C o n c e n t r a t i o n s corresponding to the maximum heig h t of the p o l a r o g r a p h i c prewave and the p o t e n t i a l d i f f e r e n c e between main wave and prewave 92 XII D i f f u s i o n c o e f f i c i e n t s of the p y r i d i n i u m ions estimated from the I l k o v i c equation I l l XIII Reduction p o t e n t i a l s f o r the p y r i d i n i u m / dihydropyr i d i n e h a l f - c e l l s 133 XIV R e s u l t s from the o x i d a t i v e t i t r a t i o n of NADH. . 139 XV Second order r a t e constants f o r the o x i d a t i o n of d i h y d r o p y r i d i n e s by f l a v i n s 158 XVI Summary of r e a c t i o n constants of r e d u c t i o n r e a c t i o n s of p y r i d i n i u m ions and analogous r e a c t i o n s 174 v i i i L I S T OF TABLES Page XVII P r e d i c t e d and o b s e r v e d p o l a r o g r a p h i c r e d u c t i o n p o t e n t i a l s o f t h e 1,1 ' - a l k y l e n e b i s ( 3 -c a r b a m o y l p y r i d i n i u m ) i o n s 197 + + X V I I I S e l e c t e d p r o p e r t i e s o f NAD , NMN and 1 - m e t h o x y m e t h y l - 3 - c a r b a m o y l p y r i d i n i u m c h l o r i d e . 203 XIX A n a x and e x t i n c t i o n c o e f f i c i e n t s o f compounds used i n t h i s work 260 i x L I S T OF FIGURES Page 1. A sample p o l a r o g r a m 19 2. A t y p i c a l p o l a r o g r a m o f a p y r i d i n i u m s a l t i n a w e a k l y b a s i c s o l u t i o n 25 3. S t a n d a r d r e a c t i o n s o f some commonly met s u b s t i t u e n t c o n s t a n t s 32 4. P s e u d o - f i r s t o r d e r d e c o m p o s i t i o n o f 1 - m e t h y 1 - 3 - c a r b a m o y 1 - 1 , 4 - d i h y d r o p y r i d i n e . . . . . . 40 5. P l o t o f s e c o n d o r d e r r a t e c o n s t a n t s f o r t h e a c e t i c a c i d c a t a l y z e d d e c o m p o s i t i o n o f d i h y d r o p y r i d i n e s ( T a b l e IV) a g a i n s t 0* ( T a b l e V) 46 6. P l o t o f s e c o n d o r d e r r a t e c o n s t a n t s f o r t h e h y d r o g e n i o n c a t a l y z e d d e c o m p o s i t i o n o f d i h y d r o p y r i d i n e s a g a i n s t 0* 47 7. S p e c t r o p h o t o m e t r i c d e t e r m i n a t i o n o f t h e pKa o f t h e 1 - c a r b o x y m e t h y 1 - 3 - c a r b a m o y l p y r i d i n i u m i o n . . . . 63 8. The e f f e c t o f c h a n g i n g c o n c e n t r a t i o n on t h e p o t e n t i a l o f t h e p o l a r o g r a p h i c r e d u c t i o n o f 1 - c a r b a m o y l m e t h y 1 - 3 - a c e t y l p y r i d i n i u m c h l o r i d e . . . . 68 9. The c o n c e n t r a t i o n dependence o f the p o l a r o g r a p h i c h a l f - w a v e p o t e n t i a l s o f l - m e t h y l - 3 - c a r b a m o y l -p y r i d i n i u m i o d i d e 69 10a P o l a r o g r a p h i c a p p l i e d p o t e n t i a l v e r s u s l o g ( i d - i ) / ( i ) f o r l - m e t h y l - 3 - c a r b a m o y l p y r i d i n i u m c h l o r i d e . . . . . 7 1 10b P o l a r o g r a p h i c a p p l i e d p o t e n t i a l v e r s u s l o g ( i d - i ) / ( i ) 2 / 3 f o r 1 - m e t h y l - 3 - c a r b a m o y l p y r i d i n i u m c h l o r i d e 72 10c P o l a r o g r a p h i c a p p l i e d p o t e n t i a l v e r s u s l o g ( i - i ) / ( i ) f o r 1-me t h y 1-3-carbamoy l p y r i d i n ium c h l o r i d e 73 11a P o l a r o g r a p h i c a p p l i e d p o t e n t i a l v e r s u s l o g ( i d - i ) / ( i ) f o r n i c o t i n o m i d e a d e n i n e d i n u c l e o t i d e . 74 l i b P o l a r o g r a p h i c a p p l i e d p o t e n t i a l v e r s u s l o g ( i ^ - i ) / ( i ) 2 / 3 f o r n i c o t i n a m i d e a d e n i n e d i n u c l e o t i d e 75 x L I S T OF FIGURES Page 11c P o l a r o g r a p h i c a p p l i e d p o t e n t i a l v e r s u s l o g ( i ^ - i ) / ( i ) f o r n i c o t i n a m i d e a d e n i n e d i n u c l e o t i d e 76 12. C o m p a r i s o n o f t h e i n t e r c e p t s from p l o t s o f p o l a r o g r a p h i c p o t e n t i a l v e r s u s l o g ( i d - i ) / ( i ) I / 2 and l o g ( i ( j - i ) / ( i ) 2/3 f o r compounds i n S e r i e s I and S e r i e s I I 8 8 13. The c o n c e n t r a t i o n dependence o f t h e prewave l i m i t i n g c u r r e n t f o r the p o l a r o g r a p h i c r e d u c t i o n o f 1 - c a r b o - i - p r o p o x y m e t h y l - 3 - c a r b a m o y l p y r i d i n i u m c h l o r i d e 9 0 14. P o l a r o g r a m o f a 1.8xlO~^M. s o l u t i o n o f 1 , 1 ' - m e t h y 1 e n e b i s ( 3 - c a r b a m o y l p y r i d i n i u m c h l o r i d e ) . 94 15. The e f f e c t o f t e m p e r a t u r e change on t h e p o l a r o g r a m o f a 2 . 3 x l 0 ~ 5 M . s o l u t i o n o f 1 , 1 ' - m e t h y l e n e b i s -( 3 - c a r b a m o y l p y r i d i n i u m c h l o r i d e ) 96 16. P l o t o f p o l a r o g r a p h i c h a l f - w a v e p o t e n t i a l s o f 1 0 - 2 M . s o l u t i o n s o f the S c r i e s I compounds a g a i n s t a* 101 17. P l o t o f p o l a r o g r a p h i c h a l f - w a v e p o t e n t i a l s o f 2X1Q-3M. s o l u t i o n s o f the S e r i e s I compounds a g a i n s t cr* 102 18. P l o t o f p o l a r o g r a p h i c h a l f - w a v e p o t e n t i a l s o f 10 - J !M. s o l u t i o n s o f t h e S e r i e s I compounds a g a i n s t n* 103 19. P l o t o f p o l a r o g r a p h i c r e d u c t i o n p o t e n t i a l s f.° o f the S e r i e s I compounds a g a i n s t a * . 117 20. P l o t o f p o l a r o g r a p h i c r e d u c t i o n p o t e n t i a l s e° o f t h e S e r i e s I I compounds a g a i n s t m e t a - s u b s t i t u e n t c o n s t a n t ff. 124 m 21. P l o t o f p o l a r o g r a p h i c r e d u c t i o n p o t e n t i a l s £° o f t h e S e r i e s I I compounds n i i n s t p a r a -s u b s t i t u e n t c o n s t a n t s O 130 P 22. P l o t o f measured c e l l p o t e n t i a l s a g a i n s t l o g [ P y + ] / [Pyll ] f o r the p y r i d i n i u m - d i h y d r o p y r i d i n e h a l f - c e l l o f 1 - m e t h o x y m e t h y 1 - 3 - c a r b a m o y 1 p y r i d i n i u m c h l o r i d e 130 x i L I S T OF FIGURES Page 23. P l o t o f measured c e l l p o t e n t i a l s a g a i n s t l o g [ P y + ] / [ P y H ] f o r the p y r i d i n i u m - d i h y d r o p y r i d i n e h a l f - c e l l o f 1 - ( 2 ' - h y d r o x y e t h y l ) - 3 - c a r b a m o y 1 -p y r i d i n i u m c h l o r i d e 2 4 . The pH depende nee o f the r e d u c t i o n p o t e n t i a l EpJJ o f 1 - c a r b a m o y l m e t h y 1 - 3 - c a r b a m o y 1 p y r i d i n i u m c h l o r i d e . . 1 3 6 2 5 . P l o t o f s t a n d a r d r e d u c t i o n p o t e n t i a l s f o r t h e p y r i d i n i u m - d i h y d r o p y r i d i n e h a l f - c e l l s o f t h e S e r i e s I compounds a g a i n s t 0* 1 4 6 2 6 . D e t e r m i n a t i o n o f the second o r d e r r a t e c o n s t a n t f o r t h e o x i d a t i o n o f NADH by r i b o f l a v i n from a p l o t o f ( l / a 0 - b 0 ) l o g ( a 0 - x ) / ( b 0 - x ) a g a i n s t t i m e 1 5 2 2 7 . D e t e r m i n a t i o n o f the s e c o n d o r d e r r a t e c o n s t a n t f o r the o x i d a t i o n o f l - c a r b a m o y l m e t h y l - 3 - c y a n o - l , 4 -d i h y d r o p y r i d i n e by FMN from a p s e u d o - f i r s t o r d e r p l o t o f l n ( A - A c o ) a g a i n s t time 1 5 4 2 8 . P l o t o f the l o g a r i t h m o f the s e c o n d o r d e r r a t e c o n s t a n t s f o r t h e o x i d a t i o n o f the S e r i e s I d i h y d r o p y r i d i n e s by f l a v i n s a g a i n s t O* 1 6 1 2 9 . Dry-box and a c c e s s o r i e s 2 1 7 3 0 . S y r i n g e and a b s o r b a n c e c e l l a p p a r a t u s f o r o x y g e n - f r e e work 2 4 1 3 1 . Sample p o l a r o g r a m s 2 4 7 3 2 . P o t e n t i o m e t r y c e l l 2 4 9 3 3 . T y p i c a l s p e c t r a o f a p y r i d i n i u m i o n and a 1 , 4 - d i h y d r o p y r i d i n e 2 6 1 3 4 . U.V. a b s o r b a n c e s p e c t r u m o f 1 - c a r b a m o y l m e t h y l - 3 -f l u o r o - 1 , 4 - d i H y d r o p y r i d i n e 2 6 2 x i i A C KNOWLEDGE ME NT I w i s h t o e x p r e s s my s i n c e r e a p p r e c i a t i o n t o P r o f . Ross S t e w a r t f o r h i s g u i d a n c e i n t h i s work and h e l p f u l s u g g e s t i o n s f o r improvements i n t h i s t h e s i s . I a l s o w i s h to thank Dr. A d d i s o n , Dr. S p i t z e r , Dr. Cox, and Dr. Gyama f o r many h e l p f u l d i s c u s s i o n s . I would l i k e t o thank 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 and t h e N a t i o n a l R e s e a r c h C o u n c i l f o r f i n a n c i a l a s s i s t a n c e . A s p e c i a l thank you i s f o r my w i f e , V i k k i , f o r her s u p p o r t t h r o u g h o u t t h i s work and f o r t y p i n g t h e r o u g h d r a f t o f t h i s t h e s i s . x i i i D e d i c a t e d t o V i k k i x i v INTRODUCTION Nicotinamide-adenine d i n u c l e o t i d e ( l a , NAD) and n i c o t i n -amide-adenine d i n u c l e o t i d e phosphate ( l b , NADP) are coenzymes i n v o l v e d i n o x i d a t i o n - r e d u c t i o n r e a c t i o n s w i t h i n l i v i n g systems. (a) : R=H (NAD ) (b) : R=P0 3 = (NADP+) The coenzymes are a l s o r e f e r r e d to by s e v e r a l other names. Throughout t h i s work, the nomenclature and a b b r e v i a t i o n s pro-posed by the Commission on Enzymes of the I n t e r n a t i o n a l Union HO OH (ID of Biochemistry w i l l be used. The p y r i d i n e r i n g was i d e n t i f i e d 2 by Warburg, et a l . as the r e a c t i v e p o r t i o n of the molecule and has l e d to the general term p y r i d i n e n u c l e o t i d e s being used 2 f o r t h e s e compounds and f o r the m o n o n u c l e o t i d e ( I I , NMN). I n the o x i d i z e d s t a t e , a b b r e v i a t e d MAD +, t h e p y r i d i n e r i n g e x i s t s as t h e p y r i d i n i u m s a l t ( I I I ) . R e d u c t i o n o f t h e coenzymes i n v o l v e s the a d d i t i o n o f two e l e c t r o n s and one p r o t o n t o form a d i h y d r o p y r i d i n e . I t was o r i g i n a l l y t h o u g h t t h a t t h e r e d u c e d form o f NAD, a b b r e v i a t e d NADH, was a 1,2- o r a 1 , 6 - d i h y d r o -p y r i d i n e . I t has s i n c e been shown t h a t e n z y m a t i c r e d u c t i o n + 3 4 o f MAD y i e l d s o n l y t h e 1 , 4 - d i h y d r o p y r i d i n e ( I V ) . ' The c h e m i c a l and b i o l o g i c a l p r o p e r t i e s o f NAD +, NADH and model compounds have been r e v i e w e d . ^  ' ^  ' ^  ' 6 ' "*"0 O n l y m a t e r i a l w h i c h i s p e r t i n e n t t o t h i s s t u d y w i l l be reviev/ed h e r e . 3 1.1 A c i d D e c o m p o s i t i o n o f 1 , 4 - D i h y d r o p y r i d i n e s : The c h a r a c t e r i s t i c u l t r a - v i o l e t a b s o r p t i o n o f 1 , 4 - d i h y d r o -p y r i d i n e c e n t e r e d n e a r 340 nanometers i s r a p i d l y l o s t i n t h e 7 p r e s e n c e o f a c i d and r e p l a c e d by a peak a t 290 n a n o m e t e r s . I f l e f t i n a c i d , t h i s compound w h i c h has been c a l l e d " p r i m a r y a c i d m o d i f i c a t i o n compound" i s f u r t h e r a l t e r e d w i t h a c o r r e s -8 12 p o n d i n g d e c r e a s e i n t h e a b s o r p t i o n a t 290 n a n o m e t e r s . ' 9,10 The s u g g e s t e d mechanism i n v o l v e s p r o t o n a t i o n o f V a t C-5 t o g i v e VI, f o l l o w e d by a n u c l e o p h i l i c a t t a c k a t C-6 o f the (VI I ) p y r i d i n e r i n g . In aqueous s o l u t i o n s , the n u c l e o p h i l e would g e n e r a l l y be a w a t e r m o l e c u l e , w i t h s u b s e q u e n t d e p r o t o n a t i o n y i e l d i n g t h e 6 - h y d r o x y - l , 4 , 5 , 6 - t e t r a h y d r o p y r i d i n e ( V I I ) , w h i c h i s the " p r i m a r y a c i d m o d i f i c a t i o n compound". The r e a c t i o n i s 11 12 s u b j e c t t o g e n e r a l a c i d c a t a l y s i s . ' I n aqueous m e d i a , the p r o t o n a t i o n s t e p i s r a t e - d e t e r m i n i n g . ^ ^ I t i s t h i s s t e p w hich c a u s e s the s h i f t i n t h e u l t r a - v i o l e t a b s o r p t i o n maximum 10 t o 290 n a n o m e t e r s . The f u r t h e r r e a c t i o n o f t h e " p r i m a r y a c i d m o d i f i c a t i o n compound" ( V I I ) , which i s much s l o w e r , i s s u s p e c t e d to i n v o l v e p r o t o n a t i o n a t C-3 w i t h s u b s e q u e n t n u c l e o p h i l i c a t t a c k a t C-2. 5 1.2 N u c l e o p h i l i c A d d i t i o n to P y r i d i n i u m S a l t s : The e l e c t r o n d e f i c i e n c y of the p y r i d i n i u m r i n g makes i t s u s c e p t i b l e to a t t a c k by n u c l e o p h i l e s . Sund 1 l i s t s s e v e r a l anions which have formed a d d i t i o n compounds with p y r i d i n i u m s a l t s ( V I I I ) . Most of these form 1 , 4 - d i h y d r o p y r i d i n e s (IXa), but hydroxide and s u l p h o x y l a t e ions apparently?form 1,2- (IXb) or 1 , 6 - d i h y d r o p y r i d i n e s . 1 H X 3 or • N I R1 (VIII) (IX) The a d d i t i o n of cyanide has r e c e i v e d c o n s i d e r a b l e a t t e n t i o n . The a d d i t i o n to NAD+, which i s r a p i d and q u a n t i t a -13 t i v e i n 1 M. cyanide, g i v e s a product with an a b s o r p t i o n band near 325 nanometers. T h i s r e a c t i o n has been used as an assay +13,14 f o r NAD since i t allows a simple spectrophotometric d e t e r m i n a t i o n to be made. The d i r e c t spectrophotometric d e t e r m i n a t i o n of NAD+ i s impossible i f other aromatic com-pounds are prese n t . Both r a t e and e q u i l i b r i u m c o n s t a n t s have been determined f o r the a d d i t i o n of cyanide i on to a "~ 15 16 58 number of p y r i d i n i u m s a l t s ' ' (see a l s o s e c t i o n 1.6, S u b s t i t u e n t E f f e c t s ) . I t has been suggested t h a t the e q u i -l i b r i u m c onstants f o r cyanide a d d i t i o n measures the ease of r e d u c t i o n , and t h e r e f o r e the redox p o t e n t i a l s , of p y r i d i n i u m s a l t s . 1,17,58 1.3 O x i d a t i o n of 1,4-Dihydropyridines by F l a v i n s : A v a r i e t y of compounds are capable of o x i d i z i n g NADH and d i h y d r o p y r i d i n e models to the corresponding p y r i d i n i u m s a l t s . 1 One b i o l o g i c a l l y important group with t h i s c a p a b i l i t y are the f l a v i n s (X), r i b o f l a v i n (Xa) and r i b o f l a v i n adenosine pyrophosphate (Xc). The coenzymes are a l s o r e f e r r e d to as f l a v i n mononucleotide (FMN) f o r (Xb) and f l a v i n adenine d i n u c l e o t i d e (FAD) f o r (Xc). Within m i t r o c h o n d r i a , much of the NADH i s o x i d i z e d by a flavoenzyme, NADH dehydrogenase. The e l e c t r o n s are t r a n s -f e r r e d from the f l a v i n through the va r i o u s enzymes of the r e s p i r a t o r y c h a i n ( a l s o r e f e r r e d to as the e l e c t r o n t r a n s f e r chain) to u l t i m a t e l y reduce oxygen. During t h i s p r o c e s s , three molecules of high energy compound adenosine t r i p h o s p h a t e are formed. The importance of flavoenzyme o x i d a t i o n s of NADH has spawned c o n s i d e r a b l e i n t e r e s t i n the mechanism of the r e a c t i o n between r i b o f l a v i n and 1 , 4 - d i h y d r o p y r i d i n e s . Singer and 1 9 Kearney found t h a t r i b o f l a v i n r e a d i l y c a t a l y z e d the o x i d a -t i o n of NADH by oxygen or cytochromes (heme molecules which are p a r t of the r e s p i r a t o r y c h a i n ) . The r e a c t i o n has s i n c e 2 0 2 1 2 2 23-27 been st u d i e d both a e r o b i c a l l y ' ' and a n a e r o b i c a l l y . 23 24 2 5 The mechanism which has been suggested ' ' i s shown i n scheme 1 using FMN as the oxidant. A d i r e c t hydrogen t r a n s f e r occurs from the 4-carbon of the NADH p y r i d i n e r i n g to the 27 5 - p o s i t i o n of a 5 - d e a z a - f l a v i n . The hydrogen t r a n s f e r step i s preceded by the formation of a flavin-NADH complex f o r w h i c h , by a n a l o g y w i t h some o t h e r f l a v i n r e a c t i o n s , a co-v a l e n t i n t e r m e d i a t e has been p r o p o s e d . 6 2 ' 6 3 o t h e r e v i d e n c e NH R= CH„OH I 2 CHOH I CHOH I CHOH I CHOH 1 (a) R= C H „ 0 P 0 o CHOH I CHOH I CHOH I CHOH I (b) 2 5 2 6 has been p r e s e n t e d i n f a v o u r o f a c h a r g e t r a n s f e r complex ' ' as has a l s o been p r o p o s e d i n the o x i d a t i o n o f NADH models by 65 t r i f l u o r o a c e t o p h e n o n e . A p o s s i b l e c o v a l e n t i n t e r m e d i a t e i s X I , w h i c h c o u l d decompose to p r o d u c t s by e l e c t r o n and p r o t o n m i g r a t i o n s as shown i n scheme 2a. H + NADH + FMN NAD + FMNH. FMNH ^  + FMN (FMNH ^  - FMN) . ^  2FMNH scheme 1. I n a c h a r g e t r a n s f e r complex the h y d r o g e n c o u l d be t r a n f e r r e d as e i t h e r a h y d r i d e (one s t e p mechanism) o r a h y d r o g e n atom (two s t e p mechanism) w i t h i n t e r m e d i a t e f r e e - r a d i c a l f o r -m a t i o n (scheme 2 ) . F l a v i n r a d i c a l s a r e formed d u r i n g the r e a c t i o n b u t t h e s e a r e b e l i e v e d t o be formed i n s e c o n d a r y r e a c t i o n s r a t h e r t h a n i n t h e i n i t i a l o x i d a t i o n - r e d u c t i o n s t e p . No e v i d e n c e has been found f o r t h e f o r m a t i o n o f NAD ,. . 23,24 , , r a d i c a l s d u r i n g t h e r e a c t i o n . The p r o t o n a t e c t p y r i d i n e r a d i c a l (XIV) has been o b s e r v e d i n e l e c t r o c h e m i c a l o x i d a t i o n s 2 9 o f NADH m o d e l s . I t i s known t o undergo r a p i d d e p r o t o n a t i o n to t h e r a d i c a l ( X I I I ) o r r a p i d d i s p r o p o r t i o n a t i o n t o t h e p y r i d i n i u m ( I I I ) and d i h y d r o p y r i d i n e (IV) compounds. The r a t e o f o x i d a t i o n o f t h e p r o t o n a t e d r a d i c a l (XIV) by r i b o -f l a v i n i s unknown, b u t i t i s u n l i k e l y t h a t t h e h y d r o g e n -atom t r a n s f e r s t e p would be r a t e d e t e r m i n i n g i n pathway (d) . S i n c e d e u t e r i u m i s o t o p e e f f e c t s i n d i c a t e the i n v o l v e m e n t o f 2 0 h y d r o g e n bond b r e a k i n g and f o r m a t i o n i n the t r a n s i t i o n s t a t e , pathway (d) i s u n l i k e l y to be o p e r a t i v e . The r a t e c o n s t a n t f o r the o x i d a t i o n o f the n e u t r a l NAD r a d i c a l ( X I I I ) by 9 9 r i b o f l a v i n has been e s t i m a t e d as between 4 x 10 and 5 x 10 -1 -1 29 M - s e c . . I t i s u n l i k e l y t h a t t h e o x i d a t i o n o f ( X I I I ) by the f l a v i n r a d i c a l would be v e r y d i f f e r e n t from t h i s r a t e . The o v e r a l l o x i d a t i o n - r e d u c t i o n r e a c t i o n i s t e n o r d e r s o f m a g n i t u d e s l o w e r t h a n t h i s r a d i c a l o x i d a t i o n . Such an enormous d i f f e r e n c e i n t h e s e two r a t e s would seem to l e a v e no p r a c t i c a l d i f f e r e n c e between pathways (b) and (c) i n 22 scheme 2. McCormick e t a l . , have measured the r a t e s o f (c) c h a r g e t r a n s f e r complex H t r a n s f e r ( X I I I ) ON H, + FMNH e l e c t r o n >• I I I t r a n s f e r x i r c h a r g e . .,, t r a n s f e r e l e c t r o n (d) . — _ > complex t r a n s f e r CONH, .+ FMN H t r a n s f e r I I I XII, (XIV) Scheme 2 10 o x i d a t i o n o f NADH by a s e r i e s o f r i b o f l a v i n a n a l o g u e s . They c o n c l u d e d t h a t , i n the t r a n s i t i o n s t a t e , a n e g a t i v e l y c h a r g e d s p e c i e s , p r o b a b l y a h y d r i d e i o n , i s a p p r o a c h i n g t h e f l a v i n , b u t t h e i r r e s u l t s may a l s o be e x p l i c a b l e by the d e v e l o p m e n t o f a n e g a t i v e c h a r g e a t N-5 o f the f l a v i n m o l e c u l e as i n pathway ( a ) . 1.4 Oxidation-Reduction.,Potenti§is. o f P y r i d i n i u m - - 7-D i h y d r o p y r i d i n e S y s t e m s : The redox p o t e n t i a l o f t h e NAD +/NADH sy s t e m , r e p r e s e n t e d by e q u a t i o n 1, has been d e t e r m i n e d p o t e n t i o m e t r i c a l l y and by NAD + + H + + 2e==-=_NADH (1) e q u i l i b r a t i o n w i t h s y stems o f known r e d o x p o t e n t i a l . 3 ° The measured c e l l p o t e n t i a l i s r e l a t e d t o t h e c o n c e n t r a t i o n o f o x i d i z e d and r e d u c e d s p e c i e s by the N e r n s t e q u a t i o n ( e q u a t i o n 2) where E i s t h e measured c e l l p o t e n t i a l , E° i s t h e s t a n d a r d „ RT , r e d , 0 , E E - I n (2) nF a ox c e l l p o t e n t i a l , R i s the gas c o n s t a n t , T i s the a b s o l u t e t e m p e r a t u r e , n i s the number o f e l e c t r o n s t r a n s f e r r e d , F i s the F a r a d a y , and a , and a a r e t h e a c t i v i t i e s o f t h e r e d ox r e d u c e d and o x i d i z e d s p e c i e s r e s p e c t i v e l y . F o r c o n v e n i e n c e , i n d i l u t e s o l u t i o n s , t h e a c t i v i t i e s c a n be r e p l a c e d by c o n c e n -t r a t i o n s . F o r t h e NAD +/NADH system t h e c e l l p o t e n t i a l i s g i v e n by e q u a t i o n 3. A t any g i v e n pH, t h e m i d p o i n t p o t e n t i a l , E = E » - H l n i^Hf - 2^22* ( PH) (3) 2F [NAD] , the p o t e n t i a l a t w h i c h t h e c o n c e n t r a t i o n o f NADH e q u a l s the c o n c e n t r a t i o n o f N;AD.+', i s g i v e n by e q u a t i o n 4. The r e d u c t i o n p o t e n t i a l s o f b i o l o g i c a l l y i m p o r t a n t compounds a r e o f t e n q u o t e d a t pH 7, a pH w h i c h i s much more m e a n i n g f u l p h y s i o l o g i c a l l y t h a n i s pH 0. T h i s p o t e n t i a l c a n be c a l c u l a t e d from t h e m i d p o i n t p o t e n t i a l a t any o t h e r pH by e q u a t i o n 5. 2 . -3 0 3 RT E 7 ! - E P H " I - f t f J ' ( P H _ 7 ) ( 5 ) The c e l l p o t e n t i a l o f t h e NAD +/NADH s y s t e m i s n o t 14 3 2 d i r e c t l y m e a s u r a b l e . Rodkey ' has u t i l i z e d a m e d i a t o r s y s t e m to measure t h e s e p o t e n t i a l s . The NAD +/NADH s y s t e m i s e q u i l i b r a t e d w i t h an e l e c t r o m o t i v e l y a c t i v e i n d i c a t o r s u c h as b e n z y l v i o l o g e n . A f t e r e q u i l i b r i u m has been r e a c h e d , the p o t e n t i a l o f the i n d i c a t o r c a n be measured w i t h a p l a t i -num e l e c t r o d e . S i n c e e q u i l i b r i u m has been a t t a i n e d , t h e measured p o t e n t i a l c o r r e s p o n d s to the p o t e n t i a l o f the NAD +/NADH s y s t e m . Rodkey has d e t e r m i n e d t h e v a l u e o f -318 14 m i l l i v o l t s f o r E ° ' a t 30°. A f t e r d e t e r m i n i n g a t e m p e r a t u r e c o e f f i c i e n t o f 1.31 m i l l i v o l t s p e r d e g r e e , he c a l c u l a t e d a 3 2 v a l u e o f -311 m i l l i v o l t s f o r E ° ' a t 25°. The r e d u c t i o n p o t e n t i a l (E°') o f t h e NADP +/NADPH system has been d e t e r m i n e d 31 i n a s i m i l a r manner as -316 m i l l i v o l t s a t 30°. By e q u i l i b r a t i n g the unknown r e d o x s y s t e m w i t h a known one, t h e unknown s t a n d a r d r e d u c t i o n p o t e n t i a l c a n be found from the o v e r a l l e q u i l i b r i u m c o n s t a n t . F o r t h e r e a c t i o n 0 X A + RED B:-=f_ RED A + 0X B the e q u i l i b r i u m c o n s t a n t i n d i l u t e s o l u t i o n s i s g i v e n by e q u a t i o n 6. [ R E D , ] [ 0 X 1 _ A o " " [OX ] f RED ] ( 6 ) A 13 K i s r e l a t e d t o E° t h r o u g h the N e r n s t e q u a t i o n w i t h c e i l E = 0. c e l l E 0 = I n K (7) c e l l nF w ' E c e l l ^ s r e - ' - a t e ^ t o t n e s t a n d a r d r e d u c t i o n p o t e n t i a l s o f A and B by e q u a t i o n 8. E c e l l = E°A- E°B <8> C o m b i n i n g (7) and (8) a l l o w s one t o d e t e r m i n e E ^ from know-l e d g e o f the e q u i l i b r i u m c o n s t a n t and E° . RT E ° = E ° + — 1 n K ( 9) A B nF K ' The main d i f f i c u l t y w i t h t h i s a p p r o a c h f o r t h e d e t e r -m i n a t i o n o f E° f o r t h e NAD +/NADH system i s the knowledge o f 14 E° f o r r e f e r e n c e s y s tems which can be e q u i l i b r a t e d w i t h NAD. C l a r k 3 0 d i s c u s s e s t h e p r o b l e m s r e l a t e d to t h i s and c a l c u l a t e s v a l u e s f o r E° ' f o r t h e NAD +/NADH system s p a n n i n g a p o t e n t i a l r a n g e from -314 t o -322 m i l l i v o l t s . Of t h e s e , C l a r k s u g g e s t s the v a l u e o f -315 m i l l i v o l t s as the most r e l i a b l e . The NADP /NADPH sys t e m has been e q u i l i b r a t e d w i t h t h e NAD /NADH 30 s y s t e m . The r e d u c t i o n p o t e n t i a l was e s t i m a t e d t o be a b o u t 5 m i l l i v o l t s more n e g a t i v e t h a n the p o t e n t i a l f o r t h e NAD +/NADH sy s t e m . P o t e n t i o m e t r i c measurements on t h e NADP sys t e m gave 31 t h e same v a l u e f o r E ° 1 as was found f o r the NAD s y s t e m . L i t t l e a t t e n t i o n has been g i v e n to t h e red o x p o t e n t i a l s 3 3 3 4 o f model s y s t e m s . K a r r e r and c o w o r k e r s ' have measured the r e d u c t i o n p o t e n t i a l o f 1 - m e t h y l - , 1 - e t h y l - , 1 - p r o p y l - , and 1 - b u t y l n i c o t i n a m i d e s p o t e n t i o m e t r i c a l l y w i t h o u t t h e use 3 0 of a p o t e n t i a l m e d i a t o r . C l a r k has us e d t h e i r d a t a t o c a l c u l a t e t h e v a l u e s o f E° ' g i v e n i n T a b l e 1. These v a l u e s a r e s u s p e c t s i n c e no m e d i a t o r s were used and the p o t e n t i a l s 3 5 were r e p o r t e d l y u n s t a b l e . L e a c h , B a x e n d a l e , and Evans d e t e r m i n e d t h e r e d u c t i o n p o t e n t i a l o f 1 - m e t h y l n i c o t i n a m i d e u s i n g b e n z y l v i o l o g e n as a m e d i a t o r . They p r e p a r e d t h e d i -h y d r o p y r i d i n e e l e c t r o c h e m i c a l l y , a method w h i c h p r o d u c e s 3 5 5 p y r i d i n e d i n e r s as w e l l as d i h y d r o p y r i d i n e . ' (See a l s o s e c t i o n 1.5, E l e c t r o c h e m i s t r y . ) The di m e r i s known to r e a c t r a p i d l y w i t h b e n z y l v i o l o g e n , " 1 ' whereas NADH, a 1 , 4 - d i h y d r o -1 14 p y r i d i n e , r e a c t s v e r y s l o w l y . ' I t i s u n c e r t a i n w h e t h e r the y were a c t u a l l y m e a s u r i n g the r e d u c t i o n p o t e n t i a l o f the p y r i d i n i u m / d i h y d r o p y r i d i n e system o r t h e p y r i d i n i u m / d i m e r TABLE I Redox P o t e n t i a l s o f NAD Mo d e l s 15 1 CH. C 2 H 5 3 7 4 9 a-TAG 6 -TAG C H 2 O C H 2 C 6 H 5 C H „ C H _ O C ^ H c 2 2 b o C H 2 C 6 H 5 C H 2 C 6 H 3 C 1 2 C H 2 C 6 H 3 C 1 2 C H 2 C 6 n 3 C l 2 C H 2 C 6 H 3 C 1 2 C H 2 C 6 H 3 C 1 2 AcO R 3 CONH. CON II. CONH CONH. CONH CONH. CONH. CONH . CONH. CONH. CO.CH. COOC 2H 5 CON(CH COO 3 2 E° ' (mv.) -419, -417 -29 0 -427 -430 -337 -412 -267 -267 -300 -361 -361 -34 9 -237 -354 -391 -404 r e f e r e n c e 30 35 30 30 58 30 59 58 58 58 58 53 58 58 58 58 OAc T a b l e I ( c o n t . ) RPPRA RPPRA RPPRA RPPRA RPPRA RPPRA RPPRA RPPRA R 3 COCH CONHOH CONH NH. CHNOH COC H _ 6 5 C O C H ( C H 3 ) 0 CSNH„ CHCHCONH E°'(nv.) -257 -315 -339 -342 -242 -243 -280 -342 r e f e r e n c e 58 17 17 17 17 17 17 17 R P P R A = 17 s y s t e m . The p o t e n t i a l s o f a g r o u p o f NAD a n a l o g u e s , c o n t a i n i n g v a r i o u s 3 - s u b s t i t u t e n t s , have been d e t e r m i n e d by A n d e r s o n and 17 K a p l a n by e n z y m a t i c e q u i l i b r a t i o n w i t h t h e NAD s y s t e m . These a u t h o r s u s e d a s t h e i r r e f e r e n c e t h e v a l u e o f -320 m i l l i v o l t s d e t e r m i n e d by B u r t o n and W i l s o n f o r E° ' o f NAD. The v a l u e s l i s t e d i n T a b l e 1 were c a l c u l a t e d u s i n g t h e v a l u e o f -315 m i l l i v o l t s s u g g e s t e d by C l a r k 3 0 f o r t h e NAD +/NADII s y s t e m . 58 W a l l e n f e l s and c o - w o r k e r s have c a l c u l a t e d t h e r e -d u c t i o n p o t e n t i a l s o f s e v e r a l NAD model compounds from t h e e q u i l i b r i u m c o n s t a n t s f o r c y a n i d e a d d i t i o n by a s s u m i n g t h a t the r a t i o o f t h e s e e q u i l i b r i u m c o n s t a n t s f o r two p y r i d i n i u m s a l t s w i l l be t h e same as the r a t i o o f t h e r e d u c t i o n e q u i l i -b r i u m c o n s t a n t s . They u s e d a r e d u c t i o n p o t e n t i a l o f -315 m i l l i v o l t s f o r NAD as an a n c h o r f o r t h e s e r i e s . 1.5 E l e c t r o c h e m i c a l R e d u c t i o n o f P y r i d i n i u m S a l t s : An example o f a c u r r e n t - p o t e n t i a l c u r v e o b t a i n e d from p o l a r o g r a p h i c r e d u c t i o n a t a d r o p p i n g m e r c u r y e l e c t r o d e i s shown i n F i g u r e 1. A l o n g the p o r t i o n o f t h e c u r v e from A t o B, the a p p l i e d p o t e n t i a l i s t o o p o s i t i v e f o r r e d u c t i o n o f the e l e c t r o a c t i v e s p e c i e s t o o c c u r and o n l y t h e base l i n e c u r r e n t i s o b s e r v e d . As t h e p o t e n t i a l becomes more n e g a t i v e , the e l e c t r o a c t i v e s p e c i e s i s r e d u c e d c a u s i n g t h e i n c r e a s e i n c u r r e n t between B and C i n F i g u r e 1. A t p o t e n t i a l s more n e g a t i v e t h a n p o i n t C, the e l e c t r o a c t i v e s p e c i e s i s b e i n g r e d u c e d as r a p i d l y as i t d i f f u s e s t o the e l e c t r o d e s u r f a c e . Thus, the p l a t e a u c u r r e n t i s known as the d i f f u s i o n c u r r e n t , a F o r most e l e c t r o d e p r o c e s s e s , w hether o x i d a t i o n s or r e d u c t i o n s , t h e c u r r e n t - p o t e n t i a l c u r v e can be d e s c r i b e d by e q u a t i o n 10, where E, E^/2' d n d ^d a r e ^ e s c r ^ - k e d f i g u r e 1, R i s t h e u n i v e r s a l gas c o n s t a n t , T i s t h e t e m p e r a -t u r e , F i s t h e F a r a d a y , n i s the number o f e l e c t r o n s t r a n s -f e r r e d i n t h e e l e c t r o d e p r o c e s s , and a i s a number between 0 and 1 c a l l e d t h e t r a n s f e r c o e f f i c i e n t . E° i s s i m p l y t h e RT U d " 1 5 E = £ ° + ^ 7 F l n 1 ( 1 0 ) E ° = E l / 2 p o t e n t i a l a t w h i c h the r i g h t hand term o f e q u a t i o n ( 1 0 ) , the l o g - c u r r e n t f u n c t i o n , e q u a l s z e r o and i s d e f i n e d i n d i f f e r e n t ways f o r d i f f e r e n t r e d u c t i o n mechanisms. I f t h e e l e c -t r o d e p r o c e s s i s r e v e r s i b l e , i . e . i f b o t h th e f o r w a r d and backward F i g u r e 1. A sample p o l a r o g r a m . 20 e l e c t r o n t r a n s f e r s are s u f f i c i e n t l y r a p i d that the e q u i l i b r i u m c o n c e n t r a t i o n s of o x i d i z e d and reduced species are a t t a i n e d a t the e l e c t r o d e s u r f a c e , a has a value of 1. At 25°C, a p l o t of E versus l o g ( i d ~ i ) / ( i ) w i l l be l i n e a r with a slope of 59/n m i l l i v o l t s . The half-wave p o t e n t i a l i s r e l a t e d to the standard r e d u c t i o n p o t e n t i a l , E°, of the e l e c t r o a c t i v e s p e c i e s by equation 12 where D and D r e d a r e t n e d i f f u s i o n (12) c o e f f i c i e n t s of the o x i d i z e d and reduced s p e c i e s r e s p e c t i v e l y . For an i r r e v e r s i b l e process, the e l e c t r o n t r a n s f e r process i s slow and the e q u i l i b r i u m c o n c e n t r a t i o n s are not a t t a i n e d at the e l e c t r o d e s u r f a c e . The half-wave p o t e n t i a l i s a f u n c t i o n of the r a t e constant of the e l e c t r o n t r a n s f e r r e a c t i o n , RT , 0.886T ^ , E, , = E° + - In — — r — — k (13) 1/2 anF 1/2 o where x i s the drop time, D i s the d i f f u s i o n c o e f f i c i e n t , and k Q i s the rate of the e l e c t r o d e r e a c t i o n at the p o t e n t i a l E ° . The t r a n s f e r c o e f f i c i e n t , a, i s l e s s than 1. In the absence of any c o m p l i c a t i n g f a c t o r s , such as a d s o r p t i o n , chemical r e a c t i o n , e t c . , a p l o t of p o t e n t i a l , E, versus l o g ( i ^ - i ) / ( i ) should be l i n e a r with a slope g r e a t e r than 59/n m i l l i v o l t s . Vlcek has summarized a l i s t of c r i t e r i a f o r determining the 3 7 r e v e r s i b i l i t y of an e l e c t r o d e p r o c e s s . 21 The c a s e o f a o n e - e l e c t r o n r e d u c t i o n w i t h s u b s e q u e n t d i m e r i z a t i o n o f t h e r e d u c t i o n p r o d u c t has been c o n s i d e r e d c a s e w i l l , i n g e n e r a l , r e s u l t i n a n o n - l i n e a r p l o t o f p o t e n -t i a l v e r s u s l o g ( i - i ) / ( i ) . T h e i r t r e a t m e n t i n c l u d e s f i v e p a r t i c u l a r s i t u a t i o n s and, f o r e a c h , the l o g a r i t h m i c f u n c t i o n s o f t h e c u r r e n t w h i c h s h o u l d g i v e r i s e t o a s t r a i g h t l i n e when p l o t t e d a g a i n s t t h e a p p l i e d p o t e n t i a l . (1) I f the e l e c t r o d e p r o c e s s i s i t s e l f i r r e v e r s i b l e , t h e d i m e r i z a t i o n r e a c t i o n w i l l have no e f f e c t . I n t h e f o u r r e m a i n i n g i n d i v i d u a l c a s e s the e l e c t r o d e p r o c e s s i s assumed r e v e r s i b l e . (2) I f the d i m e r i z a t i o n r a t e i s much s l o w e r t h a n t h e e l e c t r o d e p r o c e s s i t w i l l n o t a f f e c t the c o n c e n t r a t i o n o f r e d u c t i o n p r o d u c t a t the e l e c t r o d e s u r f a c e and, t h u s , w i l l have no e f f e c t on t h e p o l a r o g r a p h i c wave. (3) I f t h e d i m e r i z a t i o n r a t e i s f a s t e r t h a n t h e e l e c t r o d e r e a c t i o n , t h e e l e c t r o d e r e a c t i o n w i l l a p p e a r to be i r r e v e r -s i b l e . The c u r r e n t and p o t e n t i a l a r e r e l a t e d by e q u a t i o n 10, w i t h the h a l f - w a v e p o t e n t i a l b e i n g g i v e n by e q u a t i o n 12. (4) I f the d i m e r i z a t i o n e q u i l i b r i u m i s d i s p l a c e d t o w a r d s the d imer and i s a l m o s t as r a p i d as the e l e c t r o d e r e a c t i o n , the c u r r e n t and p o t e n t i a l a r e r e l a t e d by e q u a t i o n 14. K i s t h e o r e t i c a l l y . 38 B o n n a t e r r e and C a u q u i s p r e d i c t t h a t t h i s E = £ ° + — In ( i d - i ) / ( i ) RT 1/2 (14) the e q u i l i b r i u m c o n s t a n t o f the d i m e r i z a t i o n r e a c t i o n , and 5 i s the t h i c k n e s s o f t h e d i f f u s i o n l a y e r w h i c h , a t t h e d r o p p i n g m e r c u r y e l e c t r o d e , i s g i v e n by e q u a t i o n 16. A l l x .3 ^ .1/2 6 = ( - T T D T ) ( 1 6 ) o t h e r symbols have the same meaning as i n p r e v i o u s e q u a t i o n s The h a l f - w a v e p o t e n t i a l i s g i v e n by e q u a t i o n 17, where C i s the c o n c e n t r a t i o n o f t h e e l e c t r o a c t i v e s p e c i e s . •p rn E l / 2 = E ° + 2F l n ( K C ) < 1 7> (5) I f t h e d i m e r i z a t i o n r e a c t i o n i s i r r e v e r s i b l e , t h e c u r r e n t and p o t e n t i a l a r e r e l a t e d by e q u a t i o n 18. RT 2/3 E = e° + -L- i n ( i d - i ) / ( i ) z / J (18) R T 2 f € ° - E ° + 3F l n 3 F B " ( 1 9 ) A F = - - ~ (20) k d i s t h e r a t e c o n s t a n t o f t h e d i m e r i z a t i o n r e a c t i o n and a l l o t h e r symbols have t h e i r p r e v i o u s m e a n i n g s . The h a l f - w a v e p o t e n t i a l i s g i v e n by e q u a t i o n 21. These e q u a t i o n s a g r e e 23 (21) . w i t h t h o s e d e v e l o p e d by M a i r a n o v s k i i f o r an i r r e v e r s i b l e d i m e r -. . . 39 i z a t i o n . The e l e c t r o c h e m i c a l b e h a v i o r o f NAD + and o t h e r p y r i -5 4 0 d i n i u m s a l t s has r e c e n t l y been r e v i e w e d . ' The mechanism s u g g e s t e d f o r t h e e l e c t r o c h e m i c a l r e d u c t i o n o f t h e s e s a l t s i s shown i n scheme 3. A t a p o t e n t i a l o f a p p r o x i m a t e l y -670 m i l l i v o l t s ( v e r s u s t h e n o r m a l h y d r o g e n e l e c t r o d e , N.H.E.), NAD + (V) i s r e v e r s i b l y r e d u c e d ( r e a c t i o n 1) t o the r a d i c a l 40 45 51-53 (XV), NAD* g i v i n g r i s e t o wave I i n F i g u r e 2. ' ' The r a d i c a l r a p i d l y d i m e r i z e s ( r e a c t i o n 2) c o n v e r t i n g t h i s f i r s t r e d u c t i o n s t e p i n t o an o v e r a l l i r r e v e r s i b l e p r o c e s s . The h a l f - w a v e p o t e n t i a l s o f some NAD a n a l o g u e s a r e l i s t e d i n T a b l e I I . The p r e s e n c e o f the r a d i c a l i n t e r m e d i a t e has been shown u s i n g c y c l i c v o l t a m r n e t r y w i t h a r a p i d - r e t u r n s c a n ^ 40-42 r a t e . The h a l f - w a v e p o t e n t i a l s o f NAD and l - m e t h y l - 3 - c a r b -a m o y l p y r i d i n i u m s a l t s f o r the r e d u c t i o n p r o c e s s i n s t e p s 1 and 2 i n scheme 3, a r e i n d e p e n d e n t o f pH between pH 1 and 40 43 44 45 + 10. ' ' ' The p y r i d i n e m o n o n u c l e o t i d e , NiMN , h a l f - w a v e p o t e n t i a l s h i f t s t o a more n e g a t i v e v a l u e by a b o u t 80 m i l l i -4 0 v o l t s as t h e pH i n c r e a s e s from 5 t o 7.5. The p o t e n t i a l f o r 4 0 t h i s compound i s o t h e r w i s e i n d e p e n d e n t o f pH. I o n i c s t r e n g t h v a r i a t i o n s have no e f f e c t on the h a l f - w a v e p o t e n t i a l s o f N - a l k y l p y r i d i n i u m s a l t s . The h a l f - w a v e p o t e n t i a l s o f NAD + 24 and NMN+ a r e s h i f t e d t o more p o s i t i v e p o t e n t i a l s by 30 m i l l i -v o l t s and 100 m i l l i v o l t s , r e s p e c t i v e l y , as t h e i o n i c s t r e n g t h i s i n c r e a s e d from 0.1 t o 2.0 M. The e f f e c t s o f i o n i c s t r e n g t h and pH on the h a l f - w a v e p o t e n t i a l o f NMN+ have been e x p l a i n e d + o t h e r i s o m e r s scheme 3 as b e i n g due t o complex f o r m a t i o n between t h e n e g a t i v e l y 4 0 c h a r g e d p h o s p h a t e g r o u p ( I I ) and the p y r i d i n i u m r i n g . The complex would r e d u c e t h e p o s i t i v e c h a r g e i n t h e p y r i d i n i u m r i n g , t h u s making r e d u c t i o n more d i f f i c u l t . Above pH 7.5, the p h o s p h a t e g r o u p e x i s t s as the d i a n i o n ; below pH 5, i t e x i s t s i n the m o n o p r o t o n a t e d form, the monoanion. P r o t o n a -t i o n o f t h e p h o s p h a t e o r h i g h i o n i c s t r e n g t h s would d e s t r o y 26 TABLE I I H a l f - w a v e P o t e n t i a l s o f NAD M o d e l s R CH. C 2 H 5 C 3 H 7 4 9 C 5 H 1 1 C 6 H 1 3 C 7 H 1 5 C 3 H 1 7 C 1 0 H 1 9 CONH, M e d i um aqueous pH 1-13 aqueous pH 9.65 aqueous 32% e t h a n o l aqueous pH 9.65 aqueous 32% e t h a n o l aqueous pH 9.65 aqueous 32% e t h a n o l aqueous 32% e t h a n o l aqueous 32% e t h a n o l aqueous 3 2% e t h a n o l aqueous 32% e t h a n o l aqueous 32% e t h a n o l E 1 / 2 ( m v . ) wave I -780 t o -880 -830 -790 -790 -820 -790 -780 -810 -800 -760 -790 -750 -790 -740 -750 -730 -710 -710 -660 -690 wave I I -1.4 t o -1.5 -1410 -1410 •1410 Ref. 40 40, 60 5 5 40,60 5 5 40,60 5 5 5 5 27 E l / 2 * m V *' R Medium wave I wave I I R e f . C H 2 C 6 H 5 aqueous pH 7-9 -760 -1410 40,52,60 C I I 2 C 6 n 4 S ° 3 aqueous pH 7-9 -720 - 52 C H 2 C H 2 S 0 3 aqueous pH 7-9 -780 - 52 50% d i o x a n e -630 - 61 50% d i o x a n e -770 61 the complex w i t h a r e s u l t a n t p o s i t i v e s h i f t i n t h e h a l f -p o t e n t i a l . Below pH 5, the h a l f - w a v e p o t e n t i a l s o f NMN + NAD + a r e i d e n t i c a l . wave a net E l e c t r o l y s i s a t a p o t e n t i a l on the p l a t e a u o f wave I l e a d s to a p r o d u c t h a v i n g a m o l e c u l a r w e i g h t c o n s i s t e n t w i t h 5 4 5 an NAD d i n e r . ' The d i m e r s have g e n e r a l l y been assumed to have the 6 , 6 ' - s t r u c t u r e (XVI) on the b a s i s o f t h e u l t r a v i o l e t s p e c t r a o f the e l e c t r o l y z e d s o l u t i o n s " " ' 4 6 b u t the use o f t h i s 5 p r o c e d u r e has been q u e s t i o n e d . Underwood and B u r n e t t have i s o l a t e d t h r e e d i m e r i c p r o d u c t s from the e l e c t r o l y s i s o f l - b e n z y l - 3 - a c e t y l p y r i d i n i u m c h l o r i d e . They a s s i g n e d t o t h e s e t h e 4,4'-, 6,6'- and 4 , 6 ' - s t r u c t u r e s on t h e b a s i s o f NMR 5 s p e c t r a . They have a l s o t e n t a t i v e l y a s s i g n e d the 4,4'-s t r u c t u r e , on the same b a s i s , to an a p p a r e n t l y homogeneous d i m e r i c p r o d u c t r e c o v e r e d a f t e r e l e c t r o l y s i s o f an NAD + s o l u t i o n . ^ A f t e r e l e c t r o l y s i s o f s o l u t i o n s o f NAD + o r 1-methyl 3 - c a r b a m o l y p y r i d i n i u m s a l t s , a p o l a r o g r a p h i c o x i d a t i o n wave c o r r e s p o n d i n g to r e o x i d a t i o n o f the d i m e r s i s o b s e r v e d a t a p p r o x i m a t e l y -10 m i l l i v o l t s and -110 m i l l i v o l t s , r e s p e c t i v e -, 5,44 l y • D u r i n g the p o l a r o g r a p h y o f d i l u t e s o l u t i o n s ( l e s s t h a n - 5 + 10 M. ) o f NAD , a prewave has been o b s e r v e d a t a p o t e n t i a l 30 m i l l i v o l t s more p o s i t i v e t h a n wave I . A s i m i l a r prewave has been o b s e r v e d i n a c y c l i c v o l t a m m e t r y s t u d y o f 1 - e t h y l - , 41 1 - p r o p y l - , and l - b e n z y l - 3 - c a r b a m o y l p y r i d i n i u m c h l o r i d e s , w h ich was t e n t a t i v e l y a t t r i b u t e d to r e d u c t i o n o f p y r i d i n i u m 41,47 i o n s a d s o r b e d on the m e r c u r y s u r f a c e . The prewave 29 o b s e r v e d d u r i n g t h e r e d u c t i o n o f 3 - a c e t y l p y r i d i n e has been 48 s t u d i e d i n g r e a t e r d e t a i l . In t h i s c a s e the prewave i s due to normal r e d u c t i o n o f the p y r i d i n e m o i e t y . A f t e r a mono-l a y e r o f d i m e r has a d s o r b e d , r e d u c t i o n o f t h e r e m a i n i n g p y r i d i n e m o l e c u l e s r e q u i r e s a more n e g a t i v e p o t e n t i a l i n o r d e r to overcome t h e i n h i b i t i o n . Wave I I i n F i g u r e 2 i s due t o f u r t h e r r e d u c t i o n o f the r a d i c a l ( s t e p 4 i n scheme 3) and i s o n l y o b s e r v e d i n a l k a l i n e 4 0 s o l u t i o n s . H i g h i o n i c s t r e n g t h o r the p r e s e n c e o f t e t r a -alkylammonium s a l t s i s r e q u i r e d to s e p a r a t e Wave I I o f NAD + from the b a c k g r o u n d wave. The h a l f - w a v e p o t e n t i a l s a r e 5 d i f f i c u l t t o d e t e r m i n e f o r t h i s wave b u t a p p e a r to f a l l i n the v i c i n i t y o f -1.4 t o -1.5 v o l t s " * ' 4 0 a t pH 9. P l o t s o f E v s . l o g ( i ^ - i ) / ( i ) i n d i c a t e t h a t the r e d u c t i o n p r o c e s s i s 4 5,40 i r r e v e r s i b l e . A pH dependence o f -30 m i l l i v o l t s p e r pH u n i t has been r e p o r t e d f o r Wave I I o f l - m e t h y l - 3 - c a r b a m o y l -43,44,49 p y r i d i n i u m i o d i d e , a l t h o u g h one s t u d y r e p o r t e d o n l y a 4 0 + s l i g h t pH d e p e n d e n c e . Wave I I o f NAD i s r e p o r t e d l y i n d e -4 0 pende n t o f p l l . The p r o d u c t o f the Wave I I r e d u c t i o n p r o c e s s i s a d i h y d r o p y r i d i n e , b u t i t s s t r u c t u r e i s s u b j e c t t o c o n t r o -v e r s y . Good e v i d e n c e has been p r e s e n t e d f a v o u r i n g a nea r q u a n t i t a t i v e y i e l d o f l - m e t h y l - 3 - c a r b a m o y l - l , 4 - d i h y d r o -5,49 p y r i d i n e . I t has a l s o been p r o p o s e d t h a t the i s o m e r i c 1 , 6 - d i h y d r o p y r i d i n e i s formed, b a s e d on the u l t r a v i o l e t s p e c t r u m o f the e l e c t r o l y z e d s o l u t i o n . D u r i n g t h e e l e c t r o l -y s i s o f NAD + a t a p o t e n t i a l on the p l a t e a u o f Wave I I some 5 4 0 4 5 dimer i s formed i n a d d i t i o n to d i h y d r o p y r i d i n e s . ' ' 30 NADH measured by e n z y m a t i c a s s a y p l u s di r t i e r , m easured by t h e h e i g h t o f the r e o x i d a t i o n wave, does n o t a c c o u n t f o r the t o t a l + NAD e l e c t r o l y z e d . The f o r m a t i o n o f t h e 1 , 6 - d i h y d r o p y r i d i n e 4 0 i s o m e r o f NADH i s p o s t u l a t e d to a c c o u n t f o r t h e b a l a n c e . S i n c e the d i m e r i z a t i o n r a t e s a r e s i m i l a r f o r the MAD r a d i c a l 5 5 0 and t h e 1 - r a e t h y 1 - 3 - c a r b a m o y l p y r i d i n e r a d i c a l , ' t h e p r e s e n c e o f dirner i n the e l e c t r o l y s i s p r o d u c t o f NAD + and n o t i n the c a s e o f l - m e t h y l - 3 - c a r b a m o y l p y r i d i n i u m i o n i s u n e x p l a i n e d . In a p r o t i c s o l v e n t s , the s e c o n d wave does n o t a p p e a r u n l e s s 42 a p r o t o n donor i s p r e s e n t . 