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UBC Theses and Dissertations

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

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P O L A RO G R A P H I C , P O T E N T I O M E T R I C - K I N E T I C S T U D I E S OF NAD MODEL COMPOUNDS  AND  by  DONALD JAMES B.  A  Eng., M c G i l l  THESIS THE  SUBMITTED  University,  IN P A R T I A L  REQUIREMENTS  FOR  DOCTOR OF  in  NORRIS  THE  1970  F U L F I L M E N T OF  DEGREE  OF  PHILOSOPHY  the Department of CHEMISTRY  We  accept  required  THE  this  thesis  as c o n f o r m i n g  to the  standard  U N I V E R S I T Y OF B R I T I S H April,  1975  COLUMBIA  In p r e s e n t i n g t h i s  thesis  an advanced degree at the L i b r a r y s h a l l I  in p a r t i a l  f u l f i l m e n t o f the requirements f o r  the U n i v e r s i t y of B r i t i s h  make i t  freely available  f u r t h e r agree t h a t permission  for  Columbia,  I agree  r e f e r e n c e and  for e x t e n s i v e copying o f  this  that  study. thesis  f o r s c h o l a r l y purposes may be granted by the Head o f my Department o r by h i s of  representatives.  this  thesis  It  i s understood that copying o r p u b l i c a t i o n  f o r f i n a n c i a l gain s h a l l  written permission.  Department of  C / e m ;  s Ye*- cj  The U n i v e r s i t y o f B r i t i s h Vancouver 8, Canada  Columbia  Date  J/^^/r  /<£  /j?  not be allowed without my  ABSTRACT  Supervisor:  The NAD  +  Professor  effects  (I) m o d e l  Ross  Stewart  of varying  compounds  t h e 1- a n d  3-substituents  o n r e a c t i o n ' s ' ' . (1) t h r o u g h  ii  (4)  of  have  R  been (1)  R  investigated. was o b s e r v e d  o f +3.7 reaction  was  metrically.  p o f +11  ions  A reaction constant  p*  was  found  f o r the e f f e c t of  of the t w o - e l e c t r o n  reduction  i n r e a c t i o n (2) was m e a s u r e d p o t e n t i o -  Reaction  constants  reduction in reaction  o f 1 - s u b s t i t u e n t s and a  t o +12  The p o t e n t i a l  f o r the e f f e c t s  Reaction of  f o r the e f f e c t  constant  of p y r i d i n i u m  found  polarographically.  found  3-substituents.  The o n e - e l e c t r o n  constants  o f +2.8  and +9  o f 1- and 3 - s u b s t i t u e n t s ,  o f -1.9 and -6 were  found  to +11  respectively.  f o r the e f f e c t s  1- and 3 - s u b s t i t u e n t s on t h e r a t e s o f o x i d a t i o n o f  iii  were  1,4-dihydropyridines reaction of D  constant  echanism  m  may  +  t o -2.6  the a c i d (4).  reaction was  (3) .  found  results  complex  than  t h e commonly  sirailar  f o r the  decomposition  These  A  of  effect  1,4-dihydro-  suggest  of oxidation of 1,4-dihydropyridines  that by  accepted  the  flavins  mechanism o f  transfer. Reactions  HAD ,  on  reaction  be more  hydride  flavins,  o f -2.0  1-substituents  vridines,  by  (1) t h r o u g h  nicotinamide  (4) w e r e  mononucleotide  alkylenebis(3-carbamoylpyridinium)  also  (NliN)  investigated using  and  compounds  two  1,1'-  (II).  The  (ID  oxidation-reduction be  explained  p r o p e r t i e s of these  by c o n s i d e r i n g  four  compounds  the i n d u c t i v e e f f e c t s  1-substituents.  iv  of t  TABLE OF  CONTENTS Page  1.  INTRODUCTION  1  1.1  Acid Decomposition  of  1,4-Dihydropyridines.  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  1.3  O x i d a t i o n of  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  1,4-Dihydropyridines  1.5  E l e c t r o c h e m i c a l Reduction  1.6  Substituent Effects p y r i d i n e Systems  2.  SCOPE OF  3.  RESULTS 3.1  THE  Flavins.  3 5  .  6  11  of Pyridinium S a l t s  . 18  Pyridinium/Dihydro31 35 38  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 Dihydropyr i d i n e s Base-catalyzed  3.3  L i m i t a t i o n s on pH Model Compounds  3.5  . . .  INVESTIGATION  3.2  3.4  in  by  . .  Decomposition  38  Reactions  i n the Study of  52  NAD .56  Substituent Constants p y r i d i n i u m Group Polarography  of  o f the  3-carbamoyl61  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 o f Wave I  70  3.5.2  Prewave F o r m a t i o n  87  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 Polarographic Reduction P o t e n t i a l s  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 the Polarographic Reduction P o t e n t i a l s . .  v  97 .119  Page 3.6  Reduction  P o t e n t i a l s of Pyridinium-  Dihydropyridine 3.6.1  3.7 4.  Half-cells  Substituent  Effects  128 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 a t t h e 3 - P o s i t i o n . 147 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 . 150  DISCUSSION.  163  4.1  Polarographic  Reduction  Mechanisms  4.2  The E f f e c t s o f S u b s t i t u e n t s of P y r i d i n i u m Ions  163  on t h e  Reduction 170  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 R a t e s o f O x i d a t i o n by F l a v i n s 185  4.4  Comparisons pyridinium) Compounds  4.5  of 1,1'-Alkylenebis(3-carbamoylCompounds w i t h t h e S e r i e s I  C o m p a r i s o n s o f NAD  193 +  and NMN  +  with  the S e r i e s  I Compounds 5.  SUGGESTIONS  FOR FURTHER  6.  EXPERIMENTAL.  201 RESEARCH  210  . ";  214  6.1  Buffers  214  6.2  O x y g e n - f r e e Work  216  6.3  Synthesis  of Quaternary Pyridinium  6.4  Synthesis  o f 1, 4 - D i h y d r o p y r i d i n e s  6.5  Kinetics 6.5.1 6.5.2  S a l t s . . . 218 228 237  A c i d - c a t a l y z e d Decomposition of 1, 4 - D i h y d r o p y r i d i n e s R e a c t i o n s 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 vi  237 239  Page 6.6  Polarography.  244  6.7  Potentiometry  248  6.8  Absorbance  259  Spectra  BIBLIOGRAPHY  263  vii  L I S T OF  TABLES Page  I  Redox p o t e n t i a l s o f NAD  II  H a l f - w a v e p o t e n t i a l s o f NAD  III  Substituents  IV  Dihydropyridine acid-decomposition constants . . . . . . . . . . .  on  models  compounds  VI  S t a b i l i t y limits for pyridinium and d i h y d r o p y r i d i n e s  VIII  used  26  studied  A list  Slopes versus  values  15  V  VII  o f a*  the  models  in this  36  rate 43  work  49  salts 57  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 l o g ( i - i ) / ( i) / and l o g ( i - i ) / ( i ) 2  3  1  d  /  .  2  d  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 p l o t t i n g applied p o t e n t i a l versus log(i -i)/(i)  .  by 82  d  IX  77  Mean i n t e r c e p t s from p l o t s o f a p p l i e d p o t e n t i a l versus l o g ( i - i ) / ( i ) and l o g ( i - i ) / ( i ) . .  84  X  List  89  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 t h e maximum h e i g h t of the p o l a r o g r a p h i c prewave and t h e p o t e n t i a l d i f f e r e n c e between main wave and prewave  2  /  3  1  d  XII  XIII  o f compounds p l o t t e d i n F i g u r e  Diffusion estimated  R e d u c t i o n p o t e n t i a l s f o r the dihydropyr idine h a l f - c e l l s the  ions I l l  133  Results  oxidative titration  XV  Second o r d e r r a t e c o n s t a n t s f o r t h e 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 Summary o f r e a c t i o n c o n s t a n t s o f 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 reactions  viii  92  pyridinium/  XIV  XVI  2  12  c o e f f i c i e n t s o f the p y r i d i n i u m from t h e I l k o v i c e q u a t i o n  from  /  d  o f NADH. .  139  oxidation 158  reduction analogous 174  LIST  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 reduction 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 car bamoylpyr idinium) ions  +  +  XVIII  S e l e c t e d p r o p e r t i e s o f NAD , NMN and 1-methoxymethyl-3-carbamoylpyridinium  XIX  A used 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 i n t h i s work  ix  197  of  chloride  . 203  compounds 260  LIST  OF  FIGURES Page  1.  A  2.  A t y p i c a l polarogram of weakly b a s i c s o l u t i o n  3.  4.  5.  6.  7.  8.  9.  10a  sample  polarogram  Standard r e a c t i o n s of substituent constants  in  a 25  some c o m m o n l y  met 32  . . . . . .  40  P l o t of second order r a t e constants f o r hydrogen ion c a t a l y z e d decomposition of 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  the  Spectrophotometric d e t e r m i n a t i o n o f t h e pKa of the 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  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 the p o t e n t i a l of the p o l a r o g r a p h i c r e d u c t i o n of 1-carbamoylmethy1-3-acetylpyridinium chloride.  . . .  68  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 p o l a r o g r a p h i c half-wave p o t e n t i a l s of l-methyl-3-carbamoylpyridinium iodide Polarographic log ( i - i ) / (i) chloride  69  applied potential versus for l-methyl-3-carbamoylpyridinium . . . . . 7 1  Polarographic applied log(i -i)/(i) /3 f chloride o  r  potential versus 1-methyl-3-carbamoylpyridinium 72  Polarographic log ( i - i )/ ( i) chloride  applied potential versus f o r 1-me t h y 1 - 3 - c a r b a m o y l p y r i d i n ium  Polarographic log(i -i)/(i)  applied potential versus f o r nicotinomide adenine d i n u c l e o t i d e .  73  d  lib  salt  46  d  11a  pyridinium  P l o t of second o r d e r r a t e c o n s t a n t s f o r the acetic acid catalyzed decomposition of 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)  2  10c  a  P s e u d o - f i r s t order decomposition of 1-methy1-3-carbamoy1-1,4-dihydropyridine  d  10b  19  Polarographic applied potential versus l o g ( i ^ - i ) / ( i ) / 3 for nicotinamide adenine dinucleotide  74  2  x  75  LIST  OF  FIGURES Page  11c  12.  Polarographic applied p o t e n t i a l versus l o g ( i ^ - i ) / ( i ) f o r nicotinamide adenine dinucleotide  76  Comparison o f t h e i n t e r c e p t s from p l o t s o f polarographic p o t e n t i a l versus log(i -i)/(i)I/ and l o g ( i ( j - i ) / ( i ) / 3 f o r compounds i n S e r i e s I and S e r i e s I I  2  d  2  13.  14.  15.  88  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 p r e w a v e l i m i t i n g c u r r e n t f o r the polarographic reduction of 1-carbo-i-propoxymethyl-3-carbamoylpyrid inium chloride 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'-methy1enebis(3-carbamoylpyridinium  chloride)  90  .  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 of a 2.3xl0~ M. s o l u t i o n of 1,1'-methylenebis(3-carbamoylpyridinium chloride)  94  5  16.  96  P l o t o f polarographic half-wave p o t e n t i a l s of 1 0 M . s o l u t i o n s o f t h e S c r i e s I compounds a g a i n s t a*  101  P l o t of polarographic half-wave p o t e n t i a l s of 2X1Q-3M. s o l u t i o n s o f t h e S e r i e s I c o m p o u n d s a g a i n s t cr*  102  P l o t of polarographic half-wave p o t e n t i a l s of 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 n*  103  - 2  17.  18.  - J !  19.  20.  Plot of polarographic reduction potentials o f t h e S e r i e s I compounds a g a i n s t a * .  f.°  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 meta-substituent constant ff.  m  21.  22.  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 substituent constants O P 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 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 of 1-methoxymethy1-3-carbamoy1pyrid inium chloride  117  124  130  +  xi  130  LIST  OF  FIGURES Page  23.  Plot  of measured  cell  potentials  against  l o g [ P y ] / [ P y H ] f o r the pyridinium-dihydropyridine h a l f - c e l l of 1-(2'-hydroxyethyl)-3-carbamoy1pyridinium chloride +  24.  T h e pH d e p e n d e n e e o f t h e r e d u c t i o n p o t e n t i a l EpJJ o f 1-carbamoylmethy1-3-carbamoy1pyrid inium c h l o r i d e . . 1 3 6  25.  P l o t of standard reduction pyridinium-dihydropyridine I c o m p o u n d s a g a i n s t 0*  26.  28.  146  152  Determination of the second order 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-carbamoylmethyl-3-cyano-l,4d i h y d r o p y r i d i n e b y 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 ln(A-Aco) a g a i n s t time  154  P l o t of the l o g a r i t h m o f the second order constants f o r the o x i d a t i o n of the S e r i e s d i h y d r o p y r i d i n e s b y f l a v i n s a g a i n s t O*  161  0  29.  Dry-box  30.  S y r i n g e and oxygen-free  and  0  absorbance work  Sample  32.  Potentiometry  33.  T y p i c a l spectra of a 1, 4 - d i h y d r o p y r i d i n e U.V.  0  rate I  accessories  31.  34.  f o r the of the S e r i e s  Determination o f the second o r d e r r a t e constant for 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 f r o m a p l o t of ( l / a - b ) l o g ( a - x ) / ( b - x ) a g a i n s t time 0  27.  potentials half-cells  217 cell  apparatus f o r 241  polarograms  247  cell  absorbance  249 pyridinium  spectrum  i o n and  a 261  of  1-carbamoylmethyl-3-  fluoro-1,4-diHydropyridine  2  xii  6  2  A C KNOWLEDGE ME NT  I Ross  Stewart  suggestions to for  wish  thank many  Columbia  to express  my  sincere  appreciation  f o r h i s guidance  in this  work  f o r improvements  Dr. Addison,  Dr. S p i t z e r ,  helpful  discussions.  I  like  would  i n this  to thank  and t h e N a t i o n a l  Prof.  and h e l p f u l  thesis.  I also  wish  D r . C o x , a n d D r . Gyama  the U n i v e r s i t y  Research  to  Council  of  British  for financial  assistance. A  special  support  throughout  of  thesis.  this  thank this  y o u i s f o r my work  wife,  and f o r t y p i n g  xiii  Vikki,  f o r her  the rough  draft  Dedicated  to  x iv  Vikki  INTRODUCTION Nicotinamide-adenine amide-adenine d i n u c l e o t i d e involved  The  (a) :  R=H  (b) :  R=P0  Throughout by  phosphate  in oxidation-reduction  coenzymes a r e  posed  dinucleotide  this  the  (NAD  and  ( l b , NADP) a r e  reactions  within  nicotin-  coenzymes  living  systems.  ) (NADP )  =  +  3  also  work,  ( l a , NAD)  referred the  Commission  to  by  several  nomenclature  on  Enzymes o f  HO  and the  other  names.  abbreviations International  proUnion  OH  (ID of  Biochemistry  will  be  used.  The  pyridine  ring  was  identified  2 by has  Warburg, led  to  et a l . the  as  general  the term  reactive pyridine  portion  of  the  nucleotides  molecule  being  used  and  2  for  these  the  oxidized  as  abbreviated  the a d d i t i o n  (III) .  MAD  +  chemical compounds  only  pertinent  been  to this  NADH, been  Reduction  properties  and one p r o t o n thought  that  +  r e v i e w e d . ^ ' ^ ' ^ ' ' "*" study  will  that  0  exists  the reduced  reduction  (IV).  3 4 '  NADH  and model  Only  be r e v i e v / e d  t o form  1,6-dihydro-  enzymatic  o f NAD , 6  ring  In  o f t h e coenzymes  was a 1,2- o r a  shown  ( I I , NMN).  the pyridine  the 1,4-dihydropyridine  and b i o l o g i c a l have  +  I t was o r i g i n a l l y  I t has s i n c e  yields  MAD ,  o f two e l e c t r o n s  o f NAD, a b b r e v i a t e d  pyridine.  is  and f o r the m o n o n u c l e o t i d e  salt  dihydropyridine.  form  of  state,  the pyridinium  involves a  compounds  The  material  here.  which  3  1.1  Acid The  Decomposition  characteristic  pyridine  centered  of  1,4-Dihydropyridines:  ultra-violet  near  absorption  340 n a n o m e t e r s  of  i s rapidly  1,4-dihydrolost  i n the 7  presence If  left  acid  to  a n d r e p l a c e d by a p e a k  i n acid,  this  compound  m o d i f i c a t i o n compound"  ponding The  of acid  decrease  suggested give  which  mechanism  VI, f o l l o w e d  has been  i s further  i n the absorption 9,10  a t 290  altered  nanometers. called  "primary  with  a corres8 12 a t 290 n a n o m e t e r s . '  involves protonation  by a n u c l e o p h i l i c  attack  o f V a t C-5  a t C-6  of the  (VII)  pyridine  ring.  generally yielding is  In aqueous  be a w a t e r  solutions,  molecule,  with  subsequent  acid  m o d i f i c a t i o n compound". 11  the  protonation  which  to  to general  causes  acid  step  the s h i f t 10  '  compound"  (VII),  reaction  which  which  The r e a c t i o n i s  In aqueous  i n the u l t r a - v i o l e t  The f u r t h e r  (VII),  12  i s rate-determining.^^  290 n a n o m e t e r s .  modification  catalysis.  would  deprotonation  the 6-hydroxy-l,4,5,6-tetrahydropyridine  the "primary  subject  the n u c l e o p h i l e  i s much  media,  I t i s this absorption  step  maximum  of the "primary slower,  acid  i s suspected  to  involve  attack  at  protonation C-2.  at  C-3  with  subsequent n u c l e o p h i l i c  5  1.2  N u c l e o p h i l i c A d d i t i o n to Pyridinium The  susceptible anions salts but or  electron deficiency  Salts:  o f the pyridinium r i n g  t o a t t a c k by n u c l e o p h i l e s .  Sund  lists  1  w h i c h have formed a d d i t i o n compounds w i t h (VIII).  hydroxide  Most o f t h e s e  form  and s u l p h o x y l a t e  1,6-dihydropyridines .  makes i t  several  pyridinium  1,4-dihydropyridines  ions apparently?form  (IXa),  1,2-  (IXb)  1  H  3  X  or • N  I  1 (VIII) R  The attention.  (IX)  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  The a d d i t i o n t o NAD , w h i c h +  i s rapid  and q u a n t i t a -  13  tive  i n 1 M. c y a n i d e ,  near  325 nanometers.  for  NAD  +13,14  since  gives a product  with  an a b s o r p t i o n  T h i s r e a c t i o n has been u s e d i tallows  determination  t o be made.  determination  o f NAD  +  a simple  The d i r e c t  i s impossible  a s an a s s a y  spectrophotometric spectrophotometric  i fother  aromatic  com-  pounds a r e p r e s e n t .  B o t h 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 d e t e r m i n e d  f o r the a d d i t i o n of cyanide  "~  number o f p y r i d i n i u m Substituent librium  constants  reduction, salts.  Effects).  salts  15 16 58  '  '  I t has been  f o r cyanide  1,17,58  i o n to a  ( s e e a l s o s e c t i o n 1.6, suggested  that the equi-  a d d i t i o n measures  and t h e r e f o r e t h e r e d o x  band  potentials,  t h e ease o f of pyridinium  1.3  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 : A variety  and  dihydropyridine  salts. are  o f compounds a r e c a p a b l e o f o x i d i z i n g  One b i o l o g i c a l l y  1  the f l a v i n s  important group  with this  capability  (Xa) and r i b o f l a v i n  The coenzymes a r e a l s o  mononucleotide  dinucleotide  to the c o r r e s p o n d i n g p y r i d i n i u m  (X), r i b o f l a v i n  p y r o p h o s p h a t e (Xc). flavin  models  NADH  referred  (FMN) f o r (Xb) and f l a v i n  adenosine t o as  adenine  (FAD) f o r ( X c ) .  W i t h i n m i t r o c h o n d r i a , much o f t h e NADH i s o x i d i z e d by a flavoenzyme, ferred  from  respiratory chain)  NADH d e h y d r o g e n a s e .  the f l a v i n chain  through  (also  to u l t i m a t e l y  the v a r i o u s  referred  reduce  are trans-  enzymes o f t h e  t o as t h e e l e c t r o n  oxygen.  three m o l e c u l e s o f h i g h energy are  The e l e c t r o n s  transfer  During t h i s process,  compound a d e n o s i n e t r i p h o s p h a t e  formed. The  importance  spawned c o n s i d e r a b l e between r i b o f l a v i n  o f flavoenzyme interest  oxidations  o f NADH has  i n t h e mechanism o f t h e r e a c t i o n  and 1 , 4 - d i h y d r o p y r i d i n e s .  S i n g e r and  19 Kearney tion  found  that r i b o f l a v i n  readily  o f NADH by oxygen o r c y t o c h r o m e s  catalyzed  the o x i d a -  (heme m o l e c u l e s  which  are p a r t o f 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 h a s s i n c e 2 2 and a n a e r o b i c a l l y . 23-27 been s t u d i e d b o t h a e r o b i c a l l y 2 0'2 1 '  23 24 2 5 The  mechanism w h i c h  scheme 1 u s i n g occurs  from  h a s been s u g g e s t e d  FMN as t h e o x i d a n t .  the 4-carbon  '  '  A direct  o f t h e NADH p y r i d i n e  i s shown i n hydrogen ring  transfer  t o the  27 5-position step  of a 5-deaza-flavin.  i s preceded  The h y d r o g e n  transfer  by t h e f o r m a t i o n o f a f l a v i n - N A D H  complex  for  which,  valent  by a n a l o g y  intermediate  with  some  has been  other  flavin  proposed.  6 2  '  6 3  r e a c t i o n s , a coother  evidence  NH  CH„OH I CHOH I CHOH I CHOH I CHOH  CH„0P0  2  R=  R=  CHOH I CHOH I CHOH I CHOH I  1  (a)  has as  been  o  (b)  presented  has a l s o  been  i n favour  proposed  of a charge  transfer  i n t h e o x i d a t i o n o f NADH  complex  25  models  65 trifluoroacetophenone. is  XI, which  proton  could  migrations  H  + NADH  A possible covalent  decompose a s shown  to products  intermediate  by e l e c t r o n and  i n scheme 2 a .  + FMN  FMNH ^ + FMN  scheme  NAD  (FMNH ^  1.  + FMNH.  - FMN) . ^  2FMNH  '  by  26 '  ferred atom  In  a  as  either a  (two  mation  charge  step  but  2).  rather  step.  evidence  are  in  has  , , during  with  radical  has  been  been  hydrogen  mechanism)  intermediate  radicals  the  the  the  step  believed  than  ,. . 23,24 radicals (XIV)  (one  Flavin  these  reactions No  hydride  mechanism)  (scheme  reaction  t r a n s f e r complex  are  to  be  initial found  observed  or  a  be  formed  during  in  tran  hydrogen  free-radical  formed  for-  the  secondary  oxidation-reduction  for  reaction.  could  the  The  formation  of  protonatect  NAD  pyridine  in electrochemical  oxidations  29 of  NADH m o d e l s .  to  the  radical  pyridinium rate  of  flavin atom  (XIII)  (III)  and  oxidation  to  or  d i s p r o p o r t i o n a t i o n to  rapid  the  but  step  deuterium  i s known  undergo  dihydropyridine  of  i s unknown,  transfer  Since  It  protonated  rapid  (IV)  isotope  be  rate  effects  radical  (XIV) the  determining indicate  the  compounds.  i t is unlikely that  would  deprotonation  by  ribo-  hydrogen-  in  the  The  pathway  (d) .  involvement  of 20  hydrogen  bond  breaking  pathway  (d)  for  oxidation  the  riboflavin  -1 M by The  the  the  operative.  neutral  estimated  as  It  NAD  transition  The  radical  between  i s u n l i k e l y that  radical  would  be  very  oxidation-reduction  slower  than  difference  practical 2.  been  .  overall  scheme  of  be  in  4  rate  constant  (XIII) 9  x  10  state,  by 9  and  5  x  10  29  flavin  enormous no  has  -sec.  magnitude  formation  i s u n l i k e l y to  -1  the  and  in  this  two  oxidation  different  reaction  radical  these  the  is  rates  would  d i f f e r e n c e b e t w e e n p a t h w a y s (b) 22 McCormick et a l . , have measured  from  ten  oxidation.  of  (XIII)  this  orders Such  seem  to  rate. of  an leave  and  (c)  in  the  rates  of  (c)  charg e transfer complex  ON H, H transfer  + FMNH  elec tron >• I I I transfer  xir  (XIII)  . .,, (d)  charg e transfer . complex  CONH, electron — _ > transfer  .+ FMN  (XIV) Scheme  2  H transfer  III  XII,  10 oxidation  of  concluded  that,  species, but of  results  negative  pathway  (a).  by  a  i n the  probably  their a  NADH  a  of  riboflavin  transition state,  hydride  may  charge  series  also a t N-5  a  analogues. negatively  ion, i s approaching be of  explicable the  flavin  by  the  the  They charged  flavin,  development  molecule  as  in  1.4  Oxidation-Reduction.,Potenti§is. o f P y r i d i n i u m Dihydropyridine Systems: The  by  redox  equation  NAD  potential  1, h a s b e e n  + H  +  measured  cell  potential  oxidized  and reduced  2)  E i s t h e measured  cell  E  „  -  potential,  systems  i s related  species  red  given  species  F o r t h e NAD /NADH +  by e q u a t i o n  potential  concentration  equation  potential,  The  (equation  E° i s t h e s t a n d a r d  , , (2) 0  T i s the absolute  of electrons  transferred, F i s  respectively.  solutions, the a c t i v i t i e s  3.  