31 1.6 S u b s t i t u e n t E f f e c t s i n P y r i d i n i u n / D i h y d r o p y r i d i n e Systems S u b s t i t u e n t e f f e c t s a r e n o r m a l l y a n a l y z e d t h r o u g h Hammett's s i g m a - r h o l i n e a r f r e e - e n e r g y r e l a t i o n s h i p s . In t h e i r s t a n d a r d form k l o g = po-H X where i s the e m p i r i c a l l y d e r i v e d s u b s t i t u e n t c o n s t a n t f o r the s u b s t i t u e n t x, k and k a r e the r a t e o r e q u i l i b r i u m X H c o n s t a n t s o f a r e a c t i o n when the m o l e c u l e c o n t a i n s t h e sub-s t i t u e n t x o r h y d r o g e n , r e s p e c t i v e l y , and p i s t h e r e s u l t i n g s l o p e . The o r i g i n a l s u b s t i t u e n t c o n s t a n t s v/ere d e r i v e d from the i o n i z a t i o n o f b e n z o i c a c i d s w i t h an a s s i g n e d s l o p e , p, o f 1.0. A v a r i e t y o f s e t s o f s u b s t i t u e n t c o n s t a n t s have been d e v e l o p e d c o v e r i n g a v a r i e t y o f s t r u c t u r a l r e l a t i o n s h i p s 54 between s u b s t i t u e n t and r e a c t i o n c e n t r e . Among t h e most commonly u s e d s u b s t i t u e n t c o n s t a n t s a r e 0 , 0 , 0*, and CT . m p I The s t a n d a r d r e a c t i o n f o r each i s shown i n F i g u r e 3. H a l f - w a v e p o t e n t i a l s have g e n e r a l l y been u s e d i n p l a c e 5 5 o f l o g k i n p o l a r o g r a p h i c l i n e a r f r e e - e n e r g y r e l a t i o n s . T h i s p r o c e d u r e , a l t h o u g h s i m p l e r t h a n c a l c u l a t i n g l o g K o r l o g f o r the e l e c t r o d e r e a c t i o n , p r e v e n t s p v a l u e s d e t e r m i n e d from p o l a r o g r a p h y b e i n g compared w i t h p v a l u e s d e t e r m i n e d i n any o t h e r way. The v a l u e o f p f o r a r e a c t i o n m e a sures the change i n c h a r g e a t the r e a c t i o n c e n t r e i n g o i n g f r o m r e a c t a n t s t o t r a n s i t i o n s t a t e o r p r o d u c t s . I f p i s p o s i t i v e , the r e a c t a n t s p o s s e s s a g r e a t e r d e g r e e o f p o s i t i v e c h a r g e o r a l e s s e r d e g r e e 32 COOH OO + II CT(X)=PK(H) " P K ( X ) COOH :oo + H (x) 1 (II) F (x) a * : XCH COOR o r OH" ->- XCH COOH + ROH a, % = [ l 0 g ( _ _ ) (x) y i k 'OH- l o g ( ^ ) H + ] / 2 . 4 8 XCH 2COOH ^ XCH COO + H (x) 2 5 1 [ P K ( H ) " P K ( X ) ] F i g u r e 3 . 33 o f n e g a t i v e c h a r g e as i n the i o n i z a t i o n o f c a r b o x y l i c a c i d s . L a r g e r a b s o l u t e v a l u e s o f p i n d i c a t e a g r e a t e r d e g r e e o f c h a r g e a l t e r a t i o n a t t h e r e a c t i o n c e n t r e . In t h e c h o i c e s o f p y r i d i n e n u c l e o t i d e m o d e l s , l i t t l e a t t e n t i o n has been p a i d t o f i n d i n g a model w h i c h c l o s e l y p a r a l l e l s NAD i n i t s b e h a v i o u r . The models w h i c h have been u s e d v/ere g e n e r a l l y n i c o t i n a m i d e s s u b s t i t u t e d a t t h e 1 - p o s i t i o n w i t h s i m p l e a l k y l g r o u p s ( e . g . m e t h y l , e t h y l , e t c . ) o r b e n z y l g r o u p s . D i r e c t c o m p a r i s o n s o f r a t e c o n s t a n t s ( o r r e d u c t i o n p o t e n t i a l s ) have been made between NAD and model compounds, a p p a r e n t l y on t h e a s s u m p t i o n t h a t such q u a n t i t i e s a r e i n d e p e n -d e n t o f s u b s t i t u e n t e f f e c t s . 2 6 ' 3 ^ ' 4 1 4 0 1 E l v i n g and Sund a l l u d e to the p o s s i b l e e f f e c t s o f s u b s t i t u e n t s on t h e p r o p e r t i e s o f p y r i d i n i u m s a l t s and 1,4-5 6 d i h y d r o p y r i d i n e s . Lamberg, e_t a l . have c o n d u c t e d a q u a l i -t a t i v e s t u d y o f 3 - s u b s t i t u t e d - l - m e t h y l p y r i d i n i u m i o d i d e s and t h e i r NAD + a n a l o g u e s . They i n v e s t i g a t e d t h e c y a n i d e a d d i t i o n r e a c t i o n , t h e sodium d i t h i o n i t e r e d u c t i o n , and t h e a l d e h y d e o x i d a s e o x i d a t i o n o f e a c h compound. The h i g h e r c y a n i d e a f f i n i t y o f NAD + o v e r t h e 1-methyl a n a l o g u e v/as a t t r i b u t e d t o an i n c r e a s e i n t h e e l e c t r o n - w i t h d r a w i n g a b i l i t y o f t h e c a r b -amoyl g r o u p i n d u c e d by h y d r o g e n b o n d i n g w i t h t h e r i b o s e s i d e c h a i n . C o n s i d e r a t i o n was a l s o g i v e n t o the p o s s i b i l i t y o f an i n d u c t i v e e f f e c t o p e r a t i n g t h r o u g h the 1 - p o s i t i o n . In h i s 1 16 r e v i e w , Sund d i s c u s s e s the work o f W a l l e n f e l s and Diekmann i n w hich the c y a n i d e a f f i n i t i e s o f v a r i o u s 1- and 3 - s u b s t i t u t e d p y r i d i n i u m s a l t s were f o u n d to be l i n e a r l y r e l a t e d t o the 34 a b s o r p t i o n maxima o f the c y a n i d e a d d u c t s . No a t t e m p t v/as made to c o r r e l a t e e i t h e r s e t o f d a t a w i t h s u b s t i t u e n t c o n s t a n t s . 57 Kosower has d e t e r m i n e d a p o f 13.4 f o r t h e v a r i a t i o n o f A o f t h e c h a r g e t r a n s f e r band o f 3- and 4 - s u b s t i t u t e d - l -max e t h y l p y r i d i n i u m i o d i d e s . 17 A n d e r s o n and K a p l a n have d e t e r m i n e d t h e r e d o x p o t e n -t i a l s o f s e v e r a l 3 - s u b s t i t u t e d a n a l o g u e s o f NAD. They o b s e r v e d p o t e n t i a l s r a n g i n g from 73 m i l l i v o l t s more p o s i t i v e t h a n NAD t o 22 m i l l i v o l t s more n e g a t i v e t h a n NAD. They s u g g e s t e d t h a t a q u a l i t a t i v e c o r r e l a t i o n e x i s t s between the redo x p o t e n t i a l and t h e e q u i l i b r i u m c o n s t a n t f o r t h e a d d i t i o n o f c y a n i d e . U n f o r t u n a t e l y , s u b s t i t u e n t c o n s t a n t s a r e u n a v a i l -a b l e f o r most o f t h e s u b s t i t u e n t s w h i c h t h e y u s e d . The most t h o r o u g h i n v e s t i g a t i o n i n t h i s a r e a i s t h a t 15 o f L x n d q u i s t and C o r d e s , who s t u d i e d t h e c y a n i d e a d d i t i o n r e a c t i o n o f a s e r i e s o f l - a l k y l - 3 - c a r b a m o y l p y r i d i n i u m h a l i d e s . P l o t s o f t h e a d d i t i o n r a t e s and e q u i l i b r i u m c o n s t a n t s v e r s u s a * y i e l d e d v a l u e s o f 2.2 and 3.7, r e s p e c t i v e l y , f o r p * . No e f f e c t on t h e r a t e o r e q u i l i b r i u m c o n s t a n t s from i n t e r n a l c h a r g e t r a n s f e r complex f o r m a t i o n was o b s e r v e d . They were u n a b l e to e x p l a i n t h e a b n o r m a l l y h i g h a f f i n i t y o f NMN , a-NAD (t h e NAD i s o m e r c o n t a i n i n g an a - r i b o s i d e l i n k a g e t o t h e p y r i d i n e r i n g ) , and £ - N A D + ( t h e n a t u r a l coenzyme) t o w a r d c y a n i d e i o n , b u t t h e i r r e s u l t s p o i n t to a d i r e c t i n t e r a c t i o n between p y r i d i n e r i n g and t h e r i b o s e s i d e c h a i n , i n a d d i t i o n t o an i n d u c t i v e e f f e c t . 35 2. SCOPE OF THE INVESTIGATION. T h i s i n v e s t i g a t i o n was o r i g i n a l l y u n d e r t a k e n t o d e t e r -mine the f r e e e n e r g y l e v e l s o f a s e r i e s o f NAD model compounds r e l a t i v e to NAD, and u l t i m a t e l y to f i n d NAD models whose e q u i l i b r i u m w i t h r i b o f l a v i n and FMN c o u l d be d i r e c t l y m e a s u r e d . I t was f o u n d t h a t l i t t l e was known a b o u t t h e e f f e c t o f sub-s t i t u e n t s on the p r o p e r t i e s o f p y r i d i n i u m s a l t s and 1 , 4 - d i h y d r o -p y r i d i n e s ( o f t e n i n t h e work, the NAD + models ( V I I I ) w i l l be r e -f e r r e d to by the g e n e r a l term p y r i d i n i u m i o n s and t h e NADH models (V) by t h e g e n e r a l term o f d i h y d r o p y r i d i n e s ) . In an e f f o r t t o f i l l t h i s gap, s e v e r a l p r o p e r t i e s o f 1- and 3 - s u b s t i -t u t e d p y r i d i n e s have been i n v e s t i g a t e d . S e r i e s I c o n s i s t s o f l - s u b s t i t u t e d - 3 - c a r b a m o y l m e t h y l p y r i d i n e s and S e r i e s I I c o n s i s t s ( V I I I ) (V) of l - c a r b a m o y l m e t h y l - 3 - s u b s t i t u t e d p y r i d i n e s . F o r c o m p a r i s o n , NAD, NMN , and tv/o 1,1 ' - a l k y l e n e b i s ( 3-carbamoy l p y r i d i n i u m ) s a l t s ( X I X ) a and XlXb) have a l s o been i n v e s t i g a t e d . In v i e w o f the i m p o r t a n c e o f N A D - f l a v i n r e a c t i o n s , t h e r a t e s o f o x i d a t i o n o f the d i h y d r o p y r i d i n e s by r i b o f l a v i n o r FMN have been i n v e s t i g a t e d . D i h y d r o p y r i d i n e s t e n d to decompose 36 TABLE I I I S u b s t i t u e n t s on the Compounds S t u d i e d S e r i e s I a : b: CH COO CH. CONH. CONH. c: CH CH 2OH d: CH COCH CONH. CONH. e: CH„OCII 2 3 CONH. f : CH COOCH CONH. g: CH COOCH ( C H ) CONH. h: CH CN CONH. l : CH CONH CONH. S e r i e s I I 3 : k: 1: m: n: CH 2CONH 2 CH CONH CH 2CONH 2 CH 2CONH CH„ C O N H „ 2 2 COCH. CN F H OH 37 i n a c i d and an i n v e s t i g a t i o n o f t h i s r e a c t i o n was n e c e s s a r y i n o r d e r to d e t e r m i n e th e pH r a n g e s i n which t h e s e compounds c o u l d e f f e c t i v e l y be s t u d i e d . P o l a r o g r a p h y was u s e d t o examine the e f f e c t o f sub-s t i t u e n t s on the r e d u c t i o n o f p y r i d i n i u m s a l t s t o t h e c o r r e s -p o n d i n g p y r i d i n e r a d i c a l s . P o t e n t i o m e t r y was u s e d t o examine the e f f e c t o f s u b s t i t u e n t s on the r e d u c t i o n o f t h e s a l t s t o the c o r r e s p o n d i n g 1 , 4 - d i h y d r o p y r i d i n e s . The c h o i c e o f s u i t a b l e models f o r NAD i n v a r i o u s r e a c t i o n s s h o u l d be a i d e d by a b e t t e r u n d e r s t a n d i n g o f s u b s t i t u e n t e f f e c t s i n t h e s e a r e a s . F u r t h e r -more, l i g h t may be shed on any s p e c i a l e f f e c t s c o n f e r r e d by the p r e s e n c e o f t h e a d e n o s i n e m o i e t y i n NAD. 33 RESULTS 3.1 A c i d - c a t a l y z e d D e c o m p o s i t i o n o f D i h y d r o p y r i d i n e s The r a t e s o f the a c i d d e c o m p o s i t i o n r e a c t i o n ( e q u a t i o n 22) o f the d i h y d r o p y r i d i n e s were f o l l o w e d s p e c t r o p h o t o m e t r i c a l l y near the a b s o r b a n c e maximum o f the d i h y d r o p y r i d i n e ( V ) . The k i n e t i c a n a l y s i s f o l l o w s from the e q u a t i o n f o r the a b s o r b a n c e o f the s o l u t i o n ( e q u a t i o n 23) and the e q u a t i o n f o r a f i r s t - o r d e r H H I *1 H H _Q±L H H H H HO H 7 ^ N (22) (V) (VI) (VII) r e a c t i o n ( e q u a t i o n 2 7 ) . R e a r r a n g i n g the e q u a t i o n f o r the a b s o r b a n c e a t any g i v e n w a v e l e n g t h ( e q u a t i o n 23) t o c a l c u l a t e f o r t h e c o n c e n t r a t i o n o f r e a c t a n t l e a d s to e q u a t i o n (25) y = £ C + £ C , I r e a c r e a c p r o d p r o d (23) y = (£ - £ ) C + £ , C . . . . . x, r e a c p r o d r e a c p r o d i n i t i a l (24) 39 w h e r e i n , i f the r e a c t i o n i s i r r e v e r s i b l e , t h e n £ , C. p r o d x r n t i a i i s e q u i v a l e n t to t h e a b s o r b a n c e a t i n f i n i t e t i m e d i v i d e d by the c e l l p a t h l e n g t h ( e q u a t i o n 2 6 ) . S u b s t i t u t i o n o f e q u a t i o n ( A - A ) 00 C r e a c = Tfe ~ £ T ( 2 6 ) r e a c procl (26) i n t o t h e e q u a t i o n f o r a f i r s t - o r d e r r e a c t i o n ( e q u a t i o n 27) -dC r e a c = kC r e a c (27) g i v e s e q u a t i o n (28) w h i c h i n t e g r a t e s to e q u a t i o n ( 2 9 ) . Thus • d ( A - A ) 00 M A - A ) (28) d t °° £n(A - A O T ) = k t + c o n s t . (29) f o r a f i r s t - o r d e r r e a c t i o n g o i n g to c o m p l e t i o n , t h e r a t e c o n s t a n t c a n be d e t e r m i n e d from a p l o t o f £n(A - A ) a g a i n s t t i m e . Such a p l o t f o r the d e c o m p o s i t i o n o f l - m e t h y l - 3 -c a r b a m o y l - 1 , 4 - d i h y d r o p y r i d i n e (Vb) i s shown i n F i g u r e 4. I t H H ^ X ^ C O N H 2 N C H 3 (vb) t (min) 41 i s o b v i o u s t h a t t h e i n i t i a l d e c o m p o s i t i o n r e a c t i o n i s a c c o m p a n i e d by a much s l o w e r s e c o n d a r y r e a c t i o n . The a b s o r b a n c e a t i n f i n i t e time f o r the i n i t i a l r e a c t i o n , A , c a n n o t be d e t e r m i n e d by a l l o w i n g the a b s o r b a n c e t o become c o n s t a n t w i t h t i m e b e c a u s e of the o c c u r r e n c e o f the s e c o n d a r y r e a c t i o n . The a b s o r b a n c e a t i n f i n i t e time f o r t h e i n i t i a l r e a c t i o n c a n , however, be e s t i m a t e d by e x t r a p o l a t i n g the l i n e o f Jin (A - A ) v e r s u s t f o r the s e c o n d a r y r e a c t i o n backwards as shown i n F i g u r e 4. A r a e s t i m a t e d i n t h i s way was g e n e r a l l y f i v e t o t e n p e r c e n t of t h e i n i t i a l a b s o r b a n c e h i g h e r t h a n the a b s o r b a n c e r e c o r d e d a t the end o f the s e c o n d a r y r e a c t i o n , an amount s u f f i c i e n t t o c a u s e a t h r e e to f i v e p e r c e n t change i n t h e s l o p e o f t h e l o g (A - A ) v e r s u s t p l o t . The e x t i n c t i o n c o e f f i c i e n t , £, o f the i n i t i a l p r o d u c t from t h e d e c o m p o s i t i o n o f 1 - c a r b a m o y l m e t h y l - 3 -f l u o r o - 1 , 4 - d i h y d r o p y r i d i n e (IV&) v/as so l a r g e t h a t a p p l i c a t i o n o f t h i s method c a u s e d a f i v e - f o l d change i n t h e c a l c u l a t e d CH 2CONH 2 (IVJL) r a t e c o n s t a n t . A s i m i l a r v a l u e o f A c o u l d be e s t i m a t e d by v a r y i n g A^ u n t i l t h e f i r s t f i v e h a l f - l i v e s o f d a t a g i v e the b e s t c o r r e l a t i o n c o e f f i c i e n t . T h i s method was s u b s e q u e n t l y used t o d e t e r m i n e t h e r a t e c o n s t a n t s f o r t h e i n i t i a l 42 d e c o m p o s i t i o n r e a c t i o n s . P s e u d o - f i r s t - o r d e r r a t e c o n s t a n t s were d e t e r m i n e d p r i m a r i l y i n the p r e s e n c e o f a c e t a t e b u f f e r a t pH ' s r a n g i n g from 3.4 to 5.6 and a t b u f f e r c o n c e n t r a t i o n s o f 1.0 and 0.1 M. A t l e a s t f o u r d e c o m p o s i t i o n r e a c t i o n s were c a r r i e d o u t on most d i h y d r o p y r i d i n e s and t h e s e d a t a were u s e d t o c a l c u l a t e t h e s e c o n d - o r d e r r a t e c o n s t a n t s f o r c a t a l y s i s by H2°' H 3 ° ' a n d a c e t i c a c i d by e q u a t i o n (3 0 ) . The H O and R o b s = k H 2 0 [ H 2 0 1 + k H 3 0 + [ H 3 ° + ] + k A c O H t A C ° H j ( 3 0 ) ? 1 a c e t i c a c i d r a t e c o n s t a n t s a r e l i s t e d i n T a b l e IV. The v/ater c a t a l y z e d r a t e r e p r e s e n t e d o n l y a few p e r c e n t o f t h e r a t e due to H 0 + and a c e t i c a c i d and, as a r e s u l t , t h e r e were v e r y l a r g e v a r i a t i o n s i n t h e c a l c u l a t e d v a l u e s . The more r a p i d l y decomposed d i h y d r o p y r i d i n e s , 1 - m e t h y l -3 - c a r b a m o y l - , 1-carboxyme t h y l - 3 - c a r b a m o y l - , 1 - ( 2 ' - h y d r o x y e t h y l ) -3 - c a r b a m o y l - , and l - c a r b a m o y l m e t h y l - 3 - f l u o r o - l , 4 - d i h y d r o p y r i d i n e v/ere a l s o decomposed i n t r i s ( h y d r o x y m e t h y l ) a m i n o m e t h a n e ( h e r e -a f t e r c a l l e d T r i s ) b u f f e r . The T r i s c a t a l y z e d r a t e c o n s t a n t s a r e a l s o l i s t e d i n T a b l e IV. The T r i s c a t a l y z e d r a t e c o n s t a n t s c o u l d n o t be d e t e r m i n e d a c c u r a t e l y f o r the r e m a i n i n g d i h y d r o -p y r i d i n e s as t h e s e r e a c t i o n s had h a l f - l i v e s o f d a y s o r weeks w i t h the h y d r o g e n - i o n c a t a l y s i s g e n e r a l l y b e i n g t h e major r e a c t i o n . The d e c o m p o s i t i o n o f 1 - c a r b a m o y l m e t h y l - 3 - f l u o r o -1 , 4 - d i h y d r o p y r i d i n e i n a c e t a t e b u f f e r v/as t o o r a p i d t o be f o l l o w e d by n o r m a l s p e c t r o p h o t o m e t r i c t e c h n i q u e s . I n f a c t , t h i s compound decomposed i n 1.0 M. sodium p e r c h l o r a t e s o l u t i o n TABLE IV D i h y d r o p y r i d i n e A c i d - D e c o m p o s i t i o n Rate C o n s t a n t s R . ( M " 1 - s e c - 1 ; k _ x 10 Ac OH (M - s e c ) k m . x 10 T r i s , -1 -1 (M - s e c ) Minimum pll f o r use o f d i h y d r o p y r i d i n e ^ CH^COO CH, CH„ C H O H 2 2 CH,OCH, 2 3 CH 2COCH CH 0C00CH. CH 9 CR CH„C0NH. CH^CONH CH COHH, CH„CONH. CONH, CONH, CONH , CONH, CONH , CONH, CONH , CONH, COCH, CN F S p e c i a l Compounds: MADH NMNH t 77 1050 4 0 0+280 18 + 5 12 7 .8 + 1 . 3 0.45+. 07 10 . 5 + 3.0 0 . 3 0+.0 5 0.39+.06 14500. 10 . 2 + 3.6 7.7+0.8 33 157 6 2+_2 2 7 .1+2 . 3 7.7 4 . 8 + 0 . 7 0.3 2+.02 5.3 + 0 . 7 0.2 2+.0 3 0.23+.03 0.95 + 0 .10 0 .90+0.43 1.2 3.6+0 . 7 1 .19 + 0.11 86 9.1 ( T r i s ) 9.7 ( T r i s ) 9.3 ( T r i s ) 3.1 ( T r i s ) 8.1 ( T r i s ) 7.5 ( T r i s ) 7.0 ( A c e t a t e ) 7.8 ( T r i s ) 6.5 ( A c e t a t e ) 6.5 (Ac e t a t e ) 11.5 ( T r i s ) 7.1 ( T r i s ) 7.5 ( T r i s ) C a l c u l a t e d f o r a maximum d e c o m p o s i t i o n o f 10% o v e r a p e r i o d o f 24 h o u r s . 44 w i t h i n m i n u t e s and had a h a l f - l i f e ( c a l c u l a t e d from the r a t e c o n s t a n t s i n T a b l e I V ) a t pH 10 o f o n l y a b o u t f o u r h o u r s . The p r i m a r y p u r p o s e o f s t u d y i n g the a c i d d e c o m p o s i t i o n o f the d i h y d r o p y r i d i n e s [ r e a c t i o n ( 2 2 ) ] was t o d e t e r m i n e the H .• H H OH" H. H HO 7 ^ H N ( 2 2 minimum pH a t which each d i h y d r o p y r i d i n e shown i n T a b l e I V c o u l d be s t u d i e d . T h i s pJI l i m i t r e p r e s e n t s a 10% d e c o m p o s i t i o n o v e r 24 h o u r s i n 0.1 I i . b u f f e r . Some compounds decompose too r a p i d l y to be used i n a c e t a t e b u f f e r b u t had v e r y s l o w T r i s c a t a l y z e d d e c o m p o s i t i o n . The r a t e s o f the v e r y slow T r i s c a t a l y z e d d e c o m p o s i t i o n v/ere e s t i m a t e d from an e a p e r i c a l e q u a t i o n [ e q u a t i o n (31)] ba s e d on the a c e t a t e and T r i s c a t a l y z e d k = k / 4 0 0 T r i s A c O i r (31) r a t e s f o r 1 - c a r b o x y m e t h y l - , 1 - m e t h y l - , and 1 - ( 2 • - h y d r o x y e t h y l ) 3 - c a r b a m o y l - l , 4 - d i h y d r o p y r i d i n e s ( I V a , b , c ) . CONH- a: R 1 = CH COO b: R1 = CH c: R 1 = CH CH OH 4 5 A s e c o n d a r y r e s u l t w h i c h came o u t o f the s t u d y o f the d i h y d r o p y r i d i n e d e c o m p o s i t i o n v/as the d e t e r m i n a t i o n o f the Hammett r e a c t i o n c o n s t a n t p. T h i s q u a z i t i t y m e a sures the i n f l u -ence o f s u b s t i t u e n t s on the r e a c t i o n and c a n be d e t e r m i n e d from e c t u a t i o n ( 3 2 ) , where a r e p r e s e n t s the e l e c t r o n i c e f f e c t o f t h e t s u b s t i t u e n t . l o g X/X^ = Ptf (32) o X = r a t e c o n s t a n t , k, or e q u i l i b r i u m c o n s t a n t , K = X f o r the u n s u b s t i t u t e d compound o * The l o g a r i t h m s o f t h e a c e t i c a c i d and h y d r o g e n i o n c a t a l y z e d r a t e c o n s t a n t s o f the S e r i e s I compounds (IV) a r e H H H H CONH, H 30 or AcOH H H CONH. H H OH" N I R HO ONH, (22) (IV) p l o t t e d a g a i n s t a* i n F i g u r e s 5 and 6. The a* s u b s t i t u e n t s c a l e i s u s e d b e c a u s e o f the s i m i l a r i t y i n t h e s t r u c t u r a l f , Tiie most commonly met s u b s t i t u e n t c o n s t a n t s a r e a m and a p f o r meta- and p a r a - s u b s t i t u e n t s i n a r o m a t i c r i n g s and a* f o r sub-s t i t u e n t s a c t i n g on an a d j a c e n t r e a c t i o n c e n t r e t h r o u g h o n l y an i n d u c t i v e e f f e c t . F i g u r e 5 . P l o t o f s e c o n d o r d e r r a t e c o n s t a n t s f o r t h e a c e t i c a c i d c a t a l y z e d d e c o m p o s i t i o n o f d i h y d r o p y r i d i n e s ( T a b l e IV) a g a i n s t a* ( T a b l e V) -•3 -.1 .1 .3 . 5 .7 . 9 11.1 1.3 cr* 4 3 r e l a t i o n s h i p betv/een s u b s t i t u e n t and r e a c t i o n c e n t r e i n r e a c t i o n (22) and t h e r e a c t i o n used to d e f i n e a*, r e a c t i o n ( 3 3 ) . The R-COOR' or OH" R-COOH + R'OH (33) v a l u e s o f o* used a r e l i s t e d i n T a b l e V and were c a l c u l a t e d 7 2 f from C h a r t o n ' s v a l u e s o f a u s i n g e q u a t i o n ( 3 4 ) . S i n c e = a / 0 . 4 5 (34) 70 f The a* s c a l e , d e s i g n e d to be a measure o f p o l a r s u b s t i t u e n t e f f e c t s o n l y , i s d e f i n e d from the r a t e s o f e q u a t i o n (33) by the e q u a t i o n : CP 274? [ l o g ( k R / k C H . Oil- l o g (k /k ) +] y ' R CH 'H + J 70 The f a c t o r o f 2.48 i s i n c l u d e d to make p* v a l u e s c o m p a r a b l e t o normal Hammett p v a l u e s ( w h i c h a r e b a s e d on t h e i o n i z a t i o n o f b e n z o i c a c i d s . ) P o l a r s u b s t i t u e n t e f f e c t s have a l s o been de-s c r i b e d by a ' 7 4 o b t a i n e d from the i o n i z a t i o n o f x-(j-ROOH the e q u a t i o n : \ / 1 b v a A r . l o g ( K /K ,4 64 y x H 74 The f a c t o r o f 1.464 i s t h i s s c a l e c o m p a r a b l e an i n d u c t i v e s u b s t i t u e b u t c o r r e c t e d by a f a c to t h e o*" s c a l e o f Rob a • ( X ) 7 3 . X-CH 2C00H f a c t o r o f r T 3 7 i . e . , cr ° * ( C H a X ) = z a t i o n o f where the s c a l e o f c JL d e f i n i t i o n o f by o. t o a g a i n d e s i g n e d t o make p* v a l u e s from to n ormal Hammett p v a l u e s . T a f t 7 3 d e f i n e d n t c o n s t a n t , Oj, b a s e d on h i s a* v a l u e s t o r o f 0.45 to make the a-j-scale e q u i v a l e n t e r t s and M o r e l a n d , t h u s C7j(x) = 0.4 5 x has s i n c e been r e d e f i n e d f r o m the i o n i -the e q u a t i o n CJj 0 251 l o g ( K x / K H ) 7 ha: s u b s t i t u e n t the l i s t o f c o n s t a n t s , O* v a l u e s . 251 i s us e d to m a i n t a i n t h e o r i g i n a l make CTj c o m p a r a b l e t o a ' . T h i s l a t t e r u r t h e r expanded the l i s t o f i n d u c t i v e and, by a p p l i c a t i o n o f e q u a t i o n ( 3 4 ) , = a / 0 . 4 5 4 9 TABLE V A l i s t o f 0* v a l u e s used i n t h i s work H H 'CONhL N I R R l ( C H 2 X ) oJ(x) a * ( R 1 ) a* (R ) CH COO CH oCII o0H CII 2OCII 3 Cil^COCH CH 2COOCH CH ^ COOCII ( CH CH CONH, 3' 2 -.17 .00 .05 . 25 .29 .34 .34 C . 27 CH CN - . 38 0.00 0.11 0.55 0 . 64 0.76 0.76 0 .60 1.30 0.00 0.66 0 .62 0 .66 1 .2! i 7 2 7 3 c a l c u l a t e d from a j by a*(CH 0X) = a (XJ/0.45 O* v a l u e s from W e l l s ' c 7 2 not g i v e n by C h a r t o n b u t was e s t i m a t e d from the i d e n t i c a l v a l u e s f o r t h e carb o m e t h o x y and c a r b o e t h o x y g r o u p s Char t o n ' s d a t a g i v e s a more e x t e n s i v e l i s t o f p o l a r s u b s t i t u e n t 69 c o n s t a n t s t h a n a r e a v a i l a b l e from the o r i g i n a l a* v a l u e s . The e r r o r l i m i t s f o r each compound a r e +_1 s t a n d a r d d e v i a t i o n , as g i v e n i n T a b l e IV. Where e r r o r l i m i t s a r e n o t g i v e n , t h e s e have been a p p r o x i m a t e d by t h e w o r s t e r r o r among t h e o t h e r com-pounds and a r e shown i n F i g u r e s 5 and 6 as b r o k e n l i n e s . R e a c t i o n c o n s t a n t s p* o f -2.0 and -2.6 a r e f o u n d from the s l o p e s , A l o g k/Aa*, ( e q u a t i o n 35) o f F i g u r e s 5 and 6 r e s p e c t i v e -l y . The two v a l u e s o f p* a r e c l o s e enough t o g e t h e r t h a t , A l o g k P = —JZ~ (35) c o n s i d e r i n g the e r r o r i n many o f the r a t e c o n s t a n t s , t h e r e may be no r e a l d i f f e r e n c e between the two, p a r t i c u l a r l y s i n c e t h e l a r g e r r e a c t i o n c o n s t a n t i s f o r the r e a c t i o n c a t a l y z e d by the s t r o n g e r a c i d , H^O*. U s u a l l y the more r e a c t i v e r e a g e n t i s l e s s s e l e c t i v e and t h e r e f o r e s h o u l d have a s m a l l e r r e a c t i o n c o n s t a n t . However, a s i m i l a r r e v e r s a l o f t h i s g e n e r a l i z a t i o n 71 has been r e p o r t e d by B r u i c e and B e n k o v i c f o r t h i s r e a c t i o n i n w h i c h t h e more r a p i d l y decomposed l - p r o p y l - 3 - c a r b a m o y l - l , 4 -d i h y d r o p y r i d i n e (XX) i s a l s o more s e n s i t i v e to c h a n g e s i n t h e pK o f t h e a c i d c a t a l y s t t h a n i s NADH ( X X I ) . P o s s i b l y t h e a mechanism o f t h i s r e a c t i o n i s n o t as s i m p l e as has been p r o -, 10 p o s e u . (XXI) 52 3.2 B a s e - c a t a l y z e d D e c o m p o s i t i o n R e a c t i o n s Some p y r i d i n i u m s a l t s ( V I I I ) and d i h y d r o p y r i d i n e s (V) were found to be s u s c e p t i b l e t o a v a r i e t y o f base c a t a l y z e d decornpo-H. ,H ' N I ( V I I I ) ( V ) s i t i o n r e a c t i o n s . Among the s i m p l e r r e a c t i o n s were h y d r o l y s i s o f e s t e r and amide f u n c t i o n s . Amide h y d r o l y s i s , w h i c h has been 71 7 5 o b s e r v e d p r e v i o u s l y i n NAD and model compounds ' r e a c h e d s i g n i f i c a n t l e v e l s n e a r pH 10.5, which t h e r e f o r e r e p r e s e n t e d the u p p e r pH l i m i t f o r any e x p e r i m e n t s i n c e a l l compounds s t u d i e d have an amide gr o u p a t e i t h e r the 1- o r 3 - p o s i t i o n s . Amide g r o u p s i n t h e 1 - p o s i t i o n seemed t o be s l i g h t l y more s u s c e p t i b l e t o h y d r o l y s i s t h a n t h o s e i n the 3 - p o s i t i o n b u t no q u a n t i t a t i v e measurements were made i n t h i s r e g a r d . Two e s t e r s u s e d i n t h i s work, 1 - c a r b o m e t h o x y m e t h y l -and 1 - c a r b o - i - p r o p o x y m e t h y l - 3 - c a r b a m o y l p y r i d i n i u m c h l o r i d e ( V I I I f and g) underwent r a p i d h y d r o l y s i s i n a l k a l i n e s o l u t i o n s . The m e t h y l e s t e r h y d r o l y s i s had a h a l f - l i f e o f a p p r o x i m a t e l y 3 m i n u t e s ( d e t e r m i n e d p o l a r o g r a p h i c a l l y ) a t pH 9.2 and t h e i - p r o p y l e s t e r h y d r o l y z e d a t ab o u t 15% o f t h i s r a t e . T h e r e -f o r e t h e s e two compounds c o u l d n ot be used above pH 7.5 f o r 53 CONH 2 N R R ( V I I I ) (IV) f : R 1 CH COOCH R 1 the i - p r o p y l e s t e r and 6.5 f o r the m e t h y l e s t e r . The m e t h y l e s t e r o f the l - c a r b o m e t h o x y m e t h y l - 3 - c a r b a m o y l - l , 4 - d i h y d r o p y r i d i n ( I V f ) was h y d r o l y z e d s e v e r a l hundred f o l d more slo'wly t h a n v/as the c o r r e s p o n d i n g p y r i d i n i u m s a l t . A c i d c a t a l y z e d d e c o m p o s i t i o n o f the c o r r e s p o n d i n g d i h y d r o p y r i d i n e s p r e v e n t e d t h e i r use below pl-l 7.5 meaning p o t e n t i o m e t r i c measurements, w h i c h r e q u i r e t h e p r e s e n c e o f b o t h t h e p y r i d i n i u m s a l t s and t h e d i h y d r o p y r i d i n e s , c o u l d n o t be made on the m e t h y l e s t e r a t a l l and c o u l d o n l y be made n e a r pH 7.5 w i t h the i - p r o p y l e s t e r . The p o l a r o g r a p h y and the s t u d y o f the o x i d a t i o n o f t h e d i h y d r o p y r i d i n e s by f l a v i n s were n o t g r e a t l y a f f e c t e d by the h y d r o l y s e s . e i t h e r r e p r e s e n t e d o n l y a s m a l l f r a c t i o n o f the t o t a l decompo-s i t i o n r a t e o r the pseudo base underwent f u r t h e r r e a c t i o n s . A t l e a s t two p r o d u c t s were formed from 1 - c y a n o m e t h y l - 3 - c a r b a m o y l -p y r i d i n i u m c h l o r i d e ( I l l h ) above pH 7, t h e m a j o r one b e i n g a new p y r i d i n i u m s a l t w i t h a more n e g a t i v e r e d u c t i o n p o t e n t i a l . S i m i l a r d e c o m p o s i t i o n p r o d u c t s were o b s e r v e d w i t h S e v e r a l p y r i d i n i u m s a l t s underwent b a s e - c a t a l y z e d r e -a c t i o n s a t t h e p y r i d i n i u m r i n g . 71 P s e u d o - b a s e f o r m a t i o n 1,71 54 CONH2 ( m i l ) 1,1 * - m e t h y l e n e b i s ( 3 - c a r b a m o y l p y r i d i n i u m c h l o r i d e ) ( X l X a ) , l , l ' - e t h y l e n e b i s ( 3 - c a r b a n o y l p y r i d i n i u m c h l o r i d e ) ( XI Xb) , 1 - c a r b a m o y l m e t h y l - 3 - c y a n o p y r i d i n i u m c h l o r i d e (XXIIk) and 1 - c a r b a m o y l m e t h y 1 - 3 - a c e t y l p y r i d i n i u m c h l o r i d e ( X X I I j ) above pll ' s 5, 7, and 9.5 r e s p e c t i v e l y . No a t t e m p t was made t o R 3 COCH CN 3 CH2CONH2 (XXII) b: a : n = l n=2 + \ / + (XIX) i s o l a t e t h e d e c o m p o s i t i o n p r o d u c t s as t h i s p r e l i m i n a r y i n v e s t i -g a t i o n was d e s i g n e d o n l y to f i n d t h e h i g h e s t pH a t w h i c h the compounds c o u l d be u s e d . 55 D u r i n g p o t e n t i o m e t r i c measurements, i t v/as f o u n d t h a t MAD + ( l a ) and/or NADH decomposed a t pH 8 . 8 t o g i v e a m a t e r i a l ( l a ) w i t h v e r y s i m i l a r r e d u c t i o n p o t e n t i a l s ( b o t h p o t e n t i o m e t r i c and p o l a r o g r a p h i c ) t o NMNI On the b a s i s o f p o t e n t i o m e t r i c and p o l a r o g r a p h i c r e d u c t i o n p o t e n t i a l s , ( see s e c t i o n 3 . 6 ) the d e c o m p o s i t i o n p r o d u c t was t e n t a t i v e l y i d e n t i f i e d as the m o n o n u c l e o t i d e ( I I ) a r i s i n g from h y d r o l y s i s o f t h e p y r o p h o s p h a t e HO OH ( I D bond o f NAD. The o n l y d e c o m p o s i t i o n o f NAD p r e v i o u s l y r e p o r t e d i n w e a k l y a l k a l i n e s o l u t i o n i s the h y d r o l y s i s o f t h e r i b o s e -71 p y r i d i n e bond, a r e a c t i o n which c o u l d n o t p o s s i b l y be c a u s i n g the r e s u l t s o b s e r v e d h e r e (see s e c t i o n 3 . 6 ) . 3.3 L i m i t a t i o n s on pH i n the St u d y o f NAD Model Compounds T a b l e VI summarizes the r e s u l t s o f t h e b a s e - c a t a l y z e d d e c o m p o s i t i o n s g i v e n i n S e c t i o n 3.2 and the a c i d c a t a l y z e d d e c o m p o s i t i o n s g i v e n i n S e c t i o n 3.1. The l o w e r pH l i m i t i s the pH a t which the a c i d - d e c o m p o s i t i o n o f t h e d i h y d r o p y r i d i n e s ( r e a c t i o n 22) o c c u r s to an e x t e n t o f no more t h a n 10% o v e r a p e r i o d o f 24 h o u r s . The upper pH l i m i t i s t h e maximum pH a t wh i c h r i n g r e a c t i o n s o f the p y r i d i n i u m s a l t s u c h as r e a c t i o n (36) a r e n o t o b s e r v e d o v e r a p e r i o d o f 24 h o u r s , o r t h e pH a t wh i c h the h y d r o l y s i s r e a c t i o n s (37) t h r o u g h (39) o c c u r to an e x t e n t o f no more t h a n 10% o v e r 24 h o u r s . The minimum pH f o r l - c a r b a m o y l m e t h y l - 3 - f l u o r o - 1 , 4 - d i h y d r o p y r i d i n e (v£) was so h i g h I CH 2CONH 2 (V£) t h a t p o t e n t i o m e t r i c measurements and the d e t e r m i n a t i o n o f t h e o x i d a t i o n r a t e by f l a v i n s c o u l d n o t be made. To e n s u r e t h a t 57 TABLE VI S t a b i l i t y L i m i t s f o r P y r i d i n i u m S a l t s and and D i h y d r o p y r i d i n e s N I CH COO CH. CH 2CH OH CH 2OCH CH 2COCH CH^COOCH CH CN CH 2CONH CH COHH 2 2 CH 2CONH CH 2CONH minimum pH R 3 o f d i h y d r o -p y r i d i n e CONH 0 9.1 CONH CONH. CONH. CONH CONH CH 2COOCH(CH 3) CONH CONH. CONH, COCH. CN F 9.7 9.3 8.1 7.5 7.5£ 7.0 7.8 6.5 6 . 5 11. 5 maximum pH 10.5 10.5 10.5 10.5 10.5 5.7 6.6 7.0 10.5 9.5 7.0 10.5 Base r e a c t i o n 3 - c a r b o n o y l h y d r o l y s i s 1-carbomethox ym e t h y l h y d r o l y s i s 1 - c a r b o - i - p r o p o x y -m e t h y l h y d r o l y s i s r i n g r e a c t i o n s 3 - c a r b a m o y l h y d r o l y s i s r i n g r e a c t i o n s 1 - c a r b a m o y l m e t h y l h y d r o l y s i s T a b l e VI ( c o n t . ) S p e c i a l Compounds NAD NMN minimum pH o f d i h y d r o -pyr i d i n e 7.1 7.5 H 2 N ° C \ /} \ )>-CONH2 ^ — N N — / maxxmum pH Base r e a c t i o n 9.9 unknown 10.5 3 - c a r b a m o y l h y d r o l y s i s 7.0 r i n g r e a c t i o ns T h i s v a l u e was n o t measured b u t was assumed t o be t h e same t h a t o f 1 - c a r b o r n e t h o x y m e t h y l - 3 - c a r b a m o y l - 1 , 4 - d i h y d r o p y r i d i n e 59 C H 2 C O O R CH COO" no b a s e - c a t a l y z e d r e a c t i o n s o f l - c y a n o r a e t h y l - 3 - c a r b a m o y l -p y r i d i n i u m c h l o r i d e ( V H I h ) i n t e r f e r e d , p o t e n t i o m e t r i c ( V H I h ) (Vh) 6 0 measurements on t h i s compound were made a t pH 5 . 6 , d e s p i t e t h e h i g h e r d e c o m p o s i t i o n r a t e o f the d i h y d r o p y r i d i n e (Vh) a t t h i s pH. P o t e n t i o m e t r i c measurements were n o t a t t e m p t e d on 1-c a r b o m e t h o x y m e t h y l - 3 - c a r b a m o y l p y r i d i n i u m c h l o r i d e ( I l l f ) . P o t e n t i o m e t r i c measurements on l - c a r b o - i - p r o p o x y m e t h y l - 3 -c a r b a m o y l p y r i d i n i u m c h l o r i d e ( I l l g ) were made a t pH 7 w h i c h ( I I I ) (IV) f : R 1 = CH 2COOCH 3 g: R = C H o C 0 0 C H (CH ) was a compromise betv/een the a c i d and base c a t a l y z e d decompo-s i t i o n s . A l t h o u g h , b o t h t h e e s t e r h y d r o l y s i s o f ( I l l g ) and the a c i d - d e c o m p o s i t i o n o f (IVg) were f a s t e r t h a n d e s i r e d , p o t e n t i o m e t r i c measurements were s t i l l p o s s i b l e . I f a pll much above o r below 7 was u s e d , e i t h e r the e s t e r h y d r o l y s i s o f ( I l l g ) o r the a c i d - d e c o m p o s i t i o n o f (IVg) r e a c h e d an i n t o l e r a b l e r a t e . 61 3.4 S u b s t i t u e n t C o n s t a n t s o f the 3 - C a r b a r a o y l p y r i d i n i u m Group In o r d e r t o p r o p e r l y a n a l y z e the b e h a v i o u r o f 1,1'-n e t h y l e n e b i s ( 3 - c a r b a m o y l p y r i d i n i u m c h l o r i d e ) ( X l X a ) and 1,1'-e t h y 1 e n e b i s ( 3 - c a r b a m o y l p y r i d i n i u m c h l o r i d e ) (XlXb) the i n d u c t i v e e f f e c t o f the p y r i d i n i u m g r o u p must f i r s t be known. I n t u i t i v e l y , one would e x p e c t the i n d u c t i v e s u b s t i t u e n t c o n s t a n t f o r a p y r i d i n i u m g r o u p ( X X I I I ) to c l o s e l y r e s e m b l e t h a t o f ( X X I I I ) the t r i m e t h y l a m m o n i o g r o u p o r ammonio g r o u p . To t e s t t h i s h y p o t h e s i s , the pKa o f the s u b s t i t u t e d a c e t i c a c i d ( I l i a ) was + H C H 2 C O O H ChLjCOO" ( I l i a ) 6 2 d e t e r m i n e d s p e c t r o p h o t o m e t r i c a l l y ( F i g u r e 7) and f o u n d t o be 1.7 +0.1, from w h i c h a a o f 0.76 can be c a l c u l a t e d u s i n g C h a r t o n ' s c o r r e l a t i o n s [ e q u a t i o n ( 4 0 ) ] . T h i s v a l u e i s i n Cf = 0.251 l o g K /K I x H (40) f a c t i n good agreement w i t h a f o r t h e t r i m e t h y l a m m o n i o a r o u i I 7 2 (0.73) and a p p r o x i m a t e l y 20% l a r g e r t h a n t h e v a l u e o f a 7 3 f o r the ammino g r o u p (0^ = 0.60) so the v a l u e f o r O o f (XXIV) (XXIV) ONH, -CONH, (XXII) was assumed to be c l o s e t o 2 0 % l a r g e r t h a n a f o r t h e Cll NH + I ^ 3 7 2 g r o u p . ( a = 0.36) g i v i n g i t a v a l u e o f 0 . 