system  A t any g i v e n  a t which  the c e l l  +  convenience,  ( H)  concen-  potential,  (3)  P  o f NADH  by e q u a t i o n  by  potential i s  pH, t h e m i d p o i n t  the concentration  o f N;AD.', i s g i v e n  For  of the  c a n be r e p l a c e d  i ^[NAD] H f - 2^22* ,  E = E » - H2Fl n  the  3  , and a are the a c t i v i t i e s ox  and o x i d i z e d  trations.  potential. °  to the concentration of  R i s the gas constant,  and a  dilute  redox  RT , red In nF a ox  the  in  p o t e n t i o m e t r i c a l l y and by  by t h e N e r n s t  cell  n i s t h e number  reduced  represented  (1)  o f known  temperature, Faraday,  7-  + 2e==-=_NADH  +  with  E  system,  +  determined  equilibration  where  o f t h e NAD /NADH  -  4.  equals the The r e d u c t i o n  potentials at  of biologically  pH 7, a pH w h i c h  than  i s pH  midpoint  E  0.  7  !  The  i s much  This  potential  -  E  i m p o r t a n t compounds a r e o f t e n more m e a n i n g f u l  potential  physiologically  c a n be c a l c u l a t e d  from t h e  a t a n y o t h e r pH b y e q u a t i o n  5.  2 . -3 0 3 RT H " I-ftfJ'  ( P H _ 7 )  P  cell  potential  quoted  ( 5 )  o f t h e NAD /NADH +  system  i s  not  14 3 2 directly system is  measurable.  to measure  equilibrated  such  Rodkey  these  with  potential  num  electrode.  measured NAD /NADH +  After  Since  system.  Rodkey  system  +  active  equilibrium  indicator  has been  c a n be m e a s u r e d  equilibrium  corresponds  a mediator  T h e NAD /NADH  an e l e c t r o m o t i v e l y  o f the i n d i c a t o r  potential  has u t i l i z e d  potentials.  as benzyl viologen.  the  '  has been  with  has determined  a  attained,  to the p o t e n t i a l  reached, plati-  the  of the  the value  o f -318  14 millivolts  f o r E ° ' a t 30°.  coefficient  o f 1.31  After  millivolts  determining a  per degree,  temperature  he c a l c u l a t e d  a  32 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°. potential (E°') o f t h e NADP /NADPH s y s t e m +  The r e d u c t i o n has been d e t e r m i n e d  31 in  a similar By  known  manner  a s -316 m i l l i v o l t s  equilibrating  o n e , t h e unknown  t h e unknown  a t 30°.  redox  standard reduction  system  with a  potential  c a n be  found  from  the o v e r a l l  0X  the  equation  + RED :-=f_ RED  A  equilibrium  equilibrium  B  constant  constant.  +  A  in dilute  0X  For the r e a c t i o n  B  solutions  i s given  by  6.  [RED,][0X1  _  A  " "  o  [OX ] f RED  A K E  i s related  cell  =  cell  and  ^  s  r e  -'-  =  0  cell  a t e  E  Combining  through  ^  t  o  c e l l  (7) a n d  t  nF  the Nernst  equation  )  with  n  e  In K  E° A  The  main  w  standard  reduction potentials  (7) '  of A  8.  = °A-  E  E  °B  <> 8  (8) a l l o w s  of the e q u i l i b r i u m  mination  6  0.  B by e q u a t i o n  ledge  (  13  t o E° ceil  E  E  ]  RT = E° + — B nF  one t o d e t e r m i n e  constant  1n  difficulty  o f E ° f o r t h e NAD /NADH +  know-  and E° .  K  with  E^ from  K  this  approach  system  ( 9) '  f o r the deter-  i s the knowledge o f  14 E°  f o rreference  Clark  range the  discusses  3 0  values  which  the problems  c a n be e q u i l i b r a t e d w i t h related  f o r E ° ' f o r t h e NAD /NADH +  from  o f -315 m i l l i v o l t s  /NADPH  system  has been  to this  system  -314 t o - 3 2 2 m i l l i v o l t s .  value  NADP  systems  and c a l c u l a t e s  spanning  Of t h e s e ,  as t h e most  a potential  Clark  reliable.  e q u i l i b r a t e d with  NAD.  suggests The  t h e NAD  /NADH  30 system.  The r e d u c t i o n  5 millivolts system. the  more  p o t e n t i a l was e s t i m a t e d  negative  Potentiometric  than  about  t h e p o t e n t i a l f o r t h e NAD /NADH +  measurements  o n t h e NADP  system  gave 31  same v a l u e f o r E ° a s was f o u n d f o r t h e 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 t o t h e redox p o t e n t i a l s 1  33 of  t o be  model  systems.  Karrer  the  reduction  and  1-butylnicotinamides  of  a potential mediator.  calculate are  suspect  were  using  since  reportedly  determined  hydropyr i d i n e  pyridine  Clark  o f E° ' g i v e n  no m e d i a t o r s  unstable.  viologen  their  i n Table  1.  were  Leach,  used  data  These  to values  and the p o t e n t i a l s 35  Baxendale,  They  and Evans  prepared which 35 5  as dihydropyr i d i n e .  1.5, E l e c t r o c h e m i s t r y . )  rapidly  with  The dimer  viologen," ' whereas 1 14  very  the use  potential of 1-methylnicotinamide  section  benzyl  measured 1-propyl-,  has used  as a m e d i a t o r .  as well  have  1-ethyl-,  e l e c t r o c h e m i c a l l y , a method  diners  1  pyridine,  reacts  they  a c t u a l l y measuring  were  '  p o t e n t i o m e t r i c a l l y without 30  the reduction  benzyl  and c o w o r k e r s  p o t e n t i a l o f 1-methyl-,  the values  34  slowly.  pyridinium/dihydropyridine  '  produces  (See a l s o  i s known  NADH,  a  or the  to r e a c t  1,4-dihydro-  I t i s uncertain  the reduction  system  '  the d i -  whether  p o t e n t i a l of the pyridinium/dimer  15 TABLE Redox  1  R  CH.  Potentials  I o f NAD  Models  E° ' (mv.)  3  CONH.  -419,  -417  reference 30  -29 0  35  CON II.  -427  30  CONH  -430  30  -337  58  CONH.  -412  30  a-TAG  CONH  -267  59  6 -TAG  CONH.  -267  58  CONH.  -300  58  CONH .  -361  58  CONH.  -361  58  CONH.  -34 9  53  CO.CH.  -237  58  -354  58  -391  58  -404  58  2 5  C  H  3 7  4 9  CH OCH C H 2  2  6  5  CH„CH_OC^H 2 2 b o c  C  H  2 6 5  C  H  2  C  C  H  6  H  3  C  1  2  CH C H C1 2  6  3  2  C  H  2 6 3  C  l  2  C  H  2  C  6  H  3  C  1  2  C  H  2  C  6  H  3  C  1  2  C  n  COOC H 2  CON(CH COO  AcO  OAc  5  3 2  Table  I  (cont.)  R  E°'(nv.)  3  reference  RPPRA  COCH  -257  58  RPPRA  CONHOH  -315  17  RPPRA  CONH NH.  -339  17  RPPRA  CHNOH  -342  17  RPPRA  COC  -242  17  RPPRA  COCH(CH )  -243  17  RPPRA  CSNH„  -280  17  RPPRA  CHCHCONH  -342  17  RPPRA  H _  6 5 3  =  0  17 system.  The  various  potentials  3-substitutents,  of  a  group  have  of  been  NAD  analogues,  determined  by  containing  Anderson  and  17 Kaplan These  by  enzymatic  authors  used  as  millivolts  determined  The  listed  of  values -315  millivolts  equilibration their by  the  r e f e r e n c e the  Burton  i n Table  with  and  1 were  suggested  by  NAD  value  Wilson  3  for  0  of  -320  f o r E° ' o f  calculated  Clark  system.  using the  the  NAD. value  NAD /NADII +  system. 58 Wallenfels duction  potentials  equilibrium the  ratio  of  constants  of be  these  salts  will  brium  constants.  millivolts  and  the  f o r NAD  co-workers several  NAD  for cyanide  equilibrium same  as  They  used  as  have  an  the a  calculated  model  compounds  addition  constants ratio  of  by  f o r the  from  the  series.  that  pyridinium  reduction  reduction potential  anchor  re-  assuming  f o r two  the  the  of  equili-315  1.5  Electrochemical An  example  polarographic shown B,  in  the  Reduction  of  a  applied  1.  is  As  electroactive current  Thus,  as  B  plateau  Salts:  mercury  portion  too  of  and  only  C,  in  Figure  the  1.  line  known  to the  the in  p o t e n t i a l s more  the  as  A  current  increase  electroactive species  is  is  of  negative,  the  from  from  reduction base  more  At  i t d i f f u s e s to  current  curve  the  causing  obtained electrode  the  positive for  occur  C  r a p i d l y as  the  the  i s reduced  and  point  dropping  p o t e n t i a l becomes  species  than  reduced  to  the  between  negative  a  potential is species  observed.  at  Along  electroactive  Pyridinium  c u r r e n t - p o t e n t i a l curve  reduction  Figure  of  is  being  electrode  the  surface.  diffusion  current,  a For  most  reductions, equation figure ture,  F  ferred 0  and  the  10,  1,  R  is in  1  electrode  the  which  log-current  different trode  ways  process  °  n  is  transfer =  °  £  =  E  n  +  RT ^ 7 F  ^d  d  a  r  can ^  e  be  e s c r  the  number  electrons  and  a  is a  U  n  d  "  is  the  number  E°  by  ^-ked  T  of  or  described  constant,  coefficient. l  oxidations  gas  process,  is  temperatrans-  between  simply  the  1 5  1  (  1  0  )  l/2  the  right  function, for  d  universal  the  E  the  ^/2'  electrode  E  at  E  Faraday,  called  potential  E,  the  the  whether  c u r r e n t - p o t e n t i a l curve  where is  processes,  hand  equals  different  term zero  reduction  of and  equation is  (10),  defined  mechanisms.  i s r e v e r s i b l e , i . e . i f both  the  forward  in If  the and  elecbackward  Figure  1.  A  sample  polarogram.  20 electron  transfers  concentrations at  of the  of oxidized  the e l e c t r o d e  plot  are s u f f i c i e n t l y  of E versus  surface,  standard  species  species  a has a v a l u e  o f 1.  d  will  the e q u i l i b r i u m are attained A t 25°C, a  be l i n e a r w i t h a  The h a l f - w a v e p o t e n t i a l  reduction  by e q u a t i o n  that  and r e d u c e d  log ( i ~ i ) / ( i )  59/n m i l l i v o l t s .  rapid  slope  i s r e l a t e d to  p o t e n t i a l , E ° , o f the e l e c t r o a c t i v e  12 where D  and  D  a r  e  r  e  t  n  diffusion  e  d  (12)  coefficients For  of the o x i d i z e d  an i r r e v e r s i b l e p r o c e s s ,  is  slow and t h e e q u i l i b r i u m  at  the e l e c t r o d e  function  where Q  The of  surface.  o f the r a t e  E, , 1/2  k  and r e d u c e d  x i s the drop  i s the r a t e  the e l e c t r o n  +  time,  are not a t t a i n e d  of the e l e c t r o n  RT , 0.886T - In — — r — — anF 1/2  ^  should  transfer  k  (13)  o  D i s t h e d i f f u s i o n c o e f f i c i e n t , and reaction  a t the p o t e n t i a l E ° . In t h e a b s e n c e  f a c t o r s , s u c h as a d s o r p t i o n ,  e t c . , a p l o t o f p o t e n t i a l , E, v e r s u s  be l i n e a r w i t h a s l o p e  V l c e k has summarized  a list  greater  than  of c r i t e r i a 37  reversibility  reaction,  ,  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 t h a n 1.  reaction,  process  The h a l f - w a v e p o t e n t i a l i s a  of the e l e c t r o d e  any c o m p l i c a t i n g  respectively.  transfer  concentrations  constant  = E°  species  o f an e l e c t r o d e  process.  59/n  chemical log ( i ^ -  i)/(i)  millivolts.  f o r d e t e r m i n i n g the  21 The  case  dimerization  of the reduction  theoretically. case  will,  tial  versus  38  log ( i  (1)  product  result  should  the applied  I f the e l e c t r o d e  dimerization  Their  give  will  i n d i v i d u a l cases  subsequent considered  predict  in a non-linear  that  plot  treatment  of  to a  poten-  includes  the l o g a r i t h m i c  rise  this  five  functions  straight line  when  potential.  process  reaction  with  has been  and C a u q u i s  - i)/(i) .  which  against  remaining  reduction  s i t u a t i o n s and, f o r each,  the current  plotted  Bonnaterre  i n general,  particular of  of a one-electron  is itself  have  irreversible,  no e f f e c t .  the electrode  In the  process  the  four  i s assumed  reversible. (2)  I f the d i m e r i z a t i o n  rate  i s much  electrode  process  i twill  reduction  product  a t the electrode  surface  the polarographic  wave.  have  no e f f e c t  (3)  I f the d i m e r i z a t i o n  reaction, sible. 10, (4)  on  the electrode  The c u r r e n t  with  the  current  i s faster  reaction  will  and i s a l m o s t  equilibrium as r a p i d  than  RT  = £° + —  In  ( i  d  -  given  the  by  by e q u a t i o n  1/2  will  irrever-  equation  by e q u a t i o n  i s displaced  i)/(i)  of  electrode  t o be  as the e l e c t r o d e  and p o t e n t i a l a r e r e l a t e d  E  and, thus,  appear  p o t e n t i a l being  I f the d i m e r i z a t i o n dimer  rate  than the  the c o n c e n t r a t i o n  and p o t e n t i a l a r e r e l a t e d  the half-wave  the  not a f f e c t  slower  12.  towards reaction, 14.  K is  (14)  the is  equilibrium constant the t h i c k n e s s  dropping  of the d i f f u s i o n  mercury  electrode,  .3  x  6  other  symbols  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 5  =  the  concentration  at the  by e q u a t i o n  16. A l l  TTDT)  have  half-wave  i s given  which,  ^ .1/2  ( -  The  layer  (  t h e same m e a n i n g  potential  i s given  as i n previous  by e q u a t i o n  of the e l e c t r o a c t i v e  1  6  )  equations  17, where  C i s  species.  •p rn  l/2  E  (5)  =  E  °  +  2F  potential  are related  E  €  °  A i  (  K  C  < >  )  17  s  other  t  h  e  r  a  t  e  symbols  potential  F  by e q u a t i o n  RT = e ° + -L- i n ( i  -  E  °  +  3F  l  d  2  RT  d  n  I f the 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 ,  and  k  l  n  the current  18.  - i ) / ( i )  2/3 z  /  (18)  J  f  3 FB"  (  1  = - - ~ constant have  i s given  9  )  (20) 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  their  previous  by e q u a t i o n  meanings.  21.  These  The h a l f - w a v e  equations  agree  23 (21) .  with  those  developed  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  dimer-  . .. 39 ization. The  electrochemical behavior  o f NAD  and o t h e r  +  pyri-  5 40 dinium  salts  suggested shown  i n scheme  (XV), The  first  in  (versus  NAD*  giving  radical  The  3.  At a potential the normal  (V) i s r e v e r s i b l y  +  half-wave II.  '  The mechanism  hydrogen  t o wave  I i n Figure (reaction  an o v e r a l l  p o t e n t i a l s o f some The presence cyclic  half-wave  amoylpyridinium  salts  2.  '  '  2) c o n v e r t i n g  analogues  of the r a d i c a l  voltamrnetry  -670  1) t o t h e r a d i c a l 40 45 5 1 - 5 3  irreversible  NAD  salts i s  e l e c t r o d e , N.H.E.),  (reaction  into  o f these  of approximately  reduced  dimerizes  step  b e e n shown u s i n g ^ 40-42 rate . The  rise  rapidly  reduction  Table  reviewed.  f o r the electrochemical reduction  millivolts NAD  has r e c e n t l y been  with  p o t e n t i a l s o f NAD f o r the reduction  this  process.  are listed  intermediate  has  a rapid-return  and  scan  l-methyl-3-carb-  process  i n steps  1  and  2 i n s c h e m e 3, a r e i n d e p e n d e n t o f pH b e t w e e n pH 1 a n d 40 43 44 45 + 10. ' ' ' T h e 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  potential volts  shifts  t o a more  a s t h e pH i n c r e a s e s  negative from  value  by a b o u t  40 5 t o 7.5.  80  milli-  The p o t e n t i a l f o r 40  this  compound  variations  i s otherwise  have  no e f f e c t  N-alkylpyridinium  salts.  independent  o f pH.  on t h e h a l f - w a v e The h a l f - w a v e  Ionic  strength  potentials of  p o t e n t i a l s o f NAD  +  24 and  NMN  volts is  +  are shifted  a n d 100 m i l l i v o l t s ,  increased  and  t o more  from  other  potentials  respectively,  0.1 t o 2.0 M.  pH o n t h e h a l f - w a v e  +  positive  as the i o n i c  The e f f e c t s  potential  b y 30  o f NMN  strength  of ionic  have  +  milli-  been  strength explained  isomers scheme 3  as  being  due t o complex  formation  between  the negatively 40  charged  phosphate  complex  would  ring, the  exists tion  reduce  thus making  phosphate  group  ( I I ) and t h e p y r i d i n i u m  the positive  reduction  group  exists  more  difficult.  or high  form, ionic  The  i n the pyridinium  as the dianion;  i n the monoprotonated  of the phosphate  charge  ring.  Above below  pH 5,  the monoanion. strengths  pH 7 . 5 ,  would  i t  Protonadestroy  26 TABLE Half-wave  Potentials  II o f NAD  Models  CONH,  E R  M e d i um  CH.  aqueous  pH  1-13  -780  aqueous  pH  9.65  -830  C  C  2 5 H  3 7 H  4 9  C  C  C  C  C  5 11 H  6 13 H  7 15 H  3 17 H  10 19 H  wave  1 / 2  (mv.)  I to  wave -880  -1.4 -1410  II to  Ref. -1.5  40 4 0 , 60  aqueous  -790  5  32%  -790  5  ethanol  aqueous  pH  9.65  -820  -1410  40,60  aqueous  -790  5  32%  -780  5  ethanol  aqueous  pH  9.65  -810  aqueous  -800  32%  -760  ethanol  aqueous  -790  32%  -750  ethanol  aqueous  -790  32%  -740  ethanol  aqueous  -750  3 2%  -730  ethanol  •1410  40,60 5  aqueous  -710  32%  -710  5  aqueous  -660  5  32%  -690  5  ethanol  ethanol  27  E  Medium  R  C  H  2 6 5  C I I  C  H  2 6 4 °3 C  n  S  CH CH S0 2  2  3  wave  I  l/2 *  m  V  *' wave  II  Ref.  aqueous  pH  7-9  -760  -1410  aqueous  pH  7-9  -720  -  52  aqueous  pH  7-9  -780  -  52  -  61  50%  dioxane  -630  50%  dioxane  -770  40,52,60  61  the  complex  with  potential. NAD  a resultant  Below  pH  positive  5, t h e h a l f - w a v e  shift  i n t h e h a l f - wave  potentials  o f NMN  +  a net  are identical.  +  Electrolysis leads  to a product  at a potential having  on t h e p l a t e a u  a molecular  weight  o f wave  consistent  I  with  5 45 an  NAD  have  diner.  '  The dimers  the 6,6'-structure  spectra  has been  isolated  three  solutions""' 5  questioned.  dimeric  4,4'-,  assumed t o  but the use of  4 6  Underwood  products  l-benzyl-3-acetylpyridinium the  g e n e r a l l y been  (XVI) on t h e b a s i s o f t h e u l t r a v i o l e t  o f the e l e c t r o l y z e d  procedure  have  from  chloride.  and B u r n e t t  the e l e c t r o l y s i s They  assigned  this  have of  to these  6,6'- a n d 4 , 6 ' - s t r u c t u r e s o n t h e b a s i s o f NMR  5 spectra.  They  structure,  o n t h e same  dimeric  have  product  solution.^  also  basis,  recovered  After  tentatively  after  salts,  t h e 4,4'-  to an a p p a r e n t l y homogeneous  electrolysis  3-carbamolypyridinium  assigned  electrolysis of solutions  o f an o f NAD  NAD +  +  o r 1-methyl  a p o l a r o g r a p h i c o x i d a t i o n wave  corresponding  to r e o x i d a t i o n o f the dimers  approximately  -10 m i l l i v o l t s  i s observed  and -110 m i l l i v o l t s ,  at  respective-  , 5,44 ly • During  the polarography  -5 10  solutions  (less  than  + M. ) o f NAD  30 m i l l i v o l t s has  of dilute  been  , a prewave  more  observed  positive  has been than  i n a cyclic  wave  observed I.  voltammetry  at a  A similar study  potential prewave  of 1-ethyl-, 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 i c h was t e n t a t i v e l y a t t r i b u t e d t o 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 t h e m e r c u r y s u r f a c e . The prewave  29 observed  during  the reduction  of 3-acetylpyridine  has been  48 studied to  i n greater  normal  layer  to  reduction  o f dimer  pyridine  detail.  radical  o f the p y r i d i n e  has adsorbed,  molecules  overcome Wave  In t h i s  requires  case  moiety.  reduction a more  the prewave After  of the  negative  4 i n scheme  3) a n d i s o n l y  a mono-  remaining  p o t e n t i a l i n order  the i n h i b i t i o n . I I i n F i g u r e 2 i s due t o f u r t h e r  (step  i s due  reduction  observed  of the  i n alkaline  40 solutions.  High  alkylammonium  salts  from  ionic  strength  i s required  the background  or the presence  to separate  of  Wave  tetra-  II of  NAD  +  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 but appear to f a l l i n t h e v i c i n i t y o f -1.4 t o -1.5 v o l t s " * ' a t pH 9. Plots of E 4 0  vs.  log ( i ^ - i)/(i)  indicate that  the r e d u c t i o n  process  i s  4 5,40 irreversible. unit  has been  pyridinium slight  A pH d e p e n d e n c e reported  iodide,  f o r Wave  43,44,49  pH d e p e n d e n c e .  40  II of  although  Wave  o f -30 m i l l i v o l t s  p e r pH  l-methyl-3-carbamoyl-  one s t u d y  I I o f NAD  +  reported  only  i s reportedly  a  inde-  40 pendent is  of pll.  The p r o d u c t  a dihydropyridine,  versy.  Good  quantitative  evidence yield  of  o f t h e Wave  but i t s structure has been  presented  II reduction  i s subject favouring  to a  process contro-  near  l-methyl-3-carbamoyl-l,4-dihydro-  5,49 pyridine.  I t has a l s o  1,6-dihydropyridine spectrum ysis dimer  been  i s formed,  proposed based  +  During  a t a p o t e n t i a l on the p l a t e a u  i s formed  i n addition  the  isomeric  on t h e u l t r a v i o l e t  of the e l e c t r o l y z e d s o l u t i o n .  o f NAD  that  the e l e c t r o l -  o f Wave I I some 5 40 45 to d i h y d r o p y r i d i n e s . ' '  30 NADH  measured  height  by e n z y m a t i c  o f the r e o x i d a t i o n  assay wave,  plus does  dirtier,  measured  not account  by t h e  f o r the t o t a l  + NAD  electrolyzed.  The f o r m a t i o n  of the 1,6-dihydropyridine 40  isomer Since  and of  a  i s postulated to account  the d i m e r i z a t i o n  rates  dirner  i n the e l e c t r o l y s i s  product  proton  solvents,  donor  the second 42 i s present.  wave  f o r t h e MAD 5 50  radical, o f NAD  of l-methyl-3-carbamoylpyridinium  aprotic  f o r the balance.  are similar  the 1-raethy1-3-carbamoylpyridine  case In  o f NADH  +  '  radical  the presence  and n o t i nthe  i o n i s unexplained.  does  n o t appear  unless  31 1.6  Substituent  Effects  Substituent Hammett's their  e f f e c t s are normally  sigma-rho  standard  in Pyridiniun/Dihydropyridine  linear  free-energy  analyzed  Systems  through  relationships.  In  form  k log  where the  = po-  H  X  i s the e m p i r i c a l l y derived  substituent  x,  k  and k  stituent slope. the of  of a reaction x or hydrogen,  The o r i g i n a l  A variety  developed between  covering  used  standard  or  the molecule  contains  substituent  for  equilibrium  acids  with  i s the r e s u l t i n g v/ere  constants  derived  an a s s i g n e d  of substituent  the sub-  from  slope,  constants  p,  have  been  a variety  of structural relationships 54 and r e a c t i o n c e n t r e . Among t h e m o s t  substituent reaction  Half-wave  are the rate  r e s p e c t i v e l y , and p  of sets  substituent  commonly The  when  i o n i z a t i o n of benzoic 1.0.  constant  H  X  constants  substituent  constants  f o r each  potentials  are 0 , 0 , m p  i s shown  have  0*,  i n Figure  generally  been  a n d CT . I  3.  used  in  place  55 of  log k  i n polarographic  procedure, for  although  the e l e c t r o d e  polarography other in  way.  charge  transition possess  simpler  reaction,  being  compared  The v a l u e  a greater  than  free-energy  prevents with  centre  or products. degree  relations.  This  c a l c u l a t i n g l o g K or l o g p values  p values  of p f o r a reaction  at the r e a c t i o n state  linear  i n going  If p  determined  determined measures from  i n any  the change  reactants  i s p o s i t i v e , the  of p o s i t i v e charge  or a  from  to  reactants  lesser  degree  32  COOH  OO + II  (X)=P (H)  CT  "  K  P  (  K  )  X  COOH  :oo + H  1  (x)  a* :  XCH  (II)  COOR  or  (x)  F  ->- XCH COOH + ROH  OH" a, = (x) %  [l g(__) k 'OHy  ^ XCH COO  2  2  H +  i  XCH COOH  (x)  log(^)  0  5  1  [  P  K  (H)  Figure  "  3 .  P  K  ( X )  + H  ]  ]/2.48  33 of  negative  charge  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  Larger  absolute  charge  a l t e r a t i o n a t the reaction In  values  the choices  attention  has been  parallels  NAD  used  v/ere  generally  with  simple  groups.  Direct  apparently dent  of  paid  comparisons  have  been  methyl,  made b e t w e e n that  of substituent e f f e c t s . 40  2  6  '  3  models,  which  such  ^ '  4  have  been  a t the 1-position  ethyl,  etc.)  constants NAD  little  closely  which  substituted  of rate  on t h e a s s u m p t i o n  or  benzyl  (or reduction  and model  compounds,  quantities  are indepen-  1  1  Elving substituents  a model  The models  nicotinamides (e.g.  degree of  nucleotide  to finding  groups  a greater  centre.  