4 4 . The c o r r e s -p o n d i n g a * s u b s t i t u e n t c o n s t a n t s f o r t h e s u b s t i t u e n t s (XXV 7 3 and XXVI) have been c a l c u l a t e d from e q u a t i o n ( 3 4 ) g i v i n g ' C O N H , I 2 C H 2 (XXVI) C O N H , ( XXV) PH 64 v a l u e s o f 1.7 and 1 . 0 r e s p e c t i v e l y . H a v i n g d e t e r m i n e d the a ( x c n 2 ) = a i ( x ) / 0 - 4 5 <34> + i n d u c t i v e e f f e c t o f a s e c o n d p y r i d i n i u m r i n g , t h e p r o p e r t i e s o f (XlXa) and (XlXb) c a n be i n v e s t i g a t e d w i t h th e p u r p o s e o f d i s c o v e r i n g i f the p r o x i m i t y o f the two p y r i d i n e r i n g s has any e f f e c t i n a d d i t i o n t o the i n d u c t i v e e f f e c t . (XIX) See f o o t n o t e on page 4 8 ( S e c t i o n 3.1) 65 3.5 P o l a r o g r a p h y o f P y r i d i n i u m S a l t s The p o l a r o g r a p h i c r e s u l t s v/ere i n good a g r e e m e n t w i t h p r e v i o u s s t u d i e s . A t pH 5.6, NAD + (Ia),MMN + ( I I ) , and 1-methyl' 3 - c a r b a m o y l p y r i d i n i u m c h l o r i d e ( V H I b ) e x h i b i t e d one r e d u c t i o n v/ave w i t h h a l f - v / a v e p o t e n t i a l s n e a r -680 mv., -700 mv. , and -800 mv. v e r s u s the normal h y d r o g e n e l e c t r o d e (M.H.E.) r e s p e c -t i v e l y . T hese a r e i n good agreement w i t h p r e v i o u s l y r e p o r t e d HO OH HO OH ( l a ) ONH, CONH, ( V H I b ) h a l f - w a v e p o t e n t i a l s f o r t h e s e c o m p o u n d s . 5 ' 4 0 ' 4 4 Mo prewaves v/ere o b s e r v e d w i t h t h e s e compounds i n agreement v / i t h p r e v i o u s 5,40,44 work The r e d u c t i o n wave o f 1 , 1 ' - e t h y l e n e b i s ( 3 -c a r b a m o y l p y r i d i n i u m c h l o r i d e ) p r e v i o u s l y r e p o r t e d by t o be 66, 67 near -600 mv . -3 v/as o n l y o b s e r v e d a t c o n c e n t r a t i o n s o v e r 2 x 10 m o l a r . A t lov/er c o n c e n t r a t i o n s a s i n g l e v/ave v/as 6 6 o b s e r v e d a t a p p r o x i m a t e l y -430 mv. * B o t h t h e prewave and main wave o f 1 , 1 ' - m e t h y l e n e b i s ( 3 - c a r b a m o y l p y r i d i n i u m c h l o r i d e ) were o b s e r v e d near t h e p r e v i o u s l y r e p o r t e d p o t e n t i a l s o f -180 mv. and -420 mv. r e s p e c t i v e l y . 6 6 The o t h e r p y r i d i n i u m compounds have n o t been s t u d i e d p r e v i o u s l y . O n l y one compound, l - c a r b a m o y l m e t h y l - 3 - a c e t y l p y r i d i n i u m c h l o r i d e ( V I I I j ) , e x h i b i t e d tv/o r e d u c t i o n waves a t a l l c o n c e n -C H 2 C O N H 2 ( V I I I j ) t r a t i o n s . The f i r s t wave was i n d e p e n d e n t o f pH, as has been 5 4 0 found ' f o r the r e d u c t i o n v/ave (v/ave I i n F i g u r e 2) o f o t h e r p y r i d i n i u m s a l t s . The s e c o n d wave had a pH d e p e n d e n c e o f a b o u t 35 m i l l i v o l t s p e r pH u n i t . B a s e d on t h e pH d e p e n d e n c e , the f i r s t wave c o r r e s p o n d s to the r e d u c t i o n t o t h e r a d i c a l (wave I ) . The r e d u c t i o n o c c u r r i n g a t the s e c o n d wave i s n o t known b u t may i n v o l v e r e d u c t i o n t o a d i h y d r o p y r i d i n e . W i t h i n c r e a s i n g c o n c e n t r a t i o n , t h e s e c o n d wave s h i f t s to more n e g a t i v e p o t e n t i a l s by 41 m i l l i v o l t s p e r c o n c e n t r a t i o n d e c a d e . -3 At c o n c e n t r a t i o n s above 10 I!., the p l a t e a u o f t h e f i r s t v/ave A prewave has been r e p o r t e d f o r t h i s compound a t -380 mv. A d i f f e r e n c e o f 50 mv. betv/een r e p o r t e d h a l f - w a v e p o t e n t i a l s o f p y r i d i n i u m compounds i s n o t uncommon.40 67 s h i f t s n e g a t i v e l y by a much l a r g e r amount, u n t i l a t h i g h c o n c e n -t r a t i o n s the two waves merge as shown i n F i g u r e 8, p r e v e n t i n g an a n a l y s i s o f t h e f i r s t v/ave b e i n g made. The h a l f - w a v e p o t e n t i a l o f wave I ( F i g u r e 2, page 25) has been r e p o r t e d t o s h i f t t o more n e g a t i v e p o t e n t i a l s v/ith 4 3,44 -3 d e c r e a s i n g c o n c e n t r a t i o n below 10 M. A more complex c o n c e n t r a t i o n dependence v/as o b s e r v e d i n t h i s s t u d y as shown i n F i g u r e 9 f o r l - m e t h y l - 3 - c a r b a m o y l p y r i d i n i u m i o d i d e . S i m i l a r b e h a v i o u r v/as o b s e r v e d v/ith the o t h e r p y r i d i n i u m s a l t s s t u d i e d . A t c o n c e n t r a t i o n s below 10 3 H . the h a l f - w a v e p o t e n t i a l s h i f t s to more n e g a t i v e p o t e n t i a l s v/ i t h d e c r e a s i n g c o n c e n t r a t i o n . -3 The h a l f - w a v e p o t e n t i a l p a s s e s t h r o u g h a maximum n e a r 10 I-}. and t h e n r e t u r n s t o more n e g a t i v e v a l u e s w i t h i n c r e a s i n g c o n -c e n t r a t i o n . The u n u s u a l c o n c e n t r a t i o n dependence c o u l d be due to a change o f mechanism o f the e l e c t r o d e r e a c t i o n , one -4 mechanism o p e r a t i n g below 5 x 10 M. c o n c e n t r a t i o n s and tne -3 o t h e r o p e r a t i n g aPove 10 H. c o n c e n t r a t i o n s . O t h e r e v i d e n c e b e a r i n g on t h i s v / i l l be p r e s e n t e d i n s u b s e q u e n t s e c t i o n s ( S e c t i o n 3.5.1 and 3 . 5 . 3 ) . The dependence of the l i m i t i n g c u r r e n t on t h e h e i g h t of the m e r c u r y column was d e t e r m i n e d f o r l - c y a n o m e t h y l - 3 - c a r b a m o y l -p y r i d i n i u m c h l o r i d e . A p l o t of l o g v s . l o g h was l i n e a r w i t h a s l o p e o f 0.455, i n good agreement w i t h the t h e o r e t i c a l dependence of 0.5 f o r a d i f f u s i o n c o n t r o l l e d r e d u c t i o n p r o c e s s . T h i s r e s u l t a g r e e s w i t h t h e f i n d i n g s of p r e v i o u s w o r k e r s f o r o t h e r p y r i d i n i u m i o n s . -600 mv. -600 mv. l o g C (mM) 70 3.5.1 A n a l y s i s o f Wave I . In v i e w o f the t h e o r e t i c a l t r e a t m e n t bv S o n n a t e r r e a na 3 3 C a u q u i s , a c o m p a r i s o n was maae between p l o t s o f p o t e n t i a l , E, v e r s u s t h r e e c u r r e n t f u n c t i o n s , l o g ( i , - i ) / ( i ) , a 2/3 1/2 l o g ( i , - i ) / ( i ) , and l o g ( i , - i ) / ( i ) . These p l o t s a r e shown i n F i g u r e s 10 and 1.1 f o r 1 - m e t h y l - 3 - c a r b a m o y l -p y r i d i n i u m i o d i d e and MAD 4, r e s p e c t i v e l y . A f i r s t c r i t e r i o n f o r c o m p a r i s o n i s l i n e a r i t y . I t i s r e a d i l y a p p a r e n t t h a t none o f the c u r r e n t f u n c t i o n s a p p l y o v e r t h e e n t i r e c o n c e n t r a -- 5 - 2 -4 t i o n r ange from 10 to 10 M . Below 10 M . p l o t s o f E 2/3 1/2 v e r s u s l o g ( i , - i ) / ( i ) o r l o g ( i ., - i ) / ( i ) a r e l i n e a r . d " d - 3 A t c o n c e n t r a t i o n s above 10 M . p l o t s o f E v e r s u s l o g ( i - i ) / ( i ) have o n l y a s m a l l c u r v a t u r e n e a r t h e low-c u r r e n t end o f the l i n e . The r e m a i n i n g p l o t s a l l show c o n -s i d e r a b l e c u r v a t u r e . A s e c o n d c r i t e r i o n f o r c o m p a r i s o n i s s l o p e . (The s l o p e i s the change i n p o t e n t i a l p e r u n i t change i n t h e l o g - c u r r e n t f u n c t i o n , l o g ( i ^ - i ) / ( i ) , e t c . and has the u n i t s o f m i l l i -3 3 v o l t s . ) A s l o p e o f 59 mv. i s p r e d i c t e d f o r p l o t s o f p o t e n t i a l 1/2 2/3 v e r s u s l o g ( i , - i ) / ( i ) o r l o g ( i , - i ) / ( i ) . The r e s u l t s d d of t h e s e l o w - c o n c e n t r a t i o n p l o t s , w h i c h were the o n l y l i n e a r p l o t s , a r e l i s t e d i n T a b l e V I I . The s l o p e s o f t h e s e p l o t s a r e g e n e r a l l y h i g h e r t h a n p r e d i c t e d w h i c h may be due t o a d s o r p t i o n e f f e c t s o r may s i m p l y be r e l a t e d t o t h e d i f f i c u l t y o f g e t t i n g w e l l r e s o l v e d p o l a r o g r a m s a t c o n c e n t r a t i o n s a p p r o a c h -i n g the d e t e c t i o n l i m i t o f t h e i n s t r u m e n t . The p l o t s o f t h e 2/3 p o t e n t i a l v e r s u s l o g ( i - i ) / ( i ) have s l o p e s c l o s e r to the 'igure 10a. P o l a r o g r a p h i c a p p l i e d p o t e n t i a l v e r s u s l o g ( i d - i ) / ( i ) f o r l - m e t h y l - 3 - c a r b a m o y l p v r i d i n i u m c h l o r • r—— 1 1 : ~~| , -9 25 -950 •975 i—i u |? -1000 \ < > -1025 •1050 •1075 -1100 -1125 0.1 Tt. a c e t a t e , pH = 5.6 0.0157 m!i 0.10 mil. 2.0 mM. 10.0 mM. -1.0 •0.5 0 . 0 0.5 1.0 l o g ( i - i ) / ( i ) F i g u r e 10b. P o l a r o g r a p h i c a p p l i e d p o t e n t i a l v e r s u s 7 l o g ( i d - i ) / ( i ) 2 / 3 f o r l - m e t h y l - 3 - c a r b a m o y l p y r i d i n i u m c h l o r i d e ' ~ i — — i 1 — 1 — — — | 0.1 M a c e t a t e , pH = 5.6 •925 •950 -975 o t r \ Cn > -1000 •1025 > -1050 -1075 •1100 -112! -0.0157 mM .0.10 mM •2.0 mM 10.0 mM •1.5 -1.0 -0.5 0.0 0.5 l o g ( i d - i ) / ( i ) 2 / 3 1.0 F i g u r e 10c. P o l a r o g r a p h i c a p p l i e d p o t e n t i a l v e r s u s l o g ( i d - i ) / ( i ) I / 2 f o r l - m e t h y l - 3 - c a r b a r a o y l p y r i d i n i u r n c h l o r i d e 0.1 M. a c e t a t e , pH = 5.6 •9 25 0.0157 mM. -950 0.10 mM. 2 . 0 mil. 10.0 mM, •975 O -1000 < « -1025 > > e •1050 -1075 •1100 -1125 •1.5 -1.0 •0.5 0.0 l o g ( i d - i ) / ( i ) 1 / 2 0.5 1 . 0 7 4 F i g u r e 11a. P o l a r o g r a p h i c a p p l i e d p o t e n t i a l v e r s u s l o g ( i d - i ) / ( i ) f o r n i c o t i n a m i d e a d e n i n e d i n u c l e o t i d e u < t r < > -790 - 3 2 0 - 3 5 0 880 •910 -94 0 -970 -1000 -1030 0.1 M. a c e t a t e , pH 0.016 mM. 0 . 023 mM. — . 0.18 mM. x 1.8 mM. 9.0 mM. 5 . 6 - 1 . 5 - 1 . 0 •0.5 0.0 l o g ( i - i ) / ( i ) 0 . 5 1.0 1 . 5 75 P o l a r o g r a p h i c a p p l i e d p o t e n t i a l v e r s u s ) / f o r n i c o t i n a m i d e a d e n i n e d i n u c l e o t i d e a c e t a t e , pH 0.016 mM. 0.023 mM. 0.13 mM. = 5.6 1.8 mM. 9.0 mM. / —I , L _ 0.0 0.5 1.5 •1.0 -0.5 l o g ( i d - i ) / ( i ) 2 / 3 1.0 F i g u r e l o g ( i d 76 11c. P o l a r o g r a p h i c a p p l i e d p o t e n t i a l v e r s u s - i j / d ) 1 / 2 f o r n i c o t i n a m i d e a d e n i n e d i n u c l e o t i d e -2.0 -1. 5 -1.0 l o g ( i d -0.5 - i ) / ( i ) 0.0 1/2 0 . 5 1.0 7 7 TABLE V I I S l o p e s and I n t e r c e p t s o f P l o t s o f P o t e n t i a l v e r s u s l o g ( i d - i ) / ( i ) 2 / 3 and l o g ( i - i ) / ( i ) 1 / 2 S e r i e s I : CONH, E a g a i n s t E a g a i n s t l o g ( i d - i ) / ( i ) 2 / J l o g ( i d " 1 ) 7 R l c o n c e n t r a -X t i o n (mM) s l o p e i n t e r c e p t s l o p e i n t e r (mv) (mv) (mv) (mv CH 2CN . 0106 76 - 4 9 0 79 -455 .0511 60 -513 72 -487 .156 72 -474 78 -459 . 248 73 -497 82 -484 CH 2COOCH .0116 6 6 -612 72 -531 . 0167 63 -613 73 -579 . 0256 70 -615 83 -581 . 0438 63 -602 74 -577 . 0723 70 -6 08 80 -582 CH 2COOCH(CH ] .00932 65 -631 6 6 -601 .0141 * 63 -623 76 -581 .0141* 70 -632 61 -609 CH 2COCH .0109 59 -698 6 3 -668 . 0157 66 -692 74 -660 . 0240 70 -674 79 -656 CH OCH„ 2 3 .00977 59 -662 63 -633 .0251 72 -630 7 9 -601 .0360 75 -639 83 -615 .0475 66 -626 75 -603 1/2 * t h e same s o l u t i o n s r e c o r d e d a t d i f f e r e n t c u r r e n t s e n s i t i v i t i e i 78 T a b l e V I I ( c o n t . ) CH 2CH 2OH CH. c o n c e n t r a -t i o n (mM) .0176 . 0253 .0453 . 0157 2.0 E a g a i n s t l o g ( i - i ) / ( i ) 2/3 "d s l o p e (rav) 4 6 59 74 59 69 i n t e r c e p t (mv) -768 -773 -7 63 -833 -797 E a g a i n s t l o g ( i d - i ) / ( i ) s l o p e i n t e r c e p t 1/2 (mv) 55 34 82 67 77 (mv) -748 -751 -739 -8 09 -800 CH COO .0182 73 -842 98 •7 99 S e r i e s I I R 3 COCH. CN .00952 , 0182 ,0261 , 040 ,00952 ,0261 . 040 ,20 , 01«? 0261 20 00952 040 20 CH 2 CONH 2 4 9 62 55 5 8 64 66 62 67 55 62 66 55 60 69 -511 -505 -503 -503 -4 29 -423 -431 -422 -821 -8 2 2 -815 -1109 -1087 -1063 63 68 57 66 73 87 72 73 64 69 73 65 84 90 -479 -4 5 5 -4 8 3 -481 -3 94 -38 2 -406 -410 -797 -798 -803 -1030 -1063 -1054 79 T a b l e V I I ( c o n t . ) E a g a i n s t R. CONH. c o n c e n t r a -t i o n (mM) . 0145 .0515 . 0776 .127 0.2 0.7 2.0 10.0 l o 9 ( i d s l o p e (mv) 71 72 72 74 72 70 74 97 i ) / ( i ) V3 E a g a i n s t i n t e r c e p t (mv) -663 -651 -651 -642 -1245 -1243 -1231 -1246 l o g ( i d s l o p e (mv) 73 81 81 80 33 73 80 110 i ) / ( i ) 1/2 i n t e r c e p t (mv) -632 -626 -629 -626 -12 3 6 -1242 -1237 -127 0 S p e c i a l Compounds: Compound + pH 5.6 .016 NAD ( l a , page 65) .023 . 036 NMN+, pH 4.6 .020 ( I I , page 65) .030 .15 .018 .026 . 04 63 62 71 81 75 8 3 104 84 105 -637 -679 -666 -713 •700 •693 •718 •702 •707 72 66 82 9 3 87 9 8 -656 -657 •635 •677 •671 •680 CONH, .00952 .0182 . 0261 .04 0 .20 8 4 85 70 84 8 7 -4 2 2 -429 •448 •443 •427 7 9 83 70 94 94 -389 -401 -425 -414 •413 CONH, 80 Table V I I ( c o n t . ) E a g a i n s t E a g a i n s t Compound H 2NOC Wave I 1W a v e I c o n c e n t r a -t i o n (mM) CONH. .00952 .0102 .0261 .040 .20 .00952 .0182 .0261 . 040 .20 l o g ( i s l o p e (mv) 68 8 3 97 7 8 76 72 96 70 108 98 i ) / ( i ) J/3 i n t e r c e p t (mv) -197 -181 -18 0 -2 28 -201 -390 -390 -428 -435 -4 3 9 l o q ( i d s l o p e (mv) 72 111 110 90 86 70 104 75 120 89 i ) / ( i ) 1/2 i n t e r c e p t (mv) -167 -15 5 -145 -200 -187 -377 -361 -405 -4 08 -430 81 t h e o r e t i c a l v a l u e , i n d i c a t i n g e q u a t i o n (18) may be t h e a p p r o -p r i a t e e q u a t i o n t o u s e . T a b l e V I I I l i s t s t h e s l o p e s and + ~ fc.n(i. - i ) / ( i ) 2 / 3 (18) r Q h a l f - w a v e p o t e n t i a l s d e r i v e d from t h e l i n e a r p o r t i o n s o f p l o t s o f p o t e n t i a l v e r s u s l o g ( i ^ - i ) / ( i ) a t 2.0 mM. and 10.0 mM. c o n c e n t r a t i o n s . The s l o p e s o f most o f t h e s e p l o t s a r e l a r g e r t h a n t h e t h e o r e t i c a l v a l u e o f 60 mv., i n d i c a t i n g t h a t an i r r e v e r s i b l e e l e c t r o d e r e a c t i o n i s t a k i n g p l a c e . T h i s i r r e v e r s i b i l i t y i s n o t i n d u c e d m a i n l y by t h e d i m e r i z a t i o n 2/3 r e a c t i o n s i n c e t h e p l o t s o f p o t e n t i a l v e r s u s l o g ( i ^ - i ) / ( i ) 1/2 and l o g ( i n - i ) / ( i ) showed c o n s i d e r a b l e c u r v a t u r e and d e q u a l l y l a r g e s l o p e s . T h i r d l y , t h e o r y p r e d i c t s t h a t t h e i n t e r c e p t s i n e q u a t i o n s ( 1 0 ) , ( 1 4 ) , and (18) s h o u l d be i n d e p e n d e n t o f c o n c e n t r a t i o n . E = £° + £n(i - i ) / ( i ) (10) CtnF d E = e° + £ | £ n ( i , - i ) / ( i ) 1 / 2 (14) F a T h i s c o n d i t i o n i s a p p r o x i m a t e l y f u l f i l l e d by t h e low c o n c e n t r a -2/3 t i o n p l o t s o f p o t e n t i a l v e r s u s b o t h l o g ( i ^ - i ) / ( i ) and 1/2 ( i , - i ) / ( i ) . The means and s t a n d a r d d e v i a t i o n s o f t h e d i n t e r c e p t s o f t h e p l o t s a r e l i s t e d i n T a b l e IX. The s t a n d a r d d e v i a t i o n s r a n g e from 2 mv. up t o 27 mv. w i t h v a r i a t i o n s i n 82 TABLE V I I I S l o p e s and H a l f - w a v e P o t e n t i a l s O b t a i n e d by P l o t t i n g A p p l i e d P o t e n t i a l V e r s u s l o g ( i ^ - i ) / ( i ) S e r i e s I : R l 2 . 0 mM. 10 . 0 mM. s l o p e H a l f - w a v e s l o p e H a l f - w a v e (mv) p o t e n t i a l (mv) p o t e n t i a l (mv) (mv) CH CN 74 -554 92 -581 CH COOCH 3 74 -657 94 -692 C H 2COOCH(CH) 2 76 -668 90 -696 CH COCH 33 -699 97 -729 CH 2OCH 3 77 -657 85 -679 CH 2CH OH 6 6 -758 83 -780 58 -793 84 -813 CH 2COO~ 90 -826 101 -922 S e r i e s I I : •? R 3 C H 2 C O N H 2 CONH„ 2 74 -682 83 -710 CN 67 -509 90 -524 F 51 -8 38 67 -869 H 83 -1029 88 -1056 T a b l e V I I I ( c o n t . ) 83 2.0 mM. s l o p e H a l f - w a v e (mv) p o t e n t i a l (mv) 10.0 mM. s l o p e H a l f - w a v e (mv) p o t e n t i a l (mv) 61 -1224 75 -1211 S p e c i a l compounds: + N A D ( l a , page 65) 55 •683 64 -715 NT ( I I , page 65) 115 •810 103 -83 0 CONH 124 -593 129 90 672 •469 CONH, 84 TABLE IX Mean I n t e r c e p t s from P l o t s o f P o t e n t i a l v e r s u s l o g ( i d - i ) / ( i ) 2 / 3 and l o g ( i - i ) / ( i ) 1 / 2 S e r i e s I CONH, l o g ( i d - i ) / ( i ) 2 / 3 i n t e r c e p t , e° (mv. v s . N.H.E.) l o g ( i d - i ) / ( i ) i n t e r c e p t (mv. v s . N.H.E. 1/2 CH CN 2 CH COOCH CH.COOCH(CH,) 3' 2 CH 2COCH CH OCH CH CH OH CH. CH COO -494 + 16 -610 + 5 -629 +_ 5 -688 + 12 -639 + 16 -768 + 5 -815 + 25 -842 -471 + 17 -580 +_ 2 -605 + 6 -661 + 6 -613 + 15 -746 + 6 -805 + 5 -799 l e r i e s I I : R. C H 2 C O N H 2 CONH. CN COCH. F -652 + 8 -426 + 4 -506 + 4 -819 + 4 -6 28 + 3 -400 + 10 •480 +_ 3 •799 + 3 85 T a b l e IX ( c o n t . ) R H o" l o g ( i - i ) / ( i ) 2/3 i n t e r c e p t , e° (mv. v s . N.H.E.) -1086 + 23 -1241 + 7 l o g ( i d - i ) / ( i ) 1 / 2 i n t e r c e p t (mv. v s . N.H.E.) -1066 + 13 -1246 + 16 S p e c i a l compounds NAD + ( l a , page 65) NMN + ( I I , page 65) -677 + 10 •703 + 8 •649 + 12 •676 + 5 H 2 N O C -oo-\ = : N N = / + \ / • CONH, -434 + 11 -408 + 14 H 2 N O C Wave I • Wave I ONH. -209 + 17 -409 + 27 -185 + 17 -391 + 20 86 t h e i n t e r c e p t b e i n g random w i t h r e s p e c t to c o n c e n t r a t i o n c h a n g e s . The use o f the c h a r g i n g c u r r e n t c o m p e n s a t i o n , w h i c h i s n e c e s s a r y t o h o l d t h e b a s e l i n e c o n s t a n t a t low c o n c e n t r a t i o n s , c a u s e s s h i f t s i n t h e a p p l i e d p o t e n t i a l s o f as much as 100 mv. li e t r o h m s u p p l i e s a nomogram t o c o r r e c t f o r t h i s p o t e n t i a l s h i f t b u t the e r r o r c o u l d e a s i l y r e a c h 15 t o 20 mv. a t c o n c e n t r a t i o n s -5 a p p r o a c h i n g 10 M. B a s e d on t h e p r e c e d i n g a n a l y s i s , o n l y t h e low c o n c e n t r a -t i o n waves a r e c o n s i s t e n t w i t h any o f t h e t h e o r e t i c a l e q u a t i o n s v/hich m i g h t a p p l y f o r a r e v e r s i b l e e l e c t r o d e r e a c t i o n . The o n l y c r i t e r i o n v/hich d i s t i n g u i s h e s between p l o t s o f p o t e n t i a l 1/2 2/3 v e r s u s l o g ( i - i ) / ( i ) and l o g ( i - i ) / ( i ) i s s l o p e and the o b s e r v e d v a r i a t i o n s i n t h i s q u a n t i t y make i t i n c o n -c l u s i v e . M e c h a n i s t i c a l l y , t h e r e i s l i t t l e t o c h o o s e between e q u a t i o n s (14) and ( 1 8 ) , b o t h o f w h i c h a r e c o n s i s t e n t w i t h r e a c t i o n s (1) and (2) i n Scheme 3 r e p r o d u c e d i n e q u a t i o n ( 4 1 ) . (41) E q u a t i o n (14) was d e r i v e d a s s u m i n g a r a p i d d i m e r i z a t i o n 38 r e a c t i o n . The d i m e r i z a t i o n r e a c t i o n has been f o u n d t o be E = e° + — £ n ( i d - i ) / ( i ) (14) r a p i d ; thus i f the d i m e r i z a t i o n e q u i l i b r i u m i s d i s p l a c e d f a r enough tov;ards p r o d u c t s to make t h i s r e a c t i o n i r r e v e r s i b l e , as was assumed i n t h e d e r i v a t i o n o f e q u a t i o n ( 1 0 ) , e i t h e r e q u a t i o n c o u l d a p p l y . E = e o + R| £ n ( i ^ _ i ) / ( i ) 2 / 3 ( 1 3 ) The i n t e r c e p t s l i s t e d i n T a b l e IX a r e p l o t t e d a g a i n s t each o t h e r i n F i g u r e 12. The e x c e l l e n t c o r r e l a t i o n and s l o p e near u n i t y s u g g e s t t h a t the two s e t s o f d a t a a r e e s s e n t i a l l y the same and, t h e r e f o r e , s u b s e q u e n t d i s c u s s i o n s w i l l i n v o l v e o n l y d a t a d e r i v e d from p l o t s o f p o t e n t i a l v e r s u s l o g ( i d - i ) / ( i ) 2 / 3 . 3.5.2 Prewave F o r m a t i o n Prewaves were o b s e r v e d i n the p o l a r o g r a m s o f 1-methoxy m e t h y 1 - 3 - c a r b a m o y l p y r i d i n i u m c h l o r i d e ( V l l l e ) , 1 - c a r b o m e t h o x y m e t h y l - 3 - c a r b a m o y l p y r i d i n i u m c h l o r i d e ( V l l l f ) , 1 - c a r b o - i -p r o p o x y m e t h y l - 3 - c a r b a m o y l p y r i d i n i u m c h l o r i d e ( V H I g ) , l - c y a n o m e t h y l - 3 - c a r b a m o y l p y r i d i n i u m c h l o r i d e ( V l l l h ) , l - c y a n o m e t h y l - 3 - c a r b a m o y l p y r i d i n i u m c h l o r i d e ( V l l l h ) , l - c a r b a m o y l m e t h y l - 3 - c y a n o p y r i d i n i u m c h l o r i d e ( V H I k ) , and 1 ' 1 , - m e t h y l e n e b i s ( 3 - c a r b a m o y l p y r i d i n i u m c h l o r i d e ) ( X l X a ) . A t h i g h e r c o n c e n t r a t i o n s , t h e prewave wave h e i g h t becomes indepe: d e n t o f c o n c e n t r a t i o n as shown i n F i g u r e 13. T h i s b e h a v i o u r . t y p i c a l o f a d s o r p t i o n waves i n which the maximum d i f f u s i o n 88 F i g u r e 12. C o m p a r i s o n o f the i n t e r c e p t s from p l o t s o f p o l a r o g r a p h i c p o t e n t i a l v s . l o g ( i ^ - i ) / ( i ) a n d l o g ( i c j - i ) / ( i ) 2/3 f o r compounds i n S e r i e s I and S e r i e s I I . , 1 , , 1 7  c o r r . c o e f f . = 0.999 s l o p e = 1.01 L - 1 — — I . L _ _ 1 , -1000 -900 -800 -700 -600 -500 2 / 3 i n t e r c e p t s from p l o t s o f E v s . l o g ( i - i ) / ( i ) ' d *see T a b l e X f o r the c o r r e s p o n d i n g l i s t o f compounds 89 TABLE X L i s t o f Compounds P l o t t e d i n F i g u r e 12 R l 1. CH 2CONH 2. CH CN 3. CI! CON H 2 2 4. CH COOCH 5. CH C O O C H ( C H 3 ) 2 6. CII 2OCH 7. CH CONH 8. CH COCH 3 9. CH CH OH 10. CH CONH„ 2 2 11. CH. 12. CH COO 13. CH CONH n 3 CN CONH. COCH. CONH. CONH . CONH, CONH. CONH. CONH, 4. F CONH , CONH, F i g u r e 13. The c o n c e n t r a t i o n d e p e n d e n c e o f t h e prewave l i m i t i n g c u r r e n t f o r t h e p o l a r o g r a p h i c r e d u c t i o n o f l - c a r b o - i - p r o p o x y m e t h y l - 3 - c a r b a m o y l -p y r i d i n i u m c h l o r i d e ' i r — — — — i 1 » i , , . , _ 91 e: R 1=CH 2OCH f : g : h: k: CH„C00CI1 3 CH COOCH(CH ) CH^CN CH CONH P-3=CONH. CONH. CONH. CONH. CN ( V I I I ) H 2 NOC-<(^ / \ ^l>--CONH 2 (XlXa) c u r r e n t c o r r e s p o n d s to the f o r m a t i o n o f a m o n o l a y e r o f a d s o r b e d 3 7 3 9 m a t e r i a l on the e l e c t r o d e s u r f a c e . ' T a b l e XI g i v e s the c o n c e n t r a t i o n c o r r e s p o n d i n g to the maximum d i f f u s i o n c u r r e n t o f the prewave and the a p p r o x i m a t e d i f f e r e n c e between t h e h a l f -wave p o t e n t i a l s o f the prewave and main wave f o r e a c h compound. The maximum prewave d i f f u s i o n c u r r e n t o f l - c a r b o - _ i -p r o p o x y m e t h y l - 3 - c a r b a m o y l p y r i d i n i u m c h l o r i d e was i n d e p e n d e n t o f t e m p e r a t u r e . T h i s b e h a v i o u r i s c o n s i s t e n t w i t h a s t r o n g 3 9 a d s o r p t i o n p r o c e s s i n wh i c h c a s e t h e maximum d i f f u s i o n c u r r e n t would be a f f e c t e d o n l y by f a c t o r s w h i c h a f f e c t the s i z e o f the m e r c u r y d r o p . In agreement v/ith t h i s , o n l y t h e prewave i s o b s e r v e d a t low c o n c e n t r a t i o n s f o r a l l o f the com-pounds e x c e p t 1 , 1 ' - m e t h y l e n e b i s ( 3 - c a r b a m o y l p y r i d i n i u m c h l o r i d e ) . A t t h i s p o i n t i t s h o u l d be r e c a l l e d t h a t the p l o t s a t v e r y low 92 TABLE XI C o n c e n t r a t i o n s C o r r e s p o n d i n g to the Maximum H e i g h t o f the P o l a r o g r a p h i c Prewaves and the P o t e n t i a l D i f f e r e n c e Between Main Wave and Prewave R l p "3 C o n c e n t r a t i o n c o r r e s -p o n d i n g to the maximum d i f f u s i o n c u r r e n t (mM) P o t e n t i a l d i f f e r e n c e * (mv) CH OCH CONH „ 2 0.20 100 CH COOCH CONH„ 2 0.32 90 CH 2COOCH(CH 3) CONH, 2 0 .34 150 CH CN CONH 2 0.36 130 CII 2C0NH CN 0.4 9 150 H 2 N O C CONH, ^.1 200 I t has n o t been p r o v e n w h i c h wave (main wave o r p r e w a v e ) , i f e i t h e r , r e p r e s e n t s the t r u e r e d u c t i o n p o t e n t i a l o f t h e s e com-pounds. D e p e n d i n g on w h i c h wave does, i n f a c t , r e p r e s e n t t h e t r u e r e d u c t i o n p o t e n t i a l , e r r o r s as l a r g e as t h e p o t e n t i a l d i f f e r e n c e s c o u l d be i n t r o d u c e d . 93 c o n c e n t r a t i o n s ( F i g u r e s 10 and 11) were c o n s i s t e n t w i t h a r e v e r s i b l e r e d u c t i o n f o l l o w e d by an i r r e v e r s i b l e d i m e r i z a t i o n whereas the p l o t s a t h i g h c o n c e n t r a t i o n i n d i c a t e d an i r r e v e r -s i b l e r e d u c t i o n ( s e e S e c t i o n 3.5.1). F o r compounds e x h i b i t i n g a prewave, t h e n , i t i s t h e prewave w h i c h i s c o n s i s t e n t w i t h r e a c t i o n (41) and t h e main wave w h i c h e x h i b i t s i r r e v e r s i b l e (41) b e h a v i o u r . T h i s b e h a v i o u r i s c o n s i s t e n t w i t h the a d s o r p t i o n 48 mechanism p r o p o s e d by L a v i r o n f o r the r e d u c t i o n o f 3 - a c e t y l -p y r i d i n e s (see S e c t i o n 1.5 I n t r o d u c t i o n , E l e c t r o c h e m i s t r y ) i n which the r e d u c t i o n o f the b u l k o f the 3 - a c e t y l p y r i d i n e i s i n h i b i t e d by a l a y e r o f a d s o r b e d r e d u c t i o n p r o d u c t . The p o l a r o g r a p h i c b e h a v i o u r o f 1 ' 1 - m e t h y l e n e b i s ( 3 -c a r b a m o y l p y r i d i n i u m c h l o r i d e ) was more complex t h a n the o t h e r compounds. At low c o n c e n t r a t i o n s , b o t h a prewave and t h e main wave a r e seen as shown i n F i g u r e 14, i n c o n t r a s t to o t h e r p y r i d i n i u m compounds f o r v/hich the main wave was n o t seen a t c o n c e n t r a t i o n s l e s s t h a n t h a t c o r r e s p o n d i n g t o the prewave maximum. The s e c o n d wave v a r i e s between one and two t i m e s the h e i g h t o f the f i r s t wave i n an a p p a r e n t l y random way. T h i s v a r i a t i o n i s p r o b a b l y r e l a t e d to e x p e r i m e n t a l d i f f i c u l t i e s e n c o u n t e r e d a t s u c h low c o n c e n t r a t i o n s . The p o t e n t i a l d i f f e r e n c e F i g u r e 14. P o l a r o g r a m o f a 1.8 x 1 0 ~ 5 M. s o l u t i o n o f 1 , 1 ' - m e t h y l e n e b i s -( 3 - c a r b a m o y l p y r i d i n i u m c h l o r i d e ) wave I 0 . 0 5 y A 100 mv. -500 mv. ( v s . Ag/AgCl) between the prewave and main wave v/as a p p r o x i m a t e l y 2 0 0 mv. , a l a r g e r v a l u e t h a n f o u n d f o r any o t h e r p y r i d i n i u m compound. W i t h such a l a r g e p o t e n t i a l d i f f e r e n c e , i t becomes i m p o r t a n t t o know w h i c h wave r e p r e s e n t s the t r u e r e d u c t i o n p o t e n t i a l . 66 L o v e c c h i o has p r o p o s e d t h a t the f i r s t wave c o r r e s p o n d s t o r e d u c t i o n o f a d s o r b e d r e a c t a n t w h i l e t h e s e c o n d wave c o r r e -sponds to r e d u c t i o n o f s o l u b l e r e a c t a n t , t h e l a t t e r p r o c e s s p o s s i b l y b e i n g i n h i b i t e d by a d s o r b e d p r o d u c t . The two waves c o u l d n o t be d i f f e r e n t i a t e d on the b a s i s o f the p o t e n t i a l - l o g c u r r e n t a n a l y s e s l i s t e d i n T a b l e V I I , so p o l a r o g r a m s were r e c o r d e d a t t e m p e r a t u r e s between 13° and 50° t o see i f one wave i n c r e a s e d i n h e i g h t r e l a t i v e t o t h e o t h e r . I f the p r e s e n c e o f the two waves i s due to a p a r t i a l a d s o r p t i o n , as 6 6 s u g g e s t e d , t h e n i n c r e a s i n g the t e m p e r a t u r e s h o u l d i n c r e a s e the h e i g h t o f t h e t r u e r e d u c t i o n wave and d e c r e a s e t h e wave 3 9 which i s due t o a d s o r p t i o n e f f e c t s . The r e s u l t s o f t h e t e m p e r a t u r e s t u d y a r e shown i n F i g u r e 15. The waves a p p e a r t o merge as the t e m p e r a t u r e i s i n c r e a s e d and t h e r e s u l t s a r e t o o ambiguous to a l l o w the wave t h a t r e p r e s e n t s t h e t r u e r e d u c t i o n p o t e n t i a l t o be i d e n t i f i e d . P o s s i b l y the t r u e r e d u c t i o n po-t e n t i a l f a l l s somewhere i n between the two waves t h a t a r e o b s e r v e d . F i g u r e 15. The e f f e c t o f t e m p e r a t u r e change on t h e p o l a r o g r a m o f a 2.3 x 10 s o l u t i o n o f 1 , 1 ' - m e t h y l e n e b i s ( 3 - c a r b a m o y l p y r i d i n i u m c h l o r i d e ) 45° -350 mv. 97 3.5.3 The E f f e c t s o f 1 - S u b s t i t u e n t s on P o l a r o g r a p h y R e d u c t i o n P o t e n t i a l s . I n i t i a l l y the e f f e c t o f s u b s t i t u e n t s on t h e e l e c t r o -c h e m i c a l r e d u c t i o n p o t e n t i a l was e s t i m a t e d from p l o t s o f h a l f -t wave p o t e n t i a l s , E . , a g a i n s t Hammett s u b s t i t u e n t c o n s t a n t s , 1 / ^ a ( s e e S e c t i o n 1 . 6 ) . C o n v e n t i o n a l l y , t h e r e a c t i o n c o n s t a n t p w h i c h i s a measure o f t h e e f f e c t o f s u b s t i t u e n t s on a r e a c t i o n , i s d e f i n e d by e q u a t i o n (32) u s i n g e i t h e r t h e e q u i l i b r i u m A l o gY 77 X = the e q u i l i b r i u m c o n s t a n t , K, o r t h e r a t e c o n s t a n t , k, o f the r e a c t i o n . c o n s t a n t o r t h e r a t e c o n s t a n t f o r the r e a c t i o n . [ I n p r a c t i c e , p i s u s u a l l y f o u n d from the s l o p e o f a p l o t o f l o g K (or l o g k) a g a i n s t 0]. The r a t e c o n s t a n t , k , o r e q u i l i b r i u m c o n s t a n t , K, o f o r a p o l a r o g r a p h i c r e d u c t i o n c a n , u n d e r c e r t a i n c i r c u m s t a n c e s , be d e t e r m i n e d from t h e h a l f - w a v e p o t e n t i a l . I f t h e p o l a r o g r a p h i c wave i s d e s c r i b e d by e q u a t i o n ( 1 0 ) , w h i c h i s t h e e q u a t i o n f o r R T "3 q E = E l / 2 + anF £ n [ U d " ( 1 0 ) The s u b s t i t u e n t c o n s t a n t , a, i s an e m p e r i c a l q u a n t i t y d e f i n e d from a s t a n d a r d r e a c t i o n and measures t h e r e l a t i v e e f f e c t o f a s u b s t i t u e n t on t h e s t a n d a r d r e a c t i o n . A v a r i e t y o f a v a l u e s e x i s t d e p e n d i n g on t h e s t r u c t u r a l r e l a t i o n s h i p between the s u b s t i t u e n t and the r e a c t i o n c e n t r e and on t h e t y p e o f r e a c t i o n o c c u r r i n g . - 1 ' ' ' ' " 98 a s i m p l e p o l a r o g r a p h i c r e d u c t i o n wave, t h e n t h e h a l f - w a v e poten-t i a l can be r e l a t e d to t h e e q u i l i b r i u m c o n s t a n t , K, [ e q u a t i o n (12) and (7)] f o r a r e v e r s i b l e r e d u c t i o n ( a = l , see a l s o S e c t i o n = E° - £n(D /D J 1 / 2 ( 1 2 ) 3 7 1/2 nF ox r e d K ' 1.5) or t o t h e r e d u c t i o n r a t e c o n s t a n t , k Q [ e q u a t i o n (13)] f o r an i r r e v e r s i b l e r e d u c t i o n ( a < l ) . F o r a r e v e r s i b l e p o l a r o g r a p h i c : i / 2 = £ n f ° - 8 8 6 ( T / D ) 1 / 2 k Q ] ( 1 3 ) 5 5 r e d u c t i o n , e q u a t i o n s (7) and (12) c a n be combined w i t h e q u a t i o n (32) t o g i v e e q u a t i o n s (42) and ( 4 3 ) . S i n c e i t i s u n l i k e l y nF AE ( 2.303RT * Aa (42) P = nF A E l / 2 M n ( D o x / D r e d ) 1 / 2 ( 4 3 ) 2.3 0 3RT * Aa 2.3 03 Aa t h a t t h e r a t i o o f t h e d i f f u s i o n c o e f f i c i e n t s , D, w i l l be g r e a t l y a f f e c t e d by s u b s t i t u e n t c h a n g e s , t h e s e c o n d term o f e q u a t i o n (43) c a n be n e g l e c t e d g i v i n g e q u a t i o n ( 4 4 ) , from w h i c h the nF A E l / 2 (44) 2.303RT " Aa 9 9 r e a c t i o n c o n s t a n t p o f a r e v e r s i b l e r e d u c t i o n c a n be d e t e r m i n e d i f the e f f e c t o f s u b s t i t u e n t s on the h a l f - w a v e p o t e n t i a l , A E ^ ^ / A a , i s known. A s i m i l a r t r e a t m e n t c a n be a p p l i e d to an i r r e v e r s i b l e r e d u c t i o n by c o m b i n i n g e q u a t i o n s (13) and (32) to y i e l d e q u a t i o n ( 4 5 ) . S i n c e the d r o p t i m e T o f t h e d r o p p i n g QtnF . A E l / 2 A l o g [ 0 . 386 ( T/D) 1 / 2 ]  P 2.303RT Aa " Aa [ ' me r c u r y e l e c t r o d e i s under e x p e r i m e n t a l c o n t r o l and t h e d i f f u s i o n c o e f f i c i e n t s would n o t be e x p e c t e d t o change s i g n i f i -c a n t l y among a n a l o g o u s compounds t h e n t h e s e c o n d term o f e q u a t i o n (27) c a n be n e g l e c t e d to g i v e e q u a t i o n ( 4 6 ) . AE anF 1/2  P = 2.303RT ' ~ A o ~ ( 4 6 ) I n s p e c t i o n o f e q u a t i o n s (44) and (46) r e v e a l t h a t e q u a t i o n (44) ( r e v e r s i b l e r e d u c t i o n ) i s s i m p l y a s p e c i a l form o f e q u a t i o n (46) i n w h i c h t h e t r a n s f e r c o e f f i c i e n t a i s e q u a l to 1. The a s s u m p t i o n s u s e d i n t h e d e r i v a t i o n o f e q u a t i o n (44) and (46) were t h a t t h e p o l a r o g r a p h i c wave must be d e s c r i b e d by e q u a t i o n (10) ( i . e . a p l o t o f E v e r s u s l o g ( i ^ - i ) / ( i ) must be l i n e a r ) RT E = El/2 + £ n t ( i d " i>/<i>l do) and t h a t t h e t r a n s f e r c o e f f i c i e n t a must be a p p r o x i m a t e l y i n -de p e n d e n t o f s u b s t i t u e n t e f f e c t s . I f t h e s e c o n d i t i o n s do n o t p r e v a i l , t h e n a n o r m a l Hammett r e a c t i o n c o n s t a n t p c a n n o t be 1 0 0 c a l c u l a t e d d i r e c t l y from t h e e f f e c t o f s u b s t i t u e n t s on t h e h a l f -wave p o t e n t i a l s . The e f f e c t s o f s u b s t i t u e n t s on the h a l f - w a v e p o t e n t i a l s ( T a b l e V I I I ) o f t h e S e r i e s I compounds ( I I I ) a r e shown i n CONH, ( I I I ) F i g u r e s 16, 17, and 18. S i n c e the h a l f - w a v e p o t e n t i a l s were c o n c e n t r a t i o n d e p e n d e n t , (see S e c t i o n 3.5 and r e f e r e n c e 44) the p o l a r o g r a m s were a l l r e c o r d e d a t or n e a r a s i n g l e c o n c e n --2 t r a t x o n f o r any one c o r r e l a t i o n (10 M. i n F i g u r e 16, -3 -5 -5 2 x 10 M . i n F i g u r e 17, and 1 x 10 to 2 x 10 M . i n F i g u r e 18) . The h a l f - w a v e p o t e n t i a l s a r e p l o t t e d a g a i n s t a* s i n c e the s t a n d a r d r e a c t i o n f o r t h i s s u b s t i t u e n t c o n s t a n t [ e q u a t i o n (33)] has t h e s u b s t i t u e n t a t t a c h e d d i r e c t l y t o t h e r e a c t i o n RCOOR' H or OH r»- RCOOH + R'OII (33) R _ [ l o g (- ) q h _ - l o g ( ^ _ , ] ( 4 7 ) CH. CH. 70 : e n t r e , as i s t h e c a s e w i t h t h e S e r i e s I compounds ( I I I ) . The v a l u e s o f a* used were c a l c u l a t e d from C h a r t o n ' s 7 2 v a l u e s o f -2 F i g u r e 16. P l o t o f p o l a r o g r a p h i c h a l f - w a v e p o t e n t i a l s o f 10 M. s o l u t i o n s o f t h e S e r i e s I compounds ( T a b l e V I I I ) a g a i n s t a* ( T a b l e V, page 4 9 ) . -600 c o r r . c o e f f . = .972 AE /Aa* = 188 mv. X / £. -CH 2CN 33 > > e CN -650 -700 •750 -800 -850 -900 -CH 2OCH 3 -CH CONH 2 > -CH 2COOCH 3 -CH COOCH(CH 3) 2 -CH,CH,OH 2 2 -CH. -CH 2COCH 3 N I : O N H , l> -CH 2C00" -0.3 -0.1 0.1 0.3 0.5 0.7 0.9 1.1 a* 1.3 CO > > S F i g u r e 17. P l o t o f p o l a r o g r a p h i c h a l f - w a v e p o t e n t i a l o f 2 x i o " 3 ?i s o l u t i o n s o f t h e S e r i e s I compounds ( T a b l e V I I I ) a g a i n s t a * ( T a b l e V, page 49) . -550 -600 -650 -700 -750 •8 0 0h •85CT c o r r . c o e f f . 978 A E l / 2 / A a * = 1 6 2 m v " -Ch T 2C00 - C H „ 0 C H 2 3 ^ -CH COOCH l>-CH oC00CHTCH ) — CH CONH ^  3 2 j. ^ 2 ^•-CH o C 0 C H 3 •CH 2CH OH l> — CH CONH. -0 . 3 •0.1 0.1 0.3 0 . 5 0.7 0.9 1.1 1 . 3 a' F i g u r e 18. P l o t o f p o l a r o g r a p h i c h a l f - w a v e p o t e n t i a l s o f 10 M. s o l u t i o n s o f t h e S e r i e s I compounds a g a i n s t a * ( T a b l e V , page 4 9) 104 t a j u s i n g e q u a t i o n (34) s i n c e a more c o m p l e t e l i s t o f p o l a r a* = O-j/0.45 (34) 7 3 X-CH 2COOH 7 w X-CH COO" + H + (48) a j = 0.251 l o g ( K /K H) ( 4 9 ) 7 2 s u b s t i t u e n t c o n s t a n t s a r e a v a i l a b l e from C h a r t o n ' s work t h a n 6 9 7 6 from o r i g i n a l a* v a l u e s . ' The v a l u e s o f a* w h i c h were used a r e l i s t e d i n T a b l e V i n S e c t i o n 3.1.2 (page 4 9 ) . A 7 2 v a l u e o f a i s n o t g i v e n by C h a r t o n f o r t h e c a r b o - i - p r o p o x y s u b s t i t u e n t and so a v a l u e o f 0.34 has been assumed ( c * = . 7 6 ) , i d e n t i c a l to the v a l u e s r e p o r t e d f o r t h e c a r b o m e t h o x y and 7 2 - 2 c a r b o e t h o x y s u b s t i t u e n t s . A t c o n c e n t r a t i o n s o f 10 M. and -3 2 x 10 H., h a l f - w a v e p o t e n t i a l s were r e p r o d u c i b l e t o w i t h i n 5 mv. f o r e v e r y compound so t h e s e p o t e n t i a l s a r e p l o t t e d as - 5 s i n g l e p o i n t s i n F i g u r e s 16 and 17. Near 10 M., t h e p r e c i s i o n was n o t a l w a y s as good and so s t a t i s t i c a l e r r o r l i m i t s a r e shown f o r each compound i n F i g u r e 18. O n l y one p o l a r o g r a m o f -5 t h e l - c a r b o x y m e t h y l - 3 - c a r b a m o y l p y r i d i n i u m i o n a t 10 M. c o n c e n -t r a t i o n s was s u f f i c i e n t l y d i s t i n c t f o r the h a l f - w a v e p o t e n t i a l to be measured ( t h i s wave a p p e a r s as o n l y a s h o u l d e r on t h e b a c k g r o u n d ) , so t h e e r r o r f o r t h i s compound has been a p p r o x i -mated by t h e w o r s t e r r o r among the o t h e r compounds. t : See f o o t n o t e on page S e c t i o n 3.1.2 105 I n s p e c t i o n o f F i g u r e s 16, 17 and 18, r e v e a l s a common p a t t e r n a t a l l c o n c e n t r a t i o n s , the o n l y e x c e p t i o n b e i n g t h e -2 c a r b o x y compound a t 10 M. Two compounds c o n s i s t e n t l y f a l l o f f o f t h e b e s t l e a s t - s q u a r e s l i n e ; t h e methoxy compound ( w h i c h has a more p o s i t i v e h a l f - w a v e p o t e n t i a l t h a n v/ould be p r e -d i c t e d ) and t h e a c e t y l compound (which has a more n e g a t i v e h a l f - w a v e p o t e n t i a l t h a n would be p r e d i c t e d ) . S i m i l a r p a t t e r n s w i l l be s e e n i n t h e l i n e a r f r e e e n e r g y r e l a t i o n s h i p s o f o t h e r r e a c t i o n s o f t h e S e r i e s I p y r i d i n i u m s a l t s ( I I I ) and d i h y d r o -p y r i d i n e s ( I V ) . H H N R 1 R, ( I I I ) (IV) The s l o p e s o f t h e p l o t s i n F i g u r e s 16, 17 and 18 a r e s i m i l a r t o the s l o p e o f +220 mv. r e p o r t e d f o r a p l o t o f t h r e e 55 1 - a l k y l p y r i d i n i u m s a l t s b u t much s m a l l e r t h a n t h e s l o p e o f +300 mv. r e p o r t e d f o r the v a r i a t i o n s o f the h a l f - w a v e p o t e n t i a l o f a s e r i e s o f l - ( 3 ' o r 4 ' - s u b s t i t u t e d p h e n y l ) p y r i d i n i u m s a l t s 5 5 ( X X V I I ) . A l t h o u g h the s l o p e o f p l o t s o f h a l f - w a v e p o t e n t i a l (XVLT) 106 a g a i n s t ff* v a r y w i t h the c o n c e n t r a t i o n ( F i g u r e s 16, 17, and 18) -3 r e a c n m g a minimum nea r 2 x 10 f l . , t h i s v a r i a t i o n does n o t seem to be l a r g e enough to a c c o u n t f o r the d i f f e r e n c e betv/een the r e s u l t s o f t h i s s t u d y and the r e s u l t s f r o m the 1 - p h e n y l -p y r i d i n i u m s a l t s ( X X V I I ) . A n o t h e r p o s s i b l e e x p l a n a t i o n comes from the f a c t t h a t 0* was u s e d i n F i g u r e s 16, 17 and 18 and i n 5 5 Zuman's p l o t o f t h e t h r e e 1 - a l k y l p y r i d i n i u m s a l t s whereas Suman used O and 0" i n the p l o t s o f the p h e n v l p y r i d i n i u m s a l t s 5 5 1G (XXVII) . W e l l s has s u g g e s t e d t h a t making p* c o m p a r a b l e t o p f o r the h y d r o l y s i s o f e s t e r s does n o t a u t o m a t i c a l l y e n s u r e t h a t p* w i l l be c o m p a r a b l e t o p f o r o t h e r r e a c t i o n s . The d i f f e r ence c o u l d a l s o be due t o d i f f e r e n c e s i n e x p e r i m e n t a l c o n d i t i o n s w h i c h have o f t e n c a u s e d v a r i a t i o n s o f as much as 50 m i l l i v o l t s 40 i n t h e r e p o r t e d h a l f - w a v e p o t e n t i a l s o f p y r i d i n i u m s a l t s . In o r d e r to c a l c u l a t e the r e a c t i o n c o n s t a n t p from th e s l o p e s o f the p l o t s i n F i g u r e s 16, 17 and 18 u s i n g e q u a t i o n (44) , t h e p o l a r o g r a p h i c v/ave must be d e s c r i b e d by e q u a t i o n (10) CinF ' 1/2 .. A. P = 2 . 303RT ' ~~~Eo ( 4 4 ) RT E = E l / 2 + ^ ¥ ^ n [ ( i d - d O ) + and t h e t r a n s f e r c o e f f i c i e n t a must be i n d e p e n d e n t o f t The t r a n s f e r c o e f f i c i e n t , a, can be c a l c u l a t e d from the s l o p e o f a p l o t o f E v s . l o g ( i d - i ) / ( i ) by t h e e q u a t i o n : 107 s u b s t i t u e n t e f f e c t s . The f o r m e r r e q u i r e m e n t i s n o t met by -5 p o l a r o g r a p h i c waves r e c o r d e d a t 10 M. and i s o n l y a p p r o x i -- 3 m a t e l y met by p o l a r o g r a p h i c waves r e c o r d e d a t 2 x 10 Ii. and -2 10 M. (see F i g u r e s 10a and 1 1 a ) . The s e c o n d r e q u i r e m e n t , t h a t o f the t r a n s f e r c o e f f i c i e n t b e i n g c o n s t a n t , i s n o t met by a l l compounds a t e i t h e r 2 x 10 II. or 10 II. ( T r a n s f e r c o e f f i c i e n t s c a n n o t be c a l c u l a t e d f o r p o l a r o g r a p h i c waves a t -5 10 Ii. s i n c e t h e s e waves g i v e c u r v e d p l o t s o f E a g a i n s t t l o g ( i - i ) / ( i ) ) . D e s p i t e the f a i l u r e to meet t h e s e c o n d r e q u i r e m e n t , i f a v e r a g e v a l u e s o f the t r a n s f e r c o e f f i c i e n t s a r e c a l c u l a t e d f o r e a c h o f the two h i g h e r c o n c e n t r a t i o n s (a = 0.S a t 2 x 10 J M. and a = 0.67 a t 10 " M.) and u s e d i n e q u a t i o n (44) t h e n r e a c t i o n c o n s t a n t s p* o f +2.1 and +2.2 a r e anF 1/2  P 2.303RT ' AO [ ' - 2 -3 fou n d f o r t h e d a t a a t 10 M. and 2 x 10 M., r e s p e c t i v e l y . The agreement between the p* v a l u e s a t t h e s e two c o n c e n t r a -t i o n s i s s t r i k i n g , c o n s i d e r i n g the a p p r o x i m a t i o n s t h a t have been u s e d i n the c a l c u l a t i o n , and c o n s i d e r i n g the 15% d i f f e r -ence between the s l o p e s , A E ^ ^ / A a * , i n F i g u r e s 16 and 17, from which the p* v a l u e s v/ere c a l c u l a t e d . The p o l a r o g r a p h i c waves a t low c o n c e n t r a t i o n s (10 ^ M.) a r e n o t d e s c r i b e d by e q u a t i o n ( 1 0 ) , t h u s p r e v e n t i n g t h e use t See f o o t n o t e on p r e v i o u s page. 108 o f e q u a t i o n (44) t o d e t e r m i n e a r e a c t i o n c o n s t a n t a t t h i s R T E = E l / 2 + ~ £ n ( i d " i)/('±'> (10) A P gnF 1/2 P ~ 2.303RT ' Ta ( 4 4 ) c o n c e n t r a t i o n . However, i t was found ( S e c t i o n 3.5.1) t h a t the p o l a r o g r a p h i c waves r e c o r d e d a t low c o n c e n t r a t i o n s were q u i t e w e l l d e s c r i b e d by e q u a t i o n (18) ( i . e . p l o t s o f E a g a i n s t E = E° + £ | l n [ ( i d - i ) / ( i ) 2 / 3 ] (18) 2/3 l o g ( i - i ) / ( i ) were l i n e a r w i t h s l o p e s n e a r the t h e o r e t i c a l v a l u e o f 60 mv.), w h i c h i s the e q u a t i o n f o r a o n e - e l e c t r o n r e v e r s i b l e r e d u c t i o n f o l l o w e d by an i r r e v e r s i b l e d i m e r i z a t i o n [ r e a c t i o n ( 4 1 ) ] . The i n t e r c e p t , £ ° , o f e q u a t i o n (18) i s r e l a t e d to t h e s t a n d a r d r e d u c t i o n p o t e n t i a l In o r d e r to d e t e r m i n e the r e a c t i o n c o n s t a n t to use the s t a n d a r d r e d u c t i o n p o t e n t i a l f o r by e q u a t i o n (19) . one would l i k e e a c h compound s i n c e t h i s q u a n t i t y i s d i r e c t l y r e l a t e d t o t h e e q u i l i b r i u m c o n s t a n t by e q u a t i o n ( 7 ) . C o m b i n i n g e q u a t i o n s (32) and (7) i n t o (42) , t h e r e a c t i o n c o n s t a n t can be c a l c u l a t e d from the A l o g K 77 P = — A O — < 3 2> E ° « £ £ K ( 7 ) 5 5 nF AE°  P 2. 303RT ' Aa ( 4 2 ) e f f e c t o f s u b s t i t u e n t s on t h e s t a n d a r d r e d u c t i o n p o t e n t i a l . To c a l c u l a t e t h e s t a n d a r d r e d u c t i o n p o t e n t i a l from the i n t e r -c e p t , e ° , o f e q u a t i o n ( 1 8 ) , e q u a t i o n (19) c a n be r e a r r a n g e d and combined v/ith e q u a t i o n s (16) and (20) t o g i v e e q u a t i o n <f = (f TTDT) 1 / 2 (16) ( 5 0 ) . The l a s t term o f e q u a t i o n (50) i s a n u m e r i c a l c o n s t a n t RT £ n r 3 / 2 k d RT 2 3 1 / 2 - f r 3 / 2 _ _ - — £n 7 ( y ) x _ - (50) D 110 w i t h t h e v a l u e o f +21G m i l l i v o l t s when the c u r r e n t i n e q u a t i o n (18) i s e x p r e s s e d i n m i c r o a m p e r e s . The s e c o n d term c o n t a i n s the d r o p t i m e T o f the d r o p p i n g m e r c u r y e l e c t r o d e ( w h i c h i s under e x p e r i m e n t a l c o n t r o l ) , the d i m e r i z a t i o n r a t e c o n s t a n t , and t h e d i f f u s i o n c o e f f i c i e n t . The d i f f u s i o n c o e f f i c i e n t s , w hich can be e s t i m a t e d from the I l k o v i c e q u a t i o n [ e q u a t i o n 51)] a r e l i s t e d i n T a b l e X I I . U n f o r t u n a t e l y , d i m e r i z a t i o n i , = 607 n C D 1 / 2 T 1 / 6 m 2 / 3 (51) a n = number o f e l e c t r o n s t r a n s f e r r e d C = c o n c e n t r a t i o n o f e l e c t r o a c t i v e s p e c i e s ( ) m = m e r c u r y f l o w r a t e (mgm./sec.) D = d i f f u s i o n c o e f f i c i e n t (cm. / s e c . ) T = d r o p time (sec.) r a t e c o n s t a n t s have o n l y been measured f o r two o f t h e p y r i d i n i u r a d i c a l s , NAD ( X l l l a ) and l - m e t h y l - 3 - c a r b a m o y l p y r i d i n e ( X H I b ) . TABLE X I I D i f f u s i o n C o e f f i c i e n t s o f t h e P y r i d i n i u m I o n s E s t i m a t e d from the I l k o v i c E q u a t i o n R. CH COO CH. CH 2CH OH CII 2OCH 3 CH 2COCH CH COOCH - i 3 CH 2COOCH(CH ) ? CH CN CU CONH n CH 2CONH CH 2CONH CH CONH CH CONH CH CONH N ' I R. CONH COCH. CN F H 0~ S p e c i a l compounds: H2NOC-/ ^ VcONH 2 DxlO (crn 2 /sec . ) . 8 9 .52 .52 .42 .42 .37 .32 .51 .46 .54 .43 .49 1. 05 1.17 37 £n(D) (mv) -50 -52 -52 -53 -53 -54 -54 -52 -53 -52 -53 -52 -49 -49 . 93 . 23' •50 -56' T a b l e XII ( c o n t . ) NAD NMN DxlO 2 (cm sec .) RT 3F £n (D) (mv) 1.97 -46 .49 -52 ,21 78 -56 -50 * C a l c u l a t e d f o r a two e l e c t r o n r e d u c t i o n 113 The d i f f u s i o n c o e f f i c i e n t s o f KAD + and l - m e t h y l - 3 -c a r b o m o y l p y r i d i n i u r n i o n ( n i b ) i n v a r i o u s b u f f e r s have been CONH2 ( I I l b ) p r e v i o u s l y r e p o r t e d 4 7 ' 6 6 and a v e r a g e v a l u e s have been u s e d h e r e i n to c a l c u l a t e t h e v a l u e s o f -650 and -730 mv. ( v s . N.H.E.) r e s p e c t i v e l y , f o r t h e s t a n d a r d r e d u c t i o n p o t e n t i a l s E° o f t h e s e two compounds. S i n c e d i m e r i z a t i o n r a t e c o n s t a n t s a r e unknown f o r the r e m a i n i n g compounds and, hence, t h e i r s t a n d a r d r e d u c -t i o n p o t e n t i a l s a r e n o t a v a i l a b l e , a r e l i a b l e r e a c t i o n c o n -s t a n t p b a s e d on E° v a l u e s c a n n o t be o b t a i n e d . I f e q u a t i o n (50) i s combined w i t h e q u a t i o n (42) t h e n RT n "d RT „ 2,3,1/2 TT J¥ i n -J71 - I F l n 7 (7 } — (> 0 ) nF AE° H 2.3 03RT * Aa ( 4 2 ) the r e a c t i o n c o n s t a n t can be d e t e r m i n e d from t h e e f f e c t o f s u b s t i t u e n t s on the i n t e r c e p t e° o f e q u a t i o n (18) and on the E = e° + |I £ n ( i d - i ) / ( i ) 2 / 3 ( 1 8 ) 114 d i m e r i z a t i o n r a t e c o n s t a n t k by u s i n g e q u a t i o n ( 5 2 ) ( w h e r e the n i n e q u a t i o n (42) has been s e t e q u a l to 1 and the n u m e r i c a l . _ F A E ! i A i o g r 3 / 2 ^ o g n c d / ( D ) 1 / 2 ] ( 5 2 ) P 2 .303RT " Acr 3 Aa " Aa c o n s t a n t has been e l i m i n a t e d ) . E q u a t i o n (52) c a n be s i m p l i f i e d i n t o e q u a t i o n (53) s i n c e t h e d r o p t i m e T w i l l be c o n s t a n t f o r a l l compounds, and s i n c e the d i f f u s i o n c o n s t a n t s , D, do n o t v a r y s i g n i f i c a n t l y among the NAD + model compounds. ( I n column 4 o f T a b l e X I I , i t can be seen t h a t t h e v a r i a t i o n s i n t h e d i f f u s i o n c o e f f i c i e n t s r e s u l t i n d i f f e r e n c e s o f o n l y a few m i l l i v o l t s i n t h e d i f f u s i o n c o e f f i c i e n t term o f e q u a t i o n s (50) and (52)'. ) The s e c o n d term o f e q u a t i o n (53) , v/hich i s e q u a l t o F A £ l 1 A l ° g k d P 2.303RT * Aa " 3 Aa 1 ' o n e - t h i r d the r e a c t i o n c o n s t a n t , Q * , f o r the d i m e r i z a t i o n ^ d r e a c t i o n [ e q u a t i o n s (54) and (55)] c a n n o t be c a l c u l a t e d v e r y p r e c i s e l y b u t c a n be e s t i m a t e d from t h e two d i m e r i z a t i o n r a t e t c o n s t a n t s t h a t a r e known once a a * v a l u e i s e s t i m a t e d f o r the t U s u a l l y f a i r l y s m a l l r e a c t i o n c o n s t a n t s have been f o u n d f o r r a d i c a l r e a c t i o n s i n v/hich t h e r e i s no n e t change i n c h a r g e a t the r e a c t i o n c e n t r e ^ 7 b u t w i t h o n l y tv/o r a t e c o n s t a n t s known, i t i s i m p o s s i b l e t o know how w e l l t h e d i m e r i z a t i o n r a t e c o n s t a n t i s c o r r e l a t e d by a * and, t h e r e f o r e , w h e t h e r the e s t i m a t e d v a l u e o f p£ i s m e a n i n g f u l . CONH, A l o g Aa* 11. H2NOC CONH. N H H N ( 5 4 ) (55) 1 - s u b s t i t u e n t o f M A D + . The p o l a r o g r a p h i c h a l f - w a v e p o t e n t i a l o f I J A D + ( l a , page 128) i s s i m i l a r t o t h e h a l f - w a v e p o t e n t i a l s o f t h e 1 - c a r b a m o y l m e t h y l ( I I I i ) , 1 - a c e t o n y l - ( I I I d ) , 1 - m e t h o x y m e t h y l - ( I l l e ) , 1 - c a r bo-i_-propoxym e t h y 1-(111 g) and 1 - c a r b o m e t h o x y m e t h y l compounds ( I l l f ) o f S c r i e s I . T h e s e s u b s t i t u e n t s a l l have 0* s u b s t i t u e n t c o n s t a n t v a l u e s n e a r ONH, d: P = e : f : g: ( I I I ) CH 2COCH 3 CH.OCH 2 3 CH COOCH CH COOCH(CH) CH 2CONH + 0.7 and so t h i s v a l u e i s t e n t a t i v e l y a s s i g n e d to t h e 5*-( a d e n o s i n e p y r o p h o s p h a t e ) - r i b o s i d y l group (XXIX) a t t a c h e d t o NH, HO OH (XXIX) HO OH 116 the p y r i d i n e r i n g i n NAD +, a l l o w i n g t h e r e a c t i o n c o n s t a n t p* d o f -0.1 t o be e s t i m a t e d f o r the d i m e r i z a t i o n r e a c t i o n . T h i s v a l u e o f p* i s v e r y low, a s i t u a t i o n sometimes f o u n d w i t h 8 2 h y d r o g e n atom a b s t r a c t i o n r e a c t i o n s . E q u a t i o n (53) can now be r e w r i t t e n as e q u a t i o n (56) i n w h i c h A l o g k , / A a * has d F Ae° P = 2 . 303RT ' AcT* + ° - ° 3 <5 6> been r e p l a c e d by i t s e s t i m a t e d v a l u e o f -0.1. The r e a c t i o n c o n s t a n t f o r the e l e c t r o c h e m i c a l r e d u c t i o n t low c o n c e n t r a t i o n can now be d e t e r m i n e d from e q u a t i o n ( 5 6 ) . The i n t e r c e p t e° o f e q u a t i o n (18) can be d e t e r m i n e d from the a E = e . + i l J p l o g M i . i ) / ( 1 ) * / 3 , ( 1 8 ) 2/3 l i n e a r p l o t s o f E a g a i n s t l o g [ ( i , - i ) / ( i ) ' ] and t h i s has d been done f o r each compound (see T a b l e V I I and T a b l e IX) a t -5 -4 v a r i o u s c o n c e n t r a t i o n s between 10 M. and 2 x 10 M. The v a l u e s o f £° f o r t h e S e r i e s I compounds ( I I I ) a r e p l o t t e d a g a i n s t 0* i n F i g u r e 19 and g i v e a s l o p e , A e 0 / A c r * o f -218 mv. S u b s t i t u t i n g t h i s i n t o e q u a t i o n (56) g i v e s a r e a c t i o n c o n s t a n t p* o f +3.7 w h i c h i s s i g n i f i c a n t l y d i f f e r e n t from t h e v a l u e o f -3 +2.2 t h a t was f o u n d a t h i g h e r c o n c e n t r a t i o n s (>10 M.). To see i f t h i s d i f f e r e n c e i s due t o t h e two d i f f e r e n t methods o f c a l c u l a t i n g the r e a c t i o n c o n s t a n t , the h a l f - w a v e p o t e n t i a l s a t 10 ^ M. ( F i g u r e 12) were used [ v i a e q u a t i o n (44)] to F i g u r e 19, P l o t o f p o l a r o g r a p h i c r e d u c t i o n p o t e n t i a l s e° ( T a b l e IX) o f the S e r i e s I compounds a g a i n s t a* ( T a b l e V, page 49) -500 c o r r . c o e f f . = .978 A e ° / A c 7 * = 216 mv. P* = +3.7 -CH 2C 1/1 > > •600 •700 CHOCH. CH 2COOCH 3 -CH 2COOCH(CH 3) 2 •CH 7 C 0 N H 2 •CH 2COCH 3 •800 -CH 2C00 -CH 2CH OH •CH. (J N I •0.3 -0.1 0.1 0.3 0 . 5 0.7 0.9 1.1 1.3 a 118 (III) AF D = « n F 1/2 • 2.303RT ' ~Ko (44) e s t i m a t e t h e r e a c t i o n c o n s t a n t f o r the r e d u c t i o n a t low c o n -c e n t r a t i o n s , u s i n q an e s t i m a t e d t r a n s f e r c o e f f i c i e n t a o f t u n i t y . The s l o p e , AE 2 / A a * , o f F i g u r e 18 i s -214 mv. from w h i c h a r e a c t i o n c o n s t a n t o f +3.7 c a n be c a l c u l a t e d [ u s i n g e q u a t i o n ( 4 4)] i n good a g r e e m e n t w i t h t h e v a l u e o f p* c a l c u -l a t e d from e q u a t i o n ( 5 6 ) . A p* v a l u e o f +3.7 f o r t h e F A £ ° P * = 2.303RT ' A u ^ + * 0 3 (56) p o l a r o g r a p h i c r e d u c t i o n o f p y r i d i n i u m s a l t s [ r e a c t i o n (41)] i s a l s o i n good a g r e e m e n t w i t h the p* v a l u e o f +3.8 r e p o r t e d f o r t h e a c i d d i s s o c i a t i o n o f p r o t o n a t e d m e t h y l a m i n e s 7 6 [ e q u a t i o n ( 5 7 ) ] . RNH 3 + ^=r±r RNH + H + ( 5 7 ) Unambiguous v a l u e s o f t h e t r a n s f e r c o e f f i c i e n t , a, c a n n o t be c a l c u l a t e d from t h e s l o p e s o f p l o t s o f E v s . log(i„ - i ) / ( i ) ; i n i n - t , ! S e P l ° t S C U r V G d f ° r P ° l a r o g r a p h i c waves r e c o r d e d a t 10 j * . c o n c e n t r a t i o n s . The a v e r a g e s l o p e s o f t h e c u r v e s 119 C O N^> . ^ * ^ C O N H 0 e 1/ M 2 (41) To summarize, t h e n , a r e a c t i o n c o n s t a n t o f + 2.2 has been c a l c u l a t e d f o r t h e p o l a r o g r a p h i c r e d u c t i o n p r o c e s s a t h i g h c o n c e n t r a t i o n s w h i l e a r e a c t i o n c o n s t a n t o f +3.7 has been c a l c u l a t e d f o r the r e d u c t i o n p r o c e s s a t low c o n c e n t r a t i o n s . A l t h o u g h a p p r o x i m a t i o n s were u s e d i n t h e c a l c u l a t i o n o f b o t h v a l u e s , the d i f f e r e n c e betv/een the two a p p e a r s to be r e a l . T h ese r e s u l t s i m p l y a d u a l mechanism f o r t h e p o l a r o g r a p h i c r e d u c t i o n , a c o n c e p t w h i c h w i l l be g i v e n f u r t h e r c o n s i d e r a t i o n i n the D i s c u s s i o n . 3.5.4 The E f f e c t s o f 3 - S u b s t i t u e n t s on t h e P o l a r o g r a p h i c R e d u c t i o n P o t e n t i a l s The e q u a t i o n s d e v e l o p e d i n S e c t i o n 3.5.3 f o r t h e e f f e c t o f 1 - s u b s t i t u e n t s on the p o l a r o g r a p h i c r e d u c t i o n c a n a l s o be a p p l i e d to the 3 - s u b s t i t u e n t s . F o r the r e d u c t i o n o c c u r r i n g a t the e l e c t r o d e , [ r e a c t i o n ( 4 1 ) ] , one would e x p e c t a , w h i c h m CH2CONH2 (XXII) (41) CH2CONH2 120 i s d e f i n e d from the i o n i z a t i o n o f 3 - s u b s t i t u t e d b e n z o i c a c i d s [ r e a c t i o n (58)] t o p r o v i d e the a p p r o p r i a t e s e t o f s u b s t i t u e n t COOH + H (58) COO" c o n s t a n t s . The r e a c t i o n c o n s t a n t can be c a l c u l a t e d f r o m t h e e f f e c t o f s u b s t i t u e n t s on t h e h a l f - w a v e p o t e n t i a l s o f t h e S e r i e s I I compounds (XXII) u s i n g e q u a t i o n (44) i f two r e q u i r e ' ments a r e met: (a) the p o l a r o g r a p h i c waves must be d e s c r i b e d P = anF A E 2 . 3 03RT 1/2 Aa (44) by e q u a t i o n (10) and (b) the t r a n s f e r c o e f f i c i e n t s a must be E = E 1/2 + cln¥ £ n ( i d " (10) a p p r o x i m a t e l y t h e same f o r a l l compounds i n t h e s e r i e s . A t - 5 c o n c e n t r a t i o n s n e a r 10 M., t h e f i r s t r e q u i r e m e n t i s n o t met f o r any o f the compounds, v/hich e l i m i n a t e s t h e use o f e q u a t i o n M. and (44) f o r the l o w - c o n c e n t r a t i o n d a t a . A t 2 x 1 0 - 3 -10 -2 M., the f i r s t r e q u i r e m e n t i s n o t met by t h e u n s u b s t i t u t e d 121 t 3 - f l u o r o - , or 3-hydroxy-compounds. S i n c e h a l f - w a v e p o t e n t i a l s c o u l d n o t be d e t e r m i n e d f o r t h e 3 - a c e t y l compound a t t h e s e h i g h e r c o n c e n t r a t i o n s ( s e e page 6 7 ) , o n l y t h e 3-cyano and 3-c a r b a m o y l compounds a r e l e f t and d i f f e r e n t v a l u e s o f the t r a n s f e r c o e f f i c i e n t were found i n t h i s work f o r t h e s e two compounds. Thus, f o r the S e r i e s I I compounds ( X X I I ) , the h a l f - w a v e p o t e n t i a l s c a n n o t be u s e d t o d e t e r m i n e t h e r e a c t i o n c o n s t a n t f o r the e f f e c t o f 3 - s u b s t i t u e n t s on the p o l a r o g r a p h i c r e d u c t i o n . CONH, CH2CONH, ( I I I ) (XXII) T The p h e n o l i c h y d r o g e n o f l - c a r b a m o y l m e t h y l - 3 - h y d r o x y p y r i d i n i u m c h l o r i d e was f o u n d t o have a pKa o f 4.7. No p o l a r o g r a p h i c wave was o b s e r v e d f o r the p r o t o n a t e d compound ( n i n ) b u t a wave c a n be o b s e r v e d f o r the u n p r o t o n a t e d compound (XXX) above pH 7. I t i s t h i s form o f the 3-hydroxy compound wh i c h i s b e i n g d i s c u s s e d . CH2CONH2 CH2CONH2 ( I l l n ) (XXX) 122 As w i t h t h e S e r i e s I compounds ( I I I ) , p o l a r o g r a m s r e c o r d --4 ed a t c o n c e n t r a t i o n s below 2 x 10 M. a r e d e s c r i b e d by e q u a t i o n ( 1 8 ) , which i s t h e e x p r e s s i o n f o r a r e v e r s i b l e r e d u c t i o n f o l l o w e d E = E° + —— A n ( i . - i ) / ( i ) 2 / 3 (18) F d by an i r r e v e r s i b l e d i m e r i z a t i o n . I t has been shown i n S e c t i o n 3.5.3 t h a t t h e r e a c t i o n c o n s t a n t c a n be c a l c u l a t e d from the e f f e c t o f s u b s t i t u e n t s on t h e i n t e r c e p t C° o f e q u a t i o n (18) and on the d i m e r i z a t i o n r a t e c o n s t a n t by e q u a t i o n ( 5 3 ) . F A e ^ 1 A l o g k d P 2.3 03RT ' Aa " 3 Aa ( 3 ) A l o g k P d = ~ A a ( 5 5 ) The e f f e c t o f 3 - s u b s t i t u e n t s on the d i m e r i z a t i o n r a t e c o n s t a n t i s n o t known, b u t a r e a c t i o n c o n s t a n t p* o f -0.1 c a n be e s t i -mated from t h e e f f e c t o f 1 - s u b s t i t u e n t s on t h i s r e a c t i o n . T h i s v a l u e w i l l be t e n t a t i v e l y u s e d f o r A l o g k,/A0 , b u t d m w i t h the r e s e r v a t i o n t h a t r e a c t i o n c o n s t a n t s as h i g h as 4 have 8 2 been o b s e r v e d i n h y d r o g e n a b s t r a c t i o n s from p h e n o l s . A c c e p t i n g t h e v a l u e o f -0.1 f o r A l o g k ^ / A a ^ , e q u a t i o n (53) c a n be c o n v e r t e d i n t o e q u a t i o n (59) and t h i s e q u a t i o n c a n F Ae° Pm ~ 2.303RT * Ao~ + ' 0 3 < 5 9> m 123 now be used to d e t e r m i n e the r e a c t i o n c o n s t a n t f o r t h e e f f e c t o f 3 - s u b s t i t u e n t s on t h e p o l a r o g r a p h i c r e d u c t i o n p o t e n t i a l . The v a l u e s o f t h e i n t e r c e p t c° o f e q u a t i o n (18) w h i c h a r e l i s t e d i n T a b l e IX (page 8 4 ), a r e p l o t t e d a g a i n s t 0 i n F i g u r e m 20, w i t h t h e s t a t i s t i c a l e r r o r l i m i t s f o r e a c h compound shown. The s l o p e , Ae°/Aa , o f +745 mv. g i v e s a r e a c t i o n c o n s t a n t p m rm o f 10.6 u s i n g e q u a t i o n (59) a v a l u e w h i c h i s so l a r g e t h a t a r e a c t i o n c o n s t a n t o f 4 f o r the d i m e r i z a t i o n r e a c t i o n would s t i l l n o t be v e r y s i g n i f i c a n t s i n c e t h e c o r r e c t i o n term i s l / 3 p ^ o r ^ 1 . 3 . T h i s r e a c t i o n c o n s t a n t i s much l a r g e r t h a n t h e p* v a l u e o f +3.7 f o r t h e e f f e c t o f 1 - s u b s t i t u e n t s on t h i s r e a c t i o n and a l s o l a r g e r t h a n the f a i r l y s u b s t a n t i a l v a l u e o f 7 8 +5.9 r e p o r t e d f o r t h e a c i d d i s s o c i a t i o n c o n s t a n t s o f CH2CONH2 P = +10.8 (41) CH2CONH2 + I • N ( 63 ) 79 C 2H 5 C 2H 5 124 F i g u r e 20. P l o t o f the p o l a r o g r a p h i c r e d u c t i o n p o t e n t i a l s e° o f the S e r i e s I I compounds a g a i n s t m e t a - s u b s t i t u e n t c o n s t a n t s rr f! 1 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 am 125 p r o t o n a t e d p y r i d i n e s . I t i s , however, c o m p a r a b l e t o t h e p 79 o f 13.4 o b t a i n e d by Kosower f o r the v a r i a t i o n o f t h e c h a r g e -t r a n s f e r a b s o r b a n c e maximum o f 3- and 4 - s u b s t i t u t e d - 1 -a l k y l p y r i d i n i u m i o d i d e s . I n t e r e s t i n g l y , i f t h e S e r i e s I I p o l a r o g r a p h i c p o t e n t i a l s e° a r e p l o t t e d a g a i n s t a , as i n F i g u r e 21, i n s t e a d o f a g a i n s t a m t h . c o r r e l a t i o „ i . JrMly a .li5htly larger r e a c t i o n c o n s t a n t p i s f o u n d i n t h i s c a s e , 12.4 r a t h e r t h a n 10.6. The i m p r o v e d c o r r e l a t i o n would seem t o i n d i c a t e t h a t t h e r e i s a r e s o n a n c e i n t e r a c t i o n i n v o l v i n g t h e 3 - s u b s t i t u e n t . S i n c e r e s o n a n c e between 3 - s u b s t i t u e n t s and t h e r i n g i n the p y r i d i n i u m i o n (XXII) i s n o t p o s s i b l e , t h e r e s o n a n c e i n t e r -a c t i o n must be o c c u r r i n g i n the r a d i c a l p r o d u c t (XXXI) . CH 2CONH 2 CH 2CONH 2 (XXII) (XXXI) A s i m i l a r i n t e r a c t i o n has been p r o p o s e d 8 0 between t h e l o n e e l e c t r o n and the 3 - c a r b a m o y l group o f the l - m e t h y l - 3 -c a r b a m o y l p y r i d i n e r a d i c a l (XXXII) p r e p a r e d by p u l s e r a d i o l y s i s t The s t a n d a r d r e a c t i o n f o r a s u b s t i t u e n t c o n s t a n t s has a r e s o n a n c e i n t e r a c t i o n between the s u b s t i t u e n t and t h e r e a c t i o n c e n t r e w h i c h i s n o t p r e s e n t w i t h the a m s t a n d a r d r e a c t i o n . 126 a p 127 A s t a b i l i z a t i o n o f t h e r a d i c a l p r o d u c t s by e l e c t r o n w i t h d r a w i n g 3 - s u b s t i t u e n t s c o u p l e d w i t h the d e s t a b i l i z a t i o n o f t h e p o s i t i v e -l y c h a r g e d p y r i d i n i u m i o n may be s u f f i c i e n t t o e x p l a i n the e x t r a o r d i n a r i l y l a r g e p f o r t h e e f f e c t o f t h e 3 - s u b s t i t u e n t s on the p o l a r o g r a p h i c r e d u c t i o n . 128 3.6 R e d u c t i o n P o t e n t i a l s o f P y r i d i n i u m - D i h y d r o p y r i d i n e H a l f - c e l l s R e d u c t i o n p o t e n t i a l s o f p y r i d i n i u m - d i h y d r o p y r i d i n e systems were d e t e r m i n e d p o t e n t i o m e t r i c a l l y u s i n g t h e method 14 d e v e l o p e d by Rodkey. ( s e e S e c t i o n 5.8.) The r e l a t i o n s h i p between the c e l l p o t e n t i a l and the c o n c e n t r a t i o n s o f o x i d i z e d and r e d u c e d compounds i s g i v e n by the H e r n s t e q u a t i o n [ e q u a t i o n (2)] where E° i s the s t a n d a r d r e d u c t i o n p o t e n t i a l o f t h e h a l f -RT E = E° £n[Red]/[Ox] n F (2) 69 c e l l . F o r the r e d u c t i o n o f NAD + ( I a ) , shown i n e q u a t i o n ( 1 ) , CONH, H O OH •o-(Ia) HO OH + + NAD + H + 2e ^ — NADU (1) a t 2 5 ° , e q u a t i o n (2) can be r e w r i t t e n as i n e q u a t i o n (62) The v a l i d i t y o f e q u a t i o n s (1) and (62) f o r the NAD +-NADH h a l f - c e l l has been amply p r o v e n . 3 0 E q u a t i o n (62) c a n be 30 E = E° - 29.6 l o g [ N A D H ] / [ N A D + ] [ H + ] (62) 129 r e a r r a n g e d to e q u a t i o n (63) and t h e n i n t o e q u a t i o n ( 6 5 ) , t h u s a l l o w i n g the r e d u c t i o n p o t e n t i a l a t any pH, E ° ' to be d e t e r -pH E = E° - 29.6(pH) - 29.6 l o g [NADH ] / [NAD +] (63) E ° ' = E° - 29.6(pH) (64) pti E = E^ j - 29.6 l o g [NADH] / [NAD + ] (65) mined as t h e i n t e r c e p t o f a p l o t o f c e l l p o t e n t i a l , E, a g a i n s t l o g [ N A D H ] / [ N A D + ] . The v a l i d i t y o f e q u a t i o n (63) has been + t e s t e d f o r the HAD model compounds u s e d i n t h i s s t u d y by p l o t t i n g the measured c e l l p o t e n t i a l , E, a g a i n s t + t l o g f P y ]/[PyH] as shown i n F i g u r e s 22 and 23 f o r 1 - n e t h o x y -m e t h y l - 3 - c a r b a m o y l p y r i d i n i u m c h l o r i d e ( H i e ) and 1 - ( 2 ' - h y d r o x y -e t h y l ) - 3 - c a r b a m o y l p y r i d i n i u m c h l o r i d e ( I I I c ) , r e s p e c t i v e l y . ( I l l ) The symbols Py and PyH r e f e r to the p y r i d i n i u m i o n and d i -h y d r o p y r i d i n e r e s p e c t i v e l y . F i g u r e 22. P l o t o f measured c e l l p o t e n t i a l s a g a i n s t l o g [ P y + ] / [ P y H ] f o r the p y r i d i n i u m - d i h y d r o p y r i d i n e h a l f - c e l l o f 1-m e t h o x y m e t h y 1 - 3 - c a r b a m o y l p y r i d i n i u m c h l o r i d e •0.4 -0.2 -0.1 0.1 0.3 0.5 0.7 + l o g [ P y ]/[PyH] F i g u r e 23. P l o t o f m e a s u r e d c e l l p o t e n t i a l s a g a i n s t l o g [ P y + ] / [ P y H ] f o r t h e p y r i d i n i u m - d i h y d r o p y r i d i n e h a l f - c e l l o f 1 - ( 2 • - h y d r o x y e t h y l ) -3 -c a r b a m o y l p y r i d i n i u m c h l o r i d e . pH = 9.7, 0.1 M. g l y c i n e b u f f e r c o r r . c o e f f = .967 s l o p e = 29.7 mv. = -441 mv. C H 2 C H 2 O H • ° - 6 - ° - 4 "0-2 0.0 0.2 0.4 0.6 0.8 1.0 l o g [ P y + ] / [ P y H ] 132 The s l o p e s o f t h e s e p l o t s , l i s t e d i n the t h i r d column o f T a b l e X I I I , a r e i n most c a s e s q u i t e c l o s e t o t h e t h e o r e t i c a l s l o p e o f 29.6 mv., t h u s c o n f i r m i n g t h a t e q u a t i o n (66) a p p l i e s to t he NAD + model compounds as w e l l as to NAD +. The i n t e r -E = E ° ' - 29.6 l o g [ p y H ] / [ P y + ] (66) c e p t s o f p l o t s o f E a g a i n s t l o g [ P y + ] / [ P y H ] g i v e t h e r e d u c t i o n p o t e n t i a l s E ^ l i s t e d i n the f i f t h column o f T a b l e X I I I . The f o u r t h column o f T a b l e X I I I l i s t s t he pH's a t w h i c h t h e p o t e n -t i a l measurements were made w i t h each compound. R e d u c t i o n p o t e n t i a l s o f b i o l o g i c a l l y i m p o r t a n t compounds a r e o f t e n q u o t e d a t pii 7 ( p h y s i o l o g i c a l pH) r a t h e r t h a n a t pH 0 ( u n i t a c t i v i t y o f h y d r o g e n ion) b u t the r e d u c t i o n p o t e n t i a l s o f many p y r i d i n i u m s a l t s c a n n o t be d i r e c t l y m e asured a t pii 7 ( d e c o m p o s i t i o n r e a c t i o n s o f p y r i d i n i u m i o n s and d i h y d r o p y r i d i n e have been d i s c u s s e d i n S e c t i o n s 3.1 t h r o u g h 3.3) . R e d u c t i o n p o t e n t i a l s m e asured a t a pH o t h e r t h a n 7 c a n t h e o r e t i c a l l y be c o r r e c t e d t o pH 7 by e q u a t i o n (67) wh i c h i s a m o d i f i e d form E V = F ° ' - 29.6(pH-7) (67) 7 pH o f e q u a t i o n ( 6 4 ) . The v a l i d i t y o f e q u a t i o n s (64) and (67) f o r E ° ' = E° - 29.6(pH) (64) pH the MAD + model compounds u s e d i n t h i s s t u d y was t e s t e d u s i n g TABLE X I I I R e d u c t i o n P o t e n t i a l s f o r P y r i d i n i u m / D i h y d r o p y r i d i n e H a l f - c e l l s I R1 s l o p e o f R l R 3 o f E vs l o g [ P y H ] / [ P y + ] (mv) pH E° ' PH (mv v s N . H . E . ) TP O 1 L 7 (mv v s N . H . E . ) E° (mv vs N . H . E . ) s t d . dev-(mv) CH COO* CONH „ 2 -34 . 6 10 . -43 9 -350 -143 5 . 0 CH 3 CONH -22.3 10 . -463 -374 -167 7.4 CHjCH OH CONH -29.7 9.7 -441 -361 -154 4 . 1 CH OCH 2 3 CONH 2 -29 . 5 8 .8 -357 -304 - 97 1.1 CH COCH. 3 CONH 2 -26.2 8 .8 -381 -328 -121 4 .5 CH COOCH(CH ) CONH -24 . 7 7.0 -318 -318 -111 2 .9 CH CN CONH -28 . 3 5 . 6 -22 3 -263 - 56 1.7 CH CONH 2 2 CONH„ 2 -29.2 9 . 2 -385 -320 -113 1 . 6 CH CONH„ 2 2 COCff 3 -27.3 8 .8 -331 -278 - 71 1.6 CH 2CONH CN -31.9 7 . 0 -225 -225 - 18 3 . 7 CO T a b l e X I I I ( c o n t . ) s l o p e o f E vs l o g [ P y H ] / [ P y + ] (mv) PH NAD ( I a , page 128) NMN+ ( I I , page 128) > N N = / 30.3 O N H 2 -8.8 8.9 6.9 E ° £ E ° * E ° S t d (mv v s (mv v s (mv v s d e v N . H . E . ) N . H . E . ) N . H . E . ) (mv -365 -312 -105 5.7 -358 -302 - 95 4.5 -279 -282 - 15. 135 l - c a r b a m o y l m e t h y l - 3 - c a r b a m o y l p y r i d i n i u m c h l o r i d e ( I l l i ) between pH 7.7 and 9.6 as shown i n F i g u r e 24. The t r i a n g l e s O N H , C H 2 C O N H 2 ( I l l i ) r e p r e s e n t p o t e n t i a l s measured i n g l y c i n e b u f f e r , t h e diamonds p o t e n t i a l s m e asured i n T r i s b u f f e r . P o t e n t i a l s measured a t pH ' s g r e a t e r t h a n 9.8 t e n d e d to be much l e s s r e p r o d u c i b l e t h a a t l o w e r pH's and were t h e r e f o r e n o t i n c l u d e d . ( T h i s p r o b l e m was e n c o u n t e r e d a g a i n i n the measurement o f t h e r e d u c t i o n p o t e n t i a l s o f 1 - c a r b o x y m e t h y 1 - ( I I I a ) , 1 - m e t h y l - ( I l l b ) , and 1-(2 ' - h y d r o x y e t h y l ) - 3 - c a r b a m o y l p y r i d i n i u m i o n s w h i c h had to be made between pll 9.7 and 1 0 . 4 ) . The r e d u c t i o n p o t e n t i a l s • N I R : O N H , a: R 1 = CH COO b: = CH. ( I l l ) CH 9CH OH a t e ach pH, E^ J , were c a l c u l a t e d from e q u a t i o n (63) and p l o t t e d a g a i n s t pH i n F i g u r e 24, from which a pH d e p e n d e n c e , 136 . 0 -I a. 5 9 . 0 9.5 — l _ _ F i g u r e 24. The pH dependence pH o f the r e d u c t i o n p o t e n t i a l , E o f l - c a r b a m o y l m e t h y l - 3 - c a r b a m o y l p y r i d i n i u m c h l o r i d e pH ' 137 E ° * = E - 29.6 l o g [ P y H ] / [ P y + ] (66) p i i A E ^ / A p H , o f 30.5 mv. p e r pH u n i t was f o u n d , i n good a g r e e -ment w i t h the t h e o r e t i c a l dependence o f 29.6 mv. per pH u n i t . H a v i n g c o n f i r m e d t h a t the p'l dependence o f t h e p o t e n t i o -m e t r i c r e d u c t i o n p o t e n t i a l s i s as e x p e c t e d , e q u a t i o n s (64) and (67) c a n be used t o c a l c u l a t e the s t a n d a r d r e d u c t i o n p o t e n t i a l , • O _ n O I E 0 ' + 2 9.6(pH) (64) E° = E^' + 29.6(pH-7) (67) E ° , and the r e d u c t i o n p o t e n t i a l a t pH 7, E ° ' . These v a l u e s a r e l i s t e d i n the s e v e n t h and e i g h t h columns o f T a b l e X I I I r e s p e c t i v e l y . The l a s t c olumn o f T a b l e X I I I l i s t s t h e s t a n d a r d d e v i a -t i o n s o f t h e r e d u c t i o n p o t e n t i a l s o f each compound d e t e r m i n e d i n t h e f o l l o w i n g way. E q u a t i o n (66) was u s e d to c a l c u l a t e a v a l u e o f E ° ' from e a c h measured c e l l p o t e n t i a l , E, and a pii EP H = E " 2 9 , 6 l o g [ p y H ] / [ p y + ] (66) s t a n d a r d d e v i a t i o n was t h e n c a l c u l a t e d from t h e c o l l e c t i o n o f E ° 1 v a l u e s . T h i s method n i v e s a b e t t e r e s t i m a t e o f t h e e r r o r pH i n t h e r e d u c t i o n p o t e n t i a l s t h an would be o b t a i n e d from t h e s t a n d a r d d e v i a t i o n s o f the measured c e l l p o t e n t i a l s a b o u t the 138 b e s t l e a s t - s q u a r e s l i n e f o r each compound. The r e d u c t i o n p o t e n t i a l o f tiAD + ( I I I ) has been d e t e r -14 3 2 mined p r e v i o u s l y ' so t h i s compound was n o t s t u d i e d e x t e n -s i v e l y . The p o t e n t i a l was f i r s t d e t e r m i n e d i n t h i s s t u d y by H H -CONH, + HT+ 2e — 4> 1 R 1 (ill) -fir CONH, ( IV) O •o — P — O — P • w II II \_J o o HO OH HO OH NH2 <'"XJ an o x i d a t i v e t i t r a t i o n o f NADU ( I V ) , s i m i l a r t o t h e method u s e d 14 by Rodkey. T h r o u g h o u t the o x i d a t i v e t i t r a t i o n t h e c a l c u l a t e d r e d u c t i o n p o t e n t i a l ( u s i n g e q u a t i o n (65)) become more p o s i t i v e , E^J = E - 2 9.6 log [NADH] / [NAD ] (65) as shown i n T a b l e XIV and when t h e f i n a l s o l u t i o n was a s s a y e d f o r NAD + p o l a r o g r a p h i c a l l y , i t v/as fo u n d t h a t t h e s o l u t i o n c o n t a i n e d two p y r i d i n i u m s a l t s i n a p p r o x i m a t e l y a 50:50 mix-t u r e w i t h t h e s e c o n d p y r i d i n i u m i o n a p p e a r i n g a t a s l i g h t l y more n e g a t i v e p o l a r o g r a p h i c r e d u c t i o n p o t e n t i a l t h a n NAD +. TABLE XIV R e s u l t s from the O x i d a t i v e T i t r a t i o n o f NADH (mM) (NAD4") (mM) 0. 40 0 .93 1. 54 2.02 E (mv .) -3 92 -368 -355 -334 E° ' (mv.) Ph -370 -3 61 -3 5 9 -3 51 E ° • ( m v . -320 -311 -309 -301 14 0 S i n c e t h e NAD + sample was found t o be homogeneous i n p o l a r o -g r a p h i c s t u d i e s a t pH 5.6, i t was c o n c l u d e d t h a t t h e NAD + and/or t h e NADH must be deco m p o s i n g a t t h e h i g h e r pH o f 8.7, where the p o t e n t i o m e t r i c measurements were made. I n an a t t e m p t to measure t h e NAD +/NADH p o t e n t i a l b e f o r e d e c o m p o s i -t i o n became s i g n i f i c a n t , s i n g l e s o l u t i o n s were p r e p a r e d c o n -t a i n i n g b o t h NAD + and NADH (method o f m i x t u r e s ) and t h e c o n t e n t s were a n a l y z e d as soon as p o s s i b l e a f t e r t h e measured c e l l p o t e n t i a l had r e a c h e d a c o n s t a n t v a l u e . The p o l a r o g r a p h i c + + a s s a y f o r NAD showed t h a t a p p r o x i m a t e l y 15% o f t h e NAD had a g a i n been c o n v e r t e d to a n o t h e r p y r i d i n i u m compound i n the a p p r o x i m a t e l y 2 h o u r s t h a t t h e c r y s t a l s had been d i s s o l v e d i n t the b u f f e r . V a l u e s o f -309 mv. and -312 mv. were c a l c u l a t e d f o r t h e r e d u c t i o n p o t e n t i a l a t pH 7, E ° ' , from t h e measured c e l l p o t e n t i a l s o f t h e s e s o l u t i o n s b u t s i n c e o n l y t h e t o t a l d i h y d r o p y r i d i n e c o n c e n t r a t i o n and n o t the c o n c e n t r a t i o n o f NADH can be d e t e r m i n e d t h e s e v a l u e s c a n be c o n s i d e r e d as no b e t t e r t h a n r o u g h e s t i m a t e s . D e s p i t e t h i s t h e v a l u e s a r e i n f a i r a g reement w i t h p r e v i o u s l y r e p o r t e d p o t e n t i a l s f o r t h i s s y s t e m . When s o l u t i o n s c o n t a i n i n g NAD + and NADH were l e f t o v e r -n i g h t , the c e l l p o t e n t i a l s s l o w l y d r i f t e d t o more p o s i t i v e S i n c e t h e c o n c e n t r a t i o n o f NADH i t s e l f c o u l d n o t be d e t e r m i n e d , the t o t a l p y r i d i n i u m and d i h y d r o p y r i d i n e c o n c e n t r a t i o n s were used in e q u a t i o n (63) to c a l c u l a t e E ° ' . 141 p o t e n t i a l s and, a f t e r t h e s o l u t i o n s were a s s a y e d f o r p y r i d i n i u m and d i h y d r o p y r i d i n e compounds, r e d u c t i o n p o t e n t i a l s , E ° ' , o f a p p r o x i m a t e l y -300 mv. were c a l c u l a t e d [ e q u a t i o n s (65) and ( 6 7 ) ] . S i n c e the p o l a r o g r a p h i c a s s a y showed t h a t t h e s o l u t i o n c o n t a i n -ed o n l y one p y r i d i n i u m i o n , t h i s r e d u c t i o n p o t e n t i a l p r o b a b l y r e p r e s e n t s the v a l u e o f E ° ' f o r t h e d e c o m p o s i t i o n p r o d u c t o f NAD. T h i s p o t e n t i o m e t r i c r e d u c t i o n p o t e n t i a l o f -300 mv. and the p o l a r o g r a p h i c r e d u c t i o n p o t e n t i a l b e i n g s l i g h t l y more n e g a t i v e t h a n f o r NAD + a r e c o n s i s t e n t w i t h t h e d e c o m p o s i t i o n p r o d u c t b e i n g t h e m o n o n u c l e o t i d e , NMN ( I I ) , a r i s i n g from h y d r o l y s i s o f the p y r o p h o s p h a t e bond i n NAD + ( l a ) . NH HO OH HO OH ( l a ) H 90, pH 8.7 HO (II) OH HO OH 14 2 S i n c e t h e d e c o m p o s i t i o n of HAD + ( l a ) a t pH 3 . 8 c a u s e s the measured c e l l p o t e n t i a l to d r i f t t o more p o s i t i v e p o t e n -t i a l s , t h e n the e s t i m a t e d v a l u e o f -312 mv. f o r E ° 1 i s p r o b a b l y a few m i l l i v o l t s more p o s i t i v e t h an the t r u e r e d u c t i o n p o t e n -t i a l o f NAD +. T h i s p u t s E ° ' v e r y c l o s e to t h e r e d u c t i o n p o t e n t i a l s d e t e r m i n e d f o r 1 - c a r b o - i - p r o p o x y m e t h y l - and 1-c a r b a m o y l m e t h y l - 3 - c a r b a m o y l p y r i d i n i u m c h l o r i d e s . The r e d u c t i o n p o t e n t i a l o f n i c o t i n a m i d e m o n o n u c l e o t i d e ( I I ) i s a b o u t 10 t o 15 mv. more p o s i t i v e t h a n t h a t f o r NAD +, f a l l i n g v e r y c l o s e to t he r e d u c t i o n p o t e n t i a l o f l - m e t h o x y m e t h y l - 3 - c a r b a m o y l -n y r i d i n i u m c h l o r i d e . The r e d u c t i o n p o t e n t i a l o f 1 , 1 1 - e t h y l e n e b i s ( 3 -c a r b a m o y l p y r i d i n i u m c h l o r i d e ) (XlXb) has been e s t i m a t e d i n t h i s work f o r t h e two e l e c t r o n r e d u c t i o n o f one r i n g . T h e r e i s H2NOC + H 2€ CONH, \ /* ( X X X I I ) 143 some doubt as to the accuracy of the value found s i n c e compound XXXII could not be recovered during attempted syntheses. For the p o t e n t i o m e t r i c d e t e r m i n a t i o n s , XXXII was prepared _in s i t u by r e d u c t i o n of XlXb v/ith sodium d i t h i o n i t e . The c o n c e n t r a t i o n s of both XlXb and XXXII v/ere measured s p e c t r o p h o t o m e t r i c a l l y , but i n order to do t h i s , e x t i n c t i o n c o e f f i c i e n t s of both com-pounds had to be determined. An e x t i n c t i o n c o e f f i c i e n t f o r -1 -1 XlXb of 9.02 mtl -cm a t 265 nm. was found v/hich i s i n good 9 0 agreement with the value of 8.99 r e p o r t e d by C r a i g e_t a l . A s o l u t i o n of XlXb was prepared and p a r t i a l l y reduced by the a d d i t i o n of small amounts of sodium d i t h i o n i t e . The concen-t r a t i o n of XXXII was determined a f t e r each a d d i t i o n of d i t h i o n i t e from the decrease i n absorbance a t 265 nm. The concurrent i n c r e a s e i n absorbance at 340 nm. was then used to c a l c u l a t e an e x t i n c t i o n c o e f f i c i e n t of 12.5 f o r XXXII at t h i s wavelength. Since there was no n o t i c e a b l e absorbance peak near 290 nm. i n these s o l u t i o n s , i t was assumed t h a t the a c i d c a t a l y z e d decomposition (see s e c t i o n 3.1) of the d i h y d r o -p y r i d i n e r i n g of XXXII was not o c c u r i n g to a s i g n i f i c a n t •1 -1 ex t e n t . The e x t i n c t i o n c o e f f i c i e n t of 12.5 mM. -cm. f o r XXXII i s a value of 6.25 mM. "*"-cm. ^ per p y r i d i n e r i n g which i s comparable to the e x t i n c t i o n c o e f f i c i e n t s found i n t h i s work f o r other 3-carbamoy1-1,4-dihydropyridines a t t h i s wave-length (5.5 to 6.5 mM. ^-cm. ) . U n f o r t u n a t e l y , there i s no way of d i s t i n g u i s h i n g between XXXII and the f u l l y reduced compound (XXXIII) although i t i s probably s i g n i f i c a n t t h a t during d i t h i o n i t e t i t r a t i o n s of XlXb the a c i d c a t a l y z e d 144 H2NOC N N CONH 2 \ / (XXXIII) d e c o m p o s i t i o n o f t h e d i h y d r o p y r i d i n e r i n g became n o t i c e a b l e o n l y a f t e r a p p r o x i m a t e l y 1 e q u i v a l e n t o f d i t h i o n i t e had been added. On t h e b a s i s o f i n d u c t i v e e f f e c t s ( s e e s e c t i o n 3.1) one would e x p e c t X X X I I I to undergo a c i d d e c o m p o s i t i o n more r e a d i l y t h a n th e 1 , 4 - d i h y d r o p y r i d i n e r i n g o f XXXII so i t seems t h a t r e d u c t i o n o f the s econd r i n g d o e s n o t r e a c h s i g -n i f i c a n t l e v e l s u n t i l t h e f i r s t r i n g has been a l m o s t f u l l y r e d u c e d . Thus t h e o b s e r v e d p o t e n t i a l s a r e p r o b a b l y r e a s o n a b l y c l o s e t o t h e t r u e r e d u c t i o n p o t e n t i a l o f X l X b . 