pyridine  i n i t s behaviour.  alkyl  potentials)  of p indicate  acids.  a n d Sund  allude  on t h e p r o p e r t i e s  to the p o s s i b l e  of pyridinium  effects of  salts  a n d 1,4-  56 dihydropyridines. tative their  study NAD  reaction, oxidase  an  analogues.  +  t h e sodium  o f NAD  increase  amoyl  group  chain.  e_t a l .  have  conducted  of 3-substituted-l-methylpyridinium  oxidation  affinity  Lamberg,  +  They  investigated  over  compound.  the 1-methyl  The h i g h e r analogue  induced  with  Consideration  was a l s o  given  cyanide  v/as a t t r i b u t e d t o  ability  bonding  addition  and t h e a l d e h y d e  i n the electron-withdrawing by hydrogen  quali-  i o d i d e s and  the cyanide  dithionite reduction,  o f each  a  of the  carb-  the ribose  side  to the p o s s i b i l i t y  o f an  inductive review, in  which  effect operating through the 1 - p o s i t i o n . In h i s 1 16 Sund discusses t h e work o f W a l l e n f e l s a n d D i e k m a n n the cyanide  pyridinium  salts  were  affinities found  of various  t o be l i n e a r l y  1- a n d 3 - s u b s t i t u t e d related  to the  34 absorption to  maxima  correlate  o f the cyanide  either  s e t of data  adducts. with  No  attempt  substituent  v/as made  constants.  57 Kosower A max  a p o f 13.4 f o r t h e v a r i a t i o n o f  has determined  of the charge  ethylpyridinium  transfer  band  o f 3- a n d  4-substituted-l-  iodides. 17  Anderson tials  and K a p l a n  of several  3-substituted  observed  potentials  than  t o 22 m i l l i v o l t s  NAD  suggested redox of  that  able  ranging  analogues  from  more  determined  negative  and t h e e q u i l i b r i u m  Unfortunately,  the redox  o f NAD.  73 m i l l i v o l t s than  a qualitative correlation  potential  cyanide.  have  NAD.  constant  substituent  They  more  exists  poten-  positive They  between the  f o r the addition  constants  are unavail-  f o r most o f t h e s u b s t i t u e n t s which they used. 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 that  15 of  Lxndquist  reaction Plots a*  and C o r d e s ,  of a series  values  effect  on t h e r a t e  charge  transfer  unable  to explain  (the  studied  the cyanide  rates  and e q u i l i b r i u m  constants  or equilibrium  complex  formation  the abnormally  constants  from  was o b s e r v e d . high  affinity  o f NMN  containing  an a - r i b o s i d e  linkage  pyridine  ring),  and £-NAD  (thenatural  coenzyme)  but their  pyridine inductive  ring  results  point  and t h e r i b o s e  effect.  versus No  internal They  isomer  +  halides.  f o r p*.  o f 2.2 a n d 3.7, r e s p e c t i v e l y ,  NAD  ion,  addition  of l-alkyl-3-carbamoylpyridinium  of the addition  yielded  who  were , a-NAD  to the toward  cyanide  to a d i r e c t  interaction  between  side  i n addition  t o an  chain,  35 2.  SCOPE  OF T H E This  mine  i n v e s t i g a t i o n was o r i g i n a l l y  the free  relative  energy  levels  with  was f o u n d  riboflavin  that  little  on the p r o p e r t i e s  pyridines  (often  (V)  effort  to f i l l  tuted  term  by t h e g e n e r a l this  pyridines  been  could about  t h e NAD  term  model  whose  be d i r e c t l y the e f f e c t salts  ions  measured.  o f sub-  and  (VIII)  properties  investigated.  1,4-dihydrowill  be r e -  a n d t h e NADH  o f 1- a n d  Series  In an 3-substi-  I consists of  and S e r i e s  (VIII)  deter-  compounds  of dihydropyridines).  l-substituted-3-carbamoylmethylpyridines  of  to  models  models  +  pyridinium  gap, s e v e r a l  have  NAD  of pyridinium  i n t h e work,  t o by t h e g e n e r a l  models  a n d FMN  undertaken o f NAD  to f i n d  was known  stituents  ferred  of a series  t o NAD, a n d u l t i m a t e l y  equilibrium It  INVESTIGATION.  II consists  (V)  l-carbamoylmethyl-3-substituted  pyridines.  For comparison,  NAD, NMN , a n d tv/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 ) salts  (XIX)a In  rates FMN  and XlXb)  view  been  also  o f the importance  of oxidation  have  have  been  investigated.  of NAD-flavin  of the dihydropyridines  investigated.  reactions, the  by r i b o f l a v i n o r  Dihydropyridines  tend  t o decompose  36 TABLE I I I Substituents  Series  on t h e Compounds  I  Series II  a:  CH  COO  b:  CH.  c:  CH  d:  CH COCH  CONH.  e:  CH„OCII 2 3  CONH.  f:  CH  CONH.  g:  CH COOCH ( C H )  CONH.  h:  CH CN  CONH.  l :  CH  CONH.  3:  CH CONH  k:  CH  1:  CH CONH  m:  CH CONH  H  n:  CH„CONH„ 2 2  OH  CONH. CONH.  CH OH 2  COOCH  CONH  2  2  CONH 2  CONH.  COCH. CN  2  2  F  Studied  37 in  acid  and  an  i n v e s t i g a t i o n of  order  to  determine  could  effectively  the  be  Polarography stituents ponding  on  radicals.  corresponding for  of  substituents  NAD  understanding  the  used  pyridine  the  more,  was  reduction  effect  light  presence  may of  in  which  to  of  examine  various  the  necessary  in  compounds  on  the  shed  on  to  was  used  reduction  reactions effects  any  adenosine  effect  salts  Potentiometry  substituent be  was  these  the  pyridinium  1,4-dihydropyridines.  in of  ranges  reaction  studied.  the  the  models  pH  this  special moiety  The  should in  in  of  sub-  the  corres-  to  examine  the  salts  to  choice  of  suitable  aided  by  a  be  these effects NAD.  of  areas.  better  Further-  conferred  by  33 RESULTS 3.1  Acid-catalyzed The  22)  of  the a c i d  of the d i h y d r o p y r i d i n e s  near  the absorbance  kinetic of  rates  Decomposition  analysis  the s o l u t i o n H  maximum  follows  were of  followed  reaction  and H  (equation  spectrophotometrically  the d i h y d r o p y r i d i n e the e q u a t i o n  23)  H  Dihydropyridines  decomposition  from  (equation  of  (V).  f o r the  the equation  H  absorbance  for a  H HO H  I  first-order  H  H  H  _Q±L  The  7^N (22)  *1  (VI)  (V)  reaction  (equation  absorbance for  a t any  27). given  the c o n c e n t r a t i o n  y I  = £  y = x,  reac  (£  reac  C  - £  Rearranging wavelength  of reactant  reac  (VII)  + £  prod  prod  ) C reac  C  the e q u a t i o n (equation  leads  23)  f o r the to  to equation  , prod  + £ , C . . .. . prod initial  calculate (25)  (23)  (24)  39 wherein, is  i f the  equivalent  the  cell  reaction  to  path  the  length  (A C  (26)  reac  into  the  -dC  gives  Tfe  =  -  kC  (28)  A  00  for  a  - A  O  T  ) =  first-order  constant  can  -  be  A  at  then  infinite  26).  £  prod  time  , C. xrntiai  divided  Substitution  of  by  equation  )  00  ~£  T procl  for a  (  first-order  reaction  2  6  )  (equation  reac  (27)  which  i n t e g r a t e s to  MA  A  equation  (29).  Thus  )  dt  £n(A  (equation  reac  =  equation  •d(A  absorbance  equation  reac  is irreversible,  kt  -  +  °°  )  (28)  const.  reaction  determined  (29)  going from  to a  completion,  plot  of  £n(A  the -  A  rate )  against  time. Such  a  plot  for  the  decomposition  carbamoyl-1,4-dihydropyridine  (Vb)  of  i s shown  H  H  ^ X ^ C O N H  N  CH  (vb)  3  2  l-methyl-3in Figure  4.  It  27)  t  (min)  41 is  obvious  by  a much  time  for  allowing  that slower  the  absorbance  at  infinite  occurrence  estimated  estimated  a (A  time  by  of  three - A  initial  of  absorbance the to  the  reaction way  this  the  reaction,  of  as  the  five  an  ) versus  t plot.  The  from  decomposition  method  caused  a  i n the  (IV&)  v/as  five-fold  CH CONH 2  -  A  be  ) versus  recorded  of  the  to  t for A  percent  sufficient  of  change  because  absorbance  ra  of at  the the  cause  log  coefficient,  so  by  however,  ten  slope  extinction  infinite  i n F i g u r e 4. to  amount  at  time  The  absorbance  percent  the  Jin (A  accompanied  determined  can,  shown  five  product  change  be  reaction.  line  is  absorbance  constant with  generally than  The  reaction  backwards was  reaction  , cannot  become  initial  f luoro-1,4-dihydropyridine of  A  secondary  higher  secondary  reaction.  to  f o r the  in this  decomposition  reaction,  extrapolating  secondary  end  secondary  initial  the  initial  initial  the  of  the  the  £,  of  1-carbamoylmethyl-3  large in  that  the  constant.  varying  A^  A  until  best  correlation  used  to  determine  similar the  first  value  calculated  2  five  coefficient. the  rate  of  A  could  half-lives This  constants  method  be of was  f o r the  estimated data  -  application  (IVJL)  rate  the  give  by the  subsequently initial  42 decomposition were  reactions.  determined  pH ' s r a n g i n g 1.0  and  0.1  carried  by  H2°'  3°  H  R  H 0  3.4  At  =  a  k  n  H 0  rate  rate  and  variations  t o 5.6  least  four  decomposition  [  H  2  0  1  +  k  constants  represented  acetic  acid  H  more  rapidly  by  3  0  +  [  H  3 °  +  ]  +  these  i n Table  only  percent  few  and, as a r e s u l t ,  decomposed  are  also  could  n o t be  pyridines with  listed  The  1,4-dihydropyr followed  by  normal  compound  IV.  The  H  and  j  (  IV.  The  3  0  were  )  ?  1  v/ater  of the rate very  due large  1-methyl-  1-(2'-hydroxye t h y l ) -  catalysis  i n 1.0  rate  of days  generally being  (here-  constants  f o r the remaining  the  constants dihydro-  or  weeks  major  1-carbamoylmethyl-3-fluorob u f f e r v/as t o o r a p i d  spectrophotometric  decomposed  catalyzed  half-lives  of  i n acetate  catalyzed rate  Tris  accurately  decomposition idine  The T r i s  r e a c t i o n s had  the hydrogen-ion  reaction.  this  buffer.  i n Table  these  °  C  HO  i n tris(hydroxymethyl)aminomethane  determined  as  used  l-carbamoylmethyl-3-fluoro-l,4-dihydropyridine  decomposed Tris)  were  dihydropyridines,  and  called  were  values.  3-carbamoyl-,  after  A  there  1-carboxyme t h y l - 3 - c a r b a m o y l - ,  also  t  are l i s t e d a  data  of  for catalysis  A c O H  3-carbamoyl-,  v/ere  reactions  (3 0 ) . T h e  k  buffer at  concentrations  constants  equation  constants  of acetate  at buffer  rate  acid  i n the c a l c u l a t e d  The  and  d i h y d r o p y r i d i n e s and  acetic  d  2  acid  +  i n the presence  the second-order '  obs  catalyzed to  from  o u t on m o s t  calculate  acetic  primarily  M.  to  Pseudo-first-order rate  M.  techniques.  sodium  t o be In  perchlorate  fact, solution  TABLE Dihydropyridine R.  Acid-Decomposition k  (M" -sec 1  - 1  ;  _ Ac OH  (M  CONH,  77  33  CH,  CONH,  1050  157  CH„ C H O H 2 2  CONH ,  4 0 0+280  CH,OCH, 2 3  CONH,  CH COCH  CONH ,  CH C 0 0 C H .  CONH,  CH  CR  CONH ,  0.45+. 07  CH„C0NH.  CONH,  10 . 5 + 3.0  CH^CONH  COCH,  0 . 3 0+.0 5  CH  CN  0.39+.06  F  14500.  2  0  9  COHH,  CH„CONH. Special  )  k  Rate  Constants  . x 10 Tris , -1 -1 (M -sec ) m  Minimum pll f o r u s e of d i h y d r o p y r i d i n e ^  1.2  9.1  (Tris)  3.6+0 . 7  9.7  (Tris)  1 .19 + 0.11  9.3  (Tris)  3.1  (Tris)  8.1  (Tris)  7.5  (Tris)  7.0  (Acetate)  7.8  (Tris)  0.2 2+.0 3  6.5  (Acetate)  0.23+.03  6.5  (Ac e t a t e )  6 2+_2 2 7 .1+2 . 3  12 7 .8 + 1 . 3  x 10  -sec  CH^COO  18 + 5  IV  7.7 4 . 8+ 0 . 7 0.3 2+.02 5.3 + 0 . 7  86  11.5  (Tris)  Compounds:  MADH  10 . 2 + 3.6  0.95 + 0 .10  7.1  (Tris)  NMNH  7.7+0.8  0 .90+0.43  7.5  (Tris)  t  Calculated  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 period  o f 24  hours.  44 within  minutes  constants  IV)  i n Table  The of  and had a h a l f - l i f e  primary  a t pH  purpose  the d i h y d r o p y r i d i n e s  (calculated  10 o f o n l y  about  of studying  [reaction  H  from four  the a c i d  (22)]  was  to determine  H  H  OH"  7^  (22  H  could over  pH a t w h i c h  be s t u d i e d . 24  rapidly  hours  each This  decomposition.  catalyzed  decomposition [equation  k  rates  pJI l i m i t  i n acetate  catalyzed  equation  d i h y d r o p y r i d i n e shown  i n 0.1 I i . b u f f e r .  t o be u s e d  Tris  =  Some  a 10%  compounds  estimated  from  an  on t h e a c e t a t e  decomposition  decompose too slow  slow  Tris  Tris  eaperical and T r i s  / 400 AcOir  f o r 1-carboxymethyl-,  IV  i n Table  b u f f e r b u t had v e r y  (31)] based  k  represents  N  The r a t e s o f t h e v e r y v/ere  the  H.  HO  minimum  hours.  decomposition  H .•  the r a t e  catalyzed  (31)  1-methyl-,  3-carbamoyl-l,4-dihydropyridines  CONH-  (IV  1-(2•-hydroxyethyl)  and  a,b,c).  a:  R  b:  R  c:  R  1  1  1  = CH  COO  = CH = CH CH  OH  45 A  secondary  dihydropyr idine Hammett ence  r e s u l t which  decomposition  reaction  constant  of substituents  ectuation  (32),  where  p.  came  out of the study  v/as t h e d e t e r m i n a t i o n This  a represents  of the  quazitity measures  on t h e r e a c t i o n  of the  the i n f l u -  a n d c a n be d e t e r m i n e d  the e l e c t r o n i c  from  e f f e c t of the  t substituent.  l  X  X/X^ = Ptf o  = rate =  o  The catalyzed H  g  o  (32)  constant,  k, o r e q u i l i b r i u m  X f o r the unsubstituted  logarithms rate  of the acetic  constants  H  acid  3  N  or  I compounds H  CONH.  H 0  and hydrogen i o n  H  CONH,  K  compound *  o f the S e r i e s H  constant,  OH"  (IV)  are  H  H ONH,  H HO  I  AcOH  (22)  R  (IV)  plotted scale  against  i s used  a* i n F i g u r e s  because  5 a n d 6.  of the s i m i l a r i t y  T h e a*  substituent  i n the s t r u c t u r a l  f , Tiie m o s t c o m m o n l y m e t s u b s t i t u e n t c o n s t a n t s are a and a f o r m e t a - a n d 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 a n d a* f o r s u b 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 centre through only an i n d u c t i v e effect. m  p  Figure 5. P l o t of second order r a t e constants f o r the a c e t i c a c i d d e c o m p o s i t i o n of 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  catalyzed  1.3  cr*  43 relationship (22)  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  and t h e r e a c t i o n used  R-COOR'  R-COOH  or  a*,  to define  reaction  i n reaction  ( 3 3 ) . The  + R'OH  (33)  OH" values from  o f o* u s e d 72  Charton's  =  a  are listed  i n Table  of a  values  using  V and were  equation  calculated  f (34).  Since  /0.45  70  (34)  f The a * s c a l e , d e s i g n e d t o b e a m e a s u r e e f f e c t s o n l y , i s d e f i n e d from t h e r a t e s the equation: CP  274?  [  l  o  g  (  k  R  /  k  CH.  Oil-  log  y  of polar substituent of e q u a t i o n (33) b y  (k / k ) +] ' R CH 'H  70  +J  T h e f a c t o r o f 2.48 i s i n c l u d e d t o make p* v a l u e s c o m p a r a b l e t o n o r m a l 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 benzoic acids.) 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 d e s c r i b e d by a ' 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 b v the e q u a t i o n : \ / 7  4  1  a  . ,4 64 A r  l o g ( K /K x H  74  y  T h e f a c t o r o f 1.464 i s a g a i n d e s i g n e d t o make p* v a l u e s from t h i s s c a l e c o m p a r a b l e to n o r m a l Hammett p v a l u e s . Taft defined an i n d u c t i v e s u b s t i t u e nt c o n s t a n t , Oj, b a s e d o n h i s a * v a l u e s b u t c o r r e c t e d by a f a c t o r o f 0.45 t o make t h e a - j - s c a l e e q u i v a l e n t to t h e o*" s c a l e o f Rob e r t s a n d 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 t h e i o n i °*(CH X)= a • ( X ) z a t i o n o f X - C H C 0 0 H b y t h e e q u a t i o n CJj 0 251 log(K /K ) w h e r e t h e f a c t o r o f o. 251 i s u s e d t o m a i n t a i n t h e o r i g i n a l i . e . , t o make CTj c o m p a r a b l e t o a ' . T h i s s c a l e o f cr latter JL cr ha: u r t h e r expanded the l i s t o f i n d u c t i v e definition of s u b s t i t u e n t c o n s t a n t s , 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 ) , t h e l i s t o f O* v a l u e s . 7  3  =  7 3  a  7  2  3  7  T  =  a / 0 . 4 5  x  H  49 TABLE A  o f 0*  list  V  v a l u e s used H  i n this  work  H  'CONhL  N  I  R R  l  (  C  H  X  )  CH  o (x)  a * ( R  -.17  - . 38  J  2  COO  1  )  a * (R )  .00  0.00  CH CII 0H  .05  0.11  CII OCII  . 25  0.55  0.66  Cil^COCH  .29  0 . 64  0 .62  CH COOCH  .34  0.76  0 .66  CH ^COOCII ( CH 3' 2  .34  CH  CONH,  . 27  CH  CN  o  o  2  3  2  i  calculated O* v a l u e s  c  0.76  C  0 .60 1.30  from from  a  72 j  Wells  by a * ( C H X ) 0  0.00  = a  (XJ/0.45  1 .2!  73  '  72 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 f r o m t h e values f o r the carbomethoxy and c a r b o e t h o x y groups  identical  Char ton's  data  gives  a more  extensive  list  of polar  substituent 69  constants  than  The  limits  as  error given  have  Reaction  ly.  a r e shown  constants  compound  Where by  the o r i g i n a l  error  i n Figures  35)  of Figures  be  no  real  the  larger  the  stronger  i n many  o f the r a t e  d i f f e r e n c e between  reaction constant acid,  selective  constant.  com-  lines. from  5 and  the  6 respective-  together  that,  (35)  the e r r o r  may  enough  these  the other  6 as b r o k e n a r e found  deviation,  k  —JZ~  considering  e r r o r among  -2.6  o f p* a r e c l o s e  Alog  are not given,  and  values  =  limits  p* o f -2.0  two  values.  standard  5 and  (equation  a*  a r e +_1  the worst  k/Aa*,  The  less  from  Alog  P  is  IV.  approximated  and  slopes,  f o r each  i n Table  been  pounds  are available  H^O*. and  However,  a  constants,  there  t h e two, p a r t i c u l a r l y  since  i s f o r the r e a c t i o n c a t a l y z e d  Usually  therefore similar  t h e more  should  reversal  have  reactive a  by  reagent  smaller  reaction  of t h i s g e n e r a l i z a t i o n 71  has in  been  reported  by B r u i c e  which  t h e more  rapidly  dihydropyridine pK of the acid a mechanism , 10 poseu.  and B e n k o v i c  decomposed  for this  reaction  l-propyl-3-carbamoyl-l,4-  (XX) i s a l s o m o r e s e n s i t i v e t o c h a n g e s i n t h e 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 the  of this  r e a c t i o n i s not as  simple  as has been p r o -  (XXI)  52 3.2 B a s e - c a t a l y z e d Some found  Decomposition  pyridinium  salts  t o be s u s c e p t i b l e  Reactions (VIII)  and d i h y d r o p y r i d i n e s  to a variety  o f base H.  catalyzed  (V) w e r e decornpo-  ,H  ' N  I  (VIII)  sition of  (V)  reactions.  ester  Among  and amide  the simpler  functions.  reactions  Amide  were  hydrolysis,  which  71 observed  previously  significant the  upper  studied Amide  levels  pH  have  groups  susceptible quantitative Two and  pH  an amide  10.5, which  group  at either  i n the 1-position  seemed  to hydrolysis  than  those  measurements  were  made  esters  compounds  f o r any experiment  used  in this  f a n d g) u n d e r w e n t  methyl  3 minutes i-propyl fore  near  and model  ester  hydrolysis  (determined ester  these  rapid  represented  3-positions.  t o be s l i g h t l y  two c o m p o u n d s  regard.  c hlor ide  in alkaline  had a h a l f - l i f e  could  b u t no  1-carbomethoxymethyl-  hydrolysis  a t about  more  i n the 3 - p o s i t i o n  polarographically)  hydrolyzed  reached  a l l compounds  t h e 1- o r  i n this  work,  '  therefore since  has been  75  1-carbo-i-propoxyme thyl-3-carbamoylpyr i d inium  (VIII The  limit  i n NAD  hydrolysis  of  a t pH  15% o f t h i s  n o t be u s e d  solutions.  approximately 9.2  and t h e  rate.  above  pH  There7.5 f o r  53 CONH  2  N R  R  (VIII) f : R R  the  (IV) CH  1  COOCH  1  i-propyl ester  a n d 6.5  ester  of the  (IVf)  was h y d r o l y z e d  the of  pl-l 7.5 m e a n i n g  be and  several  pyridinium  the corresponding  could  of both  near  pH  the study  flavins  were  on  7.5  ring.  only  a  two p r o d u c t s chloride  new p y r i d i n i u m  salt  were  base  formed  (Illh) with  decomposition  prevented  a more  decomposition  their  which  t h a n v/as  use below  require the  ester  a t a l l and c o u l d ester.  by t h e  underwent 71  The  underwent from  polarography by  base-catalyzed formation  of the t o t a l further  re1,71  decompo-  reactions.  At  1-cyanomethyl-3-carbamoyl-  pH 7, t h e m a j o r negative  were  only  hydrolyses.  Pseudo-base  fraction  above  products  catalyzed  of the dihydropyridines  small  or the pseudo  slo'wly  and t h e d i h y d r o p y r i d i n e s ,  affected  a t the pyridinium  pyridinium  The m e t h y l  salts  the i - p r o p y l  salts  rate  Similar  with  pyridinium  sition  Acid  more  measurements,  the methyl  not greatly  represented  fold  dihydropyridines  the pyridinium  either  least  salt.  o f the o x i d a t i o n  Several actions  hundred  potentiometric  n o t b e made  made  ester.  l-carbomethoxymethyl-3-carbamoyl-l,4-dihydropyridin  corresponding  presence  f o r the methyl  one b e i n g  reduction  observed  with  a  potential.  54  CONH  2  (mil) 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 l,l'-ethylenebis(3-carbanoylpyridinium 1-carbamoylmethyl-3-cyanopyridinium  respectively.  c h l o r i d e ) ( XI Xb) ,  chloride  1-carbamoylmethy1-3-acetylpyridinium pll ' s 5, 7, a n d 9.5  c h l o r i d e ) (XlXa) ,  chloride  No a t t e m p t  R  3  COCH  (XXIIk)  and  (XXIIj)  above  was made t o  3  CN  CH CONH 2  2  (XXII)  a : n=l +  /  \  b:  +  n=2  (XIX)  isolate gation  the decomposition was d e s i g n e d  compounds  could  be  only used.  products to f i n d  as t h i s  preliminary  the highest  investi-  pH a t w h i c h t h e  55 During MAD  +  (la)  potentiometric  and/or  measurements,  NADH d e c o m p o s e d  i t v/as f o u n d  a t pH 8 . 8 t o g i v e  a  that  material  (la)  with  very  similar  reduction  potentials  (both  t o NMNI  On t h e b a s i s  polarographic  reduction  potentials,  decomposition  product  and  polarographic)  and the  (II)  mononucleotide  of  from  HO  potentiometric  (seesection 3 . 6 )  was t e n t a t i v e l y  arising  potentiometric  identified  hydrolysis  as the  of the pyrophosphate  OH  (ID  bond in  o f NAD.  weakly  The o n l y  alkaline  decomposition  o f NAD p r e v i o u s l y  s o l u t i o n i s the h y d r o l y s i s  reported  of the ribose-  71 pyridine the  bond,  results  a reaction  observed  here  which  could  not possibly  (seesection 3 . 6 ) .  be  causing  3.3  L i m i t a t i o n s o n pH Table  VI  i n the Study  summarizes  o f NAD  the r e s u l t s  decompositions  given  i n Section  3.2  decompositions  given  i n Section  3.1.  the  pH  a t which  (reaction  period which (36)  22)  occurs  t o an  The u p p e r  over  pH  The  limit  o f no m o r e  than  lower  catalyzed pH  limit  is  of the d i h y d r o p y r i d i n e s than  10% o v e r  i s t h e maximum salt  o f 24  such  hours,  (37) t h r o u g h  10% o v e r  base-catalyzed  the a c i d  o f no m o r e  a period  the h y d r o l y s i s r e a c t i o n s  extent  and  r e a c t i o n s of the p y r i d i n i u m  are not observed  which  extent  Compounds  of the  the acid-decomposition  o f 24 h o u r s . ring  Model  24 h o u r s .  pH a t  as r e a c t i o n o r t h e pH a t  (39) o c c u r The minimum  l-carbamoylmethyl-3-fluoro-1,4-dihydropyridine  a  (v£)  was  t o an pH f o r so  high  I CH CONH 2  2  (V£) that  potentiometric  oxidation  rate  by  measurements  flavins  could  and  the d e t e r m i n a t i o n  n o t be m a d e .  To  ensure  of the that  57 TABLE VI Stability  Limits  f o rPyridinium  and  Salts  and  Dihydropyridines  N  I  R  CH  COO  m i n i m u m pH o f dihydropyr i d i n e  3  CONH  maximum pH  9.1  10.5  CONH  9.7  10.5  CONH.  9.3  10.5  CH OCH  CONH.  8.1  10.5  CH COCH  CONH  CH^COOCH  CONH  7.5  CH COOCH(CH )  CONH  7.5  CH  CONH.  