3.6.1 S u b s t i t u e n t E f f e c t s a t the 1 - p o s i t i o n The r e a c t i o n c o n s t a n t p i s a measure o f t h e e f f e c t o f s u b s t i t u e n t s on a r e a c t i o n and f o r a r e v e r s i b l e r e a c t i o n c a n be d e t e r m i n e d f rom t h e e f f e c t o f s u b s t i t u e n t s on t h e e q u i l i -b r i u m c o n s t a n t [ e q u a t i o n ( 3 2 ) ] . F o r an o x i d a t i o n - r e d u c t i o n P = A l o g K (32) r e a c t i o n, r e d u c t i o n t h e e q u i l i b r i u m c o n s t a n t i s r e l a t e d t o t h e p o t e n t i a l , E° , by e q u a t i o n ( 7 ) . C o m b i n i n g 145 s t a n d a r d e q u a t i o n s E° = — £n K nF (7) (32) and (7) i n t o e q u a t i o n (42) a l l o w s t h e r e a c t i o n c o n s t a n t p t o be d e t e r m i n e d from the e f f e c t o f s u b s t i t u e n t s on the nF AE° 2.303RT * Aa (42) s t a n d a r d r e d u c t i o n p o t e n t i a l . The s t a n d a r d r e d u c t i o n p o t e n t i a l s ( l i s t e d i n T a b l e X I I I ) f o r the S e r i e s I compounds ( I I I ) a r e p l o t t e d a g a i n s t a* i n F i g u r e 25. The e r r o r l i m i t s shown f o r e a c h a r e t h e s t a n d a r d ' C O N H , + H + 2e~ N I H ,H Cf N O N H , ( I I I ) (IV) d e v i a t i o n s l i s t e d i n T a b l e X I I I . As was o b s e r v e d w i t h t h e p o l a r o g r a p h i c r e d u c t i o n , t h e c a r b o x y m e t h y l - and methoxymethy1• compounds have more p o s i t i v e r e d u c t i o n p o t e n t i a l s t h a n v/ould be e x p e c t e d . The b e s t l e a s t - s q u a r e s l i n e ( e x c l u d i n g t h e S t a n d a r d R e d u c t i o n P o t e n t i a l , E° (mv. v s . N . II. E . ) o I o I o o I o o o o I M tO o I o o I o Q o u> o o o t "O r> o * M 0 Ch o H. ^ H* II \ o > » H- r t + Q 3 to * o 01 H 0 0 r t 0 Ml oo II ro t> Ml Q >< cn CO Ml * M r t U) • H- Pi & 3 3 II H- & < CD 3 0) * • c r ro M U l ro c r co JU M H H ro < Ml CD *— 1 c X 0 o 0 ro r t 1—1 H-. M 0 1 cn 3 1 o 0 C5 Ml 0 to r t O r t ro o t r 3 o ro r t 1 H-— cn P> ro i — • M cn H-ro Ml cn 0 h H r t o c r 0 ro 3 TJ -a 0 •< c Ii nd id cn 9^1 147 1-carboxymethy1 compound) has a s l o p e , A E ° / A a * , o f +83 mv. from which a p* o f +2.8 can be c a l c u l a t e d u s i n g e q u a t i o n (42) . 7 6 T h i s v a l u e i s c o n s i d e r a b l y s m a l l e r t h a n t h e p* o f +3.8 r e p o r t e d f o r t h e a c i d d i s s o c i a t i o n o f p r o t o n a t e d m e t h y l amines [ e q u a t i o n ( 5 7 ) ] , a r e a c t i o n i n which one would e x p e c t a H. H 'CONH, + H + 2e" CON H, N I ( 6 8 ) P.-N + H. R-NH + H 2 ( 57) s i m i l a r s u b s t i t u e n t e f f e c t s i n c e b o t h r e a c t i o n (68) and (57) have s u b s t i t u e n t s a t t a c h e d t o a p o s i t i v e l y c h a r g e d q u a t e r n a r y n i t r o g e n atom v/hich i s t r a n s f o r m e d i n t o an u n c h a r g e d n i t r o g e n i n the p r o d u c t s . 3.6.2 S u b s t i t u e n t E f f e c t s a t the 3 - p o s i t i o n The d a t a i s n o t r e a l l y s u f f i c i e n t t o a l l o w a p r o p e r a n a l y s i s o f the e f f e c t o f the 3 - s u b s t i t u e n t on t h e r e a c t i o n shown i n e q u a t i o n ( 6 8 ) . Of t h e o r i g i n a l s i x compounds i n S e r i e s I I ( X X I i i t o XXIIn) the Py^PyH r e d u c t i o n p o t e n t i a l s o f o n l y the 3 - c a r b a m o y l - , 3 - a c e t y l - , and 3-cyano-compounds c o u l d be m e a s u r e d . A t a pll as h i g h as 10.5, 1-carbamoylme t h y l - 3 -f l u o r o - 1 , 4-d i h y d r o p y r i d i n e v/as decomposed ( s e e S e c t i o n 3.2) 148 too r a p i d l y f o r p o t e n t i o m e t r i c measurements to be made, and the r e d u c e d forms o f the 3-hydroxy and 3 - p r o t i o compounds c o u l d n o t be p r e p a r e d . + H + 2 e " ^ C H 2 C O N H 2 H. H C H 2 C O N H 2 (68) (XXII) (XXXIV) R„ = CONH D : k: 1 : COCH. CN F m: f l OH When t h e s t a n d a r d r e d u c t i o n p o t e n t i a l s o f t h e t h r e e S e r i e s I I compounds a r e p l o t t e d a g a i n s t a ^ a s l o p e , A E ° / A a K , o f +276 mv. ( c o r r . c o e f f . = .986) i s f o u n d from w h i c h a p o f m +9.3 can be c a l c u l a t e d u s i n g e q u a t i o n ( 4 2 ) . As was f o u n d w i t h D = n F AE" 2.3 03RT * Aa < 4 2> the p o l a r o g r a p h i c r e d u c t i o n p o t e n t i a l s , t h e e f f e c t o f t h e 3-s u b s t i t u e n t s on t h e p o t e n t i o m e t r i c r e d u c t i o n p o t e n t i a l s i s b e t t e r c o r r e l a t e d by Op ( c o r r . c o e f f . = .9994) g i v i n g a s l o p e 149 of +317 mv. from w h i c h a r e a c t i o n c o n s t a n t o f 10.7 c a n be c a l c u l a t e d , a s l i g h t l y l a r g e r v a l u e t h a n i s c a l c u l a t e d from the p l o t . The v a l u e s a r e b o t h c o n s i d e r a b l y l a r g e r t h a n the 7 7 a l r e a d y s u b s t a n t i a l r e a c t i o n c o n s t a n t o f 5.9 r e p o r t e d f o r the a c i d d i s s o c i a t i o n o f p r o t o n a t e d p y r i d i n e s [ r e a c t i o n (53)] K H + H 2e~ I^T C H 2 C O N H 2 R, N C H 2 C O N H 2 (68) p = +9.3 t o +10.7 ( 60) p = +5.9 b u t a r e q u i t e s i m i l a r to the p o f +10.8 t o +12.4 f o u n d i n t h i s work f o r t h e e f f e c t o f 3 - s u b s t i t u e n t s on t h e p o l a r o g r a p h i c r e d u c t i o n o f p y r i d i n i u m i o n s , [ e q u a t i o n ( 4 1 ) ] . (41) C H 2 C O N H 2 C H 2 C O N H 2 0 = +10.8 to +12.4 150 The O x i d a t i o n o f D i h y d r o p y r i d i n e s by F l a v i n s The o x i d a t i o n by f l a v i n s [ e q u a t i o n ( 6 9 ) ] i s one o f t h e more f a c i l e o x i d a t i o n - r e d u c t i o n r e a c t i o n s o f 1 , 4 - d i h y d r o -p y r i d i n e s . The r a t e c o n s t a n t s c a n r e a d i l y be d e t e r m i n e d I (69) a n a e r o b i c a l l y by f o l l o w i n g t h e r a t e o f r e d u c t i o n o f t h e f l a v i n i n t h e p r e s e n c e o f e x c e s s d i h y d r o p y r i d i n e , o r a e r o b i c a l l y by f o l l o w i n g t h e r a t e o f d i s a p p e a r a n c e o f t h e 340 nanometer d i h y d r o p y r i d i n e a b s o r b a n c e band i n the p r e s e n c e o f a c a t a l y t i c amount o f f l a v i n . In t h e l a t t e r method, the f l a v i n i s r a p i d l y r e o x i d i z e d by o x y g e n . B o t h methods have been u s e d p r e v i o u s l y to s t u d y t h e o x i d a t i o n o f v a r i o u s d i h y d r o p y r i d i n e s by v a r i o u s 19-24 26 f l a v i n s a t v a r i o u s t e m p e r a t u r e s . ' The f o r m e r method ha been u s e d e x c l u s i v e l y i n t h i s work. 151 The r a t e c o n s t a n t s f o r the o x i d a t i o n o f most o f the d i -h y d r o p y r i d i n e s were f o u n d u s i n g e q u a t i o n (70) f o r a s e c o n d o r d e r i r r e v e r s i b l e r e a c t i o n by p l o t t i n g the q u a n t i t y on the 1 . j ( [PyH] n - X) -, k 2 t ( [PyH] - [Fin l o g l ( [ F i ] - X } = 173^3 + c o n s t - <70> o o o where [PyH] = i n i t i a l c o n c e n t r a t i o n o f d i h y d r o p y r i d i n e s [ F l ] = i n i t i a l c o n c e n t r a t i o n o f f l a v i n o X = amount o f r e a c t a n t s u s e d up a t time t = second o r d e r r a t e c o n s t a n t l e f t s i d e o f e q u a t i o n (70) a g a i n s t t i m e as shown i n F i g u r e 26. The s l o p e o f t h i s p l o t i s t h e n the s e c o n d - o r d e r r a t e c o n s t a n t , k . The q u a n t i t y X i n e q u a t i o n (70) v/as c a l c u l a t e d from the a b s o r b a n c e a t 450 n a n o m e t e r s , w h i c h i s due s o l e l y t o the o x i d i z e d and r e d u c e d f l a v i n , u s i n g e q u a t i o n ( 7 1 ) . ,, ( A 0 - A) A = Ul T ( 7 1 ) r e a c p r o d where A = a b s o r b a n c e a t time A = i n i t i a l a b s o r b a n c e o £ = p a t h l e n g t h o f the a b s o r b a n c e c e l l r e a c ' " p r o d c „ 0 = , „ , e _ „ ^ = e x t i n c t i o n c o e f f i c i e n t s o f r e a c t a n t ( o x i d i z e d f l a v i n ) and p r o d u c t ( r e d u c e d f l a v i n ) r e s p e c t i v e l y . 1 5 3 The c u r v a t u r e a t l o n g e r t i m e s i s p r o b a b l y due to r e o x i d a t i o n o f the f l a v i n by oxygen l e a k i n g i n t o the c e l l . The r a t e o f r e a c t i o n o f FMM w i t h l - c a r b a i n o y l m e t h y l - 3 -c y a n o - 1 , 4 - d i h y d r o p y r i d i n e (v~k) was d e t e r m i n e d from p s e u d o -f i r s t o r d e r k i n e t i c s , shown i n F i g u r e 27, s i n c e t h e d i h y d r o -p y r i d i n e was p r e s e n t i n an e x c e s s o f more t h a n 100 t i m e s the c o n c e n t r a t i o n o f FMM. The p s e u d o - f i r s t o r d e r r a t e c o n s t a n t , k . , c a n be f o u n d from a p l o t o f l n ( A - A-,) a g a i n s t t i m e as shown below. The c u r v a t u r e a t l o n g e r t i m e s i s p r o b a b l y due H H (Vk) to r e o x i d a t i o n o f the f l a v i n by oxygen l e a k i n g i n t o the c e l l . S i n c e the c o n c e n t r a t i o n o f d i h y d r o p y r i d i n e was so much l a r g e r t h a n t h a t o f the f l a v i n one can w r i t e e q u a t i o n ( 7 2 ) . The d [ F l ] k [ F l ] [ P y H ] = k v [ F l ] (72) d t 2 * ob where k = k-[PyH] (73) o b s 2 c o n c e n t r a t i o n o f o x i d i z e d f l a v i n , [ F l ] , c a n be f o u n d from t h e a b s o r b a n c e by r e a r r a n g i n g (23) t o g i v e (26) and ( 7 4 ) . H-3 F i g u r e 27. D e t e r m i n a t i o n o f t h e s e c o n d o r d e r r a t e c o n s t a n t f o r t h e o x i d a t i o n o f l - c a r b a m o y l m e t h y l - 3 - c y a n o - l , 4 - d i h y d r o p y r i d i n e by FUN from a p s e u d o - f i r s t o r d e r p l o t o f l n ( A - A w ) a g a i n s t t i m e . In ( A - A a , ) i i o x ro o o I 3 I O to 0 cr II in , , II o •~< cr • c * — ' U) Ln II O • 3 o H-o 3 to 1 co 1—' U) 5 155 x = e C + E c (23) I r e a c r e a c prod p r o d (A/I) - e , c.n. c 'reac e - £ r e a c p r o d prod ' i n i t i a l (25) S u b s t i t u t i o n o f e q u a t i o n (74) i n t o (62) g i v e s (28) w h i c h A = C C (2 6) oo "prod i n i t i a l A - A( C r e a c H(£ - ) r e a c p r o d or A - A, [ F l ] = * ( E F 1 ' S l H ^ (74) i n t e g r a t e s to e q u a t i o n ( 2 9 ) . The sec o n d o r d e r r a t e c o n s t a n t d[A - A ] ~ = k [A - A ] (28) d t obs 0 0 £n(A - A ) = k , t + c o n s t . (29) v oo ob s can t h e n be found from k , u s i n g e q u a t i o n ( 7 3 ) . o b s The r a t e o f o x i d a t i o n o f NADH (XXI) by f l a v i n s has been x. r, , • 20, 22-24 , 26 r , „ ^ , 20 r e p o r t e d by v a r x o u s a u t h o r s . S u e l t e r and M e t z l e r f o u n d a se c o n d o r d e r r a t e c o n s t a n t o f 0.75 M "'"-sec f o r t h e a e r o b i c o x i d a t i o n o f NADH by r i b o f l a v i n (Xa) near pH 7. Rate c o n s t a n t s f o r t h e same o x i d a t i o n u s i n g FMN (Xb) have been 156 H. H -CONH, I I r r ~ r NH, O •P —O- N ' HO OH HO OH (XXI) 2 2 2 3 2 4 d e t e r m i n e d a e r o b i c a l l y and a n a e r o b i c a l l y "' a t pH 7 and ra n g e from 0.092 M ^ - s e c " 1 to 1.1 M _ 1 - s e c 1 , a f t e r b e i n g (X) CH OH CHOH a: R = CHOH CHOH b : R CHOH CH 2OP0 3 CHOH I CHOH I CHOH I CHOH d: R = -CH. c o r r e c t e d t o 25°C. u s i n g an a c t i v a t i o n e n e r g y o f 8.3 k c a l / m o l e . -1 -1 B r u i c e f o u n d a r a t e c o n s t a n t o f 0.43 M - s e c f o r t h e o x i d a -t i o n o f NADH a t pH 7.7 by l u m i f l a v i n ( X d ) , a f l a v i n w h i c h had 157 p r e v i o u s l y been shown to r e a c t w i t h l - p r o p y l - 3 - c a r b a m o y l - l , 4 -2 0 d i h y d r o p y r i d i n e (XX) a t t h e same r a t e as does r i b o f l a v i n . CONH, (XX) The d i v e r s e r a t e c o n s t a n t s r e p o r t e d i n t h e l i t e r a t u r e do n o t f a c i l i t a t e c o m p a r i s o n s w i t h the p r e s e n t work. S e v e r a l 2 2 24 26 a u t h o r s ' ' have a l s o d e t e r m i n e d the r a t e c o n s t a n t s f o r the o x i d a t i o n o f b o t h l - p r o p y l - 3 - c a r b a m o y l - l , 4 - d i h y d r o p y r i d i n e and NADH u n d e r s i m i l a r c o n d i t i o n s b u t t h e r a t i o s o f t h e s e two r a t e c o n s t a n t s v a r y from 65 t o 160, a g a i n a d i s c o u r a g i n g l y wide r a n g e . As a r e s u l t , o n l y o r d e r - o f - m a g n i t u d e c o m p a r i s o n s can be made between t h i s s t u d y and p r e v i o u s work. The s e c o n d - o r d e r r a t e c o n s t a n t s d e t e r m i n e d i n t h i s s t u d y a r e l i s t e d i n T a b l e XV. NADH was o x i d i z e d by r i b o f l a v i n a t a - 1 - 1 r a t e o f 0.53 M - s e c , a v a l u e w h i c h i s r e a s o n a b l y c o n s i s t e n t w i t h p r e v i o u s r a t e s u s i n g r i b o f l a v i n 2 0 and l u m i f l a v i n . 2 6 A t pH 8.1, FMN and r i b o f l a v i n were e q u a l l y e f f e c t i v e a t o x i d i z i n g 1 - a c e t o n y 1 - 3 - c a r b a m o y 1 - 1 , 4 - d i h y d r o p y r i d i n e . The l i t e r a t u r e i s v e r y c o n f u s i n g on t h i s p o i n t s i n c e r e p o r t s have FMN r e a c t -2 0 19 i n g b o t h 50% f a s t e r and 40% s l o w e r t h a n r i b o f l a v i n a t pH 7. D i h y d r o n i c o t i n a m i d e m o n o n u c l e o t i d e (MMNH, XXXV) was i s a TABLE XV Second O r d e r Rate C o n s t a n t s f o r t h e O x i d a t i o n o f D i h y d r o p y r i d i n e s by F l a v i n s H H + f l a v i n ^ R 3 + r e d u c e d f l a v i n N 1 N * 1 1 1 R1 R l R 3 F l a v i n pH k (M ''"-see - 1 ) CONH 2 Rf 1 9 . 8-10 .3 64 + 13 . Ch' CH OH 2 2 CONH Rf 1 9.8 21 + 2 . 6 CH 2OCH 3 CONH Rf 1 8 . 1-8. 7 0 .607 + .019 CH COCH 3 CONH o Rf 1 8.1 3.70 + . 24 FMN 8.1 3.70 + . 28 CH COOCH 3 CONH FMN 7.8 1 .99 + .18 CH CN CONH FMN 8 . 2 0 .150 + .022 CH CONII 2 CONH FMN 7 .8 9.4 2 . 97 2 . 98 + + .36 .09 CH COHH 2 COCH Rf 1 8.1 0. 548 + .019 CH CONH CN FMN 6 . 9 .0449 + .0032 S p e c i a l Compounds: NADH (XXIV) Rf 1 8.1 .53 NMNH (XXXV) FMN 8.1 . 249 + .012 159 o x i d i z e d a t a p p r o x i m a t e l y h a l f the r a t e o f NADH a t pH 3.1. HO OH (XXXV) A l t h o u g h t h e a b s o l u t e r a t e c o n s t a n t s a r e v e r y d i f f e r e n t , 2 2 flcCor m i c k e t a l have f o u n d a s i m i l a r 2:1 r a t i o f o r the o x i -d a t i o n r a t e s o f NADH and NMMH. R i b o f l a v i n o x i d i z e s 1 - m e t h y l -3 - c a r b a m o y 1 - 1 , 4 - d i h y d r o p y r i d i n e a t 150 t i m e s f a s t e r t h a n NADH, w h i c h i s c o m p a r a b l e t o t h e 65 t o 160 f o l d r a t e enhancements of l - p r o p y l - 3 - c a r b a m o y l - l , 4 - d i h y d r o p y r i d i n e o v e r NADH r e p o r t e d 22,24,26 . . p r e v i o u s l y . The r e m a i n i n g d i h y d r o p y r l a m e s have n o t been s t u d i e d p r e v i o u s l y . The e f f e c t o f s u b s t i t u e n t s on r e a c t i o n (69) c a n be (69) 160 measured v i a t h e r e a c t i o n c o n s t a n t , p, w h i c h i s d e t e r m i n e d from e q u a t i o n (35) u s i n g a p l o t o f l o g k a g a i n s t Hammett P = A l o g K Aa (3 5) s u b s t i t u e n t c o n s t a n t s a. The l o g a r i t h m s o f t h e r a t e c o n s t a n t s f o r the S e r i e s I compounds (IV) a r e p l o t t e d a g a i n s t t h e p o l a r s u b s t i t u e n t c o n s t a n t s , a*, ( l i s t e d i n T a b l e V, page 49) i n F i g u r e 28. As was found i n the l i n e a r f r e e e n e r g y r e l a t i o n -s h i p s o f t h e p o l a r o g r a p h i c and p o t e n t i o m e t r i c r e d u c t i o n p o t e n -t i a l s , the 1-methoxymethyl compound f a l l s o f f t h e l i n e formed by the o t h e r compounds. I n t e r e s t i n g l y , t h e o x i d a t i o n r a t e o f NADH i s q u i t e c l o s e t o t h a t o f t h e 1-methoxymethyl compound. E x c l u d i n g the p o i n t f o r 1-methoxymethy1-3-carbamoy1-1,4-d i h y d r o p y r i d i n e ( V e ) , the c o r r e l a t i o n i s q u i t e good ( c o r r . H H N R. (IV) H H N F i g u r e 28. P l o t of the logarithm of the second order r a t e constants f o r the o x i d a t i o n of the s e r i e s I d i h y d r o p y r i d i n e s by f l a v i n s a g a i n s t 0* (Table IV, page Q%). -CH. c o r r . c o e f f . = .992 ( e x c l . CH 2OCH 3) = A l o j k ^ m _ 1 > g  K Aa* •CH2CH2OH -CH 2COCH 3 0 . * H H CONH, N ' I R 1 •CH CONH^-CH 2COOCH 3 -GH OCH l . t -CH^CN -0.3 -0.1 0.1 0.3 0.5 0.7 0.9 1.1 1.3 a* 162 c o e f f . = 0 . 992) , g i v i n g a s l o p e ( e q u i v a l e n t t o p * ) o f -1.9. T h i s v a l u e i s a p p r o x i m a t e l y o n e - h a l f o f t h e m a g n i t u d e o f f o u n d f o r the p o l a r o g r a p h i c ( o n e - e l e c t r o n ) r e d u c t i o n ( p * = +3.7) and a p p r o x i m a t e l y t h r e e - f o u r t h s o f t h e m a g n i t u d e o f p * f o u n d f o r t h e p o t e n t i o m e t r i c ( t w o - e l e c t r o n ) r e d u c t i o n ( p * = +2.8). The S e r i e s I I compounds f o r w h i c h r a t e c o n s t a n t s c o u l d be d e t e r m i n e d , the 3 - c a r b a m o y l / ^ 3 - a c e t y l - , and 3-cyano-compounds (XXIV i - k ) , l e a d to r e a c t i o n c o n s t a n t ( p ) v a l u e s o f -5.4 | k: CN CH 2CONH 2 (XXIV) ( c o r r . c o e f f . = .992) and -6.1 ( c o r r . c o e f f . = .997) when l o g k 2 i s p l o t t e d a g a i n s t and r e s p e c t i v e l y . As w i t h b o t h t h e p o l a r o g r a p h i c and p o t e n t i o m e t r i c r e d u c t i o n s , the p d e r i v e d from t h e p a r a - s u b s t i t u e n t c o n s t a n t s i s s l i g h t l y l a r g e r i n m a g n i t u d e and has a s l i g h t l y b e t t e r c o r r e l a t i o n c o e f f i c i e n t . D I S C U S S I O U 4.1 P o l a r o g r a p h i c R e d u c t i o n i l e c h a n i s m s T h e g e n e r a l l y a c c e p t e d j a e c h a n i s a f o r t h e e l e c t r o c h e m i -c a l r e d u c t i o n o f p y r i d i n i u m s a l t s ( V I I I ) i s shown i n S c h e m e 3. The p o r t i o n o f t h i s s c h e m e w h i c h c o r r e s p o n d s t o t h e f i r s t +• other i s o m e r s ? + other i s o m e r s ? S c h e m e 3 r e d u c t i o n w a v e ( w a v e I i n F i g u r e 2) i s r e p r o d u c e d i n r e a c t i o n ( 4 1 ) . T h e p y r i d i n i u m s a l t ( V I I I ) u n d e r g o e s a r e v e r s i b l e o n e -e l e c t r o n r e d u c t i o n t o g i v e t h e p y r i d i n e r a d i c a l (XV) w h i c h r a p i d l y d i m e r i z e s i n a n i r r e v e r s i b l e r e a c t i o n t o g i v e X V I . 1 G 4 . N I R_ -e (41) ' I I I ) (XV) (XVI) An e q u a t i o n has been d e r i v e d 3 " ' 3 5 to d e s c r i b e the p o l a r o g r a p h i c wave when a mechanism as shown i n r e a c t i o n (41) i s o p e r a t i n g ( e q u a t i o n 1 8 ) . The v a l i d i t y o f t h i s e q u a t i o n i n the p o l a r o -e° + — l n ( i i ) / ( i ) 2/1 (18) g r a p h i c r e d u c t i o n o f p y r i d i n i u m i o n s was t e s t e d i n t h i s work by p l o t t i n g the p o t e n t i a l , E, a g a i n s t l o g ( i - i ) / ( i ) " f o r p o l a r o g r a p h i c waves a t v a r i o u s c o n c e n t r a t i o n s between 10 " ii _ o and 10 * M. P o l a r o g r a p h i c waves r e c o r d e d a t c o n c e n t r a t i o n s -A below 5 x 10 K. were r e a s o n a b l y w e l l d e s c r i b e d by e q u a t i o n 2/3 ( 1 8 ) , ( P l o t s o f E v s . log(i„ - i ) / ( i ) were l i n e a r w i t h u •f s l o p e s n e a r t h e t h e o r e t i c a l v a l u e o f 60 rav. ) i n a g r e e m e n t f , Tne s l o p e s o f t h e s e p l o t s a r e change i n p o t e n t i a l p e r u n i t c i a n g e i n l o g ( i - i ) / ( i ) - / 3 a n d t h e r e f o r e have t h e " u n i t s o f m i l l i v o l t s . 1 G 5 w i t h the mechanist) shown i n e q u a t i o n (41) . Thus i t can be (41) ( V I I I ) c o n c l u d e d t h a t t h i s mechanism i s o p e r a t i n g ( i . e . the e l e c t r o d e r e a c t i o n i s r e v e r s i b l e ) a t p y r i d i n i u m i o n ( V I I I ) c o n c e n t r a t i o n s -4 l e s s t h a n 5 x 10 M. A t c o n c e n t r a t i o n s above 10 3 H.t t h e p o l a r o g r a p h i c waves a r e n o t d e s c r i b e d by e q u a t i o n ( 1 8 ) , b u t i n s t e a d g i v e n e a r -l i n e a r p l o t s v/hen the p o t e n t i a l , E, i s p l o t t e d a g a i n s t l o g t ( i , - i ) / ( i ) . The s l o p e s o f t h e s e l a t t e r p l o t s a r e s i g n i f i -E = E l / 2 + OF" £ n ( i d " ( 1 0 ) c a n t l y l a r g e r t h a n t h e t h e o r e t i c a l s l o p e f o r a r e v e r s i b l e r e d u c t i o n (60 mv.) i n d i c a t i n g an i r r e v e r s i b l e e l e c t r o d e r e a c t i o n . T h i s t r a n s i t i o n from a r e v e r s i b l e e l e c t r o d e f A l i n e a r p l o t o f t; v s . l o g ( i d - i ) / ( i ) i s i n d i c a t i v e o f a r e v e r s i b l e or i r r e v e r s i b l e e l e c t r o d e r e a c t i o n w h i c h i s un-a f f e c t e d by p r e v i o u s or s u b s e q u e n t c h e m i c a l r e a c t i o n s . 166 -A r e a c t i o n ( c o n e . < 5 x 10 M . ) to an i r r e v e r s i b l e e l e c t r o d e — 3 r e a c t i o n ( c o n e . >10 ~ M.) c o r r e s p o n d s t o t h e maximum h e i g h t o f t h e prewave ( i . e . a m o n o l a y e r o f m a t e r i a l has been a d s o r b e d on the e l e c t r o d e ) f o r compounds which e x h i b i t e d a prewave. V a r i o u s s t u d i e s ^ ' ^ ' o ( ' have d e m o n s t r a t e d t h a t t h e d i m e r i c p r o d u c t s (XVI) o f the r e d u c t i o n a r e s t r o n g l y a d s o r b e d on the e l e c t r o d e and t h a t t h i s a d s o r b e d l a y e r c a n i n h i b i t t h e e l e c -3 9 t r o d e r e a c t i o n . Thus, a f t e r a m o n o l a y e r o f d i m e r i c p r o d u c t (XVI) has been a d s o r b e d , the r e d u c t i o n o f t h e b u l k o f the p y r i d i n i u m i o n i s i n h i b i t e d s u f f i c i e n t l y t h a t the e l e c t r o d e r e a c t i o n becomes r a t e - d e t e r m i n i n g . A p p a r e n t l y , t h e d i m e r i c p r o d u c t (XVI) i s a d s o r b e d i n a m u l t i - l a y e r f a s h i o n s i n c e i n c r e a s i n g c o n c e n t r a t i o n (from 2 x 10 'I. t o 10 M. ) c a u s e s a f u r t h e r r e t a r d a t i o n o f the e l e c t r o d e r e a c t i o n , w i t h a r e s u l t -i n g i n c r e a s e i n s l o p e o f a p l o t o f a p p l i e d p o t e n t i a l F, a g a i n s t l o g ( i - i ) / ( i ) . The c o n c e p t o f a r e v e r s i b l e e l e c t r o d e r e a c t i o n a t low -4 c o n c e n t r a t i o n s ( l e s s t h a n 5 x 10 H. ) o f p y r i d i n i u m i o n and 167 an i r r e v e r s i b l e e l e c t r o d e r e a c t i o n a t h i g h c o n c e n t r a t i o n s ( g r e a t e r t h a n 10 J M.) o f p y r i d i n i u m i o n i s a l s o s u p p o r t e d by the e f f e c t s o f 1 - s u b s t i t u e n t s on the p o l a r o g r a p h i c r e d u c t i o n p o t e n t i a l . A t low c o n c e n t r a t i o n s , a r e a c t i o n c o n s t a n t , p*,' o f +3.7 was fou n d i n t h i s work. The s i m i l a r i t y between t h i s 7 6 v a l u e and the p* o f +3.8 r e p o r t e d by W e l l s f o r the a c i d d i s s o c i a t i o n o f p r o t o n a t e d m e t h y l a r a i n e s , r e a c t i o n ( 5 7 ) , i s n - N H + R — NH_ + H (57) c o n s i s t e n t w i t h a n e a r - u n i t change i n the c h a r g e on t h e r i n g n i t r o g e n i n r e a c t i o n ( 4 1 ) . However, a t h i g h c o n c e n t r a t i o n s ' C O N H , 'I R 1 ( I I I ) ( X I I I ) ( X X X V I ) ONH 2 (4 1) -3 ( g r e a t e r t h a n 10 . ) , a r e a c t i o n c o n s t a n t p* o f o n l y +2.1 The r e a c t i o n c o n s t a n t i s a measure o f the e f f e c t o f s u b s t i -t u e n t s on a r e a c t i o n and i s d e t e r m i n e d from p = (Alogk/A(J) where a i s a s e t o f e n p e r i c a l s u b s t i t u e n t c o n s t a n t s . 163 i s f o u n d f o r the p o l a r o g r a p h i c r e d u c t i o n o f p y r i d i n i u m i o n s ( I I I ) . The l o w e r r e a c t i o n c o n s t a n t i m p l i e s a change i n t h e c h a r g e on the r i n g n i t r o g e n o f l e s s t h a n one u n i t , c o n s i s t e n t w i t h t h e r a t e - c o n t r o l l i n g e l e c t r o d e r e a c t i o n shown i n r e a c t i o n (7 5) . : O N H . 4> I R1 ( I I I ) t r a n s i t i o n s t a te (75) + 2.1 To summarize, the e l e c t r o d e r e a c t i o n has been f o u n d t o be r e v e r s i b l e as shown i n r e a c t i o n (41) a t c o n c e n t r a t i o n s o f - 4 - 3 p y r i d i n i u m i o n l e s s t han 5 x 10 :'!. In t h e v i c i n i t y o f 10 a. the s u r f a c e o f t h e e l e c t r o d e becomes s a t u r a t e d by a m o n o l a y e r o f d i m e r i c p r o d u c t ('A XXVI) . T h i s a d s o r p t i o n p r o c e s s a p p e a r s to o c c u r w i t h a l l o f t h e p y r i d i n i u m s a l t s i n v e s t i g a t e d , even though- o n l y a b o u t h a l f o f the compounds e x h i b i t e d a d s o r p t i o n p r ewaves. The l a y e r o f a d s o r b e d d i m e r i c p r o d u c t (XXXVI) i n h i b i t the e l e c t r o d e r e a c t i o n s u f f i c i e n t l y t h a t t h e e q u i l i b r i u m c o n -c e n t r a t i o n s o f p y r i d i n i u m i o n ( I I I ) and p y r i d i n e r a d i c a l ( X I I I ) 16:.) a r e not a t t a i n e d a t t h e e l e c t r o d e s u r f a c e ( i . e . the e l e c t r o d e r e a c t i o n becomes i r r e v e r s i b l e ) . As the c o n c e n t r a t i o n o f p y r i -d i n i u m i o n i s i n c r e a s e d , t h e d i m e r i c p r o d u c t (XXXVI) i s a d s o r b e d i n a m u l t i l a y e r f a s h i o n , v/ith a r e s u l t i n g i n c r e a s e i n t h e i n h i b i t i o n o f t h e e l e c t r o d e r e a c t i o n . S i n c e the e l e c t r o d e r e a c t i o n i s r a t e c o n t r o l l i n g a t -3 c o n c e n t r a t i o n s above 10 M., the r e a c t i o n c o n s t a n t p* o f + 2 . 1 can be c o n s i d e r e d a measure o f the d e g r e e o f e l e c t r o n t r a n s f e r i n t h e t r a n s i t i o n s t a t e o f r e a c t i o n ( 7 5 ) . A t l o w e r c o n c e n t r a --4 t i o n s ( l e s s t h a n 5 x 10 II.) t h e r e a c t i o n c o n s t a n t p* o f +3.7 f o r the p o l a r o g r a p h i c r e d u c t i o n s u p p o r t s a u n i t change i n t h e c h a r g e on t h e r i n g n i t r o g e n , an would be e x p e c t e d f o r the r e v e r s i b l e r e d u c t i o n shown i n r e a c t i o n ( 4 1 ) . ( I l l ) ( X I I I ) (XXXVI) 1 7 0 4.2 The E f f e c t s o f S u b s t i t u e n t s on the R e d u c t i o n o f P y r i d i n i u m I o n s P y r i d i n i u m i o n s ( V I I I ) can be r e d u c e d t o b o t h one-I R 1 ( V I I I ) e l e c t r o n and t w o - e l e c t r o n p r o d u c t s . The o n e - e l e c t r o n r e d u c -t i o n shown i n r e a c t i o n (41) can be o b s e r v e d p o l a r o g r a p h i c a l l y and the r e d u c t i o n p o t e n t i a l s f o r t h i s r e a c t i o n w i l l , i n t h e e n s u i n g d i s c u s s i o n , be r e f e r r e d t o as p o l a r o g r a p h i c r e d u c t i o n p o t e n t i a l s i n o r d e r t o d i s t i n g u i s h them from the r e d u c t i o n p o t e n t i a l s o f t h e t w o - e l e c t r o n r e d u c t i o n shown i n r e a c t i o n (68) , w h i c h a r e measured p o t e n t i o m e t r i c a l l y and hence v / i l l be c a l l e d p o t e n t i o m e t r i c r e d u c t i o n p o t e n t i a l s . S u b s t i t u e n t e f f e c t s have been r e p o r t e d i n the l i t e r a t u r e f o r s e v e r a l r e a c t i o n s 171 + H + 2e H H (68) N I R„ ( V I I I ) w hich would be e x p e c t e d t o p a r a l l e l t h e s u b s t i t u e n t e f f e c t s i n r e a c t i o n s (41) and ( 6 8 ) . Two o f t h e s e a r e r e a c t i o n s (60) 76 and t h e p r e v i o u s l y d i s c u s s e d r e a c t i o n (57) , t h e a c i d d i s s o c i a t i o n r e a c t i o n o f p r o t o n a t e d p y r i d i n e s and p r i m a r y amines, r e s p e c t i v e l y . The 3 - s u b s t i t u e n t s , R^ , i n r e a c t i o n (60) a r e a n a l o g o u s t o t h e 3 - s u b s t i t u e n t s , R^, o f t h e NAD + model compounds ( V I I I ) and t h e s u b s t i t u e n t s i n r e a c t i o n (57) N I H +- H (60) R-NH3 ^ ± : R — N H g + H + a r e a n a l o g o u s to t h e 1 - s u b s t i t u e n t s o f ( V I I I ) . F u r t h e r m o r e , i n b o t h r e a c t i o n s (60) and ( 5 7 ) , t h e n i t r o g e n atom i s u n d e r -g o i n g a change i n c h a r g e from +1 t o z e r o , as i s o c c u r i n g i n b o t h r e d u c t i o n r e a c t i o n s of p y r i d i n i u m i o n s , r e a c t i o n s ( 4 1 ) and (68) . (57) 76 172 15 L i n d q u i s t and Cord e g have d e t e r m i n e d a r e a c t i o n c o n -s t a n t , p*, o f +3.7 f o r t h e e q u i l i b r i u m o f t h e a d d i t i o n o f c y a n i d e i o n t o 1 - subs t i t u t e d - 3 - c ar bjimoyl p y r i d i n ium i o n s , r e a c t i o n ( 7 6 ) , a r e a c t i o n w h i c h i s v e r y s i m i l a r to t h e two-(76) e l e c t r o n r e d u c t i o n o f p y r i d i n i u m i o n s shown i n r e a c t i o n ( 6 8 ) . Kosower lias r e p o r t e d a p o f +13.4 f o r the c h a r g e t r a n s -f e r complex f o r m a t i o n o f p y r i d i n i u m i o d i d e s , r e a c t i o n ( 6 1 ) , a r e a c t i o n w hich i s somewhat a n a l o g o u s to t h e p o l a r o g r a p h i c ( o n e - e l e c t r o n ) r e d u c t i o n . The r e a c t i o n c o n s t a n t s o f t h e f o r e g o i n g r e a c t i o n s a r e summarized i n T a b l e XVI and t h e r e a c t i o n s a r e r e p r o d u c e d h e r e f o r c o m p a r i s o n . The e f f e c t s o f the 1 - s u b s t i t u e n t s w i l l be c o n s i d e r e d f i r s t . T h r e e r e a c t i o n s , t h e a c i d d i s s o c i a t i o n o f p r i m a r y a m i n e s , 7 6 t h e a d d i t i o n o f c y a n i d e to p y r i d i n i u m i o n s , ^ and the r e v e r s i b l e p o l a r o g r a p h i c ( o n e - e l e c t r o n ) r e d u c t i o n o f p y r i d i n i u m i o n s ( t h i s work) have n e a r l y i d e n t i c a l v a l u e s o f r e a c t i o n c o n s t a n t s 0*. U n e x p e c t e d l y , the p o t e n t i o r a e t r i c ( two-e l e c t r o n ) r e d u c t i o n v/as fo u n d to have a much s m a l l e r p * - v a l u e , 17 4 ' a b l e ;•. V J. Sunmarv o f R e a c t i o n C o n s t a n t s o f R e d u c t i o n R e a c t i o n : o f p y r i d i n i u m i o n s and a n a l o g o u s r e a c t i o n s , S u b s t i t u e n t po s i t i o n S u b s t i t u e n t c o n s t a n t s c a 1 e R e a c t i o n c o n s t a n t Re f •» c i i s s o c i a t i o n o r p r i m a r y a m m o : (57) + 3 7 6 C y a n i d e a d d i t i o n t o p y r i d i n i u m i o n s (7G) ( e c u i l i b r i u m ) o * + 3 A c i d d i s s o c i a t i o n o f p y r i d i n e s (CO) P o l a r o g r a v h i e (1-e l e c t r o n ) r e d u c -t i o n o f p y r i d i n i u m i o n s (41) in O* 0 a m + 5.9 + 3.7 +1 o . a + 12.4 73 t h i s w o r k P o t e n t i o m e t r i c ( 2 - 1 e l e c t r o n ) r e d u c t i o n 3 o f p y r i d i n i u m i o n s (68) 3 a* a m a. + 2.8 + 9 . 3 + 10.7 t h i s wo r k C h a r g e - t r a n s f e r complex f o r m a t i o n (61) i i + 13.4 79 17 ii o n l y + 2 . C , compared to the v a l u e s near +3.7 f o r the e f f e c t s of 1 - s u b s t i t u e n t s on the ot h e r e q u i l i b r i a . I t was s u r p r i s i n g t o f i n d t h a t t h e p* v a l u e s f o r r e a c -t i o n s (68) and (76) d i f f e r s i n c e the s u b s t r a t e i s the sarae i n the two c a s e s . The d i f f e r e n c e i n the p* v a l u e s must r e f l e c t d i f f e r e n t e f f e c t s o f s u b s t i t u e n t s on the s t a b i l i t y o f t h e p r o d u c t s . E q u a t i o n s (68) and (76) can be combined to g i v e the h y p o t h e t i c a l r e a c t i o n shown i n e q u a t i o n ( 7 7 ) , the r e a c t i o n (77) (IV) (XXXVII) 176 c o n s t a n t o f w h i c h c a n be c o n s i d e r e d t o be a measure o f t h e r e l a t i v e e f f e c t s o f t h e 1 - s u b s t i t u e n t s on t h e s t a b i l i t i e s o f IV and XXXVII. The e q u i l i b r i u m c o n s t a n t o f r e a c t i o n ( 7 7 ) , K , i s r e l a t e d t o t h e e q u i l i b r i u m c o n s t a n t s o f r e a c t i o n ( 6 3 ) , Ex K , and r e a c t i o n ( 7 6 ) , K , by e q u a t i o n ( 7 8 ) . Hence, the R C d C14 r e a c t i o n c o n s t a n t , p_,^  , f o r r e a c t i o n (77) c a n be f o u n d f r om C /K . (78) CM R( vEx CM Red e q u a t i o n ( 7 9 ) . The v a l u e s o f +3.7 and +2.8 f o r p*., and A l o q K A l o g K Alog K „ * E X - — - = 0 * - 0 * P r x " Arr* A a * A a * C M Rec (7 9) p* , r e s p e c t i v e l y g i v e a v a l u e o f +0.9 f o r p* . The p o s i t i v e v a l u e o f p* f o r r e a c t i o n (77) i m p l i e s t h a t e l e c t r o n w i t h -Ex d r a w i n g 1 - s u b s t i t u e n t s s t a b i l i z e the 4-cyano compounds (XXXVII) w h i l e e l e c t r o n d o n a t i n g 1 - s u b s t i t u e n t s s t a b i l i z e IV. A t f i r s t g l a n c e , i t seems u n r e a s o n a b l e t h a t e l e c t r o n -w i t h d r a w i n g s u b s t i t u e n t s s h o u l d have a g r e a t e r s t a b i l i z i n g e f f e c t on t h e compound (XXXVII) w h i c h a l r e a d y c o n t a i n s an e x t r a e l e c t r o n - w i t h d r a w i n g group i n t h e r i n g . The answer may l i e i n a r e s o n a n c e i n t e r a c t i o n , shown i n e q u a t i o n (80) w h i c h i s p o s s i b l e between t h e r i n g n i t r o g e n and t h e 3 - c a r b a m o y l g r o u p o f 1 , 4 - d i h y d r o p y r i d i n e s . T h i s 177 XXXVIII XXXIX r e s o n a n c e l e a d s to the sane c h a r g e d i s t r i b u t i o n as a r i s e s from the e l e c t r o n i c t r a n s i t i o n , e q u a t i o n ( 8 1 ) , i n 1 , 4 - d i h y d r o -(81) p y r i d i n e s . ^ ' ' x The ease o f the e l e c t r o n i c t r a n s i t i o n ( i . e . the A o f t h e a b s o r b a n c e peak) i s , t h e r e f o r e , a r e a s o n a b l e max measure o f t h e c o n t r i b u t i o n XXXIX c o u l d make to t h e o v e r a l l s t a b i l i t y o f t h e 1 , 4 - d i h y d r o p y r i d i n e . In g e n e r a l , e l e c t r o n -w i t h d r a w i n g s u b s t i t u e n t s a t t h e 1 - p o s i t i o n t e n d t o s h i f t t h e 1 15 5 3 a b s o r b a n c e peak t o s h o r t e r w a v e l e n g t h s , ' ' c o r r e s p o n d i n g to a h i g h e r e n e r g y e l e c t r o n i c t r a n s i t i o n , as one would e x p e c t s i n c e such s u b s t i t u e n t s would t e n d to d e s t a b i l i z e s t r u c t u r e XXXIX. A b s o r b a n c e peaks o f 1 , 4 - d i h y d r o p y r i d i n e s (IV) a p p e a r a t a p p r o x i m a t e l y 15 nm. l o n g e r w a v e l e n g t h s t h a n do the 173 H H H CN ONH, CONH, N I N (IV) (XXXVII) a b s o r b a n c e peaks o f t h e i r 4-cyano c o u n t e r p a r t s ( X X X V I I ) , mean-i n g t h e e l e c t r o n i c t r a n s i t i o n i n e q u a t i o n (81) i s e a s i e r v/ith IV t h e n w i t h XXXVII. T h e r e f o r e , t h e r e i s p r o b a b l y a g r e a t e r c o n t r i b u t i o n from t h e r e s o n a n c e s t r u c t u r e (XXXIX) t o t h e s t a b i l i t y o f IV t h a n t o the s t a b i l i t y o f t h e 4-cyano compound: (XXXIX) ( X X X V I I ) . E l e c t r o n - d o n a t i n g s u b s t i t u e n t s a t t h e 1 - p o s i t i o n w i l l s t a b i l i s e s t r u c t u r e XXXIX and w i l l t h e r e b y s t a b i l i z e IV to a g r e a t e r e x t e n t t h a n XXXVII. The 3 - s u b s t i t u e n t s v/ere found to have a much g r e a t e r e f f e c t on r e a c t i o n s (41) and (68) t h a n would be e x p e c t e d from a c o m p a r i s o n v/ i t h r e a c t i o n (60) . In t h e s e t h r e e r e a c t i o n s 17 9 + e" • N CH2CONH2 CH2CONH2 (XL) p=11 t o 12 (41 ) + H 4- 2e CH2CONH2 H. .H CH2CONH2 (XXXIV) p = 9 t o 11 (68) N i H N (XLI ) + H p : 5.9 (60) 78 the 3 - s u b s t i t u e n t s s h o u l d a f f e c t t h e s t a b i l i t i e s o f t h e p y r i d i n i u m r e a c t a n t s to the same e x t e n t , h e n c e , t h e d i f f e r e n t r e a c t i o n c o n s t a n t s must be due to r e l a t i v e amounts o f s t a b i l i -z a t i o n o f t h e p r o d u c t s (XL, XXXIV, XLI) by 3 - s u b s t i t u e n t s . The 3 - s u b s t i t u e n t s would n o t be e x p e c t e d t o have any g r e a t e f f e c t on t h e s t a b i l i t i e s o f the p y r i d i n e s (XLI) so t h e l a r g e r r e a c t i o n c o n s t a n t s f o r r e a c t i o n s (41) and (68) must be due to a s t a b i l i z a t i o n o f the p y r i d i n e r a d i c a l (XL) and t h e 1,4-d i h y d r o p y r i d i n e s (XXXIV) by e l e c t r o n - w i t h d r a w i n g s u b s t i t u e n t s . I t i s p r o b a b l y s i g n i f i c a n t t h a t t h e e f f e c t s o f 3 - s u b s t i t u e n t s 180 on r e a c t i o n s (41) and (68) were f o u n d t o be c o r r e l a t e d b e t t e r by p a r a - s u b s t i t u e n t c o n s t a n t s t h a n by m e t a - s u b s t i t u e n t c o n -s t a n t s a whereas the a c i d d i s s o c i a t i o n o f p r o t o n a t e d p y r i d i n e s , m r e a c t i o n ( 5 8 ) , i s c o r r e l a t e d b e t t e r w i t h a . The i m p r o v e d c o r -r e l a t i o n s v / i t h CT would i n d i c a t e t h e p r e s e n c e o f a r e s o n a n c e P t i n t e r a c t i o n between the 3 - s u b s t i t u e n t and t h e r i n g o f t h e p y r i d i n e r a d i c a l (XL) and t h e 1 , 4 - d i h y d r o p y r i d i n e ( X X X I V ) , an i n t e r a c t i o n w h i c h one v/ould n o t e x p e c t t o f i n d w i t h th e p y r i d i n e s ( X L I ) . J u s t s u c h an i n t e r a c t i o n was p r o p o s e d e a r l i e r t o e x p l a i n t h e u n e x p e c t e d l y l o w e r r e a c t i o n c o n s t a n t f o r t h e e f f e c t o f 1 - s u b s t i t u e n t s on t h e p o t e n t i o m e t r i c ( t v / o - e l e c t r o n ) r e d u c t i o n o f p y r i d i n i u m i o n s r e a c t i o n ( 6 8 ) . The c o n t r i b u t i o n (68) The p a r a - s u b s t i t u e n t c o n s t a n t s , 0p, c o n t a i n a r e s o n a n c e c o n -t r i b u t i o n w h i c h i s much l e s s s i g n i f i c a n t i n m e t a - s u b s t i t u e n t c o n s t a n t s , CTm. T a f t 7 3 has p r o p o s e d a s e p a r a t i o n o f CTp and a m i n t o i n d u c t i v e and r e s o n a n c e t e r m s : Op = CTj + CTR and CTM = Oj + 1/3 OR where Oj and CTR a r e i n d u c t i v e and r e s o n a n t c o n t r i b u t i o n s r e s p e c t i v e l y . 