CH. CH CH 2  OH  2  2  0  Base  reaction  3-carbonoyl hydrolysis  10.5 5.7  1 - c a r b o m e t h o x ym e t h y l hydrolys i s  6.6  1-carbo-i-propoxymethyl hydrolysis  7.0  7.0  ring  CONH,  7.8  10.5  COCH.  6.5  9.5  CH CONH  CN  6.5  7.0  CH CONH  F  2  3  CN  CH CONH 2  CH  2  COHH  2  2  2  11. 5  £  10.5  reactions  3-carbamoyl hydrolys is ring  reactions  1-carbamoylmethyl hydrolysis  Table  VI  (cont.) m i n i m u m pH of dihydropyr i d i n e  Special  H  maxxmum pH  7.1  9.9  NMN  7.5  10.5  N  °  C  \ ^ —  This that  /} N  value of  reaction  Compounds  NAD  2  Base  \  )>-CONH  3-carbamoyl hydrolysis  2  N — /  was  unknown  n o t measured  7.0  b u t was  assumed  ring  r e a c t i o ns  t o be  t h e same  1-carbornethoxymethyl-3-carbamoyl-1,4-dihydropyridine  59  CH COOR  CH  2  no  base-catalyzed  pyridinium  reactions  chloride  (VHIh)  (VHIh)  of  COO"  l-cyanoraethyl-3-carbamoyl-  interfered,  potentiometric  (Vh)  60 measurements higher pH.  on t h i s  decomposition  compound rate  were  made  were  measurements  was  R  g:  R  the  chloride  (Illg)  1  =  CH COOCH 2  CH C00CH o  betv/een  Although, both  potentiometric  of  =  above  (Illg)  o n 1(Illf).  were  made  a t pH  (CH  which  )  the acid the ester  measurements  or below  and base  were  7 was u s e d ,  catalyzed  hydrolysis faster  rate.  decompo-  of (Illg)  and  than  desired,  still  possible.  I f a pll  either  the ester  hydrolysis  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  intolerable  7  3  a c i d - d e c o m p o s i t i o n o f ( I V g ) were  much  chloride  this  (IV)  a compromise  sitions.  (Vh) a t  on l - c a r b o - i - p r o p o x y m e t h y l - 3 -  (III) f:  despite the  not attempted  carbomethoxymethyl-3-carbamoylpyridinium  carbamoylpyridinium  5.6,  of the dihydropyridine  P o t e n t i o m e t r i c measurements  Potentiometric  a t pH  an  61 3.4  Substituent In  Constants  order  to  of  the  properly  3-Carbaraoylpyridinium analyze  nethylenebis(3-carbamoylpyridinium ethy1enebis(3-carbamoylpyridinium  inductive  effect  Intuitively, for  a  of  the  pyridinium  one  would  expect  pyridinium  group  (XXIII)  the to  the  behaviour  chloride) chloride)  group  closely  of  (XlXa) (XlXb)  must  inductive  Group  first  1,1'and  1,1'-  the  be  known.  substituent  resemble  that  constant of  (XXIII)  the  trimethylammonio  hypothesis,  the  pKa  group of  the  or  ammonio  group.  substituted acetic  +  CH COOH 2  (Ilia)  ChLjCOO"  To  test  acid  H  this  (Ilia)  was  62 determined 1.7  +0.1,  from  Charton's  for  =  I  0.251  i n good  (0.73)  72  the  which  a a  correlations  Cf  fact  spectrophotometrically  log K  x  /K  group  (0^ =  can  be  7)  (40)].  This  value  to  be  using is in  (40)  a  I  f o r the  20%  larger 73 0.60) so  trimethylammonio than  the  value  the v a l u e  for O  of  aroui a of  (XXIV)  -CONH,  (XXIV)  assumed  found  calculated  ONH,  was  and  H  with  approximately  ammino  0.76  [equation  agreement  and  of  (Figure  (XXII)  t o be  close  to  20% l a r g e r  than a  f o r the  Cll NH  ^  I  +  3  72 group. ponding and  (  a*  XXVI)  0.36)  =  a  substituent have  been  'CONH,  I  giving  CH  i t a value  constants  calculated  of  f o r the  from  The  corres-  substituents (XXV 73 equation (34) giving CONH,  2  2  (XXVI)  0.44.  ( XXV)  PH  64 values  of  a  1.7  1.0  and  (xcn )  =  a  2  inductive  effect  of  and  (XlXa)  discovering any  effect  in  i(x)  of  a  (XlXb)  i f the  respectively.  / 0  -  < > 34  be  pyridinium investigated  proximity  addition  to  the  of  the  two  inductive  (XIX)  See  footnote  on  page  determined  4 5  second can  Having  4 8 (Section  3.1)  ring,  the  with  the  pyridine effect.  the  +  properties purpose rings  has  of  65 3.5  Polarography The previous  of Pyridinium  polarographic  studies.  A t pH  3-carbamoylpyridinium v/ave w i t h -800  mv.  tively.  half-v/ave versus These  results  chloride potentials  a r e i n good  OH  v/ere  5.6, NAD  the normal  HO  Salts i n good  (Ia),MMN  +  (VHIb)  agreement  with  ( I I ) , and 1-methyl'  +  e x h i b i t e d one r e d u c t i o n  near  - 6 8 0 mv.,  - 7 0 0 mv. , a n d  hydrogen  electrode  (M.H.E.)  agreement  with  previously reported  HO  (la)  respec-  OH  ONH, CONH,  (VHIb)  half-wave v/ere work  potentials  observed 5,40,44  with The  these  - 6 0 0 mv  66, 67  . -3 2 x 10 molar.  compounds. ' 5  compounds  r e d u c t i o n wave  carbamoylpyridinium near  f o r these  4 0  '  i n agreement  of  Mo  prewaves  v/ith  previous  4 4  1,1'-ethylenebis(3-  chloride) previously reported v/as  only  A t lov/er  observed  b y t o be  at concentrations  concentrations  a single  over  v/ave v/as  66 observed wave  of  at  approximately  mv. *  Both  the  prewave  1,1'-methylenebis(3-carbamoylpyridinium  observed  near  and  mv.  -420  have  -430  not  the  respectively.  been  Only chloride  previously  studied  one  The  6 6  main  chloride)  were  p o t e n t i a l s of  other  pyridinium  -180  mv.  compounds  previously.  compound,  (VIIIj),  reported  and  l-carbamoylmethyl-3-acetylpyridinium  exhibited  tv/o r e d u c t i o n  CH CONH 2  waves  at  a l l concen-  2  (VIIIj)  trations. 5 found  The  the  wave  was  independent  of  pH,  as  has  been  40 '  for  pyridinium about  first  35  reduction  salts.  The  millivolts  first  wave  (wave  I).  known  but  The may  increasing negative  the  second  per  pH  corresponds reduction involve  v/ave  wave  41  in Figure  a  pH  Based  to  reduction  the  occurring  concentration,  had  I  unit.  reduction  p o t e n t i a l s by  (v/ave  the  to  at a  second  millivolts  on  the  2)  dependence the to  pH  wave  per  shifts  of  radical  dihydropyridine. wave  other  dependence,  the  second  of  to  is  not  With  more  concentration  decade.  -3  At  concentrations  above  10  I!., t h e  plateau  of  the  A prewave has b e e n r e p o r t e d f o r t h i s compound a t 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 o f p y r i d i n i u m c o m p o u n d s i s n o t uncommon.40  first  v/ave  -380 mv. potentials  67  shifts  negatively  trations an  b y a much  t h e two  waves merge  a n a l y s i s of the f i r s t The  has  been  l a r g e r amount,  half-wave  reported  as  shown  v/ave b e i n g  potential  to s h i f t  in Figure  I  concentration  concentration in  Figure  dependence  v/as o b s e r v e d  At  concentrations  to  more  negative  The  half-wave  and  then  mechanism  (Section  the  pyridinium  other  result  passes  through  concentration  25)  v/ith  study  as  shown  iodide.  pyridinium  Similar  salts  studied.  potential  shifts  concentration. -3  a maximum  values  complex  with  near  10  I-}.  increasing  con-  dependence  could  be  due  o f t h e e l e c t r o d e r e a c t i o n , one -4 b e l o w 5 x 10 M. c o n c e n t r a t i o n s a n d t n e -3  v/ill and  column  10 be  Other  i n subsequent  was  the l i m i t i n g  determined A plot  i n good  with  ions  .  for  of l o g  current  evidence  sections  on  the height  of  l-cyanomethyl-3-carbamoylv s . l o g h was  agreement  for a diffusion  agrees  pyridinium  concentrations.  presented  of  o f 0.455,  o f 0.5  H.  3.5.3).  chloride.  a slope  dependence This  A more  in this  decreasing  unusual  dependence  mercury  with  M.  the half-wave  negative  aPove  this  3.5.1  The  H.  t o more  operating  on  the other  3  page  o f mechanism  operating  bearing  10  potential  The  a change  other  10  v/as o b s e r v e d  p o t e n t i a l s v/ith  returns  centration.  below  2,  -3 below  v/ith  concen-  preventing  potentials  9 f o r l-methyl-3-carbamoylpyridinium  behaviour  to  8,  (Figure  negative  4 3,44 decreasing  at high  made.  o f wave  t o more  until  with  controlled  the  linear  theoretical  reduction  the f i n d i n g s of previous  process.  workers f o r  - 6 0 0 mv.  -600  mv.  log  C  (mM)  70 3.5.1  Analysis In  E,  view  3  Cauquis,  o f Wave I . o f the t h e o r e t i c a l  treatment  bv S o n n a t e r r e  3  a comparison  versus  three  current  was maae  between  plots  of potential,  log  log ( i , - i)/(i), a 2/3 1/2 ( i , - i)/(i) , and l o g ( i , - i ) / ( i ) . These  are  shown  i n Figures  pyridinium for  a na  iodide  comparison  functions,  10 a n d 1.1 f o r 1 - m e t h y l - 3 - c a r b a m o y l -  a n d MAD ,  respectively.  4  i s linearity.  none  of the current  tion  range  plots  functions  A  It i s readily apply  over  first  criterion  apparent  that  the e n t i r e  concentra-  - 5  versus  -2 -4 t o 10 M . B e l o w 10 M .plots of E 2/3 1/2 log ( i , - i)/(i) o r l o g ( i ., - i ) / ( i ) are linear. from  10  d  "  d  - 3 At log  concentrations ( i  - i)/(i)  current  have  10  only  end o f t h e l i n e .  siderable A is  above  M . plots a small  of E  versus  curvature  The r e m a i n i n g  near  plots  the low-  a l l show  con-  curvature. second  the change  function,  criterion  f o r comparison  i n potential  i n the  slope  log-current  volts.) A s l o p e o f 59 mv. 1/2 versus l o g ( i , - i ) / ( i ) d of  plots,  plots, are  - i)/(i),  change  (The  e t c .and has the u n i t s o f m i l l i 33 i s predicted for plots of potential 2/3 or l o g ( i , - i ) / ( i ) . The r e s u l t s d  these  log(i^  per unit  i s slope.  low-concentration  are listed  generally  i n Table  higher  adsorption  effects  of  well  ing  getting  versus  o r may  resolved  the d e t e c t i o n  potential  than  limit log ( i  VII.  which  The s l o p e s  predicted simply  were  which  2/3  linear plots  be d u e t o  to the d i f f i c u l t y  at concentrations  of the instrument. - i)/(i)  of these  may  be r e l a t e d  polarograms  the only  have  The p l o t s slopes  approachof the  closer  to the  'igure 10a. Polarographic applied potential versus l o g ( i - i ) / ( i ) f o r l-methyl-3-carbamoylpvridinium chlor • r—— 1 1 : ~~| , d  0.1  Tt. a c e t a t e ,  -9 25  pH  =  5.6  0.0157 m!i 0.10 m i l .  -950  2.0 10.0  mM. mM.  •975  i—i  u |? - 1 0 0 0 \  <  -1025  > •1050  •1075  -1100  -1125  -1.0  •0.5  0.0 log ( i  0.5 -  i)/ ( i)  1.0  F i g u r e 10b. Polarographic applied potential versus l o g ( i - i ) / ( i ) / for l-methyl-3-carbamoylpyridinium 2  3  d  ~i  '  0.1  —  —  1  i  M acetate,  pH  -0.0157  mM  •925  .0.10 •950  •2.0 10.0  =  —  1  7  chloride  — — — |  5.6  mM mM mM  -975  -1000  o  tr  \ Cn  •1025  > >  -1050  -1075  •1100  -112!  •1.5  -1.0  -0.5 log(i  d  0.0 -  i ) / ( i )  0.5 2  /  3  1.0  F i g u r e 10c. Polarographic applied potential versus log(i -i)/(i)I/ f o r l-methyl-3-carbaraoylpyr idiniurn chloride 2  d  0.1  M.  •9 25  acetate,  pH  0.0157 0.10  -950  =  5.6  mM.  mM.  2 . 0 mil. 10.0  mM,  •975  O  -1000  < « >  -1025  > e  •1050  -1075  •1100  -1125  •1.5  -1.0  •0.5  l o g ( i - i ) / ( i ) d  0.0 1  /  2  0.5  1 .0  74 F i g u r e 11a. Polarographic applied potential versus log(i -i)/(i) for nicotinamide adenine d i n u c l e o t i d e d  0.1  M.  -790  acetate, 0.016  —  mM.  .  0.18  x  1.8  mM.  9.0  mM.  -350  5 . 6  mM.  0 . 023 -320  pH  mM.  880 u  < tr  <  •910  > -94 0  -970  -1000  -1030  -1.5  -1.0  •0.5 log(i  0.0 -i)/(i)  0 . 5  1.0  1 . 5  75 Polarographic applied potential versus ) / for nicotinamide adenine dinucleotide  acetate,  pH  0.016  mM.  0.023  mM.  0.13  =  5.6  mM.  1.8  mM.  9.0  mM.  /  —I  1.5  •1.0 log(i  -0.5 d  -  i)/(i)  ,  0.0 2  /  3  L_  0.5  1.0  76 F i g u r e 11c. Polarographic applied potential versus log(i - i j / d ) / for nicotinamide adenine d i n u c l e o t i d e 1  2  d  -2.0  -1. 5  -1.0 log ( i  -0.5 d  -  0.0 1/2 i)/(i)  0 .5  1.0  77 TABLE V I I Slopes  and  log  Intercepts  ( i  d  -  i ) / ( i )  of Plots 2  /  and  3  of Potential  log ( i  -  versus  i ) / ( i )  1  /  2  CONH,  Series  I: E log(i  R  concentration (mM)  l  X  CH CN 2  CH COOCH 2  CH COOCH(CH 2  CH COCH 2  CH  *  2  OCH„ 3  t h e same  ]  against d  slope (mv)  -  i ) / ( i )  E 2  /  J  intercept (mv)  log  against  ( i  slope (mv)  1/2 d  "  1  )  7  inter (mv  . 0106  76  - 4 90  79  -455  .0511  60  -513  72  -487  .156  72  -474  78  -459  . 248  73  -497  82  -484  .0116  66  -612  72  -531  . 0167  63  -613  73  -579  . 0256  70  -615  83  -581  . 0438  63  -602  74  -577  . 0723  70  -6 08  80  -582  .00932  65  -631  66  -601  .0141 *  63  -623  76  -581  .0141*  70  -632  61  -609  .0109  59  -698  63  -668  . 0157  66  -692  74  -660  . 0240  70  -674  79  -656  .00977  59  -662  63  -633  .0251  72  -630  79  -601  .0360  75  -639  83  -615  .0475  66  -626  75  -603  solutions  recorded  at d i f f e r e n t  current  sensitivitiei  78 Table  VII  (cont.) concentration (mM)  E  against  log(i  "d slope  -  intercept (mv)  (rav) CH CH OH 2  2  CH.  COO  Series  d  slope (mv)  -  i)/(i)  -768  55  -748  . 0253  59  -773  34  -751  .0453  74  -7 63  82  -739  . 0157  59  -833  67  -8 09  69  -797  77  -800  73  -842  98  •7 99  .0182  II  2  2  3  COCH.  CN  1/2  intercept (mv)  46  CH CONH R  log(i  against  .0176  2.0 CH  i)/(i)  E 2/3  .00952  49  -511  63  -479  , 0182  62  -505  68  -4 5 5  ,0261  55  -503  57  -4 8 3  , 040  58  -503  66  -481  ,00952  64  -4 29  73  -3 94  ,0261  66  -423  87  -38 2  . 040  62  -431  72  -406  ,20  67  -422  73  -410  , 01«?  55  -821  64  -797  0261  62  -8 2 2  69  -798  20  66  -815  73  -803  00952  55  -1109  65  -1030  040  60  -1087  84  -1063  20  69  -1063  90  -1054  79 Table  VII  (cont.) E  R.  concentration (mM)  CONH.  Special  l  o  9 ( i  against d  slope (mv)  i)/(i)  V3  intercept (mv)  E l o g ( i  against d  slope (mv)  i) / ( i )  i n t e r c ept (mv)  . 0145  71  -663  73  -632  .0515  72  -651  81  -626  . 0776  72  -651  81  -629  .127  74  -642  80  -626  0.2  72  -1245  33  -12 3 6  0.7  70  -1243  73  -1242  2.0  74  -1231  80  -1237  10.0  97  -1246  110  -127 0  .016  63  -637  72  -656  .023  62  -679  66  -657  . 036  71  -666  82  •635  .020  81  -713  93  •677  .030  75  •700  87  •671  .15  83  •693  98  •680  .018  104  •718  .026  84  •702  . 04  105  •707  .00952  84  -4 2 2  79  -389  .0182  85  -429  83  -401  . 0261  70  •448  70  -425  .04 0  84  •443  94  -414  .20  87  •427  94  •413  Compounds:  Compound NAD  +  (la,  page  NMN ,  pH 4.6  +  (II,  pH  65)  page  5.6  65)  CONH,  CONH,  1/2  80 Table  VII  (cont.) E  Compound  concentration (mM)  slope (mv)  i)/(i)  E J/3  intercept (mv)  against  loq ( i d slope (mv)  i) / ( i )  2  I  1  Wav e I  1/2  intercept (mv)  CONH.  H NOC  Wave  log ( i  against  .00952  68  -197  72  -167  .0102  83  -181  111  -15 5  .0261  97  -18 0  110  -145  .040  78  -2 28  90  -200  .20  76  -201  86  -187  .00952  72  -390  70  -377  .0182  96  -390  104  -361  .0261  70  -428  75  -405  . 040  108  -435  120  -4 08  .20  98  -4 3 9  89  -430  81 theoretical priate  value,  equation  indicating  to use.  + ~  half-wave plots 10.0 are  potentials  of potential mM.  an  than  irreversible  reaction  since  and  log ( i d equally large n  The  2  /  1/2  the  the slopes  appro-  and  (18)  the l i n e a r i)/(i)  slopes  portions of  a t 2.0  o f most  value  mainly  i s taking  by  mM.  and  of these  o f 60 mv.,  of potential showed  be  3  electrode reaction  the p l o t s i)/(i)  ( 1 8 ) may  lists  log ( i ^-  i s not induced  plots  indicating place.  This  the d i m e r i z a t i o n  versus  considerable  log ( i ^ curvature  i)/(i)  theory  ( 1 4 ) , and  (18)  predicts  should  that  = £° +  E  = e° + £ | £ n ( i , - i ) / ( i ) F a  d  the i n t e r c e p t s  independent  E  CtnF  £n(i  be  and  in  equations  of concentration.  - i)/(i)  condition i s approximately  (10)  1  /  (14)  2  fulfilled  by  t h e low  concentra2/3  tion  plots  of potential  versus  both  log ( i ^-  i)/(i)  and  1/2 (i, d  -  i)/(i)  2/3  slopes.  Thirdly,  This  from  the t h e o r e t i c a l  irreversibility  (10),  derived  versus  VIII  - i ) / ( i )  Q  concentrations.  larger  that  Table  fc.n(i.  r  equation  .  The  means  intercepts  of the p l o t s  deviations  range  from  and  standard  are l i s t e d  2 mv.  up  d e v i a t i o n s of the  i n Table  t o 27 mv.  IX.  with  The  standard  variations  in  82 TABLE Slopes  and Half-wave Applied  Series  Potentials  Potential  Versus  Obtained log(i^  -  by  Plotting  i)/(i)  I:  l  R  VIII  slope (mv)  CH  CN  CH  COOCH  2 . 0 mM. Half-wave potential (mv)  10 . 0 slope (mv)  mM. Half-wave potential (mv)  74  -554  92  -581  74  -657  94  -692  76  -668  90  -696  CH COCH  33  -699  97  -729  CH OCH  3  77  -657  85  -679  CH CH  OH  66  -758  83  -780  58  -793  84  -813  90  -826  101  -922  3  CH COOCH(CH) 2  2  2  CH COO~ 2  2  Series 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  83 Table  VIII  (cont.) 2.0 slope (mv)  61  Special  mM. Half-wave potential (mv)  10.0 slope (mv)  mM. Half-wave potential (mv)  -1224  75  -1211  55  •683  64  -715  115  •810  103  -83 0  124  -593  129  672  90  •469  compounds:  + NAD ( l a , page  65)  NT (II,  65)  page  CONH  CONH,  84 TABLE IX Mean  Intercepts  log(i  Series  d  from  - i ) / ( i )  2  /  Plots 3  of Potential  and l o g ( i  -  versus  i ) / ( i )  1  /  2  I CONH,  log(i  d  -  i ) / ( i )  i n t e r c e p t , e° (mv. v s . N.H.E.)  2  /  3  log(i  d  -  intercept (mv. v s . N.H.E.  CH CN 2  -494  + 16  -471  + 17  CH  -610  + 5  -580  +_ 2  CH.COOCH(CH,) 3' 2  -629  +_ 5  -605  + 6  CH COCH  -688  + 12  -661  + 6  CH  OCH  -639  + 16  -613  + 15  CH  CH  -768  + 5  -746  + 6  -815  + 25  -805  + 5  COOCH  2  OH  CH. CH  COO  leries  -842  -799  II :  R.  CH CONH 2  i)/(i)  2  CONH.  -652  + 8  -6 28  + 3  CN  -426  + 4  -400  + 10  COCH.  -506  + 4  •480 +_ 3  F  -819  + 4  •799 + 3  1/2  85 Table  IX  (cont.)  R  log(i  -  i)/(i)  2/3  log(i  d  -  i ) / ( i )  i n t e r c e p t , e° (mv. v s . N.H.E.)  intercept (mv. v s . N.H.E.)  H  -1086  + 23  -1066  + 13  o"  -1241  + 7  -1246  + 16  Special NAD  compounds -677  +  (la, NMN (II,  + 10  •649 + 12  + 8  •676 + 5  p a g e 65) •703  +  H NOC 2  p a g e 65)  -oo\=:  H NOC 2  N = /  N  +  \  CONH,  -434  + 11  -408  + 14  /•  ONH.  Wave I •  -209  + 17  -185  + 17  Wave I  -409  + 27  -391  + 20  1  /  2  86 the  intercept  The  use o f the c h a r g i n g  to  hold  shifts  random  the baseline  a  error  nomogram could  with  respect  current  constant  i n the applied  supplies the  being  compensation,  to correct reach  which  o f a s much  for this  15  as  t o 20 mv.  at  causes  100 mv.  potential  changes.  i s necessary  a t low c o n c e n t r a t i o n s ,  potentials  easily  to c o n c e n t r a t i o n  lietrohm  shift  but  concentrations  -5 approaching  10  Based tion  waves  v/hich only  and  on  the preceding  are consistent  might  apply  criterion  versus  M.  log ( i  the observed  with  distinguishes  i)/(i)  1/2  clusive.  Mechanistically,  equations  (14) a n d  reactions  (1) a n d  electrode between  and l o g ( i  variations  i n this there  (18), both  only  t h e low  concentra-  any o f t h e t h e o r e t i c a l  for a reversible  v/hich -  analysis,  reaction.  plots  i)/(i)  quantity  is little  of which  (2) i n S c h e m e  -  equations The  of potential  2/3  make  i s  slope  i t incon-  to choose  between  are consistent  3 reproduced  with  i n equation (41).  (41)  Equation  ( 1 4 ) was  derived  assuming  a rapid  dimerization  38 reaction.  The d i m e r i z a t i o n  E  = e° + —  £n(i  d  -  reaction  i)/(i)  has been  found  t o be  (14)  rapid;  thus  enough  tov;ards p r o d u c t s  as  assumed  was  equation  i f the d i m e r i z a t i o n  =  The  e  o  R|  +  £  in Figure  near  unity  suggest  only  data  log(i  d  3.5.2  -  n  (  intercepts  other  same  and,  i  ^  i ) / ( i )  2  /  12.  that  from 3  _  i  )  /  listed  (  i  reaction  irreversible,  of equation  )  (10), either  2/3  (  i n Table  The  sets  subsequent  plots  IX  excellent  t h e two  therefore,  derived  Prewave  this  i s displaced far  apply.  each  the  t o make  i n the d e r i v a t i o n  could  E  equilibrium  1  3  )  are plotted correlation  of data  are  discussions  of potential  against and  slope  essentially will  involve  of  1-methoxy  versus  .  Formation  Prewaves  were  observed  i n the polarograms  methy1-3-carbamoylpyridinium  chloride  (Vllle),  1-carbomethoxy  methyl-3-carbamoylpyridinium  chloride  (Vlllf),  1-carbo-i-  propoxymethyl-3-carbamoylpyridinium  chloride  (VHIg),  l-cyanomethyl-3-carbamoylpyridinium  chloride  (Vlllh),  l-cyanomethyl-3-carbamoylpyridinium  chloride  (Vlllh),  l-carbamoylmethyl-3-cyanopyridinium  chloride  (VHIk),  1'1,-methylenebis(3-carbamoylpyridinium  chloride)  higher  height  dent  c o n c e n t r a t i o n s , t h e p r e w a v e wave  o f c o n c e n t r a t i o n as  typical  shown  o f a d s o r p t i o n waves  in Figure  i n which  13.  (XlXa).  becomes This  t h e maximum  and At  indepe:  behaviour  diffusion  .  88 F i g u r e 12. Comparison o f the i n t e r c e p t s from p l o t s of polarographic potential vs. l o g ( i ^ - i ) / ( i ) l o g ( i j - i ) / ( i ) 2/3 f compounds i n S e r i e s I and S e r i e s I I . a  c  o  ,  coeff.  slope  =  =  1  d  7  0.999  ——I  1  -1000  ,  n  1.01  -  -900  intercepts  from  *see  X  Table  ,  1  corr.  L  r  plots  .  -800  L _ _  1  -700  of E vs. l o g ( i  ,  -600 d  -500 2/3 -i)/(i) '  f o r the corresponding l i s t  of  compounds  89 TABLE X List  o f Compounds  Rl  Plotted  n  i n Figure  3  CN  1.  CH CONH  2.  CH CN  3. 4.  CI! CON H 2 2 CH COOCH  5.  CH  6.  CII OCH  CONH,  7.  CH  CONH  CONH.  8.  CH  COCH  3  CONH.  9.  CH CH OH  CONH,  2  CONH. COCH. CONH.  COOCH(CH ) 3  2  2  CONH .  4.  10.  CH  11.  CH.  12.  CH  COO  13.  CH  CONH  2  CONH„ 2  F CONH , CONH,  12  Figure  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 polarographic reduction of l-carbo-i-propoxymethyl-3-carbamoylpyridinium chloride  i r — — — — i  1  »  i  ,  ,  .  ,  _  91  e:  R =CH OCH 1  P- =CONH.  2  f:  3  CH„C00CI1  g:  CH  h:  CONH.  3  CONH.  COOCH(CH )  CONH.  CH^CN  k:  CH  CN  CONH  (VIII)  H NOC-<(^  /  2  \ ^l>--CONH  2  (XlXa)  current  corresponds  to the formation o f a monolayer 37  material  on the e l e c t r o d e  concentration of  corresponding  the prewave  wave  o f the prewave  maximum  prewave  temperature.  adsorption current size  process  would  pounds this  i s observed except point  behaviour  i n which  be a f f e c t e d  of the mercury  prewave  At  This 39  drop.  Table  only  wave  current chloride  the h a l f -  f o r each  compound.  of  In agreement  independent  with  which  v/ith  l-carbo-_i-  was  t h e maximum  by f a c t o r s  current  between  i s consistent  case  XI g i v e s t h e  diffusion  difference  and main  propoxymethyl-3-carbamoylpyridinium of  '  diffusion  adsorbed  3 9  t o t h e maximum  and t h e a p p r o x i m a t e  potentials The  surface.  of  a strong  diffusion affect  this,  the  only the  a t l o w c o n c e n t r a t i o n s f o r a l l o f t h e com-  1,1'-methylenebis(3-carbamoylpyridinium i t should  be r e c a l l e d  that  the p l o t s  chloride).  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 t o t h e Maximum H e i g h t o f t h e P o l a r o g r a p h i c Prewaves and t h e P o t e n t i a l D i f f e r e n c e B e t w e e n M a i n Wave a n d P r e w a v e  R  p  l  Concentration corresp o n d i n g t o t h e maximum diffusion current (mM)  "3  Potential difference* (mv)  CH  OCH  CONH „ 2  0.20  100  CH  COOCH  CONH„ 2  0.32  90  CH COOCH(CH )  CONH, 2  0 .34  150  CH  CONH  0.36  130  0.4 9  150  ^.1  200  2  3  CN  CII C0NH 2  H NOC 2  2  CN  CONH,  I t h a s n o t b e e n p r o v e n w h i c h wave ( m a i n 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 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 t h e s e compounds. D e p e n d i n g o n w h i c h wave d o e s , 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 concentrations reversible whereas sible a  (Figures  reduction  the p l o t s  reduction  prewave, reaction  10  and  followed  a t high  (41)  by  an  consistent  irreversible  3.5.1).  i t i s the prewave and  were  concentration  (see S e c t i o n  then,  11)  the main  with  dimerization  indicated  an  F o r compounds  which  wave w h i c h  irreverexhibiting  i s consistent exhibits  a  with  irreversible  (41)  behaviour.  This  behaviour  i s consistent  with  the  adsorption  48 mechanism  proposed  pyridines  (see S e c t i o n  which  by  the r e d u c t i o n  inhibited  by  The  a  layer  At  seen  pyridinium  maximum. the  The  height  variation  of  shown  was  f o r v/hich than  second  first  a t such  that  wave  i s probably  encountered  behaviour  in Figure  related  low  of  product.  complex  both  than  a prewave  in contrast wave  corresponding between  i n an  in  1'1-methylenebis(3-  more  14,  3-acetyl-  3-acetylpyridine is  the main  varies  wave  the  of  Electrochemistry)  reduction  concentrations,  less  the  of  of adsorbed  compounds  concentrations  Introduction,  chloride)  low as  f o r the r e d u c t i o n  the bulk  polarographic  compounds. are  1.5  of  carbamoylpyridinium  wave  Laviron  was to  one  apparently  to e x p e r i m e n t a l  concentrations.  The  and to  other  the  main  other  not  the  and  the  seen  at  prewave  two  random  times way.  This  difficulties potential  difference  Figure  14.  P o l a r o g r a m o f a 1.8 x 1 0 ~ M. s o l u t i o n (3-carbamoylpyridinium chloride) 5  of  wave  0.05  yA  100 -500  mv.  (vs.  Ag/AgCl)  mv.  1,1'-methylenebis-  I  between larger With to  the prewave value  such  know  than  found  a large  which  and main  wave  200  v/as a p p r o x i m a t e l y  f o r any o t h e r  pyridinium  compound.  p o t e n t i a l d i f f e r e n c e , i t becomes  wave  represents  the true  reduction  mv. , a  important  potential.  66 Lovecchio reduction sponds  being  that  of soluble  inhibited  analyses  recorded  listed  a t temperatures  increased  the f i r s t  reactant  i n height  while  on  wave  between  wave  the l a t t e r  product.  the b a s i s  i n Table  corresponds  the second  reactant,  by a d s o r b e d  n o t be d i f f e r e n t i a t e d  current  wave  of adsorbed  to r e d u c t i o n  possibly could  has proposed  The  relative  correprocess  two  waves  o f the p o t e n t i a l - l o g  V I I , so p o l a r o g r a m s 13° and  to  were  50° t o s e e i f one  to the other.  I f the  presence  o f t h e two w a v e s i s d u e t o a p a r t i a l a d s o r p t i o n , a s 66 suggested, then i n c r e a s i n g the temperature should increase t h e 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 a n d d e c r e a s e t h e wave  39 which  i s due  temperature merge  to adsorption study  a r e shown  as the t e m p e r a t u r e  ambiguous  to allow  potential  t o be  tential  falls  observed.  The r e s u l t s  i n Figure  15.  i s increased  and  t h e wave  identified.  somewhere  effects.  that  The waves appear the r e s u l t s  represents  Possibly  i n between  of the  the true  the true  are too reduction  reduction  t h e two w a v e s  to  that are  po-  Figure  15.  T h e e f f e c t o f t e m p e r a t u r e c h a n g e o n t h e p o l a r o g r a m o f a 2.3 x s o l u t i o n of 1,1'-methylenebis(3-carbamoylpyridinium chloride)  10  45°  -350  mv.  97  3.5.3  The E f f e c t s Potentials. Initially  chemical  of 1-Substituents  on P o l a r o g r a p h y  the e f f e c t of s u b s t i t u e n t s  reduction  potential  was  on  estimated  Reduction  the e l e c t r o -  from  plots  of half-  t wave a  potentials,  (see Section  which is  E  Hammett  ^  1.6).  i s a measure  defined  1 /  . , against  Conventionally,  substituent the r e a c t i o n  of the e f f e c t of substituents  by e q u a t i o n  (32) u s i n g  either  the  constant p  i s usually  against  found  0].  constant  from  The r a t e  on a  p  reaction,  77  t h e 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 c o n s t a n t , k, o f t h e r e a c t i o n .  or the rate  constant  equilibrium  AlogY  X =  constants,  rate  f o r the r e a c t i o n .  the slope  constant,  of a plot  k  [In practice,  of log K  , or equilibrium  ( o r l o g k)  constant,  K,  o for be  a polarographic determined  wave  from  i s described  E  =  E  reduction  the half-wave by e q u a t i o n  l / 2  +  can, under  RT anF  certain  potential.  (10), which  I f the  n  [  U  d  "  polarographic  i s the equation f o r  "3 £  circumstances,  (  1  0  q  )  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 defined from a s t a n d a r d r e a c t i o n and m e a s u r e s 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 variety of a values 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 t h e 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 and on t h e t y p e o f r e a c t i o n occurring.- ''''" 1  98 a  simple  tial (12)  polarographic  c a n be and  related  (7)] f o r a  =  1/2  1.5) an  :  i/2  reduction,  -  £ n  P  by  c a n be  J red  A  (a<l).  *  K  k  /  2  k  (43).  3 7  [equation  Q  reversible  ]  Q  (12) '  (13)  (13)]f o r polarographic  5 5  combined  Since  Section  with  equation  i t i s unlikely  (42)  M  o x r e d 2.3 03 A a  n  changes,  neglected  giving  nF 2.303RT  A  "  2  For a  1  [equation  ( a = l , see a l s o  (12) c a n be  of the d i f f u s i o n  substituent  /  K,  poten-  (  l / 2 Aa  E  1  the half-wave  constant,  constant,  (42) and  AE * Aa  nF 2.3 0 3RT  /D  f°-886(T/D)  equations  the r a t i o  affected (43)  =  ox  rate  (7) a n d  nF 2.303RT  that  £n(D  nF  equations  to give  then  reversible reduction  reduction  =  wave,  to the e q u i l i b r i u m  or to the r e d u c t i o n  irreversible  (32)  E°  reduction  l / 2 Aa  (  D  /  D  )  1  /  coefficients, the second  equation  2  (  D,  term  (44), from  E  (44)  4  3  )  will of  be  greatly  equation  which  the  99 reaction if  constant  the e f f e c t  AE^^/Aa,  i  o f s u b s t i t u e n t s on  s  irreversible to  P  yield  known.  mercury  .  A  E  l / 2 Aa  equation  treatment  Since  Alog  i s under  c a n be a p p l i e d  1  /  2  T of the  control  n o t be e x p e c t e d  ( 2 7 ) c a n be n e g l e c t e d  '  and t h e  t o change  the second  to give  dropping  ] [  compounds t h e n  t o an  ( 1 3 ) a n d (32)  time  experimental  would  analogous  the drop  determined  potential,  equations  [ 0 . 386 ( T/D) Aa  "  coefficients  among  similar  (45).  electrode  diffusion  A  r e d u c t i o n c a n be  the half-wave  r e d u c t i o n by c o m b i n i n g  equation  QtnF 2.303RT  cantly  p of a reversible  term  signifiof  equation (46).  AE P  anF 2.303RT  =  Inspection  of equations  (reversible (46)  1/2 ' ~ A o ~  reduction)  i n which  assumptions  (44) and i s simply  the t r a n s f e r  used  were  that  (10)  (i.e. a plot  of E versus  RT  and  that  dependent prevail,  E  +  l/2  £nt(i  the transfer  d  a normal  special a  6  )  that  form  must  equation  of  i s equal  of equation  i)/(i)  (44)  equation t o 1.  The  (44) a n d (46)  be d e s c r i b e d  log ( i ^-  must  by be  equation linear)  " i>/<i>l do)  coefficient  of substituent effects. then  a  coefficient  wave  4  (46) r e v e a l  i n the derivation  the polarographic  E=  (  Hammett  a must  be a p p r o x i m a t e l y i n -  I f these  reaction  c o n d i t i o n s do n o t  constant  p cannot  be  100 calculated  directly  from  the e f f e c t  of substituents  on  the  half-  wave p o t e n t i a l s . The (Table  effects  VIII)  of substituents  of the Series  on  the half-wave  I compounds  potentials  ( I I I ) a r e shown i n  CONH,  (III)  Figures  16,  17, a n d  concentration the  18.  dependent,  polarograms  were  Since  the half-wave  (see S e c t i o n  a l l recorded  3.5  potentials  and  a t or near  were  reference a  single  44)  concen-  -2 t r a t x o n f o r any one -3  the  The  half-wave  standard  (33)]  17, a n d  H  _  1 x 10  for this  has t h e s u b s t i t u e n t  or  M.  to 2 x  OH  attached  R )  [ l o g (-  q  h  _ - l  values  as  i s the case  o f a*  used  were  with  Figure  since  [equation  to the r e a c t i o n  (33)  o  g  (  ^ _ ,  ]  (  4  7  )  70  CH.  the S e r i e s  calculated  a*  constant  directly  r»- RCOOH + R'OII  16, M . in  10  against  substituent  CH.  :entre,  i n Figure -5  p o t e n t i a l s are plotted  reaction  RCOOR'  (10 -5  M . i n Figure  2 x 10 18) .  correlation  from  I compounds  (III).  Charton's  values  7 2  The of  Figure  16.  P l o t of polarographic half-wave p o t e n t i a l s 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 a* ( T a b l e V, p a g e 4 9 ) .  corr. -600  AE  X /  £.  coeff.  /Aa* =  188  -CH CN 2  -CH OCH  3  > -700  -CH  CONH  -CH COOCH 2  -CH  2  -CH COCH 2  •750  M. against  mv.  2  >  -2  = .972  -650  33  o f 10 VIII)  3  COOCH(CH ) 3  2  3  -CH,CH,OH 2 2  > e  -CH.  -800 CN  :ONH,  -850 N  I  -900 l>  -CH C00" 2  -0.3  -0.1  0.1  0.3  0.5  a*  0.7  0.9  1.1  1.3  Figure  17.  Plot of polarographic half-wave potential of 2 x solutions o f t h e S e r i e s I compounds ( T a b l e V I I I ) a* ( T a b l e V, p a g e 49) .  corr. -550  A  E  l / 2  coeff. /  A  a  *  =  1  i o " ?i against 3  978 6  2  m  v  "  -600  -CH„0CH 2 3  -650  ^ -CH COOCH l>-CH C00CHTCH — CH CONH ^ j. ^ 2 ^•-CH C 0 C H o  )  3  CO  >  -700  o  2  3  > S  -750  •CH CH OH 2  •8 0 0h  CONH.  l> — CH  -Ch C00 T  2  •85CT  -0 . 3  •0.1  0.1  0.3  0.5 a'  0.7  0.9  1.1  1 . 3  Figure  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. solutions of the S e r i e s I compounds a g a i n s t a * ( T a b l e V , p a g e 4 9)  104 a  j  using  equation  a*  =  (34)  t  substituent  complete  =  0.251  X-CH  w  COO"  log(K  constants  original  used  are  a*  listed  H  (49)  available  values.  7  of  polar  3  (48)  +  H  are  from  7 2  Charton's  work  than  76 '  i n Table  +  /K )  69 from  list  (34)  2  j  a more  O-j/0.45  X-CH COOH 7  a  since  V  The  values  in Section  of  3.1.2  a*  which  (page  were  49).  A  7 2 value  of  a  substituent identical  to  carboethoxy -3 2 x 10 H., 5 mv.  for  i s not  given  and  a  so  the  by  value  Charton of  0.34  for has  the  been  carbo-i-propoxy  assumed  (c* =  .76),  values  r e p o r t e d f o r the carbomethoxy and 72 -2 substituents. A t c o n c e n t r a t i o n s o f 10 M. half-wave  every  p o t e n t i a l s were  compound  so  these  reproducible  p o t e n t i a l s are  to  and  within  plotted  as  - 5 single was  not  shown  the  points always  for  each  as  good  compound  16  and  and so  17.  Near  10  statistical  in Figure  18.  be  was  sufficiently  measured  background),  so  (this the  wave error  distinct  Only  appears for  this  for as  at  the  a  precision  limits  are  polarogram -5 10  half-wave  compound  t 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 m S e e f o o t n o t e on p a g e Section 3.1.2  one  ion  the  only  M.,  error  l-carboxymethyl-3-carbamoylpyridinium  trations to  in Figures  M.  of  concen-  potential  shoulder  on  has  approxi-  been  compounds.  the  105 Inspection pattern  of Figures  1 6 , 17 a n d 1 8 , r e v e a l s  at a l l concentrations,  the only  exception  a  common  being  the  -2 carboxy  compound  off  of the best  has  a more  dicted)  Two  p o s i t i v e half-wave  of the Series  consistently  (which  v/ould  h a s a more  be p r e d i c t e d ) .  free  fall  t h e methoxy compound  p o t e n t i a l than  would  i n the l i n e a r  compounds  line;  compound  p o t e n t i a l than  be s e e n  reactions  M.  least-squares  and t h e a c e t y l  half-wave will  a t 10  energy  I pyridinium  be  pre-  negative  Similar  patterns  r e l a t i o n s h i p s of  salts  (which  ( I I I ) and  other  dihydro-  pyridines (IV). H  H  N 1  R,  (III)  (IV)  R  The similar  slopes  of the plots  to the slope  in Figures  o f +220 mv. 55  reported  salts  +300 mv.  f o r the v a r i a t i o n s of the half-wave  of  smaller  for a plot  1-alkylpyridinium reported  b u t much  1 6 , 17 a n d 18 a r e  a series of l - ( 3 ' or 4'-substituted  than  of  the slope  three of  potential  p h e n y l ) p y r i d inium  salts  55 (XXVII).  Although  the slope  (XVLT)  of plots  of half-wave  potential  106 a g a i n s t ff* v a r y  with  the  concentration  (Figures  16,  17,  and  18)  -3 reacnmg seem the  a  to  minimum  be  large  results  of  pyridinium from  the  Suman  salts  plot  used  of  the  and  Wells  the p*  ence  could  in  the  slopes  in  will  be  also  have  used  in  variation  the  results  from  possible Figures  plots  of  the  does  difference the  not  betv/een  1-phenyl-  explanation  16,  1-alkylpyridinium  the  has  suggested  of  esters  comparable be  often  due  to  caused  17  and 55  salts  18  comes and  in  whereas  phenvlpyridinium  half-wave  In  order  to  of  the  (44) , t h e  plots  CinF 2 . 303RT  not  for  salts  as  reactions.  much  as  50  16,  v/ave m u s t  17 be  The  and  18  p  using  described  ' 1/2 ' ~~~Eo  by  differ  conditions  millivolts 40 salts.  pyridinium  reaction constant  to  ensure  i n experimental  p o t e n t i a l s of  in Figures  comparable  automatically  other  differences  c a l c u l a t e the  polarographic  =  does p  to  p*  t h a t making  v a r i a t i o n s of  reported  P  the  this  for  Another  three  hydrolysis  that  which  and  was  0"  fl.,  1G  (XXVII) . for  0*  10  account  (XXVII).  55  p  to  study  that  O  2 x  enough  this  fact  Zuman's  near  from  the  equation equation  (10)  .. . A  (  4  4  )  RT E  and  the  =  E  l/2  transfer  +  ^¥  ^  n  [  (  i  d  coefficient  -  +  a  dO)  must  be  independent  of  t The of a  t r a n s f e r c o e f f i c i e n t , a , c a n be c a l c u l a t e d f r o m p l o t of E vs. l o g ( i - i ) / ( i ) by t h e equation: d  the  slope  107 substituent  effects.  The  former  polarographic  waves  recorded  mately  polarographic  requirement -5  a t 10  M.  i s n o t met  and  i s only  by  approxi-3  met  by  waves  recorded  at 2 x  10  Ii. and  -2 10  M.  (see F i g u r e s  10a  that  of  the t r a n s f e r  coefficient  by  a l l compounds  coefficients  and  at either  cannot  be  11a).  2 x  The  being  10  second  requirement,  constant,  II. o r 10  calculated  i s n o t met  II.  (Transfer  for polarographic  waves  at  -5 10  Ii.s i n c e  log(i  -  these t  i)/(i)).  requirement,  calculated  (a  = 0.S  f o r each  at 2 x  10  (44) t h e n  J  curved  values of  M.  '  plots  the f a i l u r e of  t h e two  and a  to meet  higher  = 0.67  The  f o r the data  agreement  tions been  used  ence  between  which  The are t  See  M.  i n the c a l c u l a t i o n ,  t h e p* v a l u e s  v/ere  polarographic  not described  footnote  on  by  and  2 x  and  used +2.2  in are  '  10  M.,  a t these  respectively.  two  the a p p r o x i m a t i o n s and  considering  AE^^/Aa*,  in Figures  concentrathat  t h e 15% 16  and  have differ17,  from  calculated. waves  equation  previous  and  -3  considering  the s l o p e s ,  second  coefficients  " M.)  [  b e t w e e n t h e p* v a l u e s  is striking,  the  p * o f +2.1  1/2 AO  a t 10  against  concentrations  a t 10  - 2 found  of E  the t r a n s f e r  reaction constants  anF 2.303RT  P  give  Despite  i f average  are  equation  waves  a t low c o n c e n t r a t i o n s ( 1 0 ) , thus  page.  preventing  (10 ^ the use  M.)  108 of  equation  (44) t o d e t e r m i n e  a reaction constant  at  this  RT E  =  E  l / 2  +  ~  £  n  (  A P  gnF ~ 2.303RT  concentration. polarographic well  described  E  by  "  i)  / ' '> (  (10)  ±  P  1/2 ' Ta  However, waves  d  i  (  i t was  recorded  equation  = E° + £ |  ln[(i  found  4  )  (Section  3.5.1)  a t low c o n c e n t r a t i o n s  (18)  d  4  -  (i.e. plots  i ) / ( i )  of E  ]  (18)  slopes  near  2  /  3  that  were  the  quite  against  2/3 log(i value  - i)/(i) o f 60 m v . ) ,  reversible [reaction  related  were which  reduction (41)].  The  order  to determine  to  use the standard  with  i s the equation  followed  by an  intercept,  to the standard  In  linear  irreversible  the r e a c t i o n  potential  by  dimerization (18) i s  equation  c o n s t a n t one  potential for  theoretical  one-electron  £°, of equation  reduction  reduction  for a  the  each  would  (19) . like  compound  since  this  constant into  quantity  by  equation  (42) , t h e  P  E  =  °  To  of  combined  <f =  (50).  Combining  constant  The  to  the  equations  can  be  (  last  1  term  RT _ _  /  )  7  AE° Aa  '  (  the  standard  reduction  (18),  equations  TTDT)  (7)  from  the  77  equation  (f  and  32  standard  v/ith  (32)  calculated  K  the  equilibrium  < >  s u b s t i t u e n t s on  e°,  related  Alog K — A O —  nF 2. 303RT  calculate  cept, and  of  (7).  reaction  « ££  P  effect  is directly  4  2  5  )  reduction  potential  equation  (19)  can  (16)  (20)  to  and  5  potential.  from  the  inter-  be  rearranged  give  equation  (16)  2  of  equation  £nr D  3  /  2  k  d  (50)  RT - —  £n  is a  7  2 3 ( y  numerical  1  /  )  2  x  -fr _ -  constant  3 / 2  (50)  110 with  the value  (18)  i s expressed  the  drop  under and  T of the dropping  experimental  control),  i ,  a  from  i n Table  = 607 n C D  1  /  T  1  /  constants  radicals,  NAD  The d i f f u s i o n  6  m  2  equation  [equation  Unfortunately, dimerization  /  (51)  3  C  = concentration of electroactive  of electrons  flow  rate  coefficient  T = drop  (sec.)  (Xllla)  been  measured  transferred species  (  (mgm./sec.)  D = diffusion  only  constant,  coefficients,  = number  have  contains  (which i s  n  time  i n equation  term  electrode  the I l k o v i c  m = mercury  rate  The second  the d i m e r i z a t i o n rate  XII.  2  the c u r r e n t  mercury  coefficient.  c a n be e s t i m a t e d are listed  when  i n microamperes.  the d i f f u s i o n  which 51)]  time  o f +21G m i l l i v o l t s  (cm. / s e c . )  f o r two o f t h e  pyridiniu  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  (XHIb).  )  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 the P y r i d i n i u m Ions E s t i m a t e d from t h e I l k o v i c Equation  N '  I  R.  R.  DxlO (crn sec . ) 2/  CH  COO  CONH  CH. CH CH 2  OH  CII OCH 2  3  CH COCH 2  CH  -i  COOCH  3  CH COOCH(CH 2  CH  CN  CU  CONH  )  ?  n  37  £n(D) (mv)  .89  -50  .52  -52  .52  -52  .42  -53  .42  -53  .37  -54  .32  -54  .51  -52  .46  -53  CH CONH  COCH.  .54  -52  CH CONH  CN  .43  -53  CH  CONH  F  .49  -52  CH  CONH  H  1. 05  -49  CH  CONH  0~  1.17  -49  2  2  Special  H NOC-/ 2  compounds:  ^  VcONH  . 93  •50  2  . 23'  -56'  Table  XII  (cont.)  DxlO 2 (cm s e c .)  1.97  RT 3F  £n (D) (mv)  -46  .49  -52  NAD  ,21  -56  NMN  78  -50  *  Calculated  for a  two  electron  reduction  113 The  diffusion  coefficients  carbomoylpyridiniurn  o f KAD  and  +  ion ( n i b ) i n various  l-methyl-3-  b u f f e r s have  been  CONH  2  (IIlb)  previously herein  reported  to c a l c u l a t e  respectively,  Since  on E° v a l u e s  E  a  nF 2.3 03RT  cannot  used  "d n  standard  reliable  reaction  reduccon-  obtained. with  RT  „  -J71 - I F  l n  n  their  equation  (42) t h e n  2 , 3 , 1 / 2 TT  7 7 (  —  }  (  >  0 )  (  c a n be d e t e r m i n e d  on t h e i n t e r c e p t  £n(i  these  a r e unknown  AE° * Aa  constant  = e ° + |I  be  (50) i s combined  i  constants  and, hence,  are not available,  J¥  substituents  dimerization rate  compounds  equation  reaction  been  E° o f  RT  the  have  reduction potentials  potentials  H  values  f o r the standard  the remaining  If  and average  6  ( v s . N.H.E.)  for  p based  6  o f - 6 5 0 a n d - 7 3 0 mv.  compounds.  stant  '  7  the values  two  tion  4  d  from  the e f f e c t  e° o f e q u a t i o n  - i ) / ( i )  2  /  (18) a n d o n t h e  3 (  of  1  8  )  4  2  )  114 dimerization n  in  equation  . _  all vary  has  using  set  i Aiogr / 3  "  eliminated).  Equation  drop  compounds,  and  since  the  diffusion  significantly  among  the  NAD  Table  diffusion in  XII,  i t can  coefficients the  time  be  seen  result  diffusion  T  and  the  D  1 / 2  the  numerical  ]  (52)  can  ( 5 2 )  will  be  be  D,  compounds.  that  the  term  do  for not  (In  variations  in differences  coefficient  simplified  constant  constants,  model  +  (52)(where  Aa  the  of  1  d  since  4  to  ^ognc /( )  2  Aa  3  equation  equal  (53)  millivolts and  by  been  " Acr  been  k  equation  column the  has  A E !  2 .303RT  constant  constant  (42)  F  P  into  rate  of  of  only  in a  equations  few (50)  (52)'. )  The  second  term  one-third reaction  * Aa  2.303RT the  reaction  [equations  precisely  but  constants  that  can  equation  A£l  F P  of  A  l  °  g  constant,Q*, ^d and  k  (55)]  estimated  from  is  equal  to  d  Aa  "3  (54)  be  1  (53) , v / h i c h  1  for  the  cannot the  be  two  ' dimerization calculated  very  dimerization  rate  t are  known  once  a  a*  value  is  estimated  for  the  t Usually f a i r l y small reaction constants have been found f o r r a d i c a l r e a c t i o n s i n v / h i c h t h e r e i s no n e t c h a n g e 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 ^ b u t w i t h o n l y tv/o r a t e constants known, i t i s i m p o s s i b l e t o know how w e l l the d i m e r i z a t i o n rate constant i s c o r r e l a t e d by a * and, t h e r e f o r e , whether 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 . 7  11.  CONH,  H NOC  CONH.  2  H H N  N  ( 54)  Alog  Aa* 1-substituent  (55)  of MAD .  The p o l a r o g r a p h i c  +  of  IJAD  of  the 1-carbamoylmethyl(IIIi),  +  ( l a , p a g e 128)  i s similar  half-wave  to the half-wave  potential  potentials  1-acetonyl-(IIId),  1 - m e t h o x y m e t h y l - ( I l l e ) , 1 - c a r b o - i _ - p r o p o x y m e t h y 1 - ( 1 1 1 g) 1-carbomethoxymethyl substituents  compounds  a l l have  0*  (Illf)  substituent  of Scries constant  d:  P  values  =  ONH,  (adenosine  value  CH COCH 2  3  CH.OCH 2 3  f:  CH  COOCH  g:  CH  COOCH(CH) 2  i s tentatively  assigned  group  t o t h e 5*-  (XXIX)  attached  NH,  OH  near  CH CONH  pyrophosphate)-ribosidyl  HO  These  e:  (III)  + 0.7 a n d s o t h i s  I.  and  (XXIX)  HO  OH  to  116 the  pyridine  of  -0.1  value  to  of  now  be  be  p*  hydrogen  The  r e w r i t t e n as  by  (56)  =  e  plots  .  i  +  of  for  of  °-°  +  E  8 2  values  can  J  against  £°  0*  for  in Figure  Substituting p*  of  +2.2 see  +3.7  that  was  i f this  10  ^ M.  this  which  calculating at  the  now  p  l  o  56  value  M  -0.1.  electrochemical  determined  (18)  g  of  the  be  can  .  i  found  between  be  i ) / ( 1 )  from  I  19  give  and  equation  determined  */3,  at  higher  and  12)  were  used  this  Table  the  has  IX)  the  M.  are  at  a  of  -218  reaction from  The  plotted  0  gives  two  10  Ae /Acr*  different  reaction constant,  (Figure  2 x  concentrations to  the  ( 1 8 )  (III)  slope,  (56)  significantly  (56).  from  2/3 ' ] and  and  compounds  equation  d i f f e r e n c e i s due the  M.  a  reduction  -4  10  Series  into is  has  < >  log [( i , - i ) / ( i ) d compound (see T a b l e V I I  concentrations of  can  k,/Aa* d  -5 various  This  with  (53)  Alog  3  against  each  found  Equation  i n which  for  equation  l  sometimes  p* d  Ae° ' AcT*  i t s estimated  e°  reaction constant  dimerization reaction.  situation  reaction constant  E  done  a  the  equation  F 2 . 