181 from the r e s o n a n c e form (XXXIX) o f the d i h y d r o p y r i d i n e p r o d u c t (IV) (XXXIX) (IV) would be enhanced by e l e c t r o n - w i t h d r a w i n g 3 - s u b s t i t u e n t s which c an p a r t i c i p a t e i n t h i s r e s o n a n c e ( i . e . CONH 9 , COCH , and, t o some e x t e n t , CN) b u t v/ould be n o n - e x i s t e n t f o r g r o u p s which a r e i n c a p a b l e o f p a r t i c i p a t i n g i n s u c h r e s o n a n c e ( i . e . F, II, or OH) . Thus t h e 3 - c a r b a m o y l , 3 - a c e t y l and 3-cyano a n a l o g u e s o f ( I V ) , by s t a b i l i z i n g s t r u c t u r e (XXXIX) o r i t s a n a l o g u e s w i l l c o n t r i b u t e t o the o v e r a l l s t a b i l i t y o f t h e 1 , 4 - d i h y d r o p y r i d i n e (IV) and make the r e d u c t i o n e a s i e r . A r e s o n a n c e s t r u c t u r e a n a l o g o u s t o XXXIX c a n a l s o be w r i t t e n f o r t h e p y r i d i n e r a d i c a l . ( I n s t r u c t u r e ( X L I I ) t h e ( XL 11) ( X L I I I ) (XLIV) 1 0 2 e l e c t r o n has been a r b i t r a r i l y p l a c e d a t the 4 - p o s i t i o n o f t h e p y r i d i n e r i n g . Resonance s t r u c t u r e s c a n be w r i t t e n w h i c h a l s o p l a c e the l o n e e l e c t r o n on C-2 and C-6) . The e x i s t a n c e o f i n t e r -a c t i o n s between t h e 3 - s u b s t i t u e n t and the l o n e e l e c t r o n as i n /^VCONH, k - ; N 1 CH3 ( X L V ) X L I V , have been p r o p o s e d by Bruhlinann and Kayon d' J f o r the 1 -m e t h y l - 3 - c a r b a r a o y l p y r i d i n e r a d i c a l ( X L V ) so t h e r e i s l i t t l e d o u b t t h a t the r e s o n a n c e s t r u c t u r e s ( X L I I I ) and ( X L I V ) do c o n -t r i b u t e t o t h e o v e r a l l s t a b i l i t y o f t h e p y r i d i n e r a d i c a l . Q u i t e a l a r g e c o n t r i b u t i o n i s p r o b a b l y made by X L I V b e c a u s e o f t h e p r e s e n c e o f t h e a r o m a t i c r i n g . However, the e f f e c t s o f 1 -s u b s t i t u e n t s on the p o l a r o g r a p h i c r e d u c t i o n were c o n s i s t e n t w i t h a low c h a r g e d e n s i t y on t h e r i n g n i t r o g e n whereas t h e c o n t r i b u t i o n o f s t r u c t u r e s X L I I I and X L I V p l a c e a p a r t i a l p o s i -t i v e c h a r g e on t h e r i n g n i t r o g e n . P r o b a b l y t h e p a r t i a l p o s i -t i v e c h a r g e i s e f f e c t i v e l y n e u t r a l i z e d by t h e i n d u c t i o n o f e l e c t r o n d e n s i t y towards the r i n g n i t r o g e n i n s t r u c t u r e s X L V I and X L V I I . To summarize t h e n , the e f f e c t s o f 1 - and 3 - s u b s t i t u e n t s on the p o l a r o g r a p h i c ( o n e - e l e c t r o n ) and p o t e n t i o m e t r i c (XLVI) ( X L V I I ) ( t w o - e l e c t r o n ) r e d u c t i o n s o f p y r i d i n i u m i o n s c a n be e x p l a i n e d by c o n s i d e r i n g r e s o n a n c e i n t e r a c t i o n s between the 3 - s u b s t i t u e n t H H + H + 2e" PJ + 2 . 8 +9 t o +11 and the r i n g i n t h e r e d u c t i o n p r o d u c t s . These r e s o n a n c e i n t e r -a c t i o n s a r e summarized i n Schemes 4 and 5. ( A l l o f t h e p o s s i b l e r e s o n a n c e s t r u c t u r e s o f t h e p y r i d i n e r a d i c a l have n o t been shown. The m i s s i n g s t r u c t u r e s s i m p l y r e s u l t i n s h i f t i n g t h e l o n e e l e c t r o n a r o u n d t h e p y r i d i n e r i n g . ) Scheme 4 Scheme 5 10 5 4.3 The E f f e c t s o f S u b s t i t u e n t s i n D i h y d r o p y r i d i n e s on t h e Rates o f O x i d a t i o n by F l a v i n s W i t h r e g a r d t o s u b s t i t u e n t e f f e c t s , t h e p v a l u e f o r the p o t e n t i o m e t r i c ( t w o - e l e c t r o n ) r e d u c t i o n o f s u b s t i t u t e d p y r i d i n i u m i o n s and f o r the e q u i l i b r i u m between a f l a v i n and s u b s t i t u t e d 1, 4 - d i h y d r o p y r i d i n e s must be i d e n t i c a l . T h i s c a n be shown to be so by the f o l l o w i n g d e r i v a t i o n , e q u a t i o n s (82) t h r o u g h (91) . P y + + Fln " V=±:PyH + F l (8 2) K [Pyii] [ F l ] [ P y + ] [ F i l l ] Py + II + 2e~ =P vIE (33) (84) K [PyH] P v + + [Py ] [ H ] (85) '1 + H + 2e ^=±:F1H (8 6) = [F1H 1 { 8 ? )  1 [ F l ] [II ] S u b s t i t u e n t s on t h e p y r i d i n e r i n g w i l l a f f e c t o n l y r e a c t i o n s (0 2) and (84) . T h e r e f o r e one can w r i t e e q u a t i o n s (83) t h r o u g h ( 9 0 ) , where p„, i s z e r o s i n c e s u b s t i t u e n t s on t h e o y r i d i n e F l r i n g w i l l n o t a f f e c t the r e d u c t i o n p o t e n t i a l o f t h e f l a v i n . 13 0 0 = " 1 C " ' ^ E c f = A l o g [PyH] / [ P y + ] A l o g [ F l ] / [ F l u ' >:'q Aa A a A a (a a) AlogK , , r , , r + + P y _ a l o g [ P y i i ] / [ p y ] [ n ] A a A a (8 9) A 1 0 q K F l _ A l o g [ F l H + ] / [ F l ] [II +] A a Aa (90) C o m b i n i n g e q u a t i o n s ( 8 8 ) , ( 8 9 ) , and (90) g i v e s the i d e n t i t y i n e q u a t i o n ( 9 1 ) . Thus t h e r e a c t i o n c o n s t a n t p* f o r t h e e f f e c t P E q P P y " P F 1 - P P y 91) of 1 - s u b s t i t u e n t s on the e q u i l i b r i u m between f l a v i n s (X) and 1 , 4 - d i h y d r o p y r i d i n e s ( V ) , r e a c t i o n ( 6 9 ) , i s -2.8, e q u a l i n Y NH O H H N I R 1 ( V) ( 69) 13 7 magnitude t o p* f o r the p o t e n t i o m e t r i c ( t w o - e l e c t r o n ) r e d u c t i o n o f p y r i d i n i u m i o n s , b u t o p p o s i t e i n s i g n s i n c e t h e r e a c t i o n v/ith f l a v i n s (69) has been w r i t t e n f o r the o x i d a t i o n o f d i -h y d r o p y r i d i n e (V) r a t h e r t h a n the r e d u c t i o n o f p y r i d i n i u m i o n ( I I I ) . The e f f e c t o f 1 - s u b s t i t u e n t s on t h e f o r w a r d r e a c -t i o n i n e q u a t i o n (69) has been found i n t h i s s t u d y t o g i v e a r e a c t i o n c o n s t a n t p*=-1.9. The r e a c t i o n c o n s t a n t s f o r t h e e q u i l i b r i u m and t h e f o r w a r d and backward r a t e s a r e r e l a t e d by e q u a t i o n (92) a r i s i n g from a r e a r r a n g e m e n t o f t h e f a m i l i a r e q u a t i o n ( 9 3 ) . U s i n g the r e a c t i o n c o n s t a n t s f o u n d i n t h i s p = P.e ~ PT-, (92) r e v f o r Eq P E g = P f o r " P r e v ( 9 3 ) work f o r t h e e q u i l i b r i u m (p* = -2.0) and t h e r a t e o f o x i d a -Eq t i o n o f d i h y d r o p y r i d i n e s by f l a v i n s ( P | o r = -1.9) i t i s p o s s i b l e t o c a l c u l a t e a r e a c t i o n c o n s t a n t p* o f +0.9 f o r r e v the r e v e r s e r e a c t i o n ( r e d u c t i o n of p y r i d i n i u m i o n s by the f l a v i n s ) . T h i s v a l u e o f p* i s q u i t e d i f f e r e n t from t h e p* 15 of +2.2 f o u n d by L i n d q u i s t and C o r d e s f o r the r a t e o f a d d i t i o n o f c y a n i d e t o p y r i d i n i u m i o n s , r e a c t i o n ( 7 6 ) , a r e a c t i o n w h i c h i s somewhat a n a l o g o u s to t h e t w o - e l e c t r o n r e -d u c t i o n o f p y r i d i n i u m i o n s . I ndeed, v e r y s i m i l a r v a l u e s o f p have been r e p o r t e d f o r the c y a n i d e a d d i t i o n t o 188 H CN ( 7 6 ) b e n z a l d e h y d e s J and t h e sodium b o r o h y d r i d e r e d u c t i o n o f a c e t o -84 p nenones. However, t h e r e a r e c e r t a i n l y m e c h a n i s t i c d i f f e r -ences between c y a n i d e a d d i t i o n and h y d r i d e t r a n s f e r n o t the l e a s t o f which i s the f a c t t h a t the h y d r i d e i o n i s n o t a f r e e i o n i n s o l u t i o n , as i s t h e c y a n i d e i o n , b u t i s t r a n s f e r r e d d i r e c t l y f r o m one m o l e c u l e t o t h e o t h e r . Thus t h e d e g r e e o f bond f o r m a t i o n i n the t r a n s i t i o n s t a t e o f a c y a n i d e a d d i t i o n r e a c t i o n i s d e p e n d e n t p a r t i a l l y on t h e s o l v a t i o n o f the f r e e 13 9 c y a n i d e i o n and p a r t i a l l y on the m o l e c u l e t o w h i c h t h e c y a n i d e i s a d d i n g , whereas t h e d e g r e e o f h y d r i d e t r a n s f e r i n t h e t r a n s i t i o n s t a t e would depend n o s t l y on t h e s t a b i l i t y o f t h e two m o l e c u l e s between w h i c h t h e h y d r i d e i s b e i n g t r a n s f e r r e d . Thus, although, i t seems s u r p r i s i n g t h a t the r e a c t i o n c o n s t a n t f o r the r a t e o f a d d i t i o n o f c y a n i d e i o n s t o p y r i d i n i u m i o n s s h o u l d be so much l a r g e r (p* = +2.2) t h a n t h e r e a c t i o n c o n -s t a n t f o r the r a t e o f r e d u c t i o n o f p y r i d i n i u m i o n s by f l a v i n s (p* = + 0 . 9 ) f o u n d i n t h i s s t u d y i t i s n o t t o t a l l y i n c o n s i s t e n t w i t h a h y d r i d e t r a n s f e r from r e d u c e d f l a v i n t o p y r i d i n i u m i o n s . Somewhat s u r p r i s i n g l y , the 1 - s u b s t i t u e n t s have a l m o s t the same e f f e c t on t h e a c i d - d e c o m p o s i t i o n o f 1 , 4 - d i h y d r o p y r i d i n e s (p* - -2.0 t o -2.G, see S e c t i o n 3.1) as t h e y do on t h e o x i d a -t i o n o f d i h y d r o p y r i d i n e s by f l a v i n s (p* = - 1 . 9 ) . In v i e w o f u aCONH, + F l a v i n - 1 R + F l a v i n - H the r e l a t i v e l y p o or agreement between the r e a c t i o n c o n s t a n t s f o r the a d d i t i o n o f c y a n i d e (p* = +2.2) to p y r i d i n i u m i o n s and t h e r e d u c t i o n by f l a v i n s (p* = +0.9) o f p y r i d i n i u m i o n s , i t i s t e m p t i n g t o f o r m u l a t e a c o v a l e n t i n t e r m e d i a t e s u c h a s ( X L V I I I ) a s an a l t e r n a t i v e t o a h y d r i d e r e d u c t i o n . A s i m i l a r i n t e r m e d i a t e v/as p r o p o s e d by Dunn f o r t h e r e d u c t i o n o f a l d e h y d e s by 1 , 4 - d i b y d r o p y r i d i n e s . The i n t e r m e d i a t e ( X L V I I I ) , u n f o r t u n a t e l y does n o t e x p l a i n t h e d e u t e r i u m i s o t o p e e f f e c t s 2 0 (k /K = 3.1G) o b s e r v e d by S u e l t e r and M e t z l e r d u r i n g t h e o x i d a t i o n o f 1 - p r o p y l - 3 - c a r b a m o y l - 4 - d e u t r o - 1 , 4 - d i h y d r o p y r i d i n e (XL IX) s i n c e t h e p r o t o n t r a n s f e r s v/ould have to t a k e p l a c e i n a f a s t r e a c t i o n a f t e r the r a t e d e t e r m i n i n g f o r m a t i o n o f t h e i n t e r m e d i a t e ( X L V I I I ) . I t i s a l s o v e r y d i f f i c u l t t o e n v i s a g e any way to form t h i s i n t e r m e d i a t e i n t h e r e v e r s e r e a c t i o n s t a r t i n g w i t h p y r i d i n i u m i o n s and r e d u c e d f l a v i n s i n c e t h i s would r e q u i r e a n u c l e o p h i l i c a t t a c k a t the 3 - p o s i t i o n o f t h e p y r i d i n i u m r i n g , a r e a c t i o n which i s v e r y u n c h a r a c t e r i s t i c 1B 8 5 o f p y r i d i n i u m i o n s . "' " The h y d r i d e - t r a n s f e r mechanism, w h i c h 2 0,22-24 nas c o n s i d e r a b l e s u p p o r t , a p p e a r s t o b e s t e x p l a i n a l l the a v a i l a b l e e v i d e n c e , a l t h o u g h the r e a c t i o n c o n s t a n t s l ' J 2 d e t e r m i n e d i n t h i s s t u d y s e e m t o i n d i c a t e t h a t t h e m e c h a n i s m n a y Le m o r e c o m p l e x t h a n t h i s . 193 4.4 C o m p a r i s o n s o f 1 , 1 ' - A l h y l e n e b i s ( 3 - c a r b a m o y l p y r i d i n i u m ) compounds w i t h t h e S e r i e s I Compounds The S e r i e s I compounds ( I I I and IV) have been used t o : O N H , ( I I I ) H. H C O N H , N R 1 IV) d e t e r m i n e the i n d u c t i v e e f f e c t e x e r t e d by 1 - s u b s t i t u e n t s on s e l e c t e d r e a c t i o n s o f p y r i d i n i u m i o n s ( I I I ) and d i h y d r o -p y r i d i n e s ( I V ) . T h e s e r e s u l t s c an now be u t i l i z e d to e v a l u a t e the e f f e c t s o f more complex s u b s t i t u e n t s a t the 1 - p o s i t i o n , p a r t i c u l a r l y s u b s t i t u e n t s w hich may be c a p a b l e o f i n t e r a c t i n g w i t h t h e p y r i d i n e r i n g by means o t h e r t h a n i n d u c t i o n t h r o u g h the r i n g n i t r o g e n . One such s u b s t i t u e n t would be a second 8 7 p y r i d i n i u m r i n g . B a t z o l d f o u n d e v i d e n c e o f c h a r g e t r a n s f e r s t a c k i n g when i n d o l e s were added to p o l y m e r s c o n t a i n i n g p y r i d i n i u m r i n g s a t t a c h e d to a p o l y s t y r e n e b a c k b o n e . The compounds u s e d i n t h i s s t u d y (XIX) c o n t a i n e d o n l y two p y r i d i n i u m r i n g s s e p a r a t e d by one or tv/o m e t h y l e n e g r o u p s . B e f o r e t h e e f f e c t s o f a s e c o n d p y r i d i n i u m r i n g on t h e r e a c t i o n s o f the f i r s t p y r i d i n i u m r i n g c a n be e v a l u a t e d , the i n d u c t i v e s u b s t i t u e n t e f f e c t s o f the g r o u p s XXV and XXVI must be known. V a l u e s o f a* o f a p p r o x i m a t e l y 1.7 and 1.0 v/ere d e t e r m i n e d 194 ( S e c t i o n 3.4) f o r XXV and XXVI r e s p e c t i v e l y . - 2 Compound X l X a , a t a c o n c e n t r a t i o n o f 10 N . , e x h i b i t e d one p o l a r o g r a p h i c r e d u c t i o n wave w i t h a h a l f - w a v e p o t e n t i a l o f -470 mv. ( v s . M.H.E.). The h a l f - w a v e p o t e n t i a l t h a t would be e x p e c t e d on t h e b a s i s o f i n d u c t i v e e f f e c t s a l o n e ( a * = 1.7) -3 i s -450 mv. A t 2 x 10 1 1 . , compound X l X a e x h i b i t e d b o t h a prewave and a main wave b u t s i n c e the c o r r e l a t i o n o f the -3 S c r i e s I compounds ( I I I ) a t 2 x 10 M. ( S e c t i o n 5.3.5) us e d the h a l f - w a v e p o t e n t i a l s o f the main v/ave, t h e n o n l y t h e main wave s h o u l d be u s e d f o r c o m p a r i s o n . The main r e d u c t i o n wave a p p e a r e d a t a p o t e n t i a l o f a p p r o x i m a t e l y -430 mv. b u t , on the b a s i s o f i n d u c t i v e e f f e c t s o n l y (O* = 1.7) would be •CONH 2 (III ) p r e d i c t e d a t a p o t e n t i a l o f -515 mv. A t c o n c e n t r a t i o n s below -4 5 x 10 I'.., the p o l a r o g r a p a i c r e d u c t i o n p o t e n t i a l has been d e t e r m i n e d i n t h i s work as the i n t e r c e p t , £ ° , o f a p l o t o f 2/3 a n o l x e d p o t e n t i a l , E, a g a x n s t l o g ( i , - i ) / ( i ) [ e a u a t i o n a (18) ] . However, a t c o n c e n t r a t i o n s below 5 x 10 4 K. t h i s E = e° + | £ £ n ( i n - i ) / ( i ) 2 / 3 (18) compound (X l X a ) e x h i b i t e d two p o l a r o g r a p h i c r e d u c t i o n waves, one o f w h i c h had a r e d u c t i o n p o t e n t i a l , c°, o f -209 mv. and the o t h e r o f w h i c h had a r e d u c t i o n p o t e n t i a l o f -409 mv. The enormous p o t e n t i a l d i f f e r e n c e (>220 mv.) between t h e f i r s t wave (e° = -209 mv.) and the r e d u c t i o n waves a t h i g h e r c o n c e n t r a t i o n s would seem to r u l e o u t the use o f t h i s r e d u c -t i o n p o t e n t i a l i n c o m p a r i s o n s w i t h the S e r i e s I c o r r e l a t i o n s s i n c e w i t h no S e r i e s I p y r i d i n i u m i o n was t h e d i f f e r e n c e be--3 tween the h a l f - w a v e p o t e n t i a l , E , a t 2 x 10 : i . and e° L / Z more t h a n 60 mv. The s e c o n d v/ave ( E ° = -409 mv.) i s a much more r e a s o n a b l e 20 mv. more p o s i t i v e t h a n th e h a l f - w a v e -3 p o t e n t i a l o f t h e p o l a r o g r a p h i c wave a t 2 x 10 M.. From t h e i n d u c t i v e e f f e c t ( a * = 1.7) a r e d u c t i o n p o t e n t i a l , e ° , o f -417 mv. v/ould be p r e d i c t e d which i s i n good a g r e e m e n t w i t h 19 the v a l u e o f -409 mv. o b s e r v e d . The p r e d i c t e d and o b s e r v e d r e d u c t i o n p o t e n t i a l s o f XIX a r e summarized i n T a b l e X V I I . a: n = 1 b: n = 2 (XIX] -3 O n l y a t a c o n c e n t r a t i o n o f 2 x 10 Ii. i s t h e r e a s i g n i f i c a n t d i f f e r e n c e between t h e o b s e r v e d r e d u c t i o n p o t e n t i a l s and t h o s e p r e d i c t e d on t h e b a s i s o f o x i d a t i o n e f f e c t s . Thus the p r o x i m i t y o f the two p y r i d i n i u m i o n s a p p e a r s to have no s p e c i a l e f f e c t on t h e p o l a r o g r a p h i c r e d u c t i o n o f XI Xa. The r e s u l t s f o r the p o l a r o g r a p h i c r e d u c t i o n p o t e n t i a l s o f "IXb, l i s t e d i n T a b l e X V I I , a r c much l e s s d e f i n i t i v e . At - p a c o n c e n t r a t i o n o f 10 " M., the o b s e r v e d r e d u c t i o n p o t e n t i a l in a p p r o x i m a t e l y 90 rav. more n e g a t i v e t h a n t h e p o t e n t i a l o f -583 mv. p r e d i c t e d f r o n i n d u c t i v e e f f e c t s ( a * = 1 . 0 ) , whereas -3 a t 2 x 10 t h e o b s e r v e d p o t e n t i a l o f - 5 9 5 rav. i s f a i r l y c l o s e to t h e p r e d i c t e d p o t e n t i a l o f -63 0 mv. A t c o n c e n t r a -t i o n s below .'5 x 10 * i i . , the o b s e r v e d r e d u c t i o n p o t e n t i a l o f -434 mv. i s 135 nv. more p o s i t i v e t h a n would be p r e d i c t e d from i n d u c t i v e e f f e c t s (CT* = 1.0). The more n e g a t i v e o b s e r v e d p o t e n t i a l a t a c o n c e n t r a t i o n o f 10 ~ i t . i s p r o b a b l y due t o an e x t r a o r d i n a r i l y l a r g e i n h i b i t i o n o f the e l e c t r o n t r a n s f e r 19 7 Tabic ' V T ' P r e d i c t e d and O b s e r v e d P o l a r o g r a p h i c R e d u c t i o n P o t e n t i a l s oi the 1,1'-a I k y l e n e b i s ( 3 - c a r b a m o y l p y r i d inium) Compound s. Compound c o n c e n t r a t i o n r e d u c t i o n o b s e r v e d ( . ) p o t e n t i a l s from i n d u c -t i v e e f f e c t s HgNOC CONH, 10 2 x 10 -3 <5 x 10 -A -4 5 0 -4 7 0 -515 -4 3 0 -417 -409 H2NOC-<^ ^ \ \~CONH 2 10 10 <5 10 -A -5 33 -672 -623 -595 -569 -4 3 4 p r o c e s s v/ith t h i s compound, w h i c h i s a l s o r e f l e c t e d i n t h e ver> l a r g e s l o p e o f 125 mv. from p l o t s o f a p p l i e d p o t e n t i a l , E, a g a i n s t l o g ( i d - i ) / ( i ) [ e q u a t i o n ( 1 0 ] . ' The more p o s i t i v e P T E = E l / 2 + ZZF £ n ( i r t " (10) — 4 p o t e n t i a l o b s e r v e d a t c o n c e n t r a t i o n s l e s s t h a n 5 x 10 : - l. may i n d i c a t e a n o n - i n d u c t i v e i n t e r a c t i o n between t h e two r i n g s w hich i s s t a b i l i z i n g the p r o d u c t and t h u s making t h e r e d u c t i o n e a s i e r . B u t t h e r e s u l t s a t the h i g h e r c o n c e n t r a t i o n s do n o t c o r r o b o r a t e t h i s and the p o t e n t i a l o b s e r v e d a t c o n c e n t r a t i o n s -4 below 5 x 10 II. may r e s u l t from a d s o r p t i o n e f f e c t s i n s t e a d . The p o l a r o g r a p h i c r e s u l t s f o r XlXb a r e t o o i n c o n c l u s i v e t o H NOC—V \ V V M _ C O N H \ = N / \ = / * N ( C H 2 ) n / + ( X I X ) d e t e r m i n e i f any n o n - i n d u c t i v e i n t e r a c t i o n s a r e o p e r a t i n g b tween the two r i n g s . The s l o p e s o f E a g a i n s t l o g ( i were betv/een 7 0 and 9 0 mv. i ) / ( i ) f o r most compounds 19' A r e d u c t i o n p o t e n t i a l has been e s t i m a t e d i n t h i s work f o r the r e d u c t i o n o f one r i n g o f X l X b to a d i h y d r o p y r i d i n e . pound XXXII was n o t i s o l a t e d b u t was p r e p a r e d i n s i t u C O N H C O N H , + 2e~ C O N H , C O N H , (XlXb) (XXXII) from XlXb by d i t h i o n i t e r e d u c t i o n and v/as a s s a y e d s p e c t r o -p h o t o m e t r i c a l l y ( t h e method by which the e x t i n c t i o n c o e f f i -c i e n t was d e t e r m i n e d i s g i v e n i n S e c t i o n 3 . 6 ) . The r e d u c t i o n p o t e n t i a l was e s t i m a t e d t o be a p p r o x i m a t e l y -280 mv. a t pH 7. A a* v a l u e o f 1.0 f o r the i n d u c t i v e e f f e c t o f t h e u n r e d u c e d p y r i d i n i u m r i n g g i v e s a p r e d i c t e d p o t e n t i o m e t r i c r e d u c t i o n p o t e n t i a l a t pH 7, E° 1 , o f -287 mv. v/hich i s i n good agreement w i t h the o b s e r v e d r e d u c t i o n p o t e n t i a l o f -280 mv. Thus i t v/ould a p p e a r t h a t the p r o x i m i t y o f t h e two p y r i d i n e r i n g s i n X l X a and X l X b has no e f f e c t on t h e oxidation-H 2 N O O \ = . N N =y \ C H 2 > i ( a : n = l o : n = Z 200 r e d u c t i o n b e h a v i o u r o f t h e s e compounds t h a t c a n n o t be a c c o u n t -ed f o r s i m p l y on t h e b a s i s o f i n d u c t i v e e f f e c t s . 201 + + 4.5 C o m p a r i s o n o f H A D and i-JMN w i t h t h e S e r i e s I Compounds B e f o r e the e f f e c t s o f the r i b o s i d y l g r o u p on t h e p r o -+ + p a r t i e s of M A D (Ia) and M H N ( I I ) c a n be e v a l u a t e d , t h e HO OH HO OH ( l a ) HO OH ( I I ) i n d u c t i v e e f f e c t o f t h e r i b o s e g r o u p must f i r s t be e s t i m a t e d . I n t u i t i v e l y , one would e x p e c t i t t o be d e t e r m i n e d p r i m a r i l y by the p r e s e n c e o f oxygen i n the r i n g and t h e r e f o r e t o be 202 q u i t e s i m i l a r t o t h e i n d u c t i v e e f f e c t o f t h e m e t h o x y m e t h y l g r o u p . The p r o p e r t i e s o f NAD , NMN and 1-methoxymethy1-3 -c a r b a m o y l p y r i d i n i u m c h l o r i d e ( V l l l e ) a r e summarized i n T a b l e X V I I I . However, t h e i n d u c t i v e e f f e c t o f t h e r i b o s e g r o u p OH -CH 2OCH 3 ' I -CHpCHpOH HO OH * * w i l l a l s o c o n t a i n a c o n t r i b u t i o n from the h y d r o x y l g r o u p on C-2' w h i c h s h o u l d e x e r t an i n d u c t i v e e f f e c t q u i t e s i m i l a r t o t h a t o f t h e h y d r o x e t h y l g r o u p . In t h i s w o r k 3 a v a l u e 'CONH2 N CH2OCH3 ( V l l l e ) o f 0.55 has been u s e d f o r t h e m e t h o x y m e t h y l s u b s t i t u e n t and 0.11 f o r the h y d r o x y e t h y l s u b s t i t u e n t . C o m b i n i n g t h e s e two v a l u e s g i v e s a p r e d i c t e d s u b s t i t u e n t c o n s t a n t o f 0.66 f o r the r i b o s e g r o u p . Hence t h e r e d u c t i o n p o t e n t i a l s and r a t e c o n s t a n t s f o r NAD and NMN a r e a l s o compared i n T a b l e X V I I I w i t h v a l u e s p r e d i c t e d from t h e c o r r e l a t i o n s o f t h e S e r i e s I compounds ( I I I ) by ass u m i n g a v a l u e o f 0.66 f o r t h e o * - v a l u e o f t h e r i b o s i d y l g r o u p . I t s h o u l d be n o t e d t h a t a d i f f e r e n t a see T a b l e V, page 49 203 T a b l e X V I I I S e l e c t e d P r o p e r t i e s o f NAD +, NMN+, and l - m e t h o x y m e t h y l - 3 -c a r b a m o y l p y r i d i n i u m c h l o r i d e . P o l a r o g r a p h i c r e d u c t i o n p o t e n t i a l a t 1 0 ~ 2 M. (mv) + + + NAD NMN MMCPy P r e d i c t e d from ( I a ) ( I I ) ( V l l l e ) 0* = .66 f o r r i b o s e g r o u p -715 •830 -679 •698 P o l a r o g r a p h i c r e d u c t i o n p o t e n t i a l a t 2 x l 0 - 3 M. (mv) •683 -810 -657 •684 P o l a r o g r a p h i c r e d u c t i o n p o t e n t i a l a t < 5 x l 0 ~ ^ M. (mv) -6777 -703 -639 •642 P o t e n t i o m e t r i c s t a n d a r d r e d u c t i o n p o t e n t i a l (mv) •105 ( t h i s work) • 1 0 4 3 2 -95 -97 • 1 0 8 3 0 •108 Rate o f o x i d a t i o n o f d i h y d r o p y r i d i n e by f l a v i n ( M _ 1 - s e c - 1 ) 53 .25 .61 2.4 Rate o f a c i d - d e c o m p o s i -t i o n o f d i h y d r o p y r i d i n e by H 3 0 + ( M - l - s e c - 1 ) 10.2 7.7 18 15 Rate o f a c i d - d e c o m p o s i -t i o n o f d i h y d r o p y r i d i n e by a c e t i c a c i d ( M - ! s e c ~ l ) x 1 0 3 .91 90 7.7 5.9 204 -CONH, " 1 ( I I I ) tfsva.iue- can -be. c a l c u l a t e d , f o r t h e iribos,e_ .group, from 'the V 6 CT-value o f 0.66 r e p o r t e d by W e l l s f o r t h e m e t h o x y m e t h y l g r o u p . In g e n e r a l , the p r o p e r t i e s o f NAD + ( l a ) and NMN +(II) i n v e s t i g a t e d i n t h i s work a r e i n f a i r a g reement w i t h t h o s e o f l - m e t h o x y m e t h y l - 3 - c a r b a m o y l p y r i d i n i u m c h l o r i d e ( V l l l e ) and w i t h t h o s e p r e d i c t e d a s s u m i n g a a * - v a l u e o f 0.66 f o r t h e r i b o s y 1 y g r o u>p i.i p. T h e i, p o ib a r o g r a p h jL c .. r e d u c t i 6 no p o t e n t i a 1 s 1 o f : -HO OH HO OH ( l a ) ( I I ) ( V l l l e ) NAD were a p p r o x i m a t e l y 30 mv. more n e g a t i v e t h a n t h o s e o f l - m e t h o x y m e t h y l - 3 - c a r b a m o y l p y r i d i n i u m c h l o r i d e a t a l l c o n c e n -t r a t i o n s and 0 t o 30 mv. more n e g a t i v e t h a n would be p r e -d i c t e d from 0* e q u a l s 0.66. The p o l a r o g r a p h i c r e d u c t i o n o f NMN+ a p p e a r s t o be i n h i b i t e d t o a g r e a t e r e x t e n t by a d s o r p t i o n o f the d i m e r i c p r o d u c t (XVI) ( p l o t s o f E a g a i n s t l o g ( i < j - i ) / ( i ) have s l o p e s g r e a t e r t h a n 100 mv., see S e c t i o n s 3.5.1 and 4.1) t h a n i s NAD +, w h i c h i s t h e p r o b a b l e c a u s e o f t h e v e r y n e g a t i v e p o l a r o g r a p h i c r e d u c t i o n p o t e n t i a l s f o u n d f o r t h i s compound a t N d H N %1 n1 (XVI) -3 -4 c o n c e n t r a t i o n s above 10 M. A t c o n c e n t r a t i o n s below 5 x 10 M the p o l a r o g r a p h i c r e d u c t i o n p o t e n t i a l o f NMN+ i s a p p r o x i m a t e l y 25 mv. more n e g a t i v e t h a n t h a t o f NAD + and a b o u t 60 mv. more n e g a t i v e t h a n would be p r e d i c t e d from e i t h e r t h e p o l a r o g r a p h i c r e d u c t i o n p o t e n t i a l o f l - m e t h o x y m e t h y l - 3 - c a r b a m o y l p y r i d i n i u m c h l o r i d e o r from a a * - v a l u e o f 0.66. The 0 t o 30 mv. d i f f e r -ence between t h e p o l a r o g r a p h i c r e d u c t i o n p o t e n t i a l s o f NAD + and t h e p r e d i c t e d p o t e n t i a l s may r e f l e c t an e f f e c t o f the 206 a d e n i n e (L) m o i e t y toward making t h e p y r i d i n i u m r i n g more d i f f i c u l t t o r e d u c e . The d i f f e r e n c e i s so s m a l l , however, t h a t i t may n o t be s i g n i f i c a n t . The p o t e n t i o m e t r i c ( t w o - e l e c t r o n ) r e d u c t i o n p o t e n t i a l o f NAD + a g r e e s q u i t e w e l l w i t h t h e v a l u e p r e d i c t e d u s i n g a 0"*-value o f 0.66, whereas t h e p o t e n t i o m e t r i c r e d u c t i o n p o t e n -t i a l o f NMN+ a g r e e s q u i t e w e l l w i t h the s l i g h t l y l o w e r r e d u c -t i o n p o t e n t i a l f o u n d f o r l - m e t h o x y m e t h y l - 3 - c a r b a m o y l p y r i d i n i u m c h l o r i d e . The agreement between NMN+ ( I I ) and 1-methoxymethyl-3 - c a r b a m o y l p y r i d i n i u m c h l o r i d e ( V l l l e ) would be e x p e c t e d on HO OH ( I I ) ( V l l l e ) t h e b a s i s o f i n d u c t i v e e f f e c t s . The p o t e n t i o m e t r i c r e d u c t i o n p o t e n t i a l o f NAD + ( l a ) i s a p p r o x i m a t e l y 10 mv. more n e g a t i v e 207 t h a n t h o s e o f NMNT and 1 - m e t h o x y m e t h y l - 3 - c a r b a m o y l p y r i d i n i u m c h l o r i d e , w h i c h a g a i n may r e f l e c t an e f f e c t o f the a d e n i n e g r o u p (L) toward making the p y r i d i n i u m r i n g more d i f f i c u l t t o r e d u c e . The r a t e s o f o x i d a t i o n o f the c o r r e s p o n d i n g d i h y d r o -p y r i d i n e s o f NAD + and NMN+ a g a i n a r e i n good a g r e e m e n t w i t h t h a t o f the 1-methoxymethyl model compound. S u r p r i s i n g l y , t h e s e r a t e s a r e a l l c o n s i d e r a b l y s m a l l e r t h a n t h e o x i d a t i o n r a t e p r e d i c t e d u s i n g a CT-value o f 0.66. A s i m i l a r e f f e c t was o b s e r v e d by L i n d q u i s t and C o r d e s ^ i n t h e i r s t u d y o f t h e r a t e s and e q u i l i b r i a o f c y a n i d e a d d i t i o n t o p y r i d i n i u m i o n s . They o b s e r v e d t h a t NAD + had a much l a r g e r a f f i n i t y f o r c y a n i d e i o n t h a n would be e x p e c t e d on t h e b a s i s o f t h e r a t e o f t h e a d d i t i o n r e a c t i o n . In t h i s work, i t has been f o u n d t h a t t h e r a t e o f o x i d a t i o n o f NADH by f l a v i n s i s much s l o w e r t h a n would be e x p e c t e d on the b a s i s o f i t s s t a n d a r d r e d u c t i o n p o t e n -t i a l . S i n c e s i m i l a r v a l u e s o f the o x i d a t i o n r a t e and s t a n -d a r d r e d u c t i o n p o t e n t i a l were a l s o f o u n d f o r NMNH (LI) and 1 - m e t h o x y m e t h y l - 3 - c a r b a m o y l - l , 4 - d i h y d r o p y r i d i n e (Ve) t h e n t h e 203 s l o w e r r a t e s a r e p r o b a b l y r e l a t e d i n some way to t h e p r e s e n c e HO OH (LI) (Ve) o f the oxygen atom on t h e a - c a r b o n o f t h e 1 - s u b s t i t u e n t . The t w o - f o l d r a t e enhancement o f 1 - m e t h o x y m e t h y l - 3 - c a r b a m o y l - 1 , 4 -d i h y d r o p y r i d i n e (Ve) o v e r NMNH (LI) may r e f l e c t s t e r i c c rowd-i n g between t h e l a r g e r r i b o s i d y l g r o u p and the a p p r o a c h i n g f l a v i n ( X ) . R e g a r d l e s s , t h e t w o - f o l d r a t e enhancement o f R (XXI) 209 NADH (XXV) o v e r NMNH (LI) p r o b a b l y r e f l e c t s a weak s t a b i l i z a -t i o n o f t h e t r a n s i t i o n s t a t e by t h e a d e n i n e m o i e t y . The o n l y r e a c t i o n i n w h i c h NAD + and NMN+ d i d n o t be-have v e r y s i m i l a r l y t o l - m e t h o x y m e t h y l - 3 - c a r b a m o y l p y r i d i n i u m c h l o r i d e was i n the a c i d d e c o m p o s i t i o n o f t h e c o r r e s p o n d i n g d i h y d r o compounds, i n whi c h NADH and NMNH r e a c t e d much more s l o w l y t h a n d i d l - m e t h o x y m e t h y l - 3 - c a r b a m o y l - l , 4 - d i h y d r o p y r i d i n e o r t h a n would be p r e d i c t e d from a a * - v a l u e o f 0.66. On t h e b a s i s o f t h e p o l a r o g r a p h i c r e d u c t i o n p o t e n t i a l s , p o t e n t i o m e t r i c r e d u c t i o n p o t e n t i a l s , and r a t e s o f o x i d a t i o n o f t h e c o r r e s p o n d i n g d i h y d r o p y r i d i n e s by f l a v i n s , t h e r e a p p e a r s to be no s i g n i f i c a n t e f f e c t from t h e a d e n i n e p o r t i o n o f t h e NAD + m o l e c u l e on t h e o x i d a t i o n - r e d u c t i o n p r o p e r t i e s o f t h i s coenzyme. T h e r e may be a v e r y s m a l l s t a b i l i z a t i o n o f t h e p y r i d i n i u m r i n g by the a d e n i n e r e s u l t i n g i n t h e s l i g h t l y more n e g a t i v e r e d u c t i o n p o t e n t i a l s o f NAD + and s l i g h t l y f a s t e r o x i d a t i o n r a t e o f NADH. The f o r m a t i o n o f a c h a r g e t r a n s f e r complex between t h e a d e n i n e and t h e p y r i d i n i u m r i n g s o f NAD + 8 8 has been p r o p o s e d by C i l e n t o and S c h r e i e r , b u t t h e r e s u l t s o f t h i s s t u d y show t h a t the e f f e c t s o f s u c h a complex on t h e o x i d a t i o n - r e d u c t i o n p r o p e r t i e s o f NAD + a r e v e r y s m a l l a t b e s t . 210 5. SUGGESTIONS FOR FURTHER RESEARCH In some a r e a s , t h i s work has po s e d as many q u e s t i o n s as i t has a n s w e r e d . A l t h o u g h t h e a d e n i n e m o i e t y o f NAD + (Ia) (Ia) was f o u n d t o have v e r y l i t t l e e f f e c t on t h e o x i d a t i o n - r e d u c -t i o n p r o p e r t i e s o f t h i s m o l e c u l e , t h e p r e s e n c e o f oxygen i n the r i b o s e r i n g a p p e a r s to be r e s p o n s i b l e f o r t h e s l o w e r r a t e s o f o x i d a t i o n o f NADH and NMNH by f l a v i n s . I t would be o f i n t e r e s t to know (1) i f t h i s s l o w e r r a t e a l s o o c c u r s v / i t h 1 - a l k o x y m e t h y l s u b s t i t u e n t s as was f o u n d w i t h t h e 1-methoxymethyl g r o u p i n t h i s s t u d y , (2) i f any o t h e r h e t e r o - a t o m s a t t a c h e d t o th e a - c a r b o n o f the 1 - s u b s t i t u e n t c an e f f e c t a s i m i l a r r a t e r e t a r d a t i o n and (3) how t h e oxygen o f t h e 1 - s u b s t i t u e n t i n t e r -a c t s w i t h the d i h y d r o p y r i d i n e r i n g o r t r a n s i t i o n s t a t e to slow the r e a c t i o n r a t e . The t w o - e l e c t r o n r e d u c t i o n o f p y r i d i n i u m i o n s shown i n r e a c t i o n ( 6 8 ) , v/as f o u n d t o have a r e a c t i o n c o n s t a n t o f o n l y +2.8 compared w i t h r e a c t i o n c o n s t a n t s o f +3.7 f o u n d i n t h i s 15 7 6 work and e l s e w h e r e ' f o r t h r e e a n a l o g o u s r e a c t i o n s . 211 CONH, + H + 2e H H - c r ONHr ( I V ) ( 6 8 ) The d i f f e r e n c e was a t t r i b u t e d to a resonance i n t e r a c t i o n between the r i n g - n i t r o g e n and the 3-carbamoyl.group of IV, H. .H 9 NH. To f u r t h e r t e s t t h i s h ypothesis, i t would be i n t e r e s t i n g to determine a r e a c t i o n constant f o r the two-electron r e d u c t i o n of a s e r i e s of d i h y d r o p y r i d i n e s such as 1 - s u b s t i t u t e d - 3 - c h l o r o -1 , 4 - d i h y d r o p y r i d i n e s ( L I l ) f o r v/hich the resonance i n t e r a c t i o n could be e l i m i n a t e d . H H •Cl N I ( L I l ) 212 F u r t h e r s t u d i e s i n t o 1 , 1 ' - a l k y l e n e b i s ( 3 - c a r b a m o y l -p y r i d i n i u m ) compounds would be u s e f u l as p o s s i b l e models o f e n z y m a t i c NAD r e a c t i o n s . The two compounds o f t h i s t y p e u s e d i n t h i s work ( X l X a and b) e x h i b i t e d no d i r e c t i n t e r a c t i o n s : n = l : n = 2 (XIX ) between t h e two r i n g s b u t t h i s may have been due t o an i n a b i l i t y o f t h e two r i n g s to a l i g n p r o p e r l y i n t h e s e two s p e c i f i c com-pounds w h i c h have v e r y s h o r t m e t h y l e n e b r i d g e s . The r e d u c t i o n p o t e n t i a l s o f XIX compounds h a v i n g l o n g e r m e t h y l e n e b r i d g e s may be a f f e c t e d by d i r e c t i n t e r a c t i o n s between t h e two r i n g s , p a r t i c u l a r l y between t h e two r i n g s o f t h e h a l f - r e d u c e d form X X X I l . The r a t e s o f o x i d a t i o n o f b o t h XXXII and XXXIII (XXXII) compounds by f l a v i n s c o u l d a l s o be o f i n t e r e s t as model e n z y m a t i c r e a c t i o n s . 214 6. EXPERIMENTAL 6.1 B u f f e r s The w a t e r u s e d t h r o u g h o u t t h i s work was d i s t i l l e d i n a Manesty OOBE g l a s s s t i l l . S o l u t i o n s w h i c h were t o be us e d i n t h e d r y box were b o i l e d and f l u s h e d w i t h n i t r o g e n w h i l e c o o l i n g . B u f f e r s were p r e p a r e d from c o m m e r i c a l l y o b t a i n e d r e a g e n t g r a d e c h e m i c a l s ; t h e f o l l o w i n g methods were u s e d . A c e t a t e b u f f e r s : The r e q u i r e d amount o f sodium a c e t a t e c r y s t a l s to make a 1.0 M. s o l u t i o n was weighed i n t o a f l a s k . The volume o f 1.0 M. p e r c h l o r i c a c i d r e q u i r e d t o g i v e t h e d e s i r e d pH was added by p i p e t t e . Water was t h e n added up t o t h e r e q u i r e d v o lume. A c e t a t e b u f f e r s were p r e p a r e d a t pH's 5.65, 4.6 and 3.4. S o l u t i o n s o f 0.1 M. b u f f e r s were p r e p a r e d by d i l u t i o n o f t h e l . 0 M . b u f f e r s . T r i s ( h y d r o x y m e t h y l ) a m i n o m e t h a n e ( T r i s ) b u f f e r s : The r e q u i r e d amount o f T r i s c r y s t a l s to make a 1.0 M. (o r 0.1 M.) s o l u t i o n was weighed i n t o a f l a s k . The amount o f sodium p e r c h l o r a t e o r sodium c h l o r i d e r e q u i r e d to p r e p a r e a s o l u t i o n o f the p r o p e r i o n i c s t r e n g t h (1.0 o r 0.1) was t h e n weighed i n t o the f l a s k . The d e s i r e d pH was o b t a i n e d by add-i n g 1.0 M. p e r c h l o r i c a c i d ( o r h y d r o c h l o r i c a c i d ) by p i p e t t e . 2 1 5 vjater was added up t o the f i n a l volume. T r i s b u f f e r s were p r e -;-.a red. a t pK * s 7.1, o . 1, 0.7, 3 . 'J a nd 9 . 4 . E t h y l e n e d i a n i n e B u f f e r s : The r e q u i r e d amount o f e t h y l o n e d i a m i n e d i h y d r o c h l o r i d e to make a 0.05 I ' . s o l u t i o n v/as weighed i n t o a f l a s k . The v o l u n e o f 0.1 o r 1.0 ?1. sodium h y d r o x i d e r e q u i r e d t o o b t a i n t h e d e s i r e d pH v/as added by p i p e t t e and water v/as added up t o the f i n a l volume. E t h y l e n e d i a m i n e b u f f e r s v/ere p r e p a r e d a t pit's o f 7.0 and 10.4. G l y c i n e b u f f e r s : The r e q u i r e d amount o f g l y c i n e c r y s t a l s t o make an 0.1 M . s o l u t i o n v/as weighed i n t o a f l a s k . The volume o f 0.1 I ' . or 1.0 M . sodium h y d r o x i d e r e q u i r e d to o b t a i n the d e s i r e d pH v/as added by p i p e t t e . Water was t h e n added up t o t h e f i n a l v o l u m e . G l y c i n e b u f f e r s were p r e p a r e d a t pii • s o f 3.3, 9 . 2 , 9.5, 9.3, and 10.3. 216 6.2 O x y g e n - f r e e Work The d r y box u s e d f o r o x y g e n - f r e e work was a Model HE-43-2 f i t t e d w i t h a Model HE-493 D r i - T r a i n p u r c h a s e d from Vacuum A t m o s p h e r e s C o r p o r a t i o n . H i g h p u r i t y h e l i u m gas was u s e d t o s u p p l y the i n e r t a t m o s p h e r e . ( H i g h p u r i t y N i t r o g e n and " o x y g e n - f r e e " N i t r o g e n were f o u n d t o be u n s u i t a b l e be-c a u s e o f t h e i r o xygen c o n t e n t ) . The gas i n l e t s y s t e m was m o d i f i e d as i n F i g u r e 29 so t h a t i n c o m i n g gas was f o r c e d t h r o u g h the D r i - T r a i n b e f o r e e n t e r i n g t h e box. Copper t u b i n g was u s e d t o c o n n e c t t h e h e l i u m t a n k to t h e gas i n l e t on t h e d r y box. M a t e r i a l s to be t r a n s f e r r e d to the d r y box were p l a c e d i n t h e antechamber and t h e antechamber e v a c u a t e d and r e f i l l e d from the d r y box t h r o u g h v a l v e A ( s e e F i g u r e 29) t w i c e b e f o r e the d o o r between th e antechamber and t h e d r y box was opened. D u r i n g t h e e v a c u a t i o n p r o c e d u r e , f r e s h h e l i u m gas was s l o w l y i n t r o d u c e d to t h e d r y box t h r o u g h v a l v e B t o r e p l e n i s h t h a t u s e d t o r e f i l l t h e a n t e c h a m b e r . D r i -T r a i n Dry Box (He atmosphere) From h e l i u m C t a n k To vacuum pump F i g u r e 29. Dry-box and a c c e s s o r i e s i — 1 218 6.3 S y n t h e s i s o f Q u a t e r n a r y P y r i d i n i u m S a l t s Q u a t e r n a r y p y r i d i n i u m s a l t s were p r e p a r e d from t h e c o r -r e s p o n d i n g p y r i d i n e s and a l k y l h a l i d e s . M e t h y l c h l o r o a c e t a t e was p r e p a r e d from c h l o r o a c e t i c a c i d and m e t h a n o l by t h e method 8 9 o f C l i n t o n and L a s k o w s k i . I s o p r o p y l c h l o r o a c e t a t e was a l s o p r e p a r e d by the method o f C l i n t o n and L a s k o w s k i by u s i n g 2-p r o p a n o l i n p l a c e o f m e t h a n o l . A l l o f t h e p y r i d i n e s and t h e o t h e r a l k y l h a l i d e s were o b t a i n e d c o m m e r c i a l l y and u s e d w i t h -o u t f u r t h e r p u r i f i c a t i o n . The g e n e r a l method f o r p r e p a r i n g the p y r i d i n i u m s a l t s c o n s i s t e d o f m i x i n g 30 m i l l i m o l e s e ach o f t h e p y r i d i n e and a l k y l h a l i d e i n 15 m i s . o f s o l v e n t ( u s u a l l y a c e t o n e o r a c e t o n e / d i m e t h y l f o r m a m i d e ) and r e f l u x i n g f o r one d a y . The c r y s t a l s were f i l t e r e d , washed w i t h a c e t o n e and d r i e d u nder a s p i r a t o r vacuum. I n d i v i d u a l v a r i a t i o n s on t h i s scheme a r e n o t e d below. l - A c e t o n y l - 3 - c a r b a m o y l p y r i d i n i u m c h l o r i d e : F o r t y - f i v e m i l l i m o l e s o f c h l o r o a c e t o n e (5.0 mis) w i t h 30 m i l l i m o l e s o f n i c o t i n a m i d e (3.66 g r a m s ) , 8.0 m i s . o f d i m e t h y l f o r m a m i d e , and 10.0 m i s . o f a c e t o n e y i e l d e d 6.3 grams (98%) o f c r u d e p r o d u c t a f t e r r e f l u x i n g f o r two d a y s . The c r u d e p r o d u c t was d i s s o l v e d i n m e t h a n o l ( 8 mis/gram) a t 50° and f i l t e r e d . A c e t o n e was added a t 50° u n t i l t h e s o l u t i o n t u r n e d c l o u d y . The p r o d u c t r e c r y s t a l l i z e d upon c o o l i n g t o -15° (m.p.: 202-3° dec.) 219 E l e m e n t a l a n a l y s i s : c a l c u l a t e d : C: 50.35; H: 5.13;. N: 13.05 f o u n d : C : 5 0 . 4 2 ; H : 5 . 1 0 ; N : 1 3 . 2 6 . 1-Carborne thoxyme t h y 1 - 3-c a r b a m o y l p y r i d i n ium c h l o r i d e : T h i r t y - s e v e n m i l l i m o l e s o f m e t h y l c h l o r o a c e t a t e (4.0 grams) , 30 m i l l i m o l e s o f n i c o t i n a m i d e (3.66 grams) , 6.0 m i s . of d i m e t h y I f o r m a m i d e and 12.0 mis o f a c e t o n e y i e l d e d 6.3 grams (91%) o f c r u d e p r o d u c t a f t e r r e f l u x i n g f o r one day. The c r u d e p r o d u c t was d i s s o l v e d i n m e t h a n o l (5 mis./gram) a t 50° and f i l t e r e d . A c e t o n e was added a t 50° u n t i l t h e s o l u t i o n t u r n e d c l o u d y . The p r o d u c t r e c r y s t a . l l i z e d upon c o o l i n g , (m.p.: 1 6 5 - 6 ° d e c ) . E l e m e n t a l a n a l y s i s : c a l c u l a t e d : C: 46.85; H: 4.77; N:12.15 f o u n d : C: 46.64; H: 5.06; N: 11.80. 