303RT  =  intercept  been  low,  concentration  linear  for  the  abstraction reactions.  replaced  low  allowing  +  estimated  atom  The at  i n NAD ,  i s very  P  been  ring  the -3  (>10  different  mv.  constant value  M.).  of  To  methods  half-wave p o t e n t i a l s  [via equation  (44)]  to  of  Figure  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 t h e S e r i e s I c o m p o u n d s a g a i n s t a * ( T a b l e V, p a g e 4 9 )  corr.  coeff.  A e ° / A c 7 *  -500  P*  =  =  IX) o f  = .978  216  mv.  +3.7 -CH C 2  1/1 >  •600  CH COOCH 2  CHOCH.  >  3  -CH COOCH(CH ) 2  •CH C 0 N H 7  •700  •800  -CH CH 2  -CH C00  N  •CH.  -0.1  0.1  3  (J  OH  2  •0.3  2  2  •CH COCH 2  3  I  0.3  0.5  a  0.7  0.9  1.1  1.3  118  (III)  AF D = •  estimate  «nF 2.303RT  1/2 ' ~Ko  the reaction  centrations,  usinq  (44)  constant  an  f o r the r e d u c t i o n a t low  estimated  transfer  coefficient  con-  a of  t unity.  The  which  a reaction  equation lated  slope,  from  *  for  the acid  [equation  agreement  *  +  0  with  with  dissociation  i s -214 mv.  calculated  the value o f +3.7  from  [using  o f p*  calcu-  f o r the  (56)  3  of pyridinium  agreement  18  c a n be  A p* v a l u e  A£° ' Au^  reduction  i n good  of Figure  o f +3.7  (56).  F 2.303RT  =  /Aa*,  constant  equation  polarographic also  2  (44)] i n good  P  is  AE  salts  [reaction  t h e p* v a l u e  of protonated  o f +3.8  methyl  (41)]  reported  amines  7 6  (57)].  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 f r o m t h e s l o p e s o f p l o t s o f E v s . l o g ( i „ - i ) / ; i  at  n  i n - t  10  , !  S  e  P  l  °  t  S  C  U  r  V  G  j * . concentrations.  d  ° P ° l a r o g r a p h i c waves r e c o r d e d The a v e r a g e slopes of the curves f  r  (  i  )  119 CON  ^>  . e  ^*^CONH M  2 0  1/  (41)  To  summarize,  then,  a reaction  been  calculated f o r the polarographic  high  concentrations  calculated Although values, These  the d i f f e r e n c e  results  which  at  has been  concentrations.  t h e two a p p e a r s  mechanism will  o f +3.7  i n the c a l c u l a t i o n  o f both  t o be  real.  f o r the polarographic  be g i v e n  The E f f e c t s o f 3 - S u b s t i t u e n t s Reduction Potentials The  equations  1-substituents  applied at  betv/een  a dual  a concept  used  a t low  process  further  consideration  the Discussion.  3.5.4  of  imply  constant  process  were  o f + 2.2 h a s  reduction  a reaction  f o r the reduction  approximations  reduction, in  while  constant  developed  i n Section  on the p o l a r o g r a p h i c  to the 3 - s u b s t i t u e n t s .  the electrode,  on t h e P o l a r o g r a p h i c  [reaction  3.5.3 f o r t h e e f f e c t  reduction  For the r e d u c t i o n  (41)],  one would  can also  be  occurring  expect  a  , which m  (41)  CH CONH 2  (XXII)  2  CH CONH 2  2  120 is  defined  from  [reaction  the i o n i z a t i o n  (58)] to provide  of 3-substituted  the a p p r o p r i a t e  set of  + COOH  constants.  The  reaction constant  of substituents  Series  I I compounds a r e met:  P  by  =  equation  E  approximately  c a n be  the half-wave using  1/2  +  substituent  H  (58)  AE  c a l c u l a t e d from  (44)  waves  i f two  must  be  t h e same  described  (44)  (b) t h e t r a n s f e r c o e f f i c i e n t s  £  require'  1/2  Aa  cln¥  the  p o t e n t i a l s o f the  equation  (a) t h e p o l a r o g r a p h i c  (10) a n d  E  on  (XXII)  anF 2 . 3 03RT  =  acids  COO"  effect  ments  benzoic  n  (  i  d  a  must  be  (10)  "  f o r a l l compounds  i n the s e r i e s .  At  -5 concentrations  near  10  M.,  the f i r s t  requirement  i s n o t met  for any o f t h e 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 (44) f o r t h e l o w - c o n c e n t r a t i o n d a t a . At 2 x 1 0 M. and - 3  10  -2  M.,  the f i r s t  requirement  i s n o t met  by  the  unsubstituted  121 t 3-fluoro-, could higher  or 3-hydroxy-compounds.  n o t be d e t e r m i n e d  f o r the 3-acetyl  c o n c e n t r a t i o n s ( s e e page  carbamoyl transfer  compounds  compounds. half-wave constant  are left  coefficient Thus,  were  67), only  found  cannot  f o r the e f f e c t  half-wave  compound  i n this  values  work  I I compounds  be u s e d  potentials  a t these  t h e 3-cyano  and d i f f e r e n t  f o r the Series  potentials  Since  a n d 3-  of the  f o r these  two  (XXII), the  to determine  the reaction  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  reduc t i o n .  CONH,  CH CONH, 2  (III)  (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 h a v e a p K a 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 t h e p r o t o n a t e d c o m p o u n d ( n i n ) b u t a wave c a n b e o b s e r v e d f o r t h e u n p r o t o n a t e d c o m p o u n d (XXX) a b o v e pH 7. I t i s t h i s f o r m o f t h e 3 - h y d r o x y compound w h i c h i s b e i n g discussed.  CH CONH 2  (Illn)  2  CH CONH 2  (XXX)  2  122 As  with  the S e r i e s  I compounds  (III),  polarograms  record-  -4 ed  at concentrations  (18),  which E  by  an  = E° + ——  F  that  2 x 10  i s the expression  irreversible  3.5.3  below  dimerization.  and  the d i m e r i z a t i o n r a t e  F 2.3 03RT  P  Alog P  The is  d  effect  from  /  l  o  g  k  C° by  (  (  on  3  )  the e f f e c t be  this  reaction constants  been  observed  a b s t r a c t i o n s from  the value  c a n be c o n v e r t e d  F ~ 2.303RT  into  Ae° * Ao~ m  used  on  the r e s e r v a t i o n that i n hydrogen  o f -0.1 equation  +  '  5  )  0  3  constant  p* o f -0.1 c a n be  of 1-substituents  tentatively  5  the d i m e r i z a t i o n r a t e  with  m  (18)  d  Aa  but a r e a c t i o n constant  Accepting  the  equation (53).  value  P  from  k  of 3-substituents  will  followed  in Section  of equation  This  (53)  shown  c a n be c a l c u l a t e d  constant  A  equation  (18)  3  the i n t e r c e p t  Ae^ 1 ' Aa " 3  by  reduction  ~ A a  =  n o t known,  mated  2  I t has been  the r e a c t i o n constant  o f s u b s t i t u e n t s on  are described  for a reversible  An(i. - i ) / ( i ) d  effect on  M.  f o r Alog  f o r Alog (59) and  esti-  reaction.  k,/A0 , b u t d m as high as 4 phenols. k^/Aa^, this  have  82 equation  equation  < > 59  can  123 now of  be u s e d  to determine  the reaction  constant  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  The  values  listed  of the intercept  i n Table  20,  with  The  slope,  IX  (page  the s t a t i s t i c a l Ae°/Aa  reduction  c° o f e q u a t i o n  84), are plotted error  limits  , o f +745 mv.  potential.  (18) w h i c h a r e 0  against  f o r each  gives  f o r the e f f e c t  i n Figure m compound shown.  a reaction  constant  p m  m of  10.6 u s i n g  reaction  equation  constant  still  n o t be v e r y  l/3p^  or ^1.3.  the  p* v a l u e  reaction  r  (59) a v a l u e  which  i s so l a r g e  of 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 significant  This  o f +3.7  and a l s o  reaction  since  constant  f o r the effect  larger  than  the c o r r e c t i o n i s much  that a would  term i s  larger  than  of 1-substituents  the f a i r l y  substantial  dissociation  constants  on  this  value of  78 +5.9  reported  f o r the acid  P  CH CONH 2  CH CONH  2  +  2  2  5  = +10.8  (41)  2  I  • N C H  of  C H 2  ( 63 )  5  79  Figure of the rr  20. Plot S e r i e s II  124 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 e° compounds a g a i n s t m e t a - s u b s t i t u e n t constants  f! 1  -0.6  -0.4  -0.2  0.0  0.2 a  m  0.4  0.6  125 protonated  pyridines.  I t i s , however,  comparable  to the p  79 of  13.4 o b t a i n e d  transfer  by Kosower  absorbance  alkylpyridinium  maximum  a  m  are plotted t  h  .  c  o  r  r  reaction  e  l  a  t  i  i .  o „  a  The improved  there  i s a resonance  Since  resonance  Jr  between  i n this  electron  interaction  larger  than  seem  to indicate  that  involving  the  3-substituent.  and t h e r i n g  i nthe  the resonance product  CH CONH 2  inter-  (XXXI) .  2  (XXXI)  has been  and t h e 3-carbamoyl  carbamoylpyridine  li5htly  against  12.4 r a t h e r  i n the r a d i c a l  (XXII)  of  case,  i s not possible,  2  potentials  .  a  3-substituents  be o c c u r r i n g  2  21, i n s t e a d  Mly  interaction  CH CONH  A similar  II polarographic  c o r r e l a t i o n would  i o n (XXII)  must  4-substituted-1-  , as i n Figure  p i s found  10.6.  action  i f the Series  against  constant  pyridinium  o f 3- a n d  of the charge-  iodides.  Interestingly, e°  f o r the v a r i a t i o n  radical  proposed  group  (XXXII)  8 0  between  the lone  of the l-methyl-3prepared  by p u l s e  radiolysis  t The standard reaction 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 resonance 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 centre which i s n o t present with the a standard reaction. m  126  a  p  127 A  s t a b i l i z a t i o n of  3-substituents ly  charged  the  radical  coupled  pyridinium  extraordinarily on  the  large  polarographic  with ion p  may  for  products  the be the  reduction.  by  electron  destabilization s u f f i c i e n t to effect  of  the  of  withdrawing  the  explain  positivethe  3-substituents  128 3.6  Reduction  Potentials of Pyridinium-Dihydropyridine  Reduction systems  were  potentials of  determined  Half-cells  pyridinium-dihydropyridine  potentiometrically using  t h e method  14 developed between and  by R o d k e y . the c e l l  reduced  (2)]  where  potential  compounds  RT  = E°  nF  5.8.)  i s given  by t h e H e r n s t reduction  o f NAD  (2)  ( I a ) , shown  +  relationship of oxidized  equation  potential  £n[Red]/[Ox]  For the r e d u c t i o n  The  and the c o n c e n t r a t i o n s  E° i s t h e s t a n d a r d  E  cell.  (seeSection  [equation  o f the half-  69  i n equation (1),  CONH,  •o-  HO  NAD  at The  +  + H  25°, equation validity  half-cell  OH  +  + 2e  ^  —  of equations amply  = E° - 29.6  OH  NADU  (1)  (2) c a n b e r e w r i t t e n  has been  E  HO  (Ia)  as i n equation  ( 6 2 ) 30  (1) a n d ( 6 2 ) f o r t h e NAD -NADH +  proven.  3 0  Equation  log[NADH]/[NAD ][H ] +  +  (62) c a n be  (62)  129 rearranged allowing  to e q u a t i o n  the r e d u c t i o n  E  = E°  E °pti '  E  mined  as  (63)  -  = E^j  the  -  -  intercept The  +  -  into  a t any  29.6  equation  pH,  E°' pH  (65),  t o be  l o g [NADH ] / [ N A D ]  (63)  +  (64)  l o g [NADH] / [ N A D ]  (65)  +  of a  thus  deter-  29.6(pH)  29.6  log[NADH]/[NAD ].  then  potential  29.6(pH)  = E°  and  plot  validity  of c e l l of  potential,  equation  (63)  has  E,  against  been  + tested  f o r t h e HAD  plotting  the measured  + logfPy  model  compounds  cell  used  potential,  E,  in this  study  by  against  t ]/[PyH]  as  shown  i n Figures  22  and  23  methyl-3-carbamoylpyridinium  chloride  (Hie)  ethyl)-3-carbamoylpyridinium  chloride  (IIIc),  for and  1-nethoxy-  1-(2'-hydroxy-  respectively.  (Ill)  T h e s y m b o l s Py a n d PyH r e f e r hydropyr idine respectively.  to  the p y r i d i n i u m  i o n and d i -  Figure  22.  •0.4  P l o t of 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 1methoxymethy1-3-carbamoylpyridinium chloride +  -0.2  -0.1  0.1  l o g [ P y + ]/[PyH]  0.3  0.5  0.7  Figure  23.  P l o t of 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 ] / [ ] 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 carbamoylpyr idinium c h l o r i d e . +  P  pH  =  9.7,  0.1  corr.  coeff  slope  = =  M.  =  29.7  glycine  y  H  buffer  .967 mv.  -441  mv.  CH CH OH 2  •°-  6  - ° -  4  "0-2  0.0  0.2  log[Py ]/[PyH] +  0.4  2  0.6  0.8  1.0  132 The  slopes  o f these  Table  XIII,  slope  o f 29.6 mv.,  to  t h e NAD  +  E  cepts  model  of  listed  quoted  activity  (decomposition been  potentials  equation  MAD  +  equation  (66) a p p l i e s  as t o NAD .  The  +  give  +  XIII  lists  made w i t h  column  a t pii 7  of Table  reactions  be d i r e c t l y  of pyridinium  i n Sections  ions  3.1 t h r o u g h  a t a pH o t h e r  than  (67) w h i c h  The v a l i d i t y  compounds  used  a t pH 0  potentials a t pii 7  dihydropyridine  3.3) .  Reduction  i s a modified  be  form  (67)  of equations  = E° - 29.6(pH)  model  than  measured and  compounds  7 can theoretically  = F ° ' - 29.6(pH-7) pH  (64).  The  the poten-  important  ion) but the reduction  cannot  XIII.  compound.  ( p h y s i o l o g i c a l pH) r a t h e r  o f hydrogen salts  the reduction  t h e pH's a t w h i c h each  inter-  (66)  log[Py ]/[PyH]  t o pH 7 b y e q u a t i o n  E°' pH  the  that  column o f  to the theoretical  potentials of biologically  measured  EV 7  of  were  discussed  corrected  close  as w e l l  i n the f i f t h  of Table  many p y r i d i n i u m  have  quite  confirming  compounds  measurements  (unit  thus  of E against  E ^  column  often  cases  i n the t h i r d  +  Reduction are  listed  = E ° ' - 29.6 l o g [ p y H ] / [ P y ]  potentials  tial  a r e i n most  of plots  fourth  plots,  (64) a n d (67)  (64)  i n this  study  was t e s t e d  using  for  TABLE Reduction  Potentials  XIII  forPyridinium/Dihydropyridine  Half-cells  I R  R  l  R  CH CH  COO* 3  CHjCH CH  2  CH  OCH  OH  COCH. 3  CH  COOCH(CH  CH  CN  CH CH  2 2  CONH  CONH„ 2  CH CONH 2  2  pH  E° ' PH (mv v s N . H. E. )  TP O 1 7 (mv v s N . H. E. ) L  E° (mv v s N. H.E. )  std. dev(mv)  CONH „ 2  -34 . 6  10 .  -43 9  -350  -143  5.0  CONH  -22.3  10 .  -463  -374  -167  7.4  CONH  -29.7  9.7  -441  -361  -154  4. 1  -29 . 5  8 .8  -357  -304  -  1.1  -26.2  8 .8  -381  -328  -121  4 .5  CONH  -24 . 7  7.0  -318  -318  -111  2 .9  CONH  -28 . 3  5.6  -22 3  -263  -  1.7  CONH„ 2  -29.2  9. 2  -385  -320  -113  1 .6  COCff  -27.3  8 .8  -331  -278  -  71  1.6  -31.9  7. 0  -225  -225  -  18  3. 7  CONH )  s l o p e ooff E v s l o g [PyH]/[Py + ] (mv)  3  CONH  3  1  CN  2 2  3  97  56  CO  Table  XIII  (cont.) slope  of  [ P y H ] /  log  E  vs [ P y  +  PH ]  (mv)  NAD NMN  +  ( I a , page  128)  ( I I , page  128)  ONH >  N  N  = /  2  E ° £  (mv  E ° *  vs  (mv  E°  vs  (mv  vs  Std dev  N . H . E . )  N . H . E . )  N . H . E . )  8.8  -365  -312  -105  5.7  30.3  8.9  -358  -302  -  4.5  -  6.9  -279  -282  95  -  (mv  15.  135 l-carbamoylmethyl-3-carbamoylpyridinium between  pH  7.7  a n d 9.6  a s shown  chloride  i n Figure  24.  (Illi)  The t r i a n g l e s  ONH,  CH CONH 2  2  (Illi)  represent  p o t e n t i a l s measured  potentials  measured  pH ' s g r e a t e r at was  lower  than  in Tris  9.8  tended  pH's and were  encountered  potentials  again  in glycine  buffer.  less  made  i n the measurement  of 1-carboxymethy1-(IIIa),  between  p l l 9.7  and 10.4).  reproducible  not included.  (This  of the  ions  reduction  which  The r e d u c t i o n  :ONH,  a:  R  1  b:  and  had t o  potentials  = CH =  tha  problem  1-methyl-(Illb),  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 be  t h e diamonds  P o t e n t i a l s measured a t  t o be much  therefore  buffer,  COO  CH.  • N  I  CH CH 9  R  OH  (Ill )  at  each  plotted  pH, E ^ J , w e r e against  pH  calculated  i n Figure  from  24, f r o m  equation which  (63) and  a pH  dependence,  136  .0 -I  9 . 0  9.5 — l _ _  pH dependence of the r e d u c t i o n p o t e n t i a l , l-carbamoylmethyl-3-carbamoylpyridinium chloride  Figure of  a. 5  24.  The  pH  E  pH '  137 E°* p ii  = E  AE^/ApH,  of  ment  the  with  (67)  29.6  30.5  log[PyH]/[Py ]  mv.  per  pH  confirmed  be  • O  E°  E°,  and  are  listed  used  to  unit  was  dependence  that  reduction potentials  can  (66)  +  theoretical  Having metric  -  the  of  29.6  p'l d e p e n d e n c e  i s as  calculate  found,  expected,  the  standard  i n good  agree-  mv.  per  pH  the  potentio-  of  equations  (64)  reduction  En O 'I 0  +  2 9.6(pH)  (64)  =  E^'  +  29.6(pH-7)  (67)  reduction potential in  the  seventh  and  at  pH  7,  E°'.  e i g h t h columns  These of  and  potential,  _  the  unit.  values  Table  XIII  respectively. The tions in  the  value  last  of  the  of  P  standard 1  in  E°' pii  from  H  E  "  way. each  2  9  deviation  values. the  =  of  Table  ,  each  the  standard  compound  (66)  was  measured  cell  potential,  used  to  log[pyH]/[py ]  6  was  then  of  calculated  nives  the  a  than  from  better would  measured  devia-  determined  calculate E,  and  a  a  (66)  +  T h i s method  deviations  of  lists  Equation  reduction potentials  standard  XIII  reduction potentials  following  E  E° pH  column  the  estimate be  cell  collection of  obtained potentials  the from  of  error the  about  the  138 best  least-squares The  line  reduction  previously  sively.  compound.  p o t e n t i a l o f tiAD  ( I I I ) has been  +  deter-  32  14 mined  f o r each  '  so t h i s  compound  T h e p o t e n t i a l was f i r s t  was n o t s t u d i e d  determined  -fir  i n this  exten-  study  by  H H  1  -CONH, + H + 2e — T  4> R  CONH,  1  (ill)  ( IV)  NH  2  <'"XJ  O •o — P — O — P •  II  w  o  \_J HO an  II o  OH  oxidative  HO  titration  o f NADU  OH  (IV), similar  t o t h e method  used  14 by  Rodkey.  reduction  Throughout potential  E^J  as  shown  for  NAD  +  the oxidative  (using  equation  titration  ( 6 5 ) ) become  the calculated more  (65)  = E - 2 9.6 log [NADH] / [NAD ]  i n Table  X I V a n d when  polarographically,  contained  two p y r i d i n i u m  ture  with  the second  more  negative  i t v/as f o u n d  salts  pyridinium  polarographic  the f i n a l  s o l u t i o n was that  i n approximately i o n appearing  reduction  positive,  assayed  the s o l u t i o n a 50:50 at a  mix-  slightly  p o t e n t i a l than  NAD . +  TABLE Results  (mM)  from  (NAD ") 4  (mM)  the  XIV  Oxidative  E (mv .)  0. 40  -3 92  0 .93  -368  1. 54  -355  2.02  -334  Titration  E° ' (mv.) Ph -370 -3 61 -3 5 9 -3 51  of  NADH  E°•(mv. -320 -311 -309 -301  14 0 Since  t h e NAD  graphic and/or where  studies t h e NADH  taining  must  be d e c o m p o s i n g  NAD  analyzed  potential  +  a n d NADH  as soon  assay  f o r NAD  again  been  +  converted  approximately  (method  a constant  showed  that  that  after  I n an  The  prepared  con-  and t h e  contents  cell  polarographic  1 5 % o f t h e NAD  pyridinium  the c r y s t a l s  decomposi-  the measured  value.  +  pH o f 8 . 7 ,  made.  of mixtures)  approximately  to another  2 hours  were  s o l u t i o n s were  as p o s s i b l e  had r e a c h e d  t h e NAD  p o t e n t i a l before  +  single  that  i n polaro-  a t the higher  measurements  t h e NAD /NADH  significant,  both  t o be h o m o g e n e o u s  a t pH 5 . 6 , i t was c o n c l u d e d  to measure  became  were  was f o u n d  the potentiometric  attempt tion  sample  +  compound  had been  in  +  had  the  dissolved in  t the  buffer.  for  the reduction  cell  Values  fair  than  rough  agreement  solutions  concentration  c a n be d e t e r m i n e d  better  were  p o t e n t i a l a t pH 7, E ° ' , f r o m  p o t e n t i a l s o f these  dihydropyridine NADH  o f - 3 0 9 mv. a n d - 3 1 2 mv.  these  the measured  only  the t o t a l  and n o t t h e c o n c e n t r a t i o n values  estimates.  with  but since  calculated  c a n be c o n s i d e r e d  Despite  previously  this  reported  the values  potentials  of  a s no are  in  for this  system. When night,  solutions  the c e l l  containing  NAD  p o t e n t i a l s slowly  +  a n d NADH  drifted  were  t o more  left  over-  positive  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 b e 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) t o c a l c u l a t e E ° ' .  141 potentials and  and, a f t e r  dihydropyridine  approximately Since ed  t h e s o l u t i o n s were  compounds,  - 3 0 0 mv. w e r e  the polarographic  only  one p y r i d i n i u m the value  NAD.  potentiometric  the  polarographic  negative product  than being  hydrolysis  showed  ion, this  represents This  reduction  calculated  assay  assayed  f o r pyridinium  potentials, E°',  [equations  that  potential  of E ° ' f o r the decomposition reduction  reduction  f o r NAD  +  are consistent  of the pyrophosphate  NMN  bond  with  i n NAD  +  (la) .  OH  (la)  H 0, 9  HO OH (II)  probably of  pH 8.7  HO  more  the decomposition  (II), arising  HO  OH  contain-  product  slightly  NH  HO  (67)].  p o t e n t i a l o f - 3 0 0 mv. a n d  p o t e n t i a l being  the mononucleotide,  (65) and  the s o l u t i o n  reduction  of  OH  from  14 2 Since the  measured  tials, a  the decomposition  then  cell  the estimated  few m i l l i v o l t s  tial  o f NAD . +  potentials  potential  more  This  determined  +  to d r i f t  value  positive  puts  o f HAD  t o more  o f -312 mv. than  E ° ' very  close  15 mv. m o r e to  positive  the reduction  nyridinium The  forE°  than  that  potential  of  +  poten-  a n d 1The  reduction  (II) i s about falling  very  10 t o close  l-methoxymethyl-3-carbamoyl-  chloride. reduction  potential  of 1,1 -ethylenebis(3-  carbamoylpyridinium  chloride)  (XlXb)  work  i s probably  to the reduction  chlorides.  f o r NAD ,  1  poten-  reduction  f o r 1-carbo-i-propoxymethyl-  o f nicotinamide mononucleotide  causes  positive  the true  carbamoylmethyl-3-carbamoylpyridinium potential  ( l a ) a t pH 3 . 8  f o r t h e two e l e c t r o n  1  reduction  H NOC  has been  o f one r i n g .  CONH,  2  +  H  2€  \  /*  (XXXII)  estimated  i n this  There i s  143  some d o u b t XXXII  as  to the  c o u l d n o t be  a c c u r a c y of  the v a l u e  found  recovered d u r i n g attempted  since  syntheses.  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  by  The  r e d u c t i o n of XlXb  of both but  XlXb  and  in order  pounds had  t o do  to be  o f 9.02  this,  A solution addition tration  An  a t 265  was  nm.  prepared  s m a l l amounts o f  was  i n c r e a s e i n absorbance  wavelength.  Since  near  290  i n these  acid  catalyzed  pyridine ex t e n t .  work f o r o t h e r  way  (5.5  to the  during  Craig  a t 340  at  nm.  o f 12.5  i t was  The  265 was  by  of  nm.  The  then  used at  absorbance  3.1)  that of  the  concen-  f o r XXXII  assumed  (see s e c t i o n  e_t a l .  reduced  mM.  "*"-cm. ^ per p y r i d i n e  extinction  coefficients  found  3-carbamoy1-1,4-dihydropyridines  t o 6.5  mM.  of d i s t i n g u i s h i n g  compound  for  to  this  peak the  the  dihydro-  r i n g o f XXXII was n o t o c c u r i n g to a s i g n i f i c a n 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 12.5 mM. •1 -cm. -1 f o r  comparable  length  com-  i s i n good 90  addition  noticeable  solutions,  XXXII i s a v a l u e o f 6.25 is  no  decomposition  each  i n absorbance  coefficient  t h e r e was  v/hich  partially  after  concurrent  extinction  found  sodium d i t h i o n i t e .  determined  the d e c r e a s e  and  from  nm.  of both  coefficient  r e p o r t e d by  dithionite  an  concentrations  coefficients  extinction  t h e v a l u e o f 8.99  o f XXXII was  calculate  _in s i t u  -1 -cm  o f XlXb  of  extinction  determined.  mtl  agreement w i t h  sodium d i t h i o n i t e .  For  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 ,  -1 XlXb  v/ith  compound  (XXXIII)  dithionite  ^-cm.  ).  the  i t i s probably  titrations  in  which this  at this  wave-  U n f o r t u n a t e l y , t h e r e i s no  between XXXII and  although  ring  o f XlXb  the  fully  reduced  significant acid  that  catalyzed  144  H NOC 2  N  \ /  CONH  2  N  (XXXIII)  decomposition only  after  added. one  On  would  readily seems  of  the  basis  expect  than  that  Thus  3.6.1  the  be  reaction on  determined  brium  the  true  substituents  constant  P  =  equivalent  inductive to  of  until  Substituent The  1  undergo  a  from  the  the  observed reduction  has  p o t e n t i a l of  constant  is a  the  (see  p and  effect  of  does  (32)].  