1 - C a r b o - i - p r o p o x y m e t h y 1 - 3 - c a r b a m o y l p y r i d i n i u m c h l o r i d e : T h i r t y m i l l i m o l e s o f n i c o t i n a m i d e (3.66 g r a m s ) , 40 m i l l i m o l e s o f i - p r o p y i e h l o r o a c e t a t e (5.5 g r a m s ) , 5.0 m i s . o f d i m e t h y 1 f o r m a m i d e , and 10.0 m i s . o f a c e t o n e were . r e f l u x e d f o r one day, y i e l d i n g 7.14 grams (92%) o f c r u d e p r o d u c t . The c r u d e p r o d u c t was d i s s o l v e d i n m e t h a n o l (5 mis/gram) a t 50-o 60 and f i l t e r e d . A c e t o n e was added u n t i l r e c r y s t a l l i z a t i o n began. The r e c r y s t a l l i z a t i o n was c o m p l e t e d upon c o o l i n g , (m.p. : 202-4° dec) 220 E l e m e n t a l a n a l y s i s : c a l c u l a t e d : C: 51.06; H: 5.80; N: 10.83 f o u n d : C: 50.89; H: 5.69; N: 10.88. l - C a r b a m o y l m e t h y l - 3 - a c e t y l p y r i d i n i u m c h l o r i d e : T h i r t y m i l l i m o l e s e a c h o f c h l o r o a c e t a m i d e (2.80 grams) and 3 - a c e t y l p y r i d i n e (3.63 grams) were mixed w i t h 5.0 m i s . o f d i m e t h y l f o r m a m i d e and 10.0 m i s . o f a c e t o n e and r e f l u x e d f o r one day. A y i e l d o f 5.2 grams (81%) o f c r u d e p r o d u c t was o b t a i n e d . The c r u d e p r o d u c t was d i s s o l v e d i n r e f l u x i n g m e t h a n o l (12 mis/gram) and f i l t e r e d . A c e t o n e was added u n t i l c r y s t a l -l i z a t i o n began (18 mis/gram) and the s o l u t i o n was a l l o w e d t o c o o l , y i e l d i n g f i n e n e e d l e - l i k e c r y s t a l s . (m.p.: 214-214.5° dec.) E l e m e n t a l a n a l y s i s : c a l c u l a t e d : C: 50.35; H: 5.13; N: 13.05 f o u n d : C: 50.33; H: 5.00; N: 12.87. l - C a r b a m o y l m e t h y l - 3 - c a r b a m o y l p y r i d i n i u m c h l o r i d e : A y i e l d o f 6.14 grams (.95%) o f c r u d e p r o d u c t was o b t a i n -ed when 3.0 m i s . o f d i m e t h y l f o r m a m i d e , 15.0 m i s . o f a c e t o n e and 30.0 m i l l i m o l e s each o f n i c o t i n a m i d e (3.66 grams) and c h l o r o a c e t a m i d e (2.80 grams) were r e f l u x e d f o r one d a y . The c r u d e p r o d u c t was d i s s o l v e d i n : w a t e r (2.5 mis/gram) a t 65° and f i l t e r e d . E t h a n o l was added u n t i l t h e s o l u t i o n became c l o u d y . On c o o l i n g , w h i t e s t a r - l i k e c r y s t a l s f o r m e d . (m.p.: 213-4° dec.) 221 E l e m e n t a l a n a l y s i s : c a l c u l a t e d : C: 44.5; H: 4.6; N: 19.5 f o u n d : C: 44.63; H: 4.85; N: 19.46. 1 - C a r b a m o y l r a e t h y 1 - 3 - f l u o r o p y r i d i n i u m c h l o r i d e : F o u r m i s . o f d i m e t h y l f o r m a m i d e , t h i r t e e n m i s . o f a c e t o n e , t h i r t y m i l l i m o l e s e a c h o f c h l o r o a c e t a m i d e (2.80 grams) and 3 - f l u o r o p y r i d i n e (2.91 g r a m s ) , and a c a t a l y t i c amount o f sodium i o d i d e were r e f l u x e d f o r t h r e e d a y s . The r e d o i l w h i c h s e p a r a t e d was c r y s t a l l i z e d by s e e d i n g o r by c o o l i n g t h e s o l u -t i o n below 0° and s t i r r i n g the o i l i n t o the a c e t o n e l a y e r . The y i e l d was 3.92 grams ( 6 8 % ) . The c r u d e p r o d u c t was d i s s o l v e d i n r e f l u x i n g e t h a n o l (5 mis/gram) and f i l t e r e d . A c e t o n e was added u n t i l t h e s o l u t i o n t u r n e d c l o u d y (8 m i s / g r a m ) . The p r o d u c t r e c r y s t a l l i z e d upon c o o l i n g . (m.p.: 160-2° d e c . ) . E l e m e n t a l a n a l y s i s : c a l c u l a t e d : C: 44.09; H: 4.20; N: 14.70 f o u n d : C: 43.80; H: 4.17; N: 14.67. 1 - C a r b a m o y l m e t h y l - 3 - c y a n o p y r i d i n i u m c h l o r i d e : T h i r t y m i l l i m o l e s e ach o f n i c o t i n o n i t r i l e (3.04 grams) and c h l o r o a c e t a m i d e (2.80 g r a m s ) , a c a t a l y t i c amount o f sodium i o d i d e , and 15 m i s . o f a c e t o n e were r e f l u x e d f o r two d a y s , y i e l d i n g 3.8 grams (64%) o f c r u d e p r o d u c t . The c r u d e p r o d u c t was r e c r y s t a l l i z e d from 100% e t h a n o l (80 m i s / g r a m ) . (m.p.: 193-6° dec.) 222 E l e m e n t a l a n a l y s i s : c a l c u l a t e d : C: 48.61; H: 4.05; N: 21.27 f o u n d : C: 48.74; H: 4.10; N: 21.23. 1 - C a r b a m o y l m e t h y l p y r i d i n i u m c h l o r i d e : T h i r t y m i l l i m o l e s o f c h l o r o a c e t a m i d e (2.80 g r a m s ) , 5.0 m i s . o f p y r i d i n e , and 15 m i s . o f a c e t o n e , a f t e r r e f l u x i n g f o r one day, y i e l d e d 4.5 grams (87%) o f c r u d e p r o d u c t . The c r u d e m a t e r i a l was d i s s o l v e d i n r e f l u x i n g m e t h a n o l (10 mis/gram) and f i l t e r e d . A c e t o n e (30 mis/gram) was added s l o w l y and the s o l u t i o n a l l o w e d to c o o l , y i e l d i n g n e e d l e - l i k e c r y s t a l s , (m.p.: 206-206.5° dec.) E l e m e n t a l a n a l y s i s : c a l c u l a t e d : C: 48.70; H: 5.22; N: 16.23 f o u n d : C: 48.82; H: 5.03; N: 15.96. l - C a r b a m o y l m e t h y l - 3 - h y d r o x y p y r i d i n i u m c h l o r i d e : T h r e e m i l l i m o l e s e a c h o f 3 - h y d r o x y p y r i d i n e and c h l o r o -a c e t a m i d e i n 15.0 m i s . o f a c e t o n e were r e f l u x e d f o r one day. The c r u d e c r y s t a l s were f i l t e r e d , washed w i t h a c e t o n e , d r i e d u n der a s p i r a t o r vacuum and r e c r y s t a l l i z e d from 100% e t h a n o l t o g i v e w h i t e n e e d l e s (m.p.: 197-9° dec.) E l e m e n t a l a n a l y s i s : c a l c u l a t e d : C: 44.56; H: 4.78; N: 14.85 f o u n d : C: 44.73, H: 4.68; N: 14.80. 223 To c o n f i r m t h a t a l k y l a t i o n o c c u r e d a t the r i n g n i t r o g e n r a t h e r t h a n a t the 3 - h y d r o x y l g r o u p , the U.V- s p e c t r u m o f t h i s p r o d u c t was compared w i t h t h e s p e c t r a o f l - m e t h y l - 3 -97 h y d r o x y p y r i d i n i u m c h l o r x d e i n a c i d i c and b a s i c s o l u t i o n s , The a g r e e m e n t does c o n f i r m N - a l k y l a t i o n , as e x p e c t e d . pH 1 - m e t h y l - 3 - h y d r o x y t h i s p y r i d i n i u m c h l o r i d e compound 1 283 289 13 245 238 322 313 1 - C a r b o x y m e t h y l - 3 - c a r b a m o y l p y r i d i n i u m m o n o h y d r a t e : T h i r t y m i l l i m o l e s e a c h o f n i c o t i n a m i d e (3.66 grams) and sodium c h l o r o a c e t a t e (3.50 g r a m s ) ( o r sodium i o d a c e t a t e ) were a l l o w e d to r e a c t i n 20 m i s . o f r e f l u x i n g m e t h a n o l f o r one d a y . A f t e r c o o l i n g the r e a c t i o n m i x t u r e t o below 0 ° , the c r u d e p r o d u c t was f i l t e r e d and washed w i t h a c e t o n e . The c r u d e p r o d u c t was d i s s o l v e d i n water (5 m i s / g r a m ) , w h i c h was h e a t e d on a steam b a t h . T h i s s o l u t i o n was t h e n f i l t e r e d , e t h a n o l (15 mis/gram) was added and r e c r y s t a l l i z a t i o n took p l a c e upon 90 91 c o o l i n g . (m.p.: 200-3° d e c , l i t . m.p.: 2 0 4 - 6 ° , 206-8° ) 1 - C y a n o m e t h y l - 3 - c a r b a m o y l p y r i d i n i u m c h l o r i d e : S i x m i s . o f d i m e t h y l f o r m a m i d e , 12 m i s . o f a c e t o n e , 30 m i l l i m o l e s o f n i c o t i n a m i d e (3.66 g r a m s ) , and 44 m i l l i m o l e s o f c h l o r o a c e t o n i t r i l e (3.32 grams) y i e l d e d 5.1 grams (86%) o f 224 c r u d e p r o d u c t a f t e r r e f l u x i n g f o r 2 d a y s . The c r u d e c r y s t a l s were d i s s o l v e d i n water (5 mis/gram) , w h i c h v/as h e a t e d on a steam b a t h and e t h a n o l was added u n t i l t h e s o l u t i o n t u r n e d c l o u d y (4 m i s / g r a m ) . Upon c o o l i n g , the p r o d u c t r e c r y s t a l l i z e d as a f i n e , y e l l o w , c r y s t a l l i n e powder. (m.p.: 236-8° dec.) E l e m e n t a l a n a l y s i s : c a l c u l a t e d : C: 48.6; H: 4.1; N: 21.3 f o u n d : C: 48 .42, H: 4.14; N: 21.03. 1, l ' - E t h y l e n e b i s ( 3 - c a r b a m o y l p y r i d i n i u m c h l o r i d e ) : T h i r t y m i l l i m o l e s o f n i c o t i n a m i d e (3.66 g r a m s ) , 10.0 m i s . o f e t h y l e n e d i c h l o r i d e , a c a t a l y t i c amount o f sodium i o d i d e , 15.0 m i s . o f a c e t o n e and 5.0 m i s . o f d i m e t h y l f o r m a m i d e were h e a t e d to 90-100° i n a p r e s s u r e b o t t l e f o r s i x d a y s . The r e s u l t i n g c r y s t a l s were f i l t e r e d , washed w i t h a c e t o n e and d r i e d u nder a s p i r a t o r vacuum. The c r u d e p r o d u c t was d i s s o l v e d i n t o r e f l u x i n g w a t e r , c o n t a i n i n g d e c o l o u r i z i n g c a r b o n and the s o l u t i o n f i l t e r e d and a l l o w e d t o c o o l . The c r y s t a l s were r e c o v e r e d and r e c r y s t a l l i z e d a s e c o n d time from water to g i v e v e r y p a l e b e i g e c r y s t a l s (m.p.: 297° dec.) l i t . m.p.: 9 6 9 0 ' 2 8 5 ° , 3 0 0 ° ; A (E) = 265 (9090); l i t . A (£) = 265 max max 90 ( 8 9 9 0 ) . 1 - ( 2 ' - H y d r o x y e t h y l ) - 3 - c a r b a m o y l p y r i d i n i u m c h l o r i d e : T h i r t y m i l l i m o l e s o f n i c o t i n a m i d e (3.66 g r a m s ) , 40 m i l l i m o l e s o f 2 - c h l o r o e t h a n o l (3.22 g r a m s ) , 5 m i s . o f d i m e t h y l f o r m a m i d e and 10.0 m i s . o f a c e t o n e were r e f l u x e d f o r two d a y s , y i e l d i n g 2.4 grams (40%) o f c r u d e p r o d u c t . The c r u d e p r o d u c t was r e c r y s t a l l i z e d from 100% e t h a n o l to g i v e 9 2 f l u f f y w h i t e c r y s t a l s (m.p.: 1 9 5 ° ; l i t . m.p.: 1 9 5 . 5 - 1 9 6 ° , 93 182° ) . 1 - M e t h o x y m e t h y l - 3 - c a r b a m o y l p y r i d i n i u m c h l o r i d e : T h i r t y m i l l i m o l e s o f n i c o t i n a m i d e (3.66 grams) were d i s s o l v e d i n 110 m i s . o f a c e t o n e a t room t e m p e r a t u r e . T h r e e m i s . o f c h l o r o m e t h y l m e t h y l e t h e r were added d r o p w i s e to the s t i r r e d s o l u t i o n and s t i r r i n g c o n t i n u e d f o r two h o u r s . The r e c o v e r e d p r o d u c t needed no f u r t h e r p u r i f i c a t i o n . (m.p. 123-5°) E l e m e n t a l a n a l y s i s : c a l c u l a t e d : C: 47.41; H: 5.43; N: 13.83 f o u n d : C: 47.20, H: 5.61; N: 14.00. A l l a t t e m p t s a t r e c r y s t a l l i z a t i o n r e s u l t e d i n d e c o m p o s i t i o n t o n i c o t i n a m i d e . l - M e t h y l - 3 - c a r b a m o y l p y r i d i n i u m i o d i d e : The p r e p a r a t i o n f o l l o w s the method u s e d by S u e l t e r 20 and M e t z l e r f o r 1 - n - p r o p y l n i c o t i n a m i d e i o d i d e . S i x t y - f o u r m i l l i m o l e s o f n i c o t i n a m i d e (7.80 g r a m s ) , 4.0 m i s . o f m e t h y l i o d i d e , and 27 m i s . o f m e t h a n o l were r e f l u x e d f o r f o u r h o u r s y i e l d i n g 13.7 grams (81%) o f c r u d e p r o d u c t . The c r u d e mater i a l v/as r e c r y s t a l l i z e d from 100% e t h a n o l , y i e l d i n g f i n e 226 f e a t h e r - l i k e c r y s t a l s . (m.p.: 207-210° d e c . ; l i t m.p.: 33 204° ) i 1 , 1 - M e t h y l e n e b i s ( 3 - c a r b a m o y l p y r i d i n i u m ) c h l o r i d e i o d i d e : T h i r t y m i l l i m o l e s e a c h o f n i c o t i n a m i d e (3.66 grams) and c h l o r o i o d o m e t h a n e (5.30 grams) i n 15.0 mis o f a c e t o n e were r e f l u x e d f o r two d a y s . The r e s u l t i n g c r y s t a l s were f i l t e r e d , washed w i t h a c e t o n e and d r i e d u n d e r a s p i r a t o r vacuum. The c r u d e p r o d u c t v/as r e c r y s t a l l i z e d from water to g i v e b r i g h t y e l l o w c r y s t a l s (m.p.: 218-21° dec.) E l e m e n t a l a n a l y s i s : c a l c u l a t e d : C: 3'7.10; H: 3.33; N: 13.32, C l : 8.41; I : 30.20 f o u n d : C: 37.25; H: 3.26; N: 12.99, C l : 8.23, I : 29.97. T h i s compound was the o n l y p r o d u c t r e c o v e r e d d u r i n g a t t e m p t s t o s y n t h e s i z e 1 - c h l o r o m e t h y l - 3 - c a r b a m o y l p y r i d i n i u m i o d i d e , even when a f i v e - f o l d e x c e s s o f c h l o r o i o d o m e t h a n e was u s e d . r 1 ,'1-Me t h y l e n e b i s ( 3 - c a r b a m o y l p y r i d i n i u m c h l o r i d e ) : T h i r t y m i l l i m o l e s o f n i c o t i n a m i d e (3.66 g r a m s ) , 10.0 m i s . o f m e t h y l e n e c h l o r i d e , a c a t a l y t i c amount o f sodium i o d i d e , 12.0 m i s . o f a c e t o n e and 5.0 m i s . o f d i m e t h y l f o r m a m i d e were h e a t e d i n a p r e s s u r e b o t t l e a t 90-95° f o r f i v e d a y s . The 45% y i e l d o f c r u d e c r y s t a l s were f i l t e r e d , washed w i t h a c e t o n e and d r i e d under a s p i r a t o r ; -vacuum . The c r u d e p r o d u c t was d i s s o l v e d i n r e f l u x i n g w a t e r , t r e a t e d w i t h d e c o l o u r i z i n g 227 c a r b o n and r e c r y s t a l l i z e d . The c r y s t a l s were r e c r y s t a l l i z e d a second t i m e from water (m.p.: 192-3° dec.) E l e m e n t a l a n a l y s i s : c a l c u l a t e d f o r m o n o h y d r a t e : C: 44.95; H: 4.61; N: 16.13 f o u n d : C: 44.78; H: 4.70; N: 15.66. The p r o t o n r e s o n a n c e s p e c t r u m o f t h i s compound was a l m o s t i d e n t i c a l v / i t h t h a t o f 1,1 ' -me t h y l e n e b i s ( 3 - c a r b a m o y l p y r i d i n i u m ) c h l o r i d e i o d i d e , t h u s c o n f i r m i n g the s t r u c t u r e a s s i g n e d ( X l X a ) . C h e m i c a l s h i f t (no. o f hydrogen) H l H 2 H 4 H 5 H 6 c h l o r i d e i o d i d e s a l t 7.6(1) 9 ..'85(1) 9.2(1)1. .8.5(1) 9.5(1) d i c h l o r i d e s a l t 7.7(1) 9.9(1) 9.2(1) 3.5(1) 9.6(1) 228 6.4 S y n t h e s i s o f 1 , 4 - d i h y d r o p y r i d i n e s . The 1 , 4 - d i h y d r o p y r i d i n e s were p r e p a r e d by t h e c l a s s i c a l method o f r e d u c t i o n o f the c o r r e s p o n d i n g p y r i d i n i u m s a l t s 20 33 94 95 w i t h sodium d i t h i o n i t e . ' ' ' The p y r i d i n i u m s a l t s were u s e d d i r e c t l y as r e c o v e r e d from t h e i r p r e p a r a t i v e r e a c t i o n m i x t u r e s ( v i d e a n t e ) . The g e n e r a l method c o n s i s t e d o f d i s s o l v i n g 10.0 m i l l i -m o l es o f t h e p y r i d i n i u m s a l t i n 15.0 m i s . o f w a t e r . H i g h p u r i t y n i t r o g e n was b u b b l e d t h r o u g h t h i s s o l u t i o n f o r a t l e a s t t e n m i n u t e s b e f o r e t h e r e a c t i o n was begun and c o n t i n u e d t h r o u g h o u t the r e a c t i o n . Sodium d i t h i o n i t e (2.61 grams, a p p r o x i m a t e l y 15 m i l l i m o l e s ) and sodium o r p o t a s s i u m c a r b o n a t e (20 m i l l i m o l e s ) were mixed and added p o r t i o n w i s e o v e r a f i v e m i n u t e p e r i o d . F o r d i h y d r o p y r i d i n e s w h i c h a r e p a r t i c u l a r l y s e n s i t i v e t o a c i d ( e . g . l - m e t h y l - 3 - c a r b a m o y l - l , 4 - d i h y d r c - , . . p y r i d i n e ) , t h e c a r b o n a t e was r e p l a c e d by 35 m i l l i m o l e s o f sodium o r p o t a s s i u m h y d r o x i d e . T h i s was added t o the r e a c -t i o n m i x t u r e as needed to m a i n t a i n t h e pH g r e a t e r t h a n 8. A f t e r a d d i t i o n o f the sodium d i t h i o n i t e and the b a s e , th e r e a c t i o n m i x t u r e was s t i r r e d u n t i l i t t u r n e d to a b r i g h t y e l l o w c o l o u r o r u n t i l c r y s t a l l i z a t i o n o c c u r r e d . C r y s t a l s were f i l t e r e d , washed w i t h s m a l l p o r t i o n s o f w a t e r , and d r i e d u n der a s p i r a t o r vacuum. P r o d u c t s w h i c h d i d n o t c r y s t a l l i z e were r e c o v e r e d by c o n t i n u o u s e x t r a c t i o n w i t h m e t h y l e n e c h l o r i d e w h i c h had been st o r e d . - o v e r p o t a s s i u m h y d r o x i d e . The m e t h y l e n e c h l o r i d e e x t r a c t was c o n c e n t r a t e d f r o m a b o u t 200 m i s . 229 to a b o u t 20 n l s . on a r o t a r y e v a p o r a t o r . The r e m a i n i n g s o l v e n t was e v a p o r a t e d under a s p i r a t o r vacuum i n a f l a s k f i t t e d w i t h a n i t r o g e n b l e e d . The n i t r o g e n b l e e d v/as neces' s a r y i n o r d e r to r e c o v e r a c r y s t a l l i n e p r o d u c t ; w i t h o u t i t , e v a p o r a t i o n o f t h e r e m a i n i n g m e t h y l e n e c h l o r i d e l e f t o n l y a y e l l o w o i l . I n d i v i d u a l v a r i a t i o n s on t h i s g e n e r a l method a r e n o t e d below. 1 - A c e t o n y l - 3 - c a r b a m o y l - 1 , 4 - d i h y d r o p y r i d i n e : Ten m i l l i m o l e s (2.14 grams) o f c r u d e l - a c e t o n y l - 3 -c a r l a n o y l p y r i d i n i u m c h l o r i d e were d i s s o l v e d i n 15 .mis. o f v/ater. P o t a s s i u m c a r b o n a t e (1.66 grams, 12 m i l l i m o l e s ) and sodium d i t h i o n i t e (2.08 grams, 12 m i l l i m o l e s ) were mixed and added p o r t i o n w i s e . The m i x t u r e v/as s t i r r e d u n d e r n i t r o g e n f o r 30 m i n u t e s , t h e n e x t r a c t e d o v e r n i g h t w i t h m e t h y l e n e c h l o r i d e . The e x t r a c t v/as c o n c e n t r a t e d t o a b o u t 20 m i s . on a r o t a r y e v a p o r a t o r and the r e m a i n i n g s o l v e n t was e v a p o r a t e d i n vacuo i n a f l a s k f i t t e d w i t h a n i t r o g e n b l e e d . The c r u d e p r o d u c t v/as r e c r y s t a l l i z e d from e t h y l a c e t a t e (m.p. : 118-21° d e c . ) E l e m e n t a l a n a l y s i s : c a l c u l a t e d : C: 60.00 ; II: G.67; ;i : 15.5 f o u n d : C: GO. 38; FI : 6.63; N : 15.20. J 6 230 1-Carborne t h o x y m e t h y l - 3 - c a r b a m o y l - 1 , 4 - d i h y d r o p y r i d i n e : Ten m i l l i m o l e s (2.30 grams) o f c r u d e 1 -carbomethoxy-m e t h y l - 3 - c a r b a m o y l p y r i d i n i u m c h l o r i d e were d i s s o l v e d i n 20 m i s . o f w a t e r . P o t a s s i u m c a r b o n a t e (1.66 grams, 12 m i l l i -m oles) and sodium d i t h i o n i t e (2.08 grams, 12 m i l l i m o l e s ) were mixed and added p o r t i o n w i s e . The m i x t u r e was s t i r r e d u n d e r n i t r o g e n f o r 15 m i n u t e s . The r e s u l t i n g c r y s t a l s were f i l t e r e d and washed v/i t h s m a l l p o r t i o n s o f w a t e r . The c r u d e p r o d u c t was r e c r y s t a l l i z e d from benzene to g i v e y e l l o w n e e d l e s , w h i c h v/ere s t a b l e i n a i r f o r a few months. (m.p.: 128-132° dec.) E l e m e n t a l a n a l y s i s : c a l c u l a t e d : C; 55.12; H: 6.12; N: 14.29 f o u n d : C: 55.11; H: 6.19; N: 14.26. 1 - C a r b o - i - p r o p o x y m e t h y 1 - 3 - c a r b a m o y 1 - 1 , 4 - d i h y d r o p y r i d i n e : Ten m i l l i m o l e s (2.58 grams) o f 1 - c a r b o - i - p r o p o x y m e t h y l -3 - c a r b a m o y l p y r i d i n i u m c h l o r i d e were d i s s o l v e d i n 15 m i s . o f w a t e r . Sodium d i t h i o n i t e (2.61 grams, 15 m i l l i m o l e s , and p o t a s s i u m c a r b o n a t e (2.76 grams, 20 m i l l i m o l e s ) v/ere mixed and added p o r t i o n w i s e . A f t e r s t i r r i n g f o r a b o u t f i v e m i n u t e s , a r e d o i l s e p a r a t e d . The m i x t u r e was e x t r a c t e d c o n t i n u o u s l y o v e r n i g h t w i t h 150 m i s . o f m e t h y l e n e c h l o r i d e . The e x t r a c t was r e d u c e d t o a b o u t 20 m i s . on a r o t a r y e v a p o r a t o r , t h e n e v a p o r a t e d t o d r y n e s s _in v a c u o i n a f l a s k f i t t e d w i t h a n i t r o g e n b l e e d . The c r u d e p r o d u c t was r e c r y s t a l l i z e d from 231 e t h y l a c e t a t e (20 m i s / g r a m ) , b e i n g a l l o w e d t o c o o l u nder n i t r o -gen. The c r y s t a l s a p p e a r t o r e q u i r e s e v e r a l d a y s o f e x p o s u r e t o a i r f o r n o t i c e a b l e d e c o m p o s i t i o n . (m.p.: 112-4° dec.) E l e m e n t a l a n a l y s i s : c a l c u l a t e d : C: 51.06; H: 5.80; N: 10.83 f o u n d : C: 50.89; H: 5.69; N: 10.88. 1-C a r b a m o y l m e t h y l - 3 - a c e t y l - l , 4 - d i h y d r o p y r i d i n e : Ten m i l l i m o l e s (2.14 grams) o f c r u d e 1 - c a r b a m o y l m e t h y l -3 - a c e t y l p y r i d i n i u m c h l o r i d e were d i s s o l v e d i n 15 m i s . o f w a t e r . Sodium d i t h i o n i t e (2.61 grams, 15 m i l l i m o l e s ) and p o t a s s i u m c a r b o n a t e (2.76 grams, 20 m i l l i m o l e s ) were mixed and added p o r t i o n w i s e . The m i x t u r e was s t i r r e d u nder n i t r o -gen f o r 20 m i n u t e s b e f o r e f i l t e r i n g . . The f i l t r a t e was e x t r a c t e d c o n t i n u o u s l y f o r t h r e e d a ys w i t h 150 m i s . o f m e t h y l e n e c h l o r i d e , t h e e x t r a c t e v a p o r a t e d to a b o u t 20 m i s . on a r o t a r y e v a p o r a t o r , t h e n t o d r y n e s s _i_n v a c u o i n a f l a s k f i t t e d w i t h a n i t r o g e n b l e e d , y i e l d i n g 1.16 grams o f c r u d e p r o d u c t (64%) . The c r u d e m a t e r i a l was r e c r y s t a l l i z e d from e t h y l a c e t a t e (160 rals/gram), c o o l i n g u n d e r n i t r o g e n , t o g i v e f i n e y e l l o w n e e d l e s , w h i c h a r e s t a b l e f o r s e v e r a l d a ys i n a i r . (m.p.: 162-3° dec.) E l e m e n t a l a n a l y s i s : c a l c u l a t e d : C: 60.00; H: 6.67; N: 15.56 f o u n d : C: 60.31; H: 6.80; N: 15.30. 232 1 - C a r b a m o y l m e t h y 1 - 3 - c a r b a m o y l - 1 , 4 - d i h y d r o p y r i d i n e : Ten m i l l i m o l e s (2.15 grams) o f l - c a r b a m o y l m e t h y l - 3 -c a r b a m o y l p y r i d i n i u m c h l o r i d e were d i s s o l v e d i n 20 m i s . o f w a t e r . Sodium d i t h i o n i t e (3.48 grams, 20 m i l l i m o l e s ) and p o t a s s i u m c a r b o n a t e (2.76 grams, 20 m i l l i m o l e s ) were mixed and added p o r t i o n w i s e . The m i x t u r e was s t i r r e d u nder n i t r o -gen f o r t e n m i n u t e s and t h e c r u d e p p r o d u c t was f i l t e r e d , washed w i t h 30 m i s . o f c o l d w a t e r , and d r i e d under a s p i r a t o r vacuum, y i e l d i n g 1.65 grams ( 9 1 % ) . The c r u d e c r y s t a l s were r e c r y s t a l l i z e d from water (30 mis/gram) g i v i n g an o v e r a l l 66% y i e l d o f y e l l o w , r o d - l i k e c r y s t a l s , w h i c h a r e s t a b l e i n d e f i n i t e l y i n the p r e s e n c e o f a i r a t room t e m p e r a t u r e , (m.p.: 179-182° dec.) E l e m e n t a l a n a l y s i s : c a l c u l a t e d : C; 53.0; H: 6.1; N: 23.2 f o u n d : C: 52.79; H: 6.21; N: 23.05. 1 - C a r b a m o y l m e t h y l - 3 - c y a n o - 1 , 4 - d i h y d r o p y r i d i n e : F i v e m i l l i m o l e s (1.0 grams) o f c r u d e 1 - c a r b a m o y l m e t h y l -3 - c y a n o p y r i d i n i u m c h l o r i d e were d i s s o l v e d i n 7 m i s . o f water and the s o l u t i o n was f l u s h e d w i t h n i t r o g e n g a s . Sodium d i t h i o n i t e (2.0 grams) and p o t a s s i u m c a r b o n a t e (1.38 grams) powders were mixed and added p o r t i o n w i s e t o t h e s t i r r e d s o l u t i o n . C r y s t a l s formed w i t h i n a b o u t two m i n u t e s a f t e r the f i n a l a d d i t i o n o f r e a g e n t s . The m i x t u r e v/as s t i r r e d u n d e r n i t r o g e n i n a c o l d w a t e r b a t h f o r one h o u r . The 2 3 3 c r y s t a l s were f i l t e r e d , vmsJied w i t h s n a i l p o r t i o n s o f u a t s r a n d d r i e d o v e r n i g h t u n d e r a s p i r a t o r vacuum. T h e f i l t r a t e a n d w a s h i n g s w e r e e x t r a c t e d o v e r n i g h t w i t h M e t h y l e n e c h l o r i d e . The m e t h y l e n e c h l o r i d e e x t r a c t was e v a p o r a t e d t o d r y n e s s o n a r o t a r y e v a p o r a t o r . T h e f o r n o r c r y s t a l s w e r e c o n b i n c . i w i t h t h e c;: t r a c t i o n n r o d u c t a n d r e c r y s t a l l i z e d from b e n z e n e ( 1 0 0 n l $ / g r a a ) . A y i e l d o f 7 0 m i l l i g r a m s ( a p p r o x i m a t e l y 1 0 % ) o f p u r e p r o d u c t w e r e o b t a i n e d . ( n . p . = 1 3 9 - 1 3 0 . 5 ° C ) ; ; i o r i e n t a l a n a l y s i s : c a l c u l a t e d : C : 5 8 . d 0 ; i < : 5.52; : 25.77 f o u n d : C: 5 5.13; II ; 5.72; J : 2 5 . 4 5 . 1 - C a r b a n o y i n e t h y 1 - 3 - f 1 u o r o - I , 4 - d i h y : i r o p y r i d i n e : F i v e r . i l l i n o l o s ( 0 . 9 5 g r a n s ) o f 1 - c a r b a n o y l r a c t h y l - 3 -f l u o r o p y r i d i n i u r a c h l o r i d e vra s d i s s o l v e d i n 7 m i s . o f w a t e r . T h e s o l u t i o n was f l u s h e d w i t h n i t r o q e n . S o d i u m d i t h i o n i t e (1.3 gran:;) and p o t a s s i u m h y d r o x i d e ( 0 . 9 grams) were a d d e d i n a l t e r n a t e p o r t i o n s , t h e p i ! b e i n g m a i n t a i n e d g r e a t e r t h a n t e n . C r y s t a l s ; f o r m e d w i t h i n a c o u p l e o f m i n u t e s and were f i l t e r e d i m m e d i a t e l y . I f t h e c r y s t a l s were s t i r r e d i n s o l u -t i o n f o r more t h a n t e n m i n u t e s , t h e y r e d i s s o l v e d . C o n t i n u -ous e x t r a c t i o n o f t h e f i l t r a t e w i t h M e t h y l e n e c h l o r i d e y i e l d e d no p r o d u c t . P r e s u m a b l y , t h e d i h y d r o p y r i d i n e i s so 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 d e c o m p o s i t i o n t h a t t h e p r o d u c t m u s t be r e c o v e r e d i m m e d i a t e l y e v e n a t a pll a s h i g h a s t e n . The c r u d e p r o d u c t was r e c r y s t a l l i z e d f r o m b e n z e n e (40 s i i l s / g r a n ) g i v i n g a 1 0 % y i e l d o f p u r e p r o d u c t . (m.p.: 1 1 6 . 5 - 1 1 8 ° d e c . ) 234 E l e m e n t a l a n a l y s i s : c a l c u l a t e d : C: 53.85; H: 5.77; N: 17.95 f o u n d : C: 53.88; H: 5.80; N: 17.94. 1 - C y a n o m e t h y 1 - 3 - c a r b a m o y l - 1 , 4 - d i h y d r o p y r i d i n e : One gram (5 m i l l i m o l e s ) o f l - c y a n o m e t h y l - 3 - c a r b a m o y l -p y r i d i n i u m c h l o r i d e was d i s s o l v e d i n 12 m i s . o f w a t e r . P o t a s -sium c a r b o n a t e (1.38 grams, 10 m i l l i m o l e s ) and sodium d i t h i o -n i t e (1.74 grams, 10 m i l l i m o l e s ) were mixed and added p o r t i o n -w i s e . The m i x t u r e v/as s t i r r e d f o r 30 m i n u t e s b e f o r e t h e p r o -d u c t began to c r y s t a l l i z e and t h e n ; f o r a n o t h e r 30 m i n u t e s a f t e r c r y s t a l l i z a t i o n had begun. The c r y s t a l s were f i l t e r e d and washed w i t h s m a l l p o r t i o n s o f c o l d water.. The c r u d e c r y s t a l s v/ere d i s s o l v e d i n r e f l u x i n g m e t h y l e n e c h l o r i d e and f i l t e r e d . P e t r o l e u m e t h e r v/as added u n t i l c r y s t a l l i z a t i o n b e g a n . The s o l u t i o n was c o o l e d u n d e r n i t r o g e n t o y i e l d f l u f f y , p a l e y e l l o w c r y s t a l s . (m.p.: 127-130° dec.) E l e m e n t a l a n a l y s i s : c a l c u l a t e d : C: 58.89; H: 5.52; N: 25.77 f o u n d : C: 58.72; H: 5.68; N: 25.75. 1 - M e t h o x y m e t h y l - 3 - c a r b a m o y 1 - 1 , 4 - d i h y d r o p y r i d i n e : Ten m i l l i m o l e s (2.02 grams) o f l - m e t h o x y m e t h y l - 3 -c a r b a m o y l p y r i d i n i u m c h l o r i d e v/ere d i s s o l v e d i n 15 m i s . o f w a t e r . Sodium d i t h i o n i t e (2.61 grams, 15 m i l l i m o l e s ) and p o t a s s i u m c a r b o n a t e (2.76 grams, 20 m i l l i m o l e s ) were mixed and added p o r t i o n w i s e . The m i x t u r e was s t i r r e d u nder 235 n i t r o g e n f o r f i v e m i n u t e s , t h e n e x t r a c t e d c o n t i n u o u s l y o v e r -n i g h t w i t h 150 m i s . o f m e t h y l e n e c h l o r i d e . The e x t r a c t was e v a p o r a t e d t o a b o u t 20 m i s . on a r o t a r y e v a p o r a t o r . The r e -m a i n i n g s o l v e n t was e v a p o r a t e d under vacuum i n a f l a s k f i t t e d w i t h a n i t r o g e n b l e e d t o y i e l d 1.16 grams (70%) o f p a l e y e l l o w c r y s t a l s . The c r u d e c r y s t a l s were r e c r y s t a l l i z e d t w i c e from e t h y l a c e t a t e . The r e c r y s t a l l i z a t i o n f l a s k must be s c r a p e d w i t h a g l a s s r o d b e f o r e s e a l i n g t h e s o l u t i o n u nder n i t r o g e n o r r e c r y s t a l l i z a t i o n w i l l n o t o c c u r . The p u r e c r y s t a l s a r e s t a b l e f o r o n l y a few days i n t h e p r e s e n c e o f a i r . (m.p.: 106-7° dec.) E l e m e n t a l a n a l y s i s : c a l c u l a t e d : C: 57.14; H: 7.14; N: 16.67 f o u n d : C: 57.40; H: 7.07; N: 16.66. 1-(2 * - H y d r o x y e t h y l ) - 3 - c a r b a m o y l - l , 4 - d i h y d r o p y r i d i n e : Ten m i l l i m o i e s (2.02 grams) o f c r u d e 1 - ( 2 ' - h y d r o x y -e t h y l ) - 3 - c a r b a m o y l p y r i d i n i u m c h l o r i d e were d i s s o l v e d i n 15 m i s . o f w a t e r . Sodium d i t h i o n i t e (2.61 grams, 15 m i l l i m o l e s ) were added a l t e r n a t e l y . The m i x t u r e was s t i r r e d u n d e r n i t r o g e n f o r f i v e m i n u t e s , t h e n e x t r a c t e d c o n t i n u o u s l y o v e r n i g h t w i t h 150 m i s . o f m e t h y l e n e c h l o r i d e . The e x t r a c t was e v a p o r a t e d t o a b o u t 20 mis on a r o t a r y e v a p o r a t o r . The c r y s t a l s were f i l -t e r e d , d r i e d , and s t o r e d u n d e r n i t r o g e n u n t i l t h e y were t r a n s f e r r e d to t h e d r y box. L e s s t h a n a d a y ' s e x p o s u r e t o a i r i s s u f f i c i e n t to c a u s e n o t i c e a b l e d i s c o l o r a t i o n o f t h e c r y s t a l s . 236 (m.p.: 133-133.5° d e c . ; l i t . m.p.: 1 1 9 - 2 1 ° 1 0 1 ) E l e m e n t a l a n a l y s i s : c a l c u l a t e d : C: 57.14; H: 7.14; N: 16.67 f o u n d : C: 56.97; H: 6.96; N: 16.53. l - M e t h y l - 3 - c a r b a m o y l - l , 4 - d i h y d r o p y r i d i n e : Ten m i l l i m o l e s (2..64 grams) o f c r u d e l - m e t h y l - 3 - c a r b a m o y l -p y r i d i n i u m i o d i d e were d i s s o l v e d i n 15 m i s . o f water and t h e s o l u t i o n f l u s h e d w i t h n i t r o g e n . Sodium d i t h i o n i t e (2.61 grams, 15 m i l l i m o l e s ) and p o t a s s i u m h y d r o x i d e (1.9 grams, 35 m i l l i m o l e s ) were added i n a l t e r n a t e p o r t i o n s w h i l e t h e pH was m a i n t a i n e d above 8. A f t e r t h e s o l u t i o n had t u r n e d a b r i g h t y e l l o w ( a b o u t o n e - h a l f h o u r ) , i t was e x t r a c t e d c o n t i n u o u s l y o v e r n i g h t w i t h a p p r o x i m a t e l y 200 m i s . o f m e t h y l e n e c h l o r i d e . The e x t r a c t was e v a p o r a t e d t o a p p r o x i m a t e l y 20 m i s . on a r o t a r y e v a p o r a t o r and the r e m a i n i n g s o l v e n t was e v a p o r a t e d i n v acuo i n a f l a s k f i t t e d w i t h a n i t r o g e n b l e e d . A y i e l d o f 0.4 grams o f b r i g h t y e l l o w c r y s t a l s was o b t a i n e d (m.p.: 73.5-3 3 9 8 78° d e c ; l i t m.p.: 8 4 ° , 8 5 . 3>- 8 6 . 8 ° ) ; X (e) = 360 max 98 (7820); l i t A (e) = 355 (6630) ) . max A t t e m p t s t o r e c r y s t a l l i z d t h e c r u d e p r o d u c t were u n s u c c e s s f u l . The l o w e r e x t i n c t i o n c o e f f i c i e n t i n t h e l i t e r a t u r e v/as r e c o r d e d i n w a t e r , wher'e t h i s compound decomposes even i n t h e a b s e n c e o f any added a c i d as b u f f e r . The s p e c t r u m o f t h i s compound was r e c o r d e d a t pH 9.8 i n t h i s s t u d y t o slow down the decompo-s i t i o n r e a c t i o n , b u t even a t t h i s pH a d e c o m p o s i t i o n o f a few p e r c e n t an hour i s ^ o b s e r v a b l e . 6.5 K i n e t i c s K i n e t i c r u n s were f o l l o w e d by v i s i b l e or u.v. a b s o r p -t i o n s p e c t r o s c o p y . R e a c t i o n s w h i c h had h a l f - l i v e s g r e a t e r t h a n f i v e m i n u t e s were f o l l o w e d on a C a r y 16 s p e c t r o p h o t o -m e t e r . Whenever t h e a b s o r b a n c e was n o t b e i n g r e a d , t h e sample c e l l was removed from t h e p a t h o f t h e l i g h t beam. R e a c t i o n s w h i c h had h a l f - l i v e s l e s s t h a n f i v e m i n u t e s were f o l l o w e d on a Bausch and Lomb S p e c t r o n i c 505 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 . A c o n t i n u o u s r e c o r d i n g was made o f t h e a b s o r b a n c e a t a s i n g l e w a v e l e n g t h and t i m e s were marked a l o n g t h i s r e c o r d i n g . 6.5.1 A c i d - c a t a l y z e d D e c o m p o s i t i o n s o f 1 , 4 - d i h y d r o p y r i d i n e s The r a t e of d e c o m p o s i t i o n o f 1 , 4 - d i h y d r o p y r i d i n e s i n a c i d was measured by f o l l o w i n g t h e l o s s o f d i h y d r o p y r i d i n e a b s o r b a n c e i n t h e r e g i o n between 300 and 390 n a n o m e t e r s . The d e c o m p o s i t i o n i s f i r s t o r d e r and, t h e r f o r e , i n i t i a l c o n c e n -t r a t i o n s need n o t be known. The b u f f e r s u s e d were 1.0 M.. and 0.1 M. T r i s a t pH 7.1 and 1.0 M. and 0.1 M. a c e t a t e a t p H 1 s 5.65, 4.6 and 3.4. B u f f e r s o l u t i o n s were t h e r m a l l y e q u i l i -b r a t e d f o r a t l e a s t o n e - h a l f hour b e f o r e u s e . S o l u t i o n s i n 1.0 M. b u f f e r s were p r e p a r e d by d i s s o l v i n g c r y s t a l s o f t h e d i h y d r o p y r i d i n e i n t h e b u f f e r s o l u t i o n . The t i m e r was s t a r t e d . An 0.1 cm. a b s o r b a n c e c e l l was f i l l e d w i t h t h i s s o l u t i o n and p l a c e d i n t h e t h e r m o s t a t e d c e l l compartment o f t h e s p e c t r o -p h o t o m e t e r . 238 S o l u t i o n s i n 0.1 M. b u f f e r s were p r e p a r e d i n e i t h e r o f t h e two f o l l o w i n g ways. F o r s l o w e r r e a c t i o n s , 5.0 m i s . o f a 1.0 M. s o l u t i o n , p r e p a r e d as above, were i m m e d i a t e l y d i l u t e d to 50 m i s . w i t h 1.0 M. sodium p e r c h l o r a t e . A 1.0 cm. a b s o r -bance c e l l was f i l l e d w i t h t h i s d i l u t e d s o l u t i o n and p l a c e d i n a s e c o n d t h e r m o s t a t e d compartment i n t h e C a r y 16 s p e c t r o -p h o t o m e t e r . F o r r e a c t i o n s i n 1.0 M. b u f f e r w h i c h had a h a l f -l i f e o f l e s s t h a n f i v e m i n u t e s , t h e 0.1 M. s o l u t i o n was p r e -p a r e d d i r e c t l y . C r y s t a l s o f t h e d i h y d r o p y r i d i n e were d i s s o l v e d i n 1.0 M. sodium p e r c h l o r a t e . F i v e m i s . o f a 1.0 M. b u f f e r were added, th e t i m e r s t a r t e d , and t h e s o l u t i o n made up t o 50 m i s . w i t h 1.0 M. sodium p e r c h l o r a t e . An 0.1 cm. a b s o r b a n c e c e l l was f i l l e d w i t h the sample s o l u t i o n and p l a c e d i n t h e t h e r m o s t a t e d c e l l compartment o f t h e a p p r o p r i a t e s p e c t r o p h o -t o m e t e r . The r e a c t i o n m i x t u r e s were a n a l y z e d w i t h i n a few h o u r s a f t e r t h e d e c o m p o s i t i o n was begun. The pH was measured on a R a d i o m e t e r 26 pH M e t e r s t a n d a r d i z e d a t pH 4 and 7 or 7 and 10. The c o n c e n t r a t i o n o f a c e t i c a c i d was d e t e r m i n e d by t i t r a t i o n to a .phenolphthaTeinri e n d p o i n t w i t h a s t a n d a r d sodium h y d r o x i d e s o l u t i o n . The c o n c e n t r a t i o n o f p r o t o n a t e d T r i s was d e t e r m i n e d by t i t r a t i o n t o pH 10.5 w i t h a s t a n d a r d sodium h y d r o x i d e s o l u -t i o n . 239 6.5.2 R e a c t i o n Between F l a v i n s and 1 , 4 - D i h y d r o p y r i d i n e s The r a t e o f t h e r e a c t i o n between r i b o f l a v i n o r f l a v i n m o n o n u c l e o t i d e and 1 , 4 - d i h y d r o p y r i d i n e s was d e t e r m i n e d by f o l l o w i n g t h e l o s s o f a b s o r b a n c e i n t h e r e g i o n between 430 and 470 nanometers due to t h e r e d u c t i o n o f t h e f l a v i n . The d i h y d r o p y r i d i n e was p r e s e n t i n e x c e s s i n a l l c a s e s . A s t o c k s o l u t i o n o f each o f t h e r e a c t a n t s was p r e p a r e d and t h e r m a l l y e q u i l i b r a t e d i n t h e d r y box. The r e a c t a n t s were mixed i n e i t h e r o f t h e two f o l l o w i n g ways. F o r r e a c t i o n s w i t h h a l f - l i v e s g r e a t e r t h a n f i v e m i n u t e s , the r e a c t a n t s were mixed i n t h e d r y box. The t i m e r was s s t a r t e d i m m e d i a t e l y and as q u i c k l y as p o s s i b l e , a 1.0 cm. a b s o r b a n c e c e l l was f i l l e d t o o v e r f l o w i n g w i t h t h e r e a c t i o n m i x t u r e and s e a l e d w i t h a s i l i c o n e r u b b e r s t o p p e r and a r u b b e r septum. The d o u b l e s e a l was n e c e s s a r y t o keep t h e c e l l f r e e o f oxygen f o r a t l e a s t two h o u r s . The c e l l was removed from t h e d r y box and p l a c e d i n a t h e r m o s t a t e d c o m p a r t -ment o f t h e C a r y 16. A b s o r b a n c e r e a d i n g s were t a k e n u n t i l t h e a b s o r b a n c e r e m a i n e d unchanged 6OK a..t'. fea's t d p h e - h a l f e'h^our v\ ••' ,half' hoToo/'much time was r e q u i r e d t o f i l l t h e a b s o r b a n c e c e l l and remove i t from t h d r y box f o r t h i s method to be u s e d w i t h r e a c t i o n s h a v i n g h a l f - l i v e s l e s s t h a n f i v e m i n u t e s . F o r t h e s e r e a c t i o n s , t h e r e q u i r e d volume of f l a v i n s t o c k s o l u t i o n was p l a c e d i n t h e a b s o r b a n c e c e l l i n t h e d r y box and t h e c e l l was s e a l e d w i t h a s i l i c o n e r u b b e r s t o p p e r and r u b b e r septum. A s y r i n g e n e e d l e was i n s e r t e d i n t o t h e s p a c e o v e r t h e f l a v i n s o l u t i o n i n t h e c e l l . The t o p o f t h i s n e e d l e was t h e n 240 s e a l e d i n t o a t e s t tube as i n F i g u r e 30. T h i s a r r a n g e m e n t a l l o w e d t h e p r e s s u r e t o be r e l i e v e d when t h e d i h y d r o p y r i d i n e s o l u t i o n was i n j e c t e d i n t o t h e c e l l and, s i n c e t h e n e e d l e was n e v e r exposed to t h e a t m o s p h e r e , i t c o u l d n o t a c t as an avenue f o r o xygen t o d i f f u s e i n t o t h e c e l l . U s i n g t h i s method, t h e a b s o r b a n c e c e l l r e m a i n e d f r e e o f oxygen f o r a l o n g e r t i m e t h a n when t h e c e l l was f l u s h e d w i t h h i g h - p u r i t y n i t r o g e n w h i l e t h e d i h y d r o p y r i d i n e s o l u t i o n was b e i n g i n j e c t e d . The r e q u i r e d volume o f d i h y d r o p y r i d i n e s t o c k s o l u t i o n was measured i n t o a s y r i n g e i n t h e d r y box and a s m a l l volume o f d i h y d r o p y r i d i n e s t o c k s o l u t i o n was p l a c e d i n a t e s t tube to a c t as a back-up s u p p l y . The t e s t t u b e was s e a l e d w i t h a r u b b e r septum and t h e n e e d l e o f t h e s y r i n g e c o n t a i n i n g t h e d i h y d r o p y r i d i n e s o l u t i o n was i n s e r t e d t h r o u g h t h e septum. The s y r i n g e was removed from the d r y box i n t h i s way to p r e -v e n t oxygen from e n t e r i n g t h e s y r i n g e b e f o r e the s o l u t i o n s were mixed . The s y r i n g e and a b s o r b a n c e c e l l a p p a r a t u s was removed from the box. The a b s o r b a n c e c e l l was p l a c e d i n t h e thermo-s t a t e d compartment o f t h e B a u s c h and Lomb s p e c t r o p h o t o m e t e r , t h e n t h e d i h y d r o p y r i d i n e s o l u t i o n was i n j e c t e d i n t o t h e c e l l and the t i m e r was s t a r t e d as the c e l l c o n t e n t s were m i x e d . The r e c o r d i n g o f t h e a b s o r b a n c e was c o n t i n u e d u n t i l no change was e v i d e n t o v e r a p e r i o d o f a t l e a s t 15 m i n u t e s . R e o x i d a t i o n o f f l a v i n , c a u s e d by oxygen l e a k i n g i n t o t h e c e l l , was n o t i c e -a b l e w i t h i n an hour o f m i x i n g t h e s o l u t i o n s b u t t h i s was 241 J empty t e s t tube I I I I i i i i 11 11 F l a v i n s o l u t i o n s y r i n g c o n t a i n i n g d i h y d r o p y r i d i n e s o l u t i o n t e s t tube c o n t a i n -i n g a s m a l l amount o f d i h y d r o p y r i d i n e s o l u t i o n a b s o r b a n c e c e l l F i g u r e 30. S y r i n g e and a b s o r b a n c e c e l l a p p a r a t u s f o r o x y g e n - f r e e work 242 l o n g e r t h a n t h e r e a c t i o n t i m e f o r any o f t h e s o l u t i o n s p r e p a r e d i n t h i s way. S t o c k s o l u t i o n s o f r i b o f l a v i n (Xa) and f l a v i n mono-n u c l e o t i d e ^ were p r e p a r e d by d i s s o l v i n g a p p r o x i m a t e l y 7.1 CH 2OR a : R=H b: R=PO. m i l l i g r a m s o f sample i n 75 t o 100 m i s . o f b u f f e r i n a r e d c o l o u r e d Kimax " R a y - s o r b " g l a s s v o l u m e t r i c f l a s k i n t h e d r y box. The r i b o f l a v i n r e q u i r e d a t l e a s t o n e - h a l f hour o f s t i r -r i n g t o c o m p l e t e l y d i s s o l v e . The c o n c e n t r a t i o n o f t h e s t o c k s o l u t i o n was d e t e r m i n e d as soon as p o s s i b l e by m e a s u r i n g t h e a b s o r b a n c e o f the s o l u t i o n a t a; minimum,'of 't h r e e w a y e l e n g h t h s , S t o c k s o l u t i o n s o f 1 , 4 - d i h y d r o p y r i d i n e s were p r e p a r e d by d i s s o l v i n g the r e q u i r e d amount o f d i h y d r o p y r i d i n e c r y s t a l s i n 25 m i s . o f b u f f e r i n t h e d r y box. The amount o f d i h y d r o -p y r i d i n e was c h o s e n su.ch t h a t t h e r e a c t i o n s would have h a l f -l i v e s between t h r e e and t h i r t y m i n u t e s . The c o n c e n t r a t i o n 243 o f the s t o c k s o l u t i o n was d e t e r m i n e d by m e a s u r i n g t h e a b s o r -bance o f t h e s o l u t i o n a t a t l e a s t t h r e e w a v e l e n g t h s . The i n i t i a l c o n c e n t r a t i o n s o f the r e a c t a n t s were t h e n c a l c u l a t e d from th e c o n c e n t r a t i o n s o f t h e s t o c k s o l u t i o n s u s i n g t h e a p p r o p r i a t e d i l u t i o n s . 244 6.6 P o l a r o g r a p h y A l l p o l a r o g r a m s v/ere r e c o r d e d on a Metrohm E 261 P o l a -r e c o r d . The d r o p t i m e v/as r e g u l a t e d a t 0.166 s e c o n d s by a Metrohm d r o p c o n t r o l l e r a t t a c h e d to t h e d r o p p i n g m e r c u r y e l e c t r o d e . The f l o w c o n s t a n t s o f t h e c a p i l l a r y were: a m e r c u r y f l o w r a t e (m) o f 3.25 m i l l i g r a m s / s e c o n d and a n a t u r a l d r o p t i m e o f 2.5 s e c o n d s . A s c a n r a t e o f 0.1 v o l t s / m i n u t e was u s e d . The p o t e n t i a l s v/ere measured a g a i n s t a s i l v e r -s i l v e r c h l o r i d e - s a t u r a t e d p o t a s s i u m c h l o r i d e r e f e r e n c e e l e c -99 t r o d e , f o r w h i c h a v a l u e o f 222 mv. v/as u s e d t o r e f e r t h e o b s e r v e d p o l a r o g r a p h i c p o t e n t i a l s to t h e s t a n d a r d h y d r o g e n e l e c t r o d e . The P o l a r e c o r d E 261 i s f i t t e d w i t h a c h a r g i n g c u r r e n t c o m p e n s a t i o n w h i c h v/as a d j u s t e d f o r each p o l a r o g r a m to o b t a i n a base l i n e v/hich was as c l o s e as p o s s i b l e t o hor i z o n t a 1 . The b u f f e r f o r most compounds v/as 0.1 M. a c e t i c a c i d -sodium a c e t a t e a t a pH o f 5.65. The i o n i c s t r e n g t h was a d j u s t e d t o 0.1 w i t h sodium p e r c h l o r a t e . Sodium p e r c h l o r a t e was t h e p r e f e r r e d s a l t s i n c e more n u c l e o p h i l i c s a l t s , s u c h as c h l o r i d e , formed a d d i t i o n p r o d u c t s w i t h some o f t h e more e a s i l y r e d u c e d p y r i d i n i u m s a l t s . Lov/er pH b u f f e r s were u s e d f o r l - c a r b a m o y l m e t h y l - 3 - c y a n o p y r i d i n i u m c h l o r i d e (pH = 4.6) "and m e t h y l e n e b i s ( 3 - c a r b a m o y 1 - p y r i d i n i u m c h l o r i d e ) (pH = 3.4) t o p r e v e n t t h e r e a c t i o n o f t h e s e compounds w i t h h y d r o x i d e i o n . T e s t s o l u t i o n s were p r e p a r e d by e i t h e r d i s s o l v i n g a known w e i g h t o f p y r i d i n i u m s a l t or d i l u t i n g a s t o c k s o l u t i o n 245 o f p y r i d i n i u m s a l t i n a p i p e t t e d volume o f b u f f e r i n a w a t e r -j a c k e t e d p o l a r o g r a p h y v e s s e l t h e r m o s t a t e d a t 2 5 ° . These t e s t s o l u t i o n s were t h e n d e o x y g e n a t e d by b u b b l i n g h i g h p u r i t y n i -t r o g e n t h r o u g h the s o l u t i o n f o r a t l e a s t t e n m i n u t e s . The n i t r o g e n f l o w was d i v e r t e d to p a s s o v e r the s o l u t i o n v / h i l e t h e p o l a r o g r a m v/as r e c o r d e d , a p e r i o d o f f i v e to s e v e n minu t e s. In o r d e r to c a r r y o u t a c o m p l e t e a n a l y s i s o f the p o l a r o g r a p h i c waves, the c u r r e n t must be measured a t s e v e r a l p o t e n t i a l s a l o n g the wave. The f o l l o w i n g two methods were used to d e t e r m i n e th e c u r r e n t , i , and t h e l i m i t i n g c u r r e n t , i , . I f the wave was s u f f i c i e n t l y i s o l a t e d from a l l o t h e r d waves t h a t b o t h the base l i n e and p l a t e a u were l i n e a r , as i n F i g u r e 31a, t h e n t h e s e l i n e a r p o r t i o n s were e x t r a p o l a t e d to more n e g a t i v e and more p o s i t i v e p o t e n t i a l s , r e s p e c t i v e l y . A t any g i v e n p o t e n t i a l , t h e l i m i t i n g c u r r e n t ( i ^ ) was measured as the d i s t a n c e between the e x t r a p o l a t e d base and p l a t e a u l i n e s and the c u r r e n t ( i ) as the d i s t a n c e betv/een th e p o l a r o -g r a p h i c wave and the e x t r a p o l a t e d base l i n e . C u r r e n t s were measured from a p p r o x i m a t e l y 3% t o 97% o f the l i m i t i n g c u r r e n t . When a s e c o n d wave a p p e a r e d w i t h i n a b o u t 200 m i l l i -v o l t s o f the measured wave, as i n F i g u r e 31b the p l a t e a u r e g i o n c o u l d n o t be e x t r a p o l a t e d to more p o s i t i v e p o t e n t i a l s . A s i n g l e v a l u e f o r the l i m i t i n g c u r r e n t was measured a t t h e i n f l e c t i o n p o i n t betv/een th e two waves. The c u r r e n t was measured as b e f o r e . The p r e s e n c e o f the s e c o n d wave c a u s e s (b) F i g u r e 31. Sample p o l a r o g r a m s 247 a d i s t o r t i o n o f the p l a t e a u r e g i o n o f the measured wave so c u r r e n t s were o n l y measured up t o 85% o f the l i m i t i n g c u r r e n t . 243 6.7 P o t e n t i o m e t r y P o t e n t i o m e t r i c measurements were made i n t h e d r y box under a h e l i u m a t m o s p h e r e . P o t e n t i a l s were r e c o r d e d on a Beckman Model G pH m e t e r . The i n s t r u m e n t was c a l i b r a t e d p e r i o d i c a l l y w i t h an u n b u f f e r e d s o l u t i o n c o n t a i n i n g 0.1 M. each o f p o t a s s i u m f e r r o c y a n i d e and p o t a s s i u m f e r r i c y a n i d e . The c e l l p o t e n t i a l was measured w i t h a p l a t i n u m e l e c t r o d e r e f e r r e d t o a s a t u r a t e d p o t a s s i u m c h l o r i d e - c a l o m e l e l e c t r o d e ( F i g u r e 3 2 ) . The c e l l c o n t e n t s were assumed' to have r e a c h e d e q u i l i b r i u m when p o t e n t i a l s r e c o r d e d an hour a p a r t were c o n -s t a n t to w i t h i n one m i l l i v o l t . 14 3 1 3 2 Rodkey ' ' has s u c c e s s f u l l y u s e d a f l a v o e n z y m e x a n t h i n e o x i d a s e t o a c c e l e r a t e e q u i l i b r i u m between NAD and a p o t e n t i a l m e d i a t o r s u c h as b e n z y l v i o l o g e n . The same m e d i -a t o r system was u s e d i n t h i s s t u d y to measure NAD s o l u t i o n s and t r i e d w i t h some NAD m o d e l s . The x a n t h i n e o x i d a s e was p u r c h a s e d from Sigma C o r p . as a s u s p e n s i o n i n 2.3 M. ammonium s u l p h a t e . The s p e c i f i c e n z y m a t i c a c t i v i t y q u o t e d by Sigma was us e d as a g u i d e when d e t e r m i n i n g t h e volume o f s u s p e n s i o n to use e a c h t i m e . T h r e e u n i t s o f enzyme were p r e p a r e d f o r each p o t e n t i o m e t r y c e l l , where one u n i t i s the amount r e q u i r e d to c o n v e r t one m i c r o m o l e o f x a n t h i n e t o u r i c a c i d p e r m i n u t e a t pH 7.5 and 2 5 ° . The s u s p e n s i o n was c e n t r i f u g e d , the s u p e r -n a t a n t l i q u i d d i s c a r d e d and the s o l i d t r a n s f e r r e d to the d r y box. The d e s i r e d amounts o f NAD +, NADH, and b e n z y l v i o l o g e n were weighed i n t o t h e p o t e n t i o m e t r y c e l l and 25 m i s . o f b u f f e r 249 F i g u r e 32. P o t e n t i o m e t r y c e l l . v/ere added. The t o t a l c o n c e n t r a t i o n o f NAD' and NADH was be--3 -3 tween 1 x 10 and 5 x 10 M., w i t h the b e n z y l v i o l o g e n c o n -c e n t r a t i o n b e i n g f i v e t o t e n p e r c e n t o f t h i s . One t o two m i s . o f t h i s s o l u t i o n were t h e n used to d i s s o l v e the x a n t h i n e o x i d a s e and t r a n s f e r i t i n t o t h e p o t e n t i o m e t r y c e l l . The x a n t h i n e o x i d a s e - b e n z y l v i o l o g e n m e d i a t o r system worked w e l l w i t h NAD b u t gave e r r a t i c r e s u l t s w i t h NAD m o d e l s . A s o l u t i o n c o n t a i n i n g o x i d i z e d and r e d u c e d l - m e t h o x y m e t h y l - 3 -c a r b a m o y l p y r i d i n e and b e n z y l v i o l o g e n was p r e p a r e d as above f o r NAD. The s o l u t i o n was s p l i t i n t o two c e l l s and t h r e e u n i t s o f x a n t h i n e o x i d a s e were added to e a c h . A f t e r e q u i l i -b r i u m was a p p a r e n t l y r e a c h e d ( t h e c e l l p o t e n t i a l s v/ere c o n -s t a n t f o r a t l e a s t one h o u r ) , the two c e l l p o t e n t i a l s d i f f e r e d by 50 mv. In o t h e r a t t e m p t s v/ i t h l - m e t h o x y m e t h y l - 3 - c a r b a m o y l -p y r i d i n i u m , 1 - c a r b a m o y l m e t h y 1 - 3 - c a r b a m o y l p y r i d i n i u m and 1-m e t h y 1 - 3 - c a r b a m o y l p y r i d i n i u m i o n s the c e l l p o t e n t i a l s d i d n o t r e s p o n d i n a s y s t e m a t i c way w i t h c h a n g e s i n t h e p y r i d i n i u m -t o - d i h y d r o p y r i d i n e r a t i o . F l a v i n s were t e s t e d as p o s s i b l e m e d i a t o r s f o r t h e NAD models s i n c e r i b o f l a v i n and FMN a r e r e a d i l y r e d u c e d by 1,4-d i h y d r o p y r i d i n e s and a r e e l e c t r o m o t i v e l y a c t i v e . The r e d u c -t i o n p o t e n t i a l s o f f l a v i n s a r e more p o s i t i v e t h a n any o f t h e NAD models t e s t e d , i n some c a s e s by as much as 200 mv. b u t d e s p i t e t h i s , the measured p o t e n t i a l s shov/ed v e r y l i t t l e s c a t t e r . M e t h y l v i o l o g e n was a l s o added as a c o - m e d i a t o r to s o l u t i o n s o f l - m e t h y l - 3 - c a r b a m o y l p y r i d i n i u m , 1 - c a r b o x y m e t h y l -3 - c a r b a m o y l p y r i d i n i u m and 1-(2 * - h y d r o x y e t h y l ) - 3 - c a r b a m o y l -251 p y r i d i n i u m s i n c e t h e c e l l p o t e n t i a l s o f t h e s o l u t i o n s n e a r pH 10 were w i t h i n 50 mv. o f t h e r e d u c t i o n p o t e n t i a l o f m e t h y l v i o l o g e n . M e t h y l v i o l o g e n , b e n z y l v i o l o g e n , m e t h y l r e d , and a l i z a r i n b l u e S d i d n o t r e a c t d i r e c t l y w i t h 1 , 4 - d i h y d r o -p y r i d i n e s and, t h e r e f o r e , r i b o f l a v i n must s t i l l be u s e d to c a t a l y z e t h e r e d u c t i o n o f m e t h y l v i o l o g e n by 1 , 4 - d i h y d r o -p y r i d i n e s . The n e c e s s i t y o f u s i n g r i b o f l a v i n as a c a t a l y s t may n e g a t e any p o s s i b l e b e n e f i t s d e r i v e d from u s i n g t h e m e t h y l v i o l o g e n s i n c e t h e c e l l p o t e n t i a l s o f i d e n t i c a l s o l u -t i o n s were s i m i l a r whether m e t h y l v i o l o g e n was p r e s e n t or n o t . To e n s u r e t h a t t h e f l a v i n s were g i v i n g c o r r e c t p o t e n -t i a l measurements, t h e method was compared w i t h t h e x a n t h i n e o x i d a s e / b e n z y l v i o l o g e n m e d i a t o r s y s t e m i n t h e measurement o f NAD + p o t e n t i a l s . I d e n t i c a l s o l u t i o n s c o n t a i n i n g NAD + and NADH were p r e p a r e d . B e n z y l v i o l o g e n and x a n t h i n e o x i d a s e were added to one s o l u t i o n and r i b o f l a v i n was added t o t h e o t h e r . The r i b o f l a v i n s o l u t i o n r e q u i r e d o v e r t w e l v e h o u r s f o r t h e p o t e n t i a l t o r e a c h a c o n s t a n t v a l u e , a f t e r w h i c h ti m e t h e measured p o t e n t i a l s o f t h e two s o l u t i o n s a g r e e d t o w i t h i n 5 mv. In g e n e r a l , t h e o x i d a t i o n - r e d u c t i o n p o t e n t i a l s o f t h e NAD models were d e t e r m i n e d by the method o f m i x t u r e s . The d e s i r e d q u a n t i t i e s o f a s u b s t i t u t e d p y r i d i n i u m s a l t and t h e c o r r e s p o n d i n g d i h y d r o p y r i d i n e were weighed i n t o a Metrohm p o l a r o g r a p h y c e l l f i t t e d w i t h a water j a c k e t . The w e i g h t o f p y r i d i n i u m s a l t and d i h y d r o p y r i d i n e were c h o s e n to g i v e a t o t a l -3 - 2 c o n c e n t r a t i o n between 10 and 10 M. Two t o f o u r m i l l i g r a m s 252 o f r i b o f l a v i n o r FMN (and a p p r o x i m a t e l y o n e - h a l f m i l l i g r a m o f m e t h y l v i o l o g e n , when used) were weighed i n t o the c e l l . R i b o -f l a v i n was u s e d when t h e pH was g r e a t e r t h a n 7.5. A t pH's l e s s t h a n 7.5, t h e r e d u c e d r i b o f l a v i n p r e c i p i t a t e d s i g n i f i -c a n t l y , l e a v i n g t o o l i t t l e r i b o f l a v i n i n s o l u t i o n f o r i t t o a c t e f f e c t i v e l y as a p o t e n t i a l m e d i a t o r so FMN was us e d i n s t e a d . (A c o n c e n t r a t i o n o f f l a v i n o f f i v e t o t e n p e r c e n t o f the t o t a l p y r i d i n i u n v d i h y d r o p y r i d i n e c o n c e n t r a t i o n was r e q u i r e d . ) T w e n t y - f i v e m i s . o f b u f f e r were added t o t h e c e l l and t h e c r y s t a l s were d i s s o l v e d by s t i r r i n g t h e s o l u t i o n f o r 15 t o 20 m i n u t e s . B u f f e r s were c h o s e n t o p r e v e n t , where p o s s i b l e , t h e d e c o m p o s i t i o n o f p y r i d i n i u m i o n s w h i l e m i n i m i z i n g t h e a c i d c a t a l y z e d d e c o m p o s i t i o n o f the d i h y d r o p y r i d i n e . The p o t e n -t i a l s o f NAD +, NMN+, l - c a r b a m o y l m e t h y l - 3 - c a r b a m o y l p y r i d i n i u m i o n ( I l l i ) , l - a c e t o n y l - 3 - c a r b a m o y l p y r i d i n i u m i o n ( H i e ) and l - m e t h o x y m e t h y l - 3 - c a r b a m o y l p y r i d i n i u m i o n ( H i d ) c o u l d be d e t e r m i n e d betv/een pH 8 and 10. A c i d - c a t a l y z e d d e c o m p o s i t i o n o f t h e - d i h y d r o p y r i d i n e s p r e v e n t e d p o t e n t i o m e t r i c measurements b e i n g made o f 1-(2 * - h y d r o x y e t h y l ) - 3 - c a r b a m o y l p y r i d i n i u m i o n ( i i i c ) below pH 9.5 or l - m e t h y l - 3 - c a r b a m o y l p y r i d i n i u m ( I l l b ) and l - c a r b o x y m e t h y l - 3 - c a r b a m o y l p y r i d i n i u m i o n s ( I l i a ) below pH 10. The o x i d i z e d f orms o f 1 - c y a n o m e t h y l - 3 - c a r b a m o y l -p y r i d i n i u m ( I I I h ) , 1 - c a r b a m o y l m e t h y 1 - 3 - c y a n o p y r i d i n i u m ( I l l k ) and l - c a r b a m o y l m e t h y l - 3 - a c e t y l p y r i d i n i u m ( I I I j ) i o n s r e a c t s u f f i c i e n t l y r a p i d l y w i t h base t o p r e v e n t t h e i r p o t e n t i a l s 253 I I I V a : R l = C H 2 C 0 ° R 3 - CONH 2 b: CH 3 CONH 0 c : CH CH,OH CONH d : CH 2OCH 3 CONH 2 e : CH„COCH„ 2 3 CONH f : CH 2COOCH 3 CONH 2 g: CH 2COOCH(CH 3) CONH 2 h: CH 2CN CONH 2 i : CH 2CONH CONH 2 j : CH 2CONH 2 COCH 3 k : CH 2CONH CN 1 : CH 2CONH 2 F m : CH^CONH H n : CH 2CONH 2 . OH 254 from b e i n g measured a t pH's g r e a t e r t h a n 6.0, 7.5, and 9.5, r e s p e c t i v e l y . A t t h e two l o w e r pH's t h e r e i s some u n a v o i d a b l e d e c o m p o s i t i o n o f l - c y a n o m e t h y l - 3 - c a r b a m o y l - l , 4 - d i h y d r o p y r i d i n e (Vh) and 1 - c a r b a m o y l m e t h y l - 3 - c y a n o - 1 , 4 - d i h y d r o p y r i d i n e ( V k ) . The e s t e r g r o u p o f the l - c a r b o m e t h o x y m e t h y l - 3 - c a r b a m o y l -p y r i d i n i u m i o n ( I l l f ) was so r a p i d l y h y d r o l y z e d by base t h a t i t was o n l y s t a b l e i n a c i d i c media where t h e c o r r e s p o n d i n g d i h y d r o p y r i d i n e 1 - c a r b o r n e t h o x y m e t h y l - 3 - c a r b a m o y l - 1 , 4 - d i h y d r o -p y r i d i n e (Vf) underwent r a p i d a c i d - c a t a l y z e d d e c o m p o s i t i o n . Thus i t was i m p o s s i b l e to d e t e r m i n e t h e r e d u c t i o n p o t e n t i a l o f t h i s compound. The more s t e r i c a l l y h i n d e r e d e s t e r o f the l - c a r b o - i - p r o p o x y m e t h y l - 3 - c a r b a m o y l p y r i d i n i u m i o n ( I l l g ) was s u f f i c i e n t l y s t a b l e a t pH 7 t o a l l o w p o t e n t i a l d e t e r m i n -a t i o n s b u t even a t t h i s pH, 30% o f t h e e s t e r had been h y d r o -l y z e d to t h e 1 - c a r b o x y m e t h y l compound ( I l i a ) and 20-30% o f the d i h y d r o p y r i d i n e m a t e r i a l had decomposed a f t e r e i g h t h o u r s . I t was assumed t h a t o n l y one d i h y d r o p y r i d i n e compound was p r e s e n t , 1 - c a r b o - i - p r o p o x y m e t h y 1 - 3 - c a r b a m o y 1 - 1 , 4 - d i h y d r o -p y r i d i n e ( V g ) . W i t h a r e d u c t i o n p o t e n t i a l d i f f e r e n c e o f a p p r o x i m a t e l y 60 m i l l i v o l t s between the 1 - c a r b o - i - p r o p o x y m e t h y l compound and the 1 - c a r b o x y m e t h y l compound and n e a r e q u a l c o n -c e n t r a t i o n s o f t h e two p y r i d i n i u m i o n s , o n l y one t o two p e r -c e n t o f t h e t o t a l d i h y d r o p y r i d i n e c o n c e n t r a t i o n s h o u l d be i n t h e form o f l - c a r b o x y m e t h y l - 3 - c a r b a m o y l - l , 4 - d i h y d r o p y r i d i n e ( V a ) . The l a b i l i t y o f t h e e s t e r g r o u p was u t i l i z e d f o r t h e 255 p r e p a r a t i o n o f l - c a r b o x y m e t h y l - 3 - c a r b a m o y l - l , 4 - d i h y d r o p y r i d i n e s i n c e a t h i g h pH, t h e e s t e r o f l - c a r b o m e t h o x y m e t h y l - 3 - c a r b a m o y l -1 , 4 - d i h y d r o p y r i d i n e (Vf) i s r a p i d l y h y d r o l y z e d and t h e p r o d u c t 1 - c a r b o x y m e t h y l - 3 - c a r b a m o y l - 1 , 4 - d i h y d r o p y r i d i n e (Va) i s d e -composed o n l y v e r y s l o w l y . E q u i l i b r i u m as d e t e r m i n e d by a c o n s t a n t c e l l p o t e n t i a l was g e n e r a l l y r e a c h e d w i t h i n t h r e e t o e i g h t h o u r s . Those p y r i d i n i u m i o n s w i t h l e s s n e g a t i v e r e d u c t i o n p o t e n t i a l s t e n d e d to r e a c h e q u i l i b r i u m t h e q u i c k e s t . A f t e r t h e c e l l p o t e n t i a l was measured, the s o l u t i o n s were a n a l y z e d t o d e t e r m i n e th e f i n a l c o n c e n t r a t i o n s o f p y r i d i n i u m s a l t and d i h y d r o p y r i d i n e • and t h e pH. A sample o f t h e p o t e n t i o m e t r y s o l u t i o n was p l a c e d i n an 0.1 cm. a b s o r b a n c e c e l l i n t h e d r y box. The r e m a i n i n g s o l u t i o n was p l a c e d i n an e r l e n m e y e r f l a s k s e a l e d w i t h a r u b b e r septum. As q u i c k l y as p o s s i b l e a f t e r r e m o v a l from t h e d r y box, t h e a b s o r b a n c e o f the s o l u t i o n was r e a d on t h e C a r y 16 s p e c t r o -p h o t o m e t e r a t two w a v e l e n g t h s between 440 and 460 nm. and a t a minimum o f t h r e e w a v e l e n g t h s between 300 and 400 nm. T h e s e w a v e l e n g t h r e g i o n s i n c l u d e t h e a b s o r b a n c e maxima o f IV>^=^ihydropyriain'e: and oxidized.'.f l a v i n s . The c o n c e n t r a t i o n s o f d i h y d r o p y r i d i n e and o x i d i z e d and r e d u c e d f l a v i n were c a l -c u l a t e d by s o l v i n g t h e s y s t e m s o f s i m u l t a n e o u s l i n e a r e q u a t i o n s c o n t a i n i n g t h e a b s o r b a n c e d a t a . A t l e a s t f i v e d i f f e r e n t a b s o r b a n c e c o m b i n a t i o n s v/ere used and t h e r e s u l t i n g v a l u e s a v e r a g e d t o g i v e t h e c o n c e n t r a t i o n v a l u e s u s e d i n s u b s e q u e n t c a l c u l a t i o n s . When n e c e s s i t a t e d by h i g h d i h y d r o p y r i d i n e c o n --3 c e n t r a t i o n s ( g r e a t e r t h a n 5 x 10 M.), t h e p o t e n t i o m e t r i c s o l u t i o n s were d i l u t e d v / i t h b u f f e r b e f o r e t h e a b s o r b a n c e was r e a d . The c o n c e n t r a t i o n o f p y r i d i n i u m s a l t i n t h e p o t e n t i o m -e t r y s o l u t i o n was d e t e r m i n e d by p o i a r o g r a p h y . A c a l i b r a t i o n l i n e was c a l c u l a t e d by the method o f l e a s t s q u a r e s from a t l e a s t s e v e n c o n c e n t r a t i o n - d i f f u s i o n c u r r e n t p a i r s . The c a l i -. -2 -4 b r a t i o n l i n e s were g e n e r a l l y l i n e a r between 10 and 10 M. a l t h o u g h the l i n e s d i d n o t p a s s t h r o u g h t h e o r i g i n . The p o t e n t i o m e t r y s o l u t i o n was removed from t h e d r y box s e a l e d i n an e r l e n m e y e r f l a s k . The p o l a r o g r a m was r e c o r d e d v / i t h i n tv/o m i n u t e s a f t e r p o u r i n g t h e e r l e n m e y e r c o n t e n t s i n t o t h e p o l a r o g r a p h y c e l l . The a n a l y s i s must be c a r r i e d o u t q u i c k l y to m i n i m i z e t h e f l a v i n - c a t a l y z e d a i r o x i d a t i o n o f d i h y d r o -p y r i d i n e t o t h e p y r i d i n i u m i o n w h i c h i s b e i n g a s s a y e d . A f t e r r e c o r d i n g t h e p o l a r o g r a m , t h e pH o f t h e s o l u t i o n v/as d e t e r -m i n e d . E x c e p t v/hen x a n t h i n e o x i d a s e was u s e d , t h e pH o f a p a r t i c u l a r b u f f e r n e v e r v a r i e d by more t h a n .05 pH u n i t s . P y r i d i n i u m s a l t s have an a b s o r b a n c e maximum between 260 and 270 nm. and a t t e m p t s were made to use d i r e c t s p e c t r o -p h o t o m e t r y d e t e r m i n a t i o n s o f t h e s e compounds. O n l y 1-a c e t o n y l - 3 - c a r b a m o y l p y r i d i n i u m and 1 - c a r b a m o y l m e t h y l - 3 -a c e t y l p y r i d i n i u m i o n s c o u l d be a s s a y e d i n t h i s way. The r e s u l t s f o r t h e s e tv/o compounds a g r e e d w i t h the p o l a r o g r a p h i c r e s u l t s to w i t h i n t e n p e r c e n t . The p r o d u c t o f a c i d c a t a l y z e d 257 d e c o m p o s i t i o n o f 1 , 4 - d i h y d r o p y r i d i n e s e x h i b i t s a h i g h a b s o r -1 9 10 71 bance maximum near 290 nm. ' ' ' wh i c h i n t e r f e r e s w i t h the d i r e c t s p e c t r o p h o t o m e t r i c d e t e r m i n a t i o n s o f o t h e r p y r i d i n i u m compounds. 14 31 3 2 The s u c c e s s o f Rodkey ' ' w i t h the c y a n i d e a d d u c t as an a s s a y method f o r NAD + i n s p i r e d a t t e m p t s to use t h i s method f o r NAD + m o d e l s . S u c c e s s was l i m i t e d t o 1-methoxy-m e t h y l - 3 - c a r b a m o y l p y r i d i n i u m i o n s ( H i d ) and 1 - a c e t o n y 1 - 3 -c a r b a m o y l p y r i d i n i u m i o n s ( H i e ) s i n c e s u b s t i t u t e d p y r i d i n e s h a v i n g much more n e g a t i v e r e d u c t i o n p o t e n t i a l s t h a n NAD were no t c o v e r t e d q u a n t i t a t i v e l y to 4 - c y a n o - l , 4 - d i h y d r o p y r i d i n e s i n aqueous c y a n i d e s o l u t i o n s and t h e c y a n i d e a d d u c t s o f 1-c y a n o m e t h y l - 3 - c a r b a m o y l p y r i d i n i u m and 1 - c a r b a m o y l m e t h y l - 3 -a c e t y l p y r i d i n i u m i o n s , w h i c h b o t h have more p o s i t i v e r e d u c t i o n p o t e n t i a l s t h a n NAD + u n d e r w e n t s i g n i f i c a n t d e c o m p o s i t i o n o v e r a 15 m i n u t e p e r i o d a t pH 5.6 and 8.1 r e s p e c t i v e l y , t h e pH ' s a t w h i c h t h e p o t e n t i o m e t r y o f t h e s e compounds v/as c a r r i e d o u t . I n t h e few c a s e s i n w h i c h the method was use d s u c c e s s f u l l y , i t c o n f i r m e d t h e c o n c e n t r a t i o n d e t e r m i n e d by p o l a r o g r a p h y . The p o l a r o g r a p h i c d e t e r m i n a t i o n o f t h e p y r i d i n i u m i o n h e l d a n o t h e r a d v a n t a g e o v e r t h e two s p e c t r o p h o t o m e t r i c methods w h i c h were u n a b l e t o d e t e c t d e c o m p o s i t i o n s w h i c h l e f t the p y r i d i n i u m r i n g i n t a c t . I f t h e r e d u c t i o n p o t e n t i a l s o f th e o r i g i n a l p y r i d i n i u m i o n and i t s d e c o m p o s i t i o n p r o d u c t a r e s u f f i c i e n t l y d i f f e r e n t , as v/as o f t e n t h e c a s e , t h e n t h e two c o n c e n t r a t i o n s c a n be d e t e r m i n e d by p o l a r o g r a p h y . One 258 commonly met d e c o m p o s i t i o n r e a c t i o n was the h y d r o l y s i s o f t h e 3 - c a r b a m o y l g r o u p a t pH's g r e a t e r t h a n 10. The 3 - c a r b o x y p r o -d u c t had a h a l f - w a v e p o t e n t i a l betv/een 200 and 300 mv. more n e g a t i v e t h a n t h e v/ave due to. t h e 3 - c a r b a m o y l compound. The e s t e r g r o u p s o f l - c a r b o m e t h o x y m e t h y l - 3 - c a r b a m o y l p y r i d i n i u m ( I l l f ) and l - c a r b o - i - p r o p o x y m e t h y l - 3 - c a r b a m o y l p y r i d i n i u m ( I l l g ) i o n s u nderwent s i g n i f i c a n t b a s e - c a t a l y z e d h y d r o l y s i s a t a pH as low as 7 and t h e r e s u l t i n g l - c a r b o x y m e t h y l - 3 - c a r b a m o y l -p y r i d i n i u m i o n ( I l i a ) was e a s i l y d i s t i n g u i s h e d from t h e o r i g i n a l e s t e r s on t h e p o l a r o g r a m s . 259 6.8 A b s o r b a n c e S p e c t r a A b s o r b a n c e s p e c t r a were r e c o r d e d on a C a r y 16 s p e c t r o -p h o t o m e t e r between 220 and 520 nm. a t 10 nm. i n t e r v a l s . S p e c t r a l d e t e r m i n a t i o n s were r e p e a t e d u n t i l t h e e x t i n c t i o n c o e f f i c i e n t s n e a r A were r e p r o d u c i b l e t o w i t h i n 3%. The max v a l u e s o f A and t h e c o r r e s p o n d i n g e x t i n c t i o n c o e f f i c i e n t s max o f most o f t h e compounds u s e d i n t h i s s t u d y a r e l i s t e d i n T a b l e XIX. The s p e c t r a were r e c o r d e d i n w a t e r , e x c e p t t h o s e o f l - m e t h y l - 3 - c a r b a m o y l - 1 , 4 - d i h y d r o p y r i d i n e ( V b ) , l - ( 2 ' -h y d r o x y e t h y 1 ) - 3 - c a r bamoyl-1, 4-d i h y d r o p y r i d i n e (Vc) , 1 - c a r b o x y -me t h y l - 3 - c a r b a m o y l - 1 , 4 - d i h y d r o p y r i d i n e ( V a ) , and 1 - c a r b a m o y l -m e t h y 1 - 3 - f l u o r o - 1 , 4 - d i h y d r o p y r i d i n e ( V I ) , w h i c h decompose r a p i d l y i n pu r e v/ater and t h e r e f o r e were r e c o r d e d i n pH 10 a: R^=CH 2C00~ R 3=CONH 2 b: CH- CONH„ 3 2 c: CH^CH OH C0NH 2 d: CH CONH., F b u f f e r . T y p i c a l s p e c t r a o f a p y r i d i n i u m s a l t and a d i h y d r o -p y r i d i n e a r e shown i n F i g u r e 33. The s p e c t r u m o f 1 - c a r b a m o y l -m e t h y l - 3 - f l u o r o - 1 , 4 - d i h y d r o p y r i d i n e was so u n l i k e t h e o t h e r s p e c t r a t h a t i t i s shown s e p a r a t e l y i n F i g u r e 34. T a b l e XIX A and E x t i n c t i o n C o e f f i c i e n t s o f max H H CH COO CONH 2 - 356 7360 CH3 CONH 0 - - 360 7280 CH,CH 2OH CONH,, 265 4340 357 7510 CH 2OCH 3 CONH 2 264 ^4500 338 6490 CH „,COCH„ 2 3 CONH 266 4860 353 6300 CH 2COOCH 3 CONH 2 266 4750 347 6900 CH COOCH(CH ) CONH - - 349 6360 CH 2CN CONH 2 265 4900 340 5820 CH 2CONH 2 CONH 267 4800 351 6800 CH 2CONH 2 COCH 3 267 . 5 4450 370 12000 CH CONH 2 CN 269.5 4670 334 5680 CH CONH „ 2 2 F - - see F i g u r e 34 t were n o t meas u r e d . 260 Compounds Used i n t h i s Work F i g u r e 33. T y p i c a l S p e c t r a o f a P y r i d i n i u m i o n and a 1 , 4 - d i h y d r o p y r i d i n e T 1 — i 1 1 1 1 1 r —i 1 1 1 ^ i 1 , i 1 1 i I 220 240 260 280 300 320 340 360 380 400 £ t—1 w a v e l e n g t h (nanometers) F i g u r e 34. U.V. a b s o r b a n c e s p e c t r u m o f l - c a r b a m o y l m e t h y l - 3 - f l u o r o - 1 , 4 -d i h y d r o p y r i d i n e w a v e l e n g t h (nanometers) 263 BIBLIOGRAPHY 1. H. Sund i n B i o l o g i c a l O x i d a t i o n s , T.P. S i n g e r , E d . , I n t e r s c i e n c e , New Y o r k , 1968. 2. 0. Warburg, W. C h r i s t i a n , and A. G r i e s e , B i o c h e m . Z . , 282, 157 ( 1 9 3 5 ) . 3. D. M a u z e r a l l and F.H. Wes t h e i m e r , J . Amer. Chem. S o c . , 77, 2261 (1955) . 4. M. P u l l m a n , A. San P i e t r o , and S.P. C o l o w i c k , J . B i o l . Chem., 206, 129 ( 1 9 5 4 ) . 5. A.L. Underwood and R.W. B u r n e t t , E l e c t r o a n a l . Chem., 6^ , A . J . B a r d , Ed., Dekk e r , New Y o r k , 1972, p. 2. 6. U. E i s n e r and J . K u t h a n , Chem. Rev., 7 2, 1 ( 1 9 7 2 ) . 7. 0. Warburg and W. C h r i s t i a n , B i o c h e m . Z., 274 , 112 ( 1 9 3 4 ) . 8. G.W. R a f t u , S. C h a y k i n , and E.G. K r e b s , J . B i o 1 . Chem., 208, 799 ( 1 9 5 3 ) . 9. K.S. C h o i and S.G.A. A l i v i s a t o s , B i o c h e m i s t r y , 7_, 190 (1968) . 10. C.S.Y. Kim and S. C h a y k i n , B i o c h e m i s t r y , 7_, 2339 ( 1 9 6 8 ) . 11. C.C. J o h n s t o n , J . L . G a r d n e r , C H . S u e l t e r , and D.E. M e t z l e r , B i o c h e m i s t r y , /2, 689 ( 1 9 6 3 ) . 12. S.G.A. A l i v i s a t o s , F. Unger, and G.J. Abraham, . B i o c h e m i s t r y , _4, 2616 (1965) . 13. S.P. C o l o w i c k , N.D. K a p l a n , and M.M. C i o t t i , J . B i o 1 . Chem., 191, 447 (1951) . 14. F.L. Rodkey, J . B i o l . Chem., 213, 777 ( 1 9 5 5 ) . 15. R.N. L i n d q u i s t and E.H. C o r d e s , J . Amer. Chem. S o c . , 90, 1269 (1968) . 16. K. W a l l e n f e l s and H. Diekmann, Ann., 621, 166 ( 1 9 5 9 ) . 17. B.M. A n d e r s o n and N.O. K a p l a n , J . B i o l . Chem., 234, 1226 (1959) . 18. E.N. Shaw i n H e t e r o c y c l i c Compounds, V. 14, P a r t 2, K l i n g e n b e r g , Ed., I n t e r s c i e n c e , New Y o r k , 1964, p. 1. 264 19. T.P. S i n g e r and E.B. K e a r n e y , J . B i o l . Chem., 183, 409 (1950) . 20. C H . S u e l t e r and D.E. M e t z l e r , B i o c h i m . B i o p h y s . A c t a , 44, 23 (1960) . 21. B.M. A n d e r s o n and C D . A n d e r s o n , B i o c h i m . B i o p h y s . A c t a , 205, 161 (1970) . 22. F. Y-H. Wu, R.E. M a c K e n z i e , and D.B. McCormick, B i o c h e m i s t r y , 9, 2219 (1970) . 23. G.H. Radda and M. C a l v i n , B i o c h e m i s t r y , _3, 384 ( 1 9 6 4 ) . 24. J . L . Fox and G. T o l l i n , B i o c h e m i s t r y , fS, 3865 ( 1 9 6 6 ) . 25. I . I s e n b e r g , S.L. B a i r d , J r . , A. S z e n t - G y o r g y i , P r o c .  N a t . A c a d . S c i . , 47 , 245 (1961) . 26. T.C. B r u i c e , L. Ma i n , S. S m i t h , P.Y. B r u i c e , J . Amer.  Chem. S o c , 93 , 7327 ( 1 9 7 1 ) . 27. M. B r u s t l e i n and T.C. B r u i c e , J . Amer. Chem. Soc., 94, 6548 ( 1 9 7 2 ) . 28. E . J . Land and A . J . Swallow, J . B i o c h e m . S o c . , 116, 16P (1970) . 29. W.J. B l a e d e l and R.G. Haas, A n a l y t i c a l Chem., 4 2, 918 (1970) . 30. W.M. C l a r k , O x i d a t i o n - R e d u c t i o n P o t e n t i a l s o f O r g a n i c  S y s t e m s , W i l l i a m s and W i l l i a m s Co., B a l t i m o r e , 1960, p. 487. 31. F.L. Rodkey and J.A. Donovan, J r . , J . B i o l . Chem., 23 4, 677 ( 1 9 5 9 ) . 32. F.L. Rodkey, J . B i o l . Chem., 234, 188 ( 1 9 5 9 ) . 33. P. K a r r e r , G. S c h w a r z e n b a c h , F. Benz, and U. S o l m s s e n , H e l v . Chim. A c t a , 19, 811 (1936) . 34. P. K a r r e r and F . J . S t o n e , H e l v . Chim. A c t a , 20, 418 ( 1 9 3 7 ) . 35. S . J . L e a c h , J.H. B a x e n d a l e , and M.G. E v a n s , A u s t r a l i a n  J . Chem., 6, 395 ( 1 9 5 3 ) . 36. K. B u r t o n and T . W i l s o n , B i o c h e m . J . , 54, 86 ( 1 9 5 3 ) . 37. A.A. V l c e k i n P r o g r e s s i n I n o r g a n i c Chem., F.A. C o t t o n , Ed., I n t e r s c i e n c e , New Y o r k , 1963 , pT 225 . 265 38. R. B o n n a t e r r e and G. C a u q u i s , J . E l e c t r o a n a l . Chem., 3 2, 199 ( 1 9 7 1 ) . 39. S.G. M a i r a n o v s k i i , C a t a l y t i c and K i n e t i c Waves i n P o l a r - o g r a p h y , Plenum P r e s s , New Y o r k , 1968. 40. P . J . E l v i n g , J . E . O ' R e i l l y , C O . Schmakel i n Methods o f  B i o c h e m i c a l A n a l y s i s , 21, D. G l i c k , Ed., p. 287. 41. A . J . Cunningham and A.L. Underwood, B i o c h e m i s t r y , j>, 266 (1967) . 42. K.S.V. Santhanum and P . J . E l v i n g , J . Amer. Chem. Soc., 95, 5482 (1973) . 43. D. T h e v e n o t and R. B u v e t , J . E l e c t r o a n a l y t i c a l Chem.,  39, 447 ( 1 9 7 2 ) . 44. D. T h e v e n o t and G. Hammouya, E x p e r i e n t i a S u p p l . , 18, 631 (1972) . 45. J.N. B u r n e t t and A.L. Underwood, B i o c h e m i s t r y , 4_, 2060 (1965) . 46. C O . S chmakel, e_t a l . , J . E l e c t r o c h e m . S o c . , 121, 345 (1974) . 47. D.G. E p p l e and A.M. W i l s o n , B i o c h e m i s t r y , 5_, 3170 ( 1 9 6 6 ) . 48. E. L a v i r o n , B u l l . S o c . Chim. F r a n c e , 1962, No. 3, 418. 49. J.N. B u r n e t t and A.L. Underwood, J . Org• Chem., 3 0, 1154 (1965) . 50. E . J . Land and A . J . Swallow, B i o c h i m . B i o p h y s . A c t a , 162, 327 (1968) . 51. B. Ke, B i o c h e m . B i o p h y s . A c t a , 20, 547 ( 1 9 5 6 ) . 52. I . Bergmann i n P o l a r o g r a p h y 1964, G.J. H i l l s , Ed., I n t e r s c i e n c e , New Y o r k , 1966, p. 985. 53. R . C Kaye and H.I. S t o n e h i l l , J . Chem. Soc., 1952, 3244. 54. C.G. Swain & E.C. L u p t o n , J . Amer. Chem. S o c , 90, 4328 ( 1 9 6 8 ) . 55. P. Zuman, S u b s t i t u e n t E f f e c t s i n O r g a n i c P o l a r o g r a p h y , Plenum P r e s s , New Y o r k , 1957. 266 56. M.R. Lamborg, R.M. B u r t o n , and N.O. K a p l a n , J . Amer. v-Chem. Soc., 79, 6173 (1957) . 57. E.M. Kosower, I n t r o d u c t i o n to P h y s i c a l O r g a n i c C h e m i s t r y , J o h n W i l e y and Sons, New Y o r k , 1968. 58. K. W a l l e n f e l s i n S t e r i c C o u r s e o f M i c r o b i o l o g i c a l Reac- t i o n s , G.E.W. W o l s t e n h o l m e and C M . O'Connor, E d s . , J . and A. C h u r c h i l l , London, 1959, p. 10. 59. A . C L o v e s a y , J . M e d i c i n . Chem., 13, 693 ( 1 9 7 0 ) . 60. W. C i u s s a , P.M. S t r o c c h i , and G. Adamo, Ga z z . Chim. I t a l . , 80, 604 (1950) . 61. C. C a r r u t h e r s and V. S u n t z e f f , A r c h . B i o c h e m . B i o p h y s . , 45, 140 (1953) . 62. P. Hemmerich, A.P. B h a d u i r , G. B l a n k e n h o r n , M. B r u s t l e i n , W. Haas, and W.-R. Knappe i n O x i d a s e s and R e l a t e d Redox  Systerns, T.E. K i n g , H.S. Mason, and M. M o r r i s o n , E d s . , U n i v e r s i t y P a r k P r e s s , B a l t i m o r e , 1973, p. 3. 63. P. Hemmerich, S. G h i s l a , U. Hartmann, and F. M u l l e r i n F l a v i n s and F l a v o p r o t e i n s , H. Kamin, Ed., U n i v e r s i t y P a r k P r e s s , B a l t i m o r e , 1971. 64. L. Fox and G. T o l l i n , B i o c h e m i s t r y , 5_, 3873 ( 1 9 6 6 ) . 65. J . J . S t e f f e n s and D.M. Chipman, J . Amer. Chem. Soc., 93, 6694 ( 1 9 7 1 ) . 66. R.K. L o v e c c h i o , D i s s . A b s t . I n t . B, 3 0-,- 5397 ( 19 7 0 ) . 67. D.J.H. McClemmens, A.K. G a r r i s o n , A.L. Underwood, J . O r g . Chem., 34, 1867 ( 1 9 6 9 ) . 68. D.R. B r o w n i n g , E l e c t r o m e t r i c Methods, M c G r a w - H i l l , London, 1969. 69. J . H i n e , P h y s i c a l O r g a n i c C h e m i s t r y , M c G r a w - H i l l , London, 1962. 70. R^W. T a f t , J r . , J . Amer. Chem. S o c , 7 5, 4231 ( 1 9 5 3 ) . 71. T.C. B r u i c e and S . J . B e n k o v i c , B i o o r g a n i c Mechanisms, V o l . 2, W.A. B e n j a m i n , New Y o r k , 1966, p. 301-349. 72. M. C h a r t o n , J . O r g . Chem., 29, 1222 ( 1 9 6 4 ) . 73„ . R.W. T a f t , J r . , J . Amer. Chem. S o c , 79, 1045 ( 1 9 5 7 ) . 267 74. J.D. R o b e r t s and W.T. M o r e l a n d , J r . , J . Amer. Chem.  Soc . , 75, 2167 ( 1 9 5 3 ) . 75. R.M. B u r t o n and N.O. K a p l a n , A r c h . B i o c h e m . B i o p h y s . , 101, 139 ( 1 9 6 3 ) . 76. P.R. W e l l s , L i n e a r F r e e E n e r g y R e l a t i o n s h i p s , Academic P r e s s , London, 1968. 77. L.P. Hammett, P h y s i c a l O r g a n i c C h e m i s t r y , 2nd E d i t i o n , M c G r a w - H i l l , New Y o r k , 1970. 78. J . C l a r k and D.D. P e r r i n , Q u a r t . Rev., 1964, 295. 79. E.M. Kosower, P r o g r e s s i n P h y s . O r g . Chem., J3 , 81 ( 1 9 6 5 ) . 80. U. Bruhlmann and E . Hayon, J . Amer. Chem. S o c . , 96, 6169 ( 1 9 7 4 ) . 81. C.B. B a r l i n and D.D. P e r r i n , Q u a r t . Rev., 1966, 75. 82. A.F. T r o t m a n - D i c k e n s o n i n A d v a n c e s i n F r e e R a d i c a l  C h e m i s t r y , 1_, G.H. W i l l i a m s , Ed., Log o s P r e s s , London, 1965, p. 30. 83. H. Kwart and T. T a k e s h i t a , J . Amer. Chem. S o c . , 84, 2833 ( 1 9 6 2 ) . 84. J.W. B a k e r and H.B. H o p k i n s , J . Chem. S o c . , 1949, 1089. 85. M.F. Dunn i n P y r i d i n e N u c l e o t i d e Dependent D e h y d r o - g e n a s e s , H. Sund, Ed., S p r i n g e r - r V e r l a g , New Y o r k , 1970, p. 38 . 86. L.A. P a q u e t t e , P r i n c i p l e s o f Modern H e t e r o c y c l i c  C h e m i s t r y , W.A. B e n j a m i n , New Y o r k , 1968 . 87. F.H. B a t z o l d , D i s s . A b s t . I n t . B, 33, 4184 ( 1 9 7 3 ) . 88. G. C i l e n t o and S. S c h r e i e r , A r c h . B i o c h e m . B i o p h y s . , 107, 102 ( 1 9 6 4 ) . 89. R.O. C l i n t o n and S.C. L a s k o w s k i , J . Amer. Chem. Soc., 70, 3135 ( 1 9 4 8 ) . 90. J.H. C r a i g , P.C. Huang, T.G. S c o t t , N.J. L e o n a r d , J .  Amer. Chem. S o c . , 94, 5872 ( 1 9 7 2 ) . 91. K. W a l l e n f e l s , M. G e l l i c h , F. K u b o w i t z , Ann., 621, 137, (1959) . 268 92. H.G. W i n d m u e l l e r , C . J . Ackerman, H. Bakerman, O. M i c k e l s e n , J . B i o l . Chem., 234, 889 ( 1 9 5 9 ) . 93. G. D r e h f a h l and K.H. K o n i g , Chem. B e r . , 87, 1628 ( 1 9 5 4 ) . 94. K. S c h e n k e r and J . Druey, H e l v . Chim. A c t a , 42, 1960 (1959) . 95. B. J - S . Wang and E.R. T h o r n t o n , J . Amer. Chem. S o c . , 90, 1216 ( 1 9 6 8 ) . 96. H. L e t t e i , W. Haede, and R. Ruhbaum, Ann., 579, 123 ( 1 9 5 3 ) . 97. S.A. H a r r i s , T . J . Webb, and K. F o l k e r s , J . Amer. Chem.  Soc . , 62, 3198 ( 1 9 4 0 ) . 98. R.F. H u t t o n and F.H. Wes t h e i m e r , T e t r a h e d r o n , 3_, 73 ( 1 9 5 8 ) . 99. G.J. J a n z i n R e f e r e n c e E l e c t r o d e s T h e o r y and P r a c t i s e , D.J.G. I v e s and G.J. J a n z , E d s . , Academic P r e s s , New Yo r k , 1961. 100. I.M. K o l t h o f f and W.J. T o m s i c e k , J . P h y s . Chem., 39, (1935) . 101. O.M. F r i e d m a n , K. P o l l a k , and E. K h e d a u r i , J . Med. Chem., 6, 462 ( 1 9 6 3 ) . 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
https://iiif.library.ubc.ca/presentation/dsp.831.1-0061082/manifest

Comment

Related Items