section  XXXII not  been  been  more  so  i t  reach  almost  probably  3.1)  sigfully  reasonably  XlXb.  1-position  for of  had  decomposition  ring ring  noticeable  dithionite  p o t e n t i a l s are  the  reaction  became  of  acid  ring  E f f e c t s at  [equation  AlogK  second  first  ring  effects  1,4-dihydropyridine  reduction  reduced.  of  XXXIII  the  levels  to  dihydropyridine  approximately  nificant  close  the  measure a  of  effect  reversible reaction  substituents For  the  an  on  the  of can  equili-  oxidation-reduction  (32)  145 r e a c t i o n,  the  reduction  potential,  = — nF  E°  (32) p  and  t o be  equilibrium  Figure  (42) a l l o w s  the e f f e c t  the r e a c t i o n  reduction potentials  error  (listed  (III) are plotted  limits  shown  f o r each  +  H  +  2e~  N  I  against are the  XIII)  a* i n standard  ,H ONH,  N  (IV)  (III)  listed  polarographic  be  i n Table  Cf  'CONH,  compounds  constant  o f s u b s t i t u e n t s on t h e  H  deviations  equations  (42)  I compounds  The  s tandard  potential.  standard  25.  Combining  AE° * Aa  reduction  the S e r i e s  (7).  to the  (7)  from  nF 2.303RT  for  equation  equation  determined  The  E° , by  i s related  £n K  (7) i n t o  standard  constant  i n Table  reduction,  h a v e more  expected.  The  XIII.  was  observed  the carboxymethyl-  positive  best  As  reduction  least-squares  the  and methoxymethy1•  potentials  line  with  than  v/ould  (excluding the  Standard  Reduction I  I  tO  o o  M o  o  I  I  o  o  o o o I  o  Potential,  o  E°  (mv.  vs. I  o  *  r> M  II  \  +  Q  "O  o  >  o  0 H.  ^  *  o  oo  II  ro  0  Ml Ml  0  t>  Q  *  >< M  •  3  II  *  •  CD cr  Ul co  cr ro JU  CD  < Ml *— 1  u>  0  .  H-  3 0)  ro M M  ro c  HM 0 cn 3  1 1  0  Ml  to rt tr  O o  —  cn rt Pi  ro r t  1  o C5  o  Ml  0 o  1—  o  0  & 3 H- &  H H  X o  rt  o  rt  01 H  U)  <  Q  Ch  H*  H3  »  to  CO  o  N . II. E . )  1  0  rt  ro 3  ro r t  H-  cn P> i—• M cn  ro H-  ro Ml  cn  o t  H  o 0  0 h  rt cr  ro  3 TJ -a  id nd  0 •< c Ii cn  9^1  147 1-carboxymethy1  compound)  from  of  which  a  p*  +2.8  has  can  a  be  AE°/Aa*,  slope,  of  calculated using  +83  mv.  equation  (42) .  7 6 This  value  reported  is considerably  for  [equation  the  acid  (57)],  a  smaller  dissociation  reaction  than of  i n which  'CONH,  the  p*  of  protonated one w o u l d H. H  +3.8 methyl  expect  H + 2e" N  (68)  I  similar have  R-NH  substituent  substituents  nitrogen in  H.  +  the  3.6.2  atom  The  data  is  analysis  of  shown  equation  only be  effect  is  H  ( 57)  since to  a  both  reaction  positively  transformed  into  an  (68)  and  charged  (57)  quaternary  uncharged  nitrogen  products.  Substituent  in  Series  +  2  attached  v/hich  a  CON H,  +  P.-N  amines  II  the  the  (XXIii  E f f e c t s at not  effect  At  of  (68). to  a  Of  the the  high  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  to  allow  3-substituent original Py^PyH  3-acetyl-,  pll a s  3-position  sufficient  the  XXIIn)  3-carbamoyl-,  measured.  really  the  as  and 10.5,  on  six  a  the  proper reaction  compounds  reduction  in  potentials  3-cyano-compounds  could  1-carbamoylme t h y l - 3 -  decomposed  (see  Section  of  3.2)  148 too  rapidly f o rpotentiometric  the  reduced  could  forms  n o t be  m e a s u r e m e n t s t o be made, a n d  o f the 3-hydroxy  and 3 - p r o t i o  prepared. H. +  CH CONH 2  compounds  H  2e"  +  H  ^  CH CONH  2  2  (XXII)  (68) 2  (XXXIV)  R„  = CONH  D :  COCH.  k:  CN  1 :  F  m:  fl  OH  When Series of  the standard  I I compounds  +276 mv.  +9.3  (corr.  =  coeff.  =  2.3 03RT  polarographic  better  a^ a  .986) i s f o u n d equation  slope,  from  (42).  AE°/Aa , K  which  a p of m A s was f o u n d with  AE"  n F  substituents  p o t e n t i a l s of the three  are plotted against  c a n be c a l c u l a t e d u s i n g  D  the  reduction  * Aa  < > 42  reduction  p o t e n t i a l s , the e f f e c t  on t h e p o t e n t i o m e t r i c  c o r r e l a t e d by O  p  (corr.  reduction  coeff.  =  o f t h e 3-  potentials i s  .9994)  giving  a  slope  149 of  +317  mv.  from  calculated, the  slightly  plot.  already the  a  which  The  reaction  larger  values  substantial  acid  a  are  dissociation  of  value both  reaction  constant than  of  is  10.7  protonated  of  be  calculated  considerably  constant  can  5.9  larger reported  pyridines K  from than  7 7  the  for  [reaction  (53)]  H  R, +  H  2e~  I^T N  C H  2  C O N H  C H  2  p  =  +9.3  to  2  C O N H  (68) 2  +10.7  ( 60)  p  but work  are  quite  for  the  reduction  of  =  similar effect  of  +5.9  to  the  p  of  +10.8  3-substituents  pyridinium  ions,  to on  [equation  +12.4  the  found  in  this  polarographic  (41)].  (41)  C H  2  C O N H  C H  2  0  =  +10.8  to  +12.4  2  C O N H  2  150 The  Oxidation The  more  of  Dihydropyridines  oxidation  facile  by  flavins  The  rate  Flavins  [equation  oxidation-reduction  pyridines.  by  (69)]  reactions  constants  can  of  i s one  of  the  1,4-dihydro-  r e a d i l y be  determined  I  (69)  anaerobically in  the  by  following  by  presence  of  study  by  the  excess  rate  of  the  In  oxygen. oxidation  the  of  disappearance band  i n the  latter  Both of  rate  been  at  used  various  methods  various  in this  or the  have  been  work.  the  flavin  aerobically 340  presence the  of  nanometer  of  a  flavin used  dihydropyridines  temperatures.  exclusively  of  method,  19-24 flavins  reduction  dihydropyridine,  absorbance  flavin.  reoxidized to  of  the  dihydropyridine amount  following  catalytic is rapidly  previously by  various  26 '  The  former  method  ha  151 The  rate  constants  hydropyridines order  were  found  irreversible  1 ( [PyH] o  [Fi n o  -  where  o  using  ( [PyH] - X) -, = ( [ F i ] - X o j  g  l  [PyH]  =  =  The k  .  of equation  slope The  of this quantity  absorbance oxidized  a t 450  and  nanometers,  (A =  flavin,  -  0  where  „ ,„ reac 0 =  -  as  <> 70  of  of  used  flavin up  a t time t  constant  shown  the second-order  i n Figure rate  which using  i s due equation  solely  26.  constant,  ( 7 0 ) v/as c a l c u l a t e d f r o m  the  to the  (71).  ( 7 1 )  = absorbance = initial  o  , _ „ ^ '"prod  time  on the  prod  £ e  c o n s t  rate  T  A A  +  of reactants order  second  A)  Ul reac  c  i s then  i n equation  reduced  ,, A  X  second  the d i -  t  concentration  (70) a g a i n s t  plot  for a  i n i t i a l concentration d ihydropyr idines  = initial  o  2  (70)  of  the q u a n t i t y  173^3  }  X = amount  side  k  n  [Fl]  left  equation  r e a c t i o n by p l o t t i n g  . l  f o r the o x i d a t i o n o f most  = path =  at  time  absorbance  length  of the absorbance  cell  e x t i n c t i o n c o e f f i c i e n t s of reactant ( o x i d i z e d f l a v i n ) and p r o d u c t (reduced flavin) respectively.  153  The  curvature  at longer  times  of  the f l a v i n  by o x y g e n  leaking  The  rate  of reaction  cyano-1,4-dihydropyridine first  order  pyridine  kinetics,  was  present  concentration k  .  , c a n be  shown  found  below.  The  excess  curvature  cell.  27,  o f more  from  since than  order  pseudo-  the 100  dihydrotimes  rate  times  the  constant,  o f l n ( A - A-,) a g a i n s t  at longer  H  to r e o x i d a t i o n  l-carbainoylmethyl-3 -  pseudo-first plot  due  determined  i n Figure  The a  the  with  (v~k) was  i n an  from  into  o f FMM  shown  o f FMM.  i s probably  time  i s probably  as  due  H  (Vk)  to  reoxidation  Since than  of  the f l a v i n  the c o n c e n t r a t i o n that  of the f l a v i n  d [Fl] dt  where  concentration absorbance  k  k  ob s  by o x y g e n  leaking  of dihydropyridine one  can w r i t e  [Fl][PyH] 2*  = k  v  ob  was  equation  [Fl]  = k-[PyH] 2  of oxidized  by r e a r r a n g i n g  flavin,  into  the  s o much (72).  cell. larger  The  (72)  (73)  [ F l ] , c a n be  (23) t o g i v e  found  (26) and ( 7 4 ) .  from  the  F i g u r e 27. D e t e r m i n a t i o n of the second o r d e r l-carbamoylmethyl-3-cyano-l,4-dihydropyridine p l o t of ln(A-A ) a g a i n s t time.  rate constant b y FUN from a  f o r the o x i d a t i o n of p s e u d o - f i r s t order  w  In  (A-Aa,)  i I O  i  o  to  0 II  , ,  o cr  •~< *—'  II  •  o o to  H-  co U)  3  5  3  x  ro  o o I  cr  in  II  •  c U)  Ln O  3  H-  3  1  1—'  155  x I  = e  reac  C  'reac  Substitution  A  e  = C "prod  reac  C  reac  H(£  [Fl]  *  integrates  £n(A v  can  then  - A ] ~ dt  rate  a second  aerobic  - A  (  -  prod  )  (74)  (29).  = k  obs  The second  order  rate  [A - A ]  constant  (28)  0 0  (29)  k , using equation (73). obs o f o x i d a t i o n o f NADH ( X X I ) b y f l a v i n s  order  authors. rate  o x i d a t i o n o f NADH  constants  (2 6)  from  x. r, , • r e p o r t e d by v a r x o u s found  (28) w h i c h  - A ) = k , t + const. oo ob s  be f o u n d  The  (62) g i v e s  'S l H ^  F1  to equation  d[A  (25)  n  - A,  = ( E  (23)  prod  initial  reac  A  or  c  (74) i n t o  A C  prod  , c. . prod 'initial - £ prod  of equation  oo  E  e  -  (A/I)  c  +  reac  f o r t h e same  2 0 , 22-24 , 26  constant  r  Suelter  , „ ^ , 20 and M e t z l e r  o f 0.75 M "'"-sec  by r i b o f l a v i n  oxidation using  (Xa) near FMN  has been  for pH 7.  (Xb) h a v e  been  the Rate  156  H. H  NH, -CONH, O  I r  r  HO  ~  I  N  •P —O-  '  r HO  OH  OH  (XXI)  determined  aerobically  range  0.092  from  22  M^-sec"  23 24 "' a t pH 7 a n d  and a n a e r o b i c a l l y 1  t o 1.1 M  _ 1  -sec  1  ,  after  being  (X)  CH  CH OP0  OH  2  CHOH  CHOH a:  R =  3  I  CHOH  b :  R  d:  CHOH  R = -CH.  I  CHOH  CHOH  I  CHOH  CHOH corrected  t o 25°C.  using  an a c t i v a t i o n energy -1  Bruice tion  found  o f NADH  a rate  constant  o f 0.43 M  a t pH 7.7 b y l u m i f l a v i n  o f 8.3  kcal/mole.  -1 -sec  f o r the oxida-  (Xd), a f l a v i n  which had  157 previously  been  shown  l-propyl-3-carbamoyl-l,4-  to react with  2  dihydropyridine  (XX) a t t h e same  rate  as does  0  riboflavin.  CONH,  (XX)  The  diverse  facilitate  constants  comparisons  22 authors  rate  '  '  have  also  o x i d a t i o n o f both  and  NADH u n d e r  two  rate  can  the present  work.  do n o t  Several  range.  The  from  As a r e s u l t , between  i n Table  the rate  constants f o r  conditions but the ratios  vary  this  second-order  listed  determined  l-propyl-3-carbamoyl-l,4-dihydropyridine  similar  constants  be made  are  with  i n the l i t e r a t u r e  24 26  the  wide  reported  only  and p r e v i o u s  constants  NADH  these  a discouragingly  order-of-magnitude  study  rate  XV.  65 t o 1 6 0 , a g a i n  of  comparisons  work.  determined  i n this  was o x i d i z e d b y r i b o f l a v i n  study at a  - 1 - 1 rate  o f 0.53 M  with  previous  pH  8 . 1 , FMN  -sec rates  , a value  using  and r i b o f l a v i n  which  riboflavin were  2  i s reasonably 0  and l u m i f l a v i n .  equally effective  1-acetony1-3-carbamoy1-1,4-dihydropyridine. is  very  confusing  consistent  The  2 6  At  at oxidizing literature  o n t h i s p o i n t s i n c e r e p o r t s h a v e FMN r e a c t 20 19 i n g b o t h 50% f a s t e r and 40% s l o w e r than 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  isa TABLE  XV  Order Rate C o n s t a n t s for 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  Second  H  H  ^ 3 R  + flavin  +  reduced  flavin  N  N  * 1  1  1  1 1  R  R  l  R  Flavin  3  CONH  2  Rf 1  pH 9 .8-10 .3  Ch' CH OH 2 2  CONH  Rf 1  9.8  CH OCH  CONH  Rf 1  8 .1-8. 7  Rf 1  2  CH  CH  3  COCH  COOCH  3  CH CN CH  CONII  CH  COHH  CH  CONH  Special  2  2  (M ''"-see - ) 1  64  + 13 .  21  + 2. 6  0 .607  +  .019  8.1  3.70  +  . 24  FMN  8.1  3.70  +  . 28  CONH  FMN  7.8  1 .99  +  .18  CONH  FMN  8.2  0 .150  +  .022  CONH  FMN  7 .8  2 . 97  +  .36  9.4  2 . 98  +  .09  0. 548  +  .019  +  .0032  +  .012  CONH  3  k  o  COCH  Rf 1  8.1  CN  FMN  6. 9  .0449  Compounds:  NADH  (XXIV)  Rf 1  8.1  .53  NMNH  (XXXV)  FMN  8.1  . 249  159 oxidized  at approximately  half  HO  the rate  o f NADH  a t pH 3 . 1 .  OH  (XXXV)  Although  the absolute  rate  constants  a r e very  different,  22 flcCormick dation  et a l  rates  have  o f NADH  found  a similar  a n d NMMH.  3-carbamoy1-1,4-dihydropyridine which  i s comparable  2:1 r a t i o  Riboflavin  oxidizes  a t 150 t i m e s  t o t h e 65 t o 1 6 0 f o l d  f o r the o x i -  faster  rate  1-methylthan  NADH,  enhancements  of  l-propyl-3-carbamoyl-l,4-dihydropyridine o v e r NADH reported 22,24,26 . . previously. 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  studied The  previously.  effect of substituents  on r e a c t i o n  (69) c a n be  (69)  160 measured from  v i a the r e a c t i o n  equation  P  =  substituent for  (35)  Alog Aa  using  a plot  p,  which  of l o g k  i s determined  against  K  Hammett  (3 5)  constants  the S e r i e s  constant,  a.  The  I compounds  logarithms  of  (IV) a r e p l o t t e d H  the  rate  constants  against  the  V,  49)  polar  H  N R.  (IV)  substituent  constants,  Figure  As  ships  of  tials, by  28.  the  compounds. close  the p o i n t  and  dihydropyridine  i n Table  linear  free  falls  Interestingly, of the  page  energy  potentiometric  compound  to that for  (listed  i n the  1-methoxymethyl  i s quite  Excluding  found  the polarographic  the other  NADH  was  a*,  in  relation-  reduction  o f f the  line  the o x i d a t i o n  1-methoxymethyl  potenformed  rate  compound.  1-methoxymethy1-3-carbamoy1-1,4-  (Ve), the c o r r e l a t i o n i s q u i t e H  H  N  good  of  (corr.  Figure  28.  P l o t o f t h e l o g a r i t h m o f t h e second 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 t h e 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* ( T a b l e I V , page Q%). -CH.  corr. = K  c o e f f . = .992 ( e x c l . Alojk^ Aa*  m  _  1  >  CH OCH ) 2  3  g  •CH CH OH 2  2  -CH COCH 2  •CH  H  3  CONH^-CH COOCH 2  3  H  0.*  CONH, -GH OCH  N'  I  R  1  -CH^CN  l.t  -0.3  -0.1  0.1  0.3  0.5 a*  0.7  0.9  1.1  1.3  162 coeff. This  =  0 . 992) , g i v i n g  value  a  i s approximately  found  f o r the p o l a r o g r a p h i c  +3.7)  and  p*  found  (p*  =  approximately  (equivalent  one-half  of  three-fourths  to p*)  of  -1.9.  the magnitude  (one-electron)  f o r the p o t e n t i o m e t r i c  of  of (p*  reduction  the magnitude  (two-electron)  =  of  reduction  +2.8). The  be  slope  Series  determined,  (XXIV  I I compounds  the  i - k ) , lead  f o r which  rate  3-carbamoyl/^3-acetyl-,  to r e a c t i o n  constant  |  constants  and  (p)  3-cyano-compounds  values  k:  CH CONH 2  could  of  -5.4  CN  2  (XXIV)  (corr.  coeff.  log  i s plotted against  k  both  2  .992)  and  the p o l a r o g r a p h i c  derived in  =  from  magnitude  and  -6.1  (corr.  and  has  a  potentiometric  slightly  =  .997)  respectively.  the p a r a - s u b s t i t u e n t and  coeff.  As  reductions,  constants  better  when with the  is slightly  correlation  p  larger  coefficient.  D I S C U S S I O U  4.1  Polarographic  The  Reduction  generally  cal  reduction  3.  The p o r t i o n  ilechanisms  accepted  of pyridinium o f this  jaechanisa salts  (VIII)  scheme which  +• o t h e r i s o m e r s ?  reduction (41).  The  electron rapidly  wave  (wave  pyridinium  reduction dimerizes  I  to give  i s shown  other  i n Scheme t o the  f i r s t  isomers?  3  i n Figure salt  electrochemi-  corresponds  +  Scheme  f o r the  (VIII)  2)  i s reproduced  undergoes  the pyridine  i n an i r r e v e r s i b l e  a  r e v e r s i b l e one-  radical  reaction  i n reaction  (XV)  which  to give XVI.  1 G 4  -e  .N  (41)  I  R_  'III)  An  (XV)  equation  wave  when  has  18).  e°  by  derived "' 3  a mechanism  (equation  graphic  been  The  + —  reduction  plotting  of  as  shown  validity  waves  to d e s c r i b e  3 5  in reaction  of  ln(i  this  i)/(i)  pyridinium  the p o t e n t i a l ,  polarographic  (XVI)  E,  (41)  equation  polarographic  is  ions  operating  i n the  2/1  polaro-  (18)  was  against  at various  the  tested  log(i  concentrations  in this  -i)/(i)" between  work for  10  " ii  _o  and  10  * M. 10  Polarographic -A  below  5 x  (18),  (Plots of  K.  were  waves  reasonably  recorded well  at  concentrations  described  by  equation  2/3  E v s . log(i„  -  u  i)/(i)  were  linear  with  •f slopes  near  the t h e o r e t i c a l  value  f ,  of  60  rav. )  in  agreement  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 ciange i n l o g(i - i)/(i)-/3 t h e r e f o r e have t h e " u n i t s o f millivolts. a  n  d  1G5  with  the  m e c h a n i s t ) shown  in  equation  (41) .  Thus  i t can  be  (41)  (VIII)  concluded reaction less  that  than  not  mechanism  is reversible) at 5 x  At are  this  10  -4  linear  plots  (i , -  i)/(i).  by  v/hen  pyridinium  ion  ( i . e . the (VIII)  electrode  concentrations  M.  concentrations  described  is operating  above  equation  the  10  H.  3  t  (18),  potential,  E,  but  the  polarographic  instead  give  waves  near-  is plotted against  log  t  E  cantly  =  E  larger  reduction reaction.  (60  The  l/2  +  slopes  OF"  £  n  (  i  d  of  the  p l o t s are  mv.)  indicating  (  theoretical  transition  latter  "  than  This  these  an  from  slope  for  irreversible a  reversible  a  1  0  signifi-  )  reversible electrode  electrode  f A l i n e a r p l o t o f t; v s . l o g ( i - i)/(i) i s i n d i c a t i v e of 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 una f f e c t e d by p r e v i o u s o r 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 . d  166  reaction  (cone.  <5 x  10  -A  M.)  to  an  irreversible  electrode  — 3  reaction  (cone.  >10  of  the  prewave  on  the  electrode)  Various  ~  M.)  corresponds  ( i . e . a monolayer for  compounds  s t u d i e s ^ ' ^ '  products  (XVI)  of  the  electrode  and  that  o  (  '  have  maximum has  exhibited  demonstrated  are  adsorbed  the  material  which  reduction  this  of  to  strongly  layer  can  height  been a  adsorbed  prewave.  that  the  adsorbed  inhibit  dimeric  on  the  the  elec-  39 trode  reaction.  (XVI)  has  been  pyridinium reaction product  ion  (XVI)  further  is  the  inhibited  i s adsorbed  retardation  increase  log  ( i  in  slope  i ) / ( i) . concept of  concentrations  a monolayer  reduction  (less  in  the  of  a  2 x  that  5 x  10  -4  product  bulk  the  the  10  'I. t o  H. ) o f  electrode  the  10  reaction,  applied  of  dimeric  fashion  since M. )  with  potential  reversible electrode  than  dimeric  Apparently,  electrode  p l o t of  of the  a multi-layer  (from  of  a  of  sufficiently  rate-determining.  concentration  ing  The  after  adsorbed,  becomes  increasing a  Thus,  reaction  pyridinium  causes a  F,  resultagainst  at  low  ion  and  167  an  irreversible  (greater the  than  effects  10  J  M.)  r e a c t i o n a t high  of pyridinium  of 1-substituents  potential. of  electrode  i n this  ion i s also  supported  on t h e p o l a r o g r a p h i c  A t low c o n c e n t r a t i o n s ,  +3.7 was f o u n d  concentrations  work.  reduction  a reaction constant,  The s i m i l a r i t y  by  between  p*,' this  76 value  a n d t h e p * o f +3.8  dissociation  n - NH  consistent nitrogen  reported  of protonated  RR—NH_ —  +  with  methylaraines,  f o r the acid  reaction  + H  a near-unit  i n reaction  by W e l l s  (41).  (57),i s  (57)  change  i n the charge  However,  a t high  on t h e r i n g  concentrations  'CONH,  ONH  2  (4 1)  'I R  1  (XIII)  ( I I I )  (greater  than  10  -3  . ) , a reaction constant  ( X X X V I )  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 m e a s u r e 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 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  is  found  (III).  the  The  charge with  for  on  lower  the  the  polarographic reaction  ring  reduction  constant  nitrogen  of  of  pyridinium  implies  less  than  rate-controlling electrode  a  one  change  ions  in  the  unit,  consistent  shown  in  reaction  reaction  (7 5) .  :ONH.  (75)  4> I R  1  (III)  transition s ta te  + 2.1  To be  summarize,  r e v e r s i b l e as  the  shown  electrode  in  reaction  reaction  (41)  at  has  been  found  concentrations  to of  -4 pyridinium the  surface  of  dimeric  to  occur  of  prewaves.  the  The  of  than  5 x  10  :'!.  electrode  becomes  ('A XXVI)  This  a l l of  about  electrode  centrations  less  product  with  though- o n l y  the  ion  - 3  the  half  layer  of  reaction  .  the  pyridinium  ion  salts  dimeric  sufficiently (III)  vicinity  saturated  compounds  adsorbed  the  adsorption  pyridinium  of  In  that and  by  a  process  of  product the  appears even  adsorption (XXXVI)  equilibrium  pyridine  a.  monolayer  investigated,  exhibited  10  radical  inhibit con(XIII)  16:.)  are  not a t t a i n e d  reaction dinium  irreversible).  ion i s increased,  adsorbed in  becomes  a t the e l e c t r o d e  in a multilayer  the i n h i b i t i o n Since  of  surface As  the e l e c t r o d e  product  v/ith  the e l e c t r o d e reaction  electrode  the c o n c e n t r a t i o n  the dimeric fashion,  ( i . e . the  a  (XXXVI)  resulting  of  pyri-  is increase  reaction. i s rate  controlling  at  -3 concentrations can in  be  considered  (less  than  state  5 x  the p o l a r o g r a p h i c  charge  on  reversible  (Ill)  10  M.,  a measure  the t r a n s i t i o n  tions for  above  the r i n g  of  the degree  of r e a c t i o n -4  10  reduction  shown  ( X I I I )  an  constant of  (75).  II.) t h e r e a c t i o n  nitrogen,  reduction  the r e a c t i o n  At  electron lower  constant  supports  a  would  expected  be  p*  unit  in reaction (41).  (XXXVI)  of+ 2 . 1 transfer  concentra-  p*  change  of  +3.7  i n the  f o r the  17 0  4.2  The E f f e c t s o f Ions  Substituents  Pyridinium  (VIII)  ions  on  can  the  be  Reduction  reduced  of  to both  Pyridinium  one-  I R  1  (VIII)  electron tion  and  and  shown  two-electron  in reaction  the r e d u c t i o n  ensuing  potentials  i n order  potentials  of  which  are measured  potentiometric have  been  (41)  be  referred  be  two-electron  one-electron  observed  to as  reduction  p o t e n t i o m e t r i c a l l y and  i n the  potentials. literature  will,  polarographic from  reduc-  polarographically  reaction  t o d i s t i n g u i s h them  reduction  reported  can  The  potentials for this  discussion,  the  products.  the  shown  i n the reduction  reduction  in reaction  hence  v/ill  Substituent for several  be  (68) ,  called  effects reactions  171  H  H  + H  +  (68)  2e N  I  R„  (VIII)  which in  would  be e x p e c t e d  reactions  to p a r a l l e l  (41) a n d ( 6 8 ) .  the substituent  Two o f t h e s e  effects  are reactions  (60)  76 and  the previously  dissociation amines, (60)  reaction  respectively.  are analogous  model  discussed  compounds  reaction  (57)  of protonated pyridines The 3 - s u b s t i t u e n t s ,  to the 3-substituents,  (VIII)  , the acid and p r i m a r y  R^ , i n r e a c t i o n R^,  and t h e s u b s t i t u e n t s  o f t h e NAD  +  i n r e a c t i o n (57) +- H  (60)  N  I  H  R-NH3  are in  analogous both  going in and  both  +  of  H  from  reactions  +1  76 (57)  +  (VIII).  (60) and ( 5 7 ) , t h e n i t r o g e n  i n charge  reduction  (68) .  ±: R — NHg  to the 1-substituents  reactions  a change  ^  to zero,  of pyridinium  Furthermore, atom  i s under-  as i s o c c u r i n g ions,  reactions(41)  172  Lindquist stant,  and  p * , o f +3.7  cyanide  15  Cordeg  have  determined  f o r the e q u i l i b r i u m  a reaction  of the a d d i t i o n of  i o n t o 1 - s u b s t i t u t e d - 3 - c ar bjimoyl p y r i d i n ium  reaction  (76), a reaction  which  con-  i s very  similar  ions,  t o t h e two-  (76)  electron  reduction  Kosower fer  complex  reaction  (one-electron)  summarized for  the  constants  i n Table  XVI  The  and  reaction electron)  f o r the charge  iodides,  reaction  to the  trans-  (61), a  polarographic  of the foregoing  the r e a c t i o n s  effects  reactions are  are reproduced  of the 1 - s u b s t i t u e n t s  will  here be  first. reactions,  the acid of cyanide  reversible polarographic  pyridinium  i n reaction (68).  reduction.  the a d d i t i o n  7 6  shown  p o f +13.4  analogous  reaction  Three amines,  a  ions  of pyridinium  i s somewhat  comparison.  considered  reported  formation  which  The  lias  of pyridinium  ions  (this  constants reduction  0*.  work)  dissociation  to p y r i d i n i u m  (one-electron) have  nearly  Unexpectedly,  v/as f o u n d  of  t o have  primary i o n s , ^  reduction  identical  of  values  the p o t e n t ioraetr i c a much  smaller  and  of ( two-  p*-value,  17 4  'able Sunmarv  ;•. V  J.  of Reaction Constants of Reduction Reaction: 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 ti t u ent po s i t i o n  Substituent constant sca 1e  Reac t i o n constant  i ssoc i a t i o n • »ci or p r i m a r y ammo: (57)  +3  Cyanide a d d i t i o n to pyridinium ions (7G) (ecu ilibrium)  o*  Acid dissociation of p y r i d i n e s (CO)  Potentiometric (2electron) reduction o f p y r i d i n i u m i o n s (68) Charge-transfer complex formation (61)  O* 0  am 1 3 3  a* a m  a.  ii  7  f  6  +3  + 5.9  73  + 3.7  this work  + 2.8 +9 . 3 + 10.7  th i s wo r k  +  79  in  Polarogravhie (1electron) reduct i o n of pyridinium ions (41)  Re  +1 o . a + 12.4  13.4  17 ii  only + 2 . C ,  compared  1-substituents It tions  was  (68) and  the two c a s e s . different products.  t o t h e v a l u e s n e a r +3.7 f o r t h e e f f e c t s o f  on t h e o t h e r surprising (76)  equilibria.  to find  differ  t h a t t h e p* v a l u e s  for reac-  s i n c e t h e s u b s t r a t e i s t h e sarae i n  The d i f f e r e n c e i n t h e p* v a l u e s m u s t  reflect  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 s t a b i l i t y o f t h e Equations  (68)  and (76) c a n be c o m b i n e d  t h e h y p o t h e t i c a l r e a c t i o n shown  i n equation  to give  (77), the reaction  (77)  (IV)  (XXXVII)  176  constant  of  which  relative  effects  IV  and  XXXVII.  K  , is related  can of  be  the  The  considered  to  be  a  1-substituents  on  the  equilibrium constant  to  the  of  measure  of  the  stabilities  reaction  equilibrium constants  of  of  (77),  reaction  (63),  Ex K  , and r e a c t i o n ( 7 6 ) , K , by e q u a t i o n 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)  (78).  Hence,  can  found  the  R C  v  equation  C /K . CM Red CM R(  Ex  (79).  The  Aloq K „  *  P  r  p*  "  x  of  while  extra  on  and  give  a  value  reaction  equation the  of  (77)  glance,  compound  answer  may  (80)  which  3-carbamoyl  for  p*.,  0*  i t seems  +0.9  for  and  that  of  .  in  already  resonance  positive with-  compounds  greater  the  The  electron  unreasonable  stabilize  IV.  that electronstabilizing contains  an  ring. interaction,  i s p o s s i b l e between  group  Rec  1-substituents  a  (7 9)  0 *  p*  4-cyano  which  group  l i e in a  the  have  (XXXVII)  -  CM  implies  stabilize  electron-withdrawing The  =  s u b s t i t u e n t s should  the  +2.8  -  electron donating  first  withdrawing effect  for  and  Aa*  1-substituents  At  +3.7  Aa*  p* Ex  (XXXVII)  of  Alog K  —  Arr*  drawing  in  -  X  , respectively  value  values  from  (78)  Alog K  E  be  the  ring  1,4-dihydropyridines.  shown  nitrogen This  177  XXXVIII  resonance the  leads  electronic  XXXIX  t o t h e sane transition,  charge  distribution  equation  (81), i n  as a r i s e s  from  1,4-dihydro-  (81)  pyridines.^'' the  A  The  ease  of  of the absorbance  max  measure  x  of the c o n t r i b u t i o n  stability  the e l e c t r o n i c peak)  i s , therefore,  XXXIX  could  of the 1 , 4 - d i h y d r o p y r i d i n e .  withdrawing  substituents  make  to  a higher  since XXXIX. at  such  peak  a t the 1 - p o s i t i o n  to shorter  energy  electronic  substituents  Absorbance  approximately  15  wavelengths,  peaks nm.  would  to the  tend  15 '  transition, tend  a reasonable  In g e n e r a l ,  1 absorbance  transition (i.e.  overall electron-  to s h i f t  53 '  corresponding  as one would  to d e s t a b i l i z e  than  do  expect  structure  o f 1 , 4 - d i h y d r o p y r i d i n e s (IV) longer wavelengths  the  the  appear  173  H H  H CN ONH,  CONH,  N  N  I  (IV)  absorbance ing IV  peaks  (XXXVII)  of their  the e l e c t r o n i c then  with  contribution stability  transition  XXXVII. from  4-cyano  i n equation  Therefore,  the resonance  o f IV t h a n  counterparts  there  (81) i s e a s i e r  i s probably  structure  to the s t a b i l i t y  (XXXVII),  (XXXIX)  meanv/ith  a greater to the  o f the 4-cyano  compound:  (XXXIX)  (XXXVII).  will to  Electron-donating substituents  stabilise  a greater The  effect from  structure  extent  than  XXXIX  a comparison  (41) and  v/ith  thereby  1-position stabilize  IV  XXXVII.  3 - s u b s t i t u e n t s v/ere  on r e a c t i o n s  and w i l l  a t the  found  to have  (68) t h a n  reaction  (60) .  would  a much be  In these  greater  expected three  reactions  17  +  e"  9  (41 )  •N  p=11 t o 12  CH CONH 2  CH CONH (XL) 2  2  2  H. .H +  H  4- 2e  (68) p = 9 t o 11  CH CONH 2  CH CONH 2  2  2  (XXXIV)  + N  N  H  (XLI )  i  the  3-substituents  pyridinium reaction zation The  constants  must  constants  stabilization  i s probably  the s t a b i l i t i e s extent,  ( X L , XXXIV,  would  (XXXIV)  XLI) by  that  any  great  ( X L I ) so t h e l a r g e r (68) must  be due t o  (XL) a n d t h e 1,4-  by e l e c t r o n - w i t h d r a w i n g  significant  of s t a b i l i -  3-substituents.  t o have  (41) a n d radical  the d i f f e r e n t  amounts  o f the p y r i d i n e s  of the pyridine  of the  hence,  n o t be e x p e c t e d  for reactions  (60)  p : 5.9  be d u e t o r e l a t i v e  on t h e s t a b i l i t i e s  dihydropyridines It  affect  t o t h e same  of the products  reaction a  reactants  3-substituents  effect  should  H  the effects  of  substituents. 3-substituents  78  180  on  reactions  by  para-substituent  stants  a  (41)  whereas  and  (68)  were  found  to  be  than  by  meta-substituent  constants the  acid  dissociation  of  correlated  better  protonated  con-  pyridines,  m reaction  (58),  relations  v/ith  is  correlated  CT  would  better  indicate  with  the  a  .  The  presence  of  improved a  cor-  resonance  P t interaction pyridine  between  radical  interaction pyridines to  explain  effect  of  reduction  which  (XLI). the  the  (XL)  3-substituent  and  one  v/ould  Just  such  unexpectedly  1-substituents of  the  pyridinium  on ions  and  the  ring  1,4-dihydropyridine  not an  expect  to  find  interaction  lower the  reaction  was  (68).  constant  an  the  proposed  The  the  (XXXIV),  with  potentiometr ic  reaction  of  for  earlier the  (tv/o-elec tron) contribution  (68)  The para-substituent c o n s t a n t s , 0p, contain a resonance cont 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 constants, CT . Taft has p r o p o s e d a s e p a r a t i o n o f CT a n d a i n t o i n d u c t i v e a n d r e s o n a n c e t e r m s : Op = CTj + CTR a n d CT = Oj + 1/3 O R w h e r e Oj a n d CT a r e i n d u c t i v e and resonant contributions respectively. 7  3  m  M  p  R  m  181  from  the resonance  form  (XXXIX)  o f the d i h y d r o p y r i d i n e  (IV)  (IV) which and, which F,  would  (XXXIX)  be e n h a n c e d  can p a r t i c i p a t e t o some  extent,  are incapable  II, o r OH) .  Thus  by e l e c t r o n - w i t h d r a w i n g i n this  CN)  resonance  b u t v/ould  3-acetyl  will  contribute  written  resonance  structure  to the o v e r a l l  ( I V ) a n d make  and  ,  f o r groups  3-cyano  (XXXIX)  stability  t o XXXIX  (In s t r u c t u r e  (XLIII)  COCH  resonance ( i . e .  the r e d u c t i o n  s t r u c t u r e analogous  f o r the p y r i d i n e r a d i c a l .  ( XL 11)  be n o n - e x i s t e n t  the 3-carbamoyl,  analogues  A  9  i n such  o f ( I V ) , by s t a b i l i z i n g  3-substituents  ( i . e . CONH ,  of participating  analogues  1,4-dihydropyridine  product  or  i t s  o f the  easier. can also (XLII)  (XLIV)  be the  102  electron  has been  pyridine  ring.  place  arbitrarily  Resonance  c a n be w r i t t e n  a n d C-6) .  the 3-substituent  between  a t the 4 - p o s i t i o n  structures  o n C-2  the lone e l e c t r o n  actions  placed  of the  which  The e x i s t a n c e  and t h e l o n e  also  of  electron  inter-  as i n  /^VCONH,  k - ; N  1  CH  3  (XLV)  have  X L I V ,  been  proposed  by B r u h l i n a n n  methyl-3-carbaraoylpyridine doubt  that  the resonance  tribute  to the o v e r a l l  a  contribution  large  presence  substituents with  structures  density  of structures  charge  on t h e r i n g  tive  charge  is effectively  and  X L I V  However,  X L I I I  and  the r i n g  were  place  X L I V  Probably  do  ( X L I V )  con-  radical.  Quite  of the of 1 -  consistent  whereas the a partial  the p a r t i a l  by t h e i n d u c t i o n  nitrogen  1 -  i s little  because  nitrogen  neutralized  f o r the  the e f f e c t s  reduction  nitrogen.  towards  and  J  in structures  posi-  posiof X L V I  X L V I I .  To on  density  by  on t h e r i n g  tive  electron  made  on t h e p o l a r o g r a p h i c  a low charge  contribution  ring.  ( X L I I I )  of the pyridine  i s probably  of the aromatic  d  ( X L V ) so t h e r e  radical  stability  and Kayon '  summarize  the polarographic  then,  the e f f e c t s  (one-electron)  o f 1 - and  and  3-substituents  potentiometric  (XLVI)  (XLVII)  (two-electron)  reductions  by  resonance  considering  of  pyridinium  ions  can  i n t e r a c t i o n s between  H  the  actions  ring are  resonance shown. lone  i n the  summarized  structures  The  reduction  missing  electron  around  products.  i n Schemes  of  the  the  4  and  pyridine  structures  3- substituent  + 2.8  PJ  to  +9  the  explained  H  + H + 2e"  and  be  simply  pyridine  These 5.  result  ring.)  resonance  ( A l l of  radical  +11  have  the not  inter-  possible been  in shifting  the  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 R a t e s o f O x i d a t i o n by F l a v i n s  With  regard  potentiometric ions  and  to substituent  (two-electron)  f o r the e q u i l i b r i u m  1, 4 - d i h y d r o p y r i d i n e s be  so by  must  the f o l l o w i n g  Py  +  be  effects,  reduction  p value  and  identical.  c a n be  This  equations  (82)  + Fl  K  [Py  =  Substituents  (90), ring  on  (84) .  where will  =P vIE  (84)  +  +  ]  (85)  + [ H  ]  2e ^=±:F1H  [F1H  (8 6)  1  {  8  ?  )  [ F l ] [II ]  1  and  2e~  +  (91) .  (33)  [PyH] Pv  '1 + H  (0 2)  II  to  (8 2)  +  +  shown  through  [ P y ] [Fill]  Py  pyridinium  substituted  [Pyii] [ F l ]  K  the  f o r the  of substituted  between a f l a v i n  derivation,  + Fln"V=±:PyH  the  on  p„, Fl  the p y r i d i n e Therefore i s zero  not a f f e c t  one  since  ring  will  can write  affect  equations  substituents  the reduction  only  on  the  reactions (83)  through  oyridine  p o t e n t i a l of the  flavin.  13 0 = "  0  "'^  1 C  E c  Aa  >:'q  AlogK  f  A l o g [PyH] / [ P y ]  Aa  Py  _  ,,  , ,  a log [Pyii] / [ p r  Aa  Fl  Combining equation  of  y  +  ] [n  ]  +  +  (90)  Aa  a  equations  E q  P  ( 8 8 ) , ( 8 9 ) , and  Thus  P y  1-substituents  "  P  on  1,4-dihydropyridines  the r e a c t i o n  F1  -  a)  (8 9)  _ A l o g [ F l H ] / [ F l ] [II ]  (91).  P  +  r  (a  Aa  Aa  A 10 q K A  Alog [Fl] / [Flu'  +  =  P  (90) g i v e s  constant  p*  the  identity  f o r the  effect  91)  P y  the e q u i l i b r i u m (V), reaction  in  between  flavins  ( 6 9 ) , i s -2.8,  (X)  equal  and in  Y  NH  O H  H  ( 69)  N  I  R  1  ( V)  13 7  magnitude of  t o p*  pyridinium  v/ith  ions,  flavins  (III) .  tion  (V) r a t h e r  The  effect  i n equation  reaction  and  than  found  The  the forward  the r e a c t i o n  =  P  for  "  P  of dihydropyridines  by  This  found  o f p*  in  (  = -2.0)  flavins  (reduction  value  by  this  (92)  to c a l c u l a t e a r e a c t i o n  flavins).  constants  a  familiar  rev  tion  reaction  of the  T  (p* Eq  reverse  are related  P-, Eq  f o r the e q u i l i b r i u m  the  rates  rearrangement  work  possible  in this  reac-  f o r the  (93).  Eg  the forward  reaction constants  equation  P  pyridinium  to give  from  = P.e ~ for  of d i -  study  (92) a r i s i n g  rev  the r e a c t i o n  of  on  and b a c k w a r d a  reduction  f o r the o x i d a t i o n  the r e d u c t i o n  equation  p  since  of 1-substituents  p*=-1.9.  Using  (two-electron)  i n sign  written  (69) has b e e n  constant  equilibrium  but opposite  (69) h a s b e e n  hydropyridine ion  f o r the p o t e n t i o m e t r i c  and  (P|  = o  constant  r  3  )  the rate  of  -1.9)  i tis  p* rev  of p y r i d i n i u m  i s quite  9  different  oxida-  o f +0.9 f o r ions  by t h e  from  t h e p*  15 of  +2.2  found  by  Lindquist  addition  of cyanide  reaction  which  duction p  have  i s somewhat  reported  Cordes  to pyridinium  of pyridinium been  and  ions.  f o r the r a t e  ions,  analogous Indeed,  f o r the cyanide  reaction  of  (76), a  to the t w o - e l e c t r o n  re-  very  of  similar  addition  to  values  188  H  CN  (76)  benzaldehydes  and  J  the  sodium  borohydride  reduction  of  aceto-  certainly  mechanistic  differ-  transfer  the  84 pnenones. ences  between  least  of  ion  in  which  cyanide  is  solution,  directly bond  However,  from  formation  reaction  one in  the as  there  addition  fact  i s the  molecule the  i s dependent  are  and  that  the  cyanide to  the  transition partially  hydride  hydride ion,  but  other. state  on  the  of  ion  a  i s not  is  Thus  not  a  free  transferred the  cyanide  solvation  of  degree  of  addition the  free  13 9 cyanide is  i o n and p a r t i a l l y  adding,  transition two  the r a t e  stant  with  be  found  a hydride  same  (p*  - -2.0  which  n o s t l y on  larger  effect  of reduction i n this  transfer  study  from  surprisingly, on  ions  than  of dihydropyridines  transferred.  ions  i t i s not t o t a l l y  3.1)  by f l a v i n s  of the  constant ions  the r e a c t i o n  flavin  by  of  as they  inconsistent  have  +  Flavin  1,4-dihydropyridines  do  (p* = -1.9).  a -1 R  ions.  almost  on  the oxidaIn view  CONH,  u  con-  flavins  to pyridinium  the 1 - s u b s t i t u e n t s  see S e c t i o n  cyanide  i n the  to pyridinium  of pyridinium  reduced  the  the r e a c t i o n  the acid-decomposition  t o -2.G,  transfer  i s being  that  ( p * = +2.2)  to which  the s t a b i l i t y  the hydride  i t seems s u r p r i s i n g  f o r the r a t e  the  depend  of hydride  of a d d i t i o n of cyanide  Somewhat  tion  would  s o much  = +0.9)  the molecule  the degree  between  although,  should  (p*  state  molecules  Thus, for  whereas  on  +  Flavin-H  of  the  relatively  for  the  addition  and  the  reduction  it  is  poor  tempting  (XLVIII)  as  of  cyanide  by  to  an  agreement  between  (p*  flavins  formulate  alternative  (p* a  to  =  a  hydride  aldehydes  1,4-dibydropyridines. does  not  Dunn  explain  the  reaction  to  +0.9)  covalent  v/as  unfortunately  by  +2.2)  intermediate by  proposed  =  the  constants  pyridinium  of  ions  pyridinium  intermediate reduction.  for  the  The  intermediate isotope  as  such A  reduction  deuterium  ions,  similar  of (XLVIII), effects  20 (k  /K  =  oxidation  3.1G) of  observed  Suelter  and  Metzler  during  the  1-propyl-3-carbamoyl-4-deutro-1,4-dihydropyridine  (XL IX)  since  a  reaction after  fast  by  the  proton  t r a n s f e r s v/ould  the  rate  have  determining  to  take  formation  place of  the  in  intermediate any  way  starting would  to  form  with  require  pyridinium  (XLVIII). this  pyridinium a  ring,  pyridinium  is also  intermediate  ions  and  very in  a  reaction  ions.  "'  85 "  which  The  difficult  the  reverse  reduced  nucleophilic attack  1B  of  It  at  the  i s very  flavin  to  envisage  reaction  since  3-position  this of  the  uncharacteristic  hydride-transfer  mechanism,  which  2 0,22-24 nas  considerable  support,  the  a v a i l a b l e evidence,  although  appears the  to  reaction  best  explain a l l  constants  l'J2  d e t e r m i n e d n a y  Le  more  i n  this  c o m p l e x  s t u d y t h a n  seem t h i s .  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  193  4.4  Comparisons of 1,1'-Alhylenebis(3-carbamoylpyridinium) compounds w i t h t h e S e r i e s I Compounds  The  Series  I compounds  ( I I I and  H.  IV)  have  been  used  to  H  :ONH,  CONH,  N R  (III)  determine selected  the  reactions (IV).  effects  the  These  exerted  results  complex  substituents  the p y r i d i n e ring  effect  of pyridinium  o f more  particularly with  IV)  the i n d u c t i v e  pyridines  1  ring  nitrogen.  1-substituents  ( I I I ) and  c a n now  be  which  may  such  be  other  at  the  capable than  substituent  to  evaluate  1-position, of  interacting  induction  would  on  dihydro-  utilized  substituents  by means  One  ions  by  be  a  through second  87 pyridinium stacking  ring.  when  pyridinium compounds  Before of  used  attached  of a  pyridinium effects  of  found added  evidence  of charge  to polymers  to a p o l y s t y r e n e  study  separated  the e f f e c t s  substituent  were  in this  rings  the f i r s t  Values  indoles  rings  pyridinium  Batzold  one  second ring  c a n be  1.7  only  ring  evaluated,  on  The  two  tv/o m e t h y l e n e  pyridinium  the groups  of a* of a p p r o x i m a t e l y  or  containing  backbone.  (XIX) c o n t a i n e d  by  transfer  groups. the  the  reactions  inductive  XXV  and  XXVI  must  be  known.  and  1.0  v/ere  determined  194  (Section  3.4)  f o r XXV  and XXVI  respectively. - 2  Compound one  XlXa,  polarographic  at a concentration  reduction  of  - 4 7 0 mv.  ( v s . M.H.E.).  be  expected  on  wave  with  N .,  o f 10  a half-wave  The h a l f - w a v e  exhibited  potential  potential  that  would  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 - 4 5 0 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 p r e w a v e a n d a m a i n wave b u t s i n c e t h e c o r r e l a t i o n o f t h e -3 Scries the wave  I compounds  half-wave should  appeared the  basis  ( I I I ) a t 2 x 10  potentials  be u s e d  o f the main  f o r comparison.  at a potential of inductive  M.  v/ave,  then  The main  of approximately e f f e c t s only  (Section  (O*  5.3.5) only  = 1.7)  the main  reduction  - 4 3 0 mv.  used  wave  b u t , on  would  be  •CONH  2  (III ) predicted  at  a  p o t e n t i a l of  -515  mv.  At  concentrations  below  -4 5 x  10  I'.., t h e  determined anolxed (18) ] .  in  this  However,  compound  =  of  which  the  other  The  enormous  of  wave  E,  at  e°  as  agaxnst  £n(i  a  -  n  exhibited  had  the  log(i, a  two  had  a  =  -209  would  seem  tween  the  no  Series  I  2  /  and to  5 x  10  pyridinium  plot  of  [eauation K.  4  been  this  (18)  reduction  c°,  of  (>220 mv.)  -209  out  the  the ion  of  Series was  -409  waves  use  waves,  mv.  and  mv.  between  reduction  with  a  2/3  p o t e n t i a l of  the  rule  of  3  potential,  p o t e n t i a l i n comparisons with  i)/(i)  below  reduction  mv.)  -  polarographic  potential difference  (e°  p o t e n t i a l has  i n t e r c e p t , £°,  i) / ( i )  reduction  which  concentrations  since  reduction  concentrations  + |£  (XlXa)  one  tion  work  potential,  E  first  polarograpaic  the  at  higher  this  reduc-  I correlations  the  difference  be-  -3 half-wave  more  than  60  mv.  more  reasonable  potential,  The 20  second  mv.  more  E  L /  v/ave  Z  , at  (E° =  2 x  10  -409  p o s i t i v e than  :i.  mv.)  the  e°  and  is a  much  half-wave -3  potential  of  inductive  effect  -417  mv.  the  v/ould  be  polarographic (a*  =  1.7)  predicted  a  wave  at  reduction  which  is  in  2 x  10  M..  potential, good  From e°,  agreement  the  of with  19  the  value  o f - 4 0 9 mv.  reduction  observed.  p o t e n t i a l s o f XIX  The p r e d i c t e d  a r e summarized  and  observed  i n Table  XVII.  a:  n = 1  b:  n = 2  (XIX]  -3 Only  at a concentration  difference those  between  predicted  proximity special  effect  results  "IXb, l i s t e d  a concentration in a p p r o x i m a t e l y -583  mv.  on  10  the observed the basis  o f t h e two  The of  on  of 2 x  Ii. i s there  reduction  of oxidation  pyridinium  ions  the polarographic  i n Table  XVII,  effects.  reduction  less  Thus  to have  the  no  o f XIXa.  reduction  a r c much  significant  p o t e n t i a l s and  appears  f o r the p o l a r o g r a p h i c  a  potentials  definitive.  At  -p  o f 10 " M., 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 90 rav. m o r e n e g a t i v e than the p o t e n t i a l o f  predicted  fron  inductive  effects  ( a * = 1.0), whereas  -3 at  2 x 10  the observed  close  to the p r e d i c t e d  tions  below  -434 mv. from  .'5 x 10  potential  p o t e n t i a l o f -63 0 mv.  * i i . ,  i s 135 n v . m o r e  inductive  effects  the observed  p o s i t i v e than  (CT* = 1 . 0 ) .  at a concentration  extraordinarily  large  p o t e n t i a l o f - 5 9 5 rav. i s  o f 10  inhibition  At  concentra-  reduction would  The more ~ i t .  be  p o t e n t i a l of  predicted  negative  i s probably  of the e l e c t r o n  fairly  observed due  transfer  t o an  19 7  Tabic  'V  T '  P r e d i c t e d and Observed P o l a r o g r a p h i c Reduction P o t e n t i a l s oi t h e 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 i n i u m ) Compound s .  Compound  concentration (. )  10  CONH,  HgNOC  2 x 10 <5 x 10  -3 -A  10  H NOC-<^ 2  ^  \  \~CONH  reduction observed potentials from inductive effects  -4 5 0  -4 7 0  -515  -4 3 0  -417  -409  -5 33  -672  -623  -595  -569  -4 3 4  2  10 <5  10  -A  process large  v/ith  this  slope  against  compound,  o f 125  log(i  d  -  mv.  from  i)/(i)  which  i s also  plots  of a p p l i e d  potential,  (10].'  more  [equation  reflected  The  i n t h e ver> E,  positive  PT E  =  E  l / 2  ZZF  +  £  n  (  rt  i  "  (10)  —4 potential indicate which  observed  at concentrations  a non-inductive  is stabilizing  easier.  But  corroborate  interaction  the product  the r e s u l t s this  and  and  less  than  between thus  a t the higher  5 x  : - l . may  10  the  two  rings  making  the  reduction  concentrations  the p o t e n t i a l observed  at  do  not  concentrations  -4 below The  5 x  10  II. may  polarographic  result  results  H NOC—V \ = N  \  from  f o r XlXb  V  V  M_  adsorption  effects  instead.  are too i n c o n c l u s i v e to  C O N H  \ = /  /  * (CH ) N  2  /  +  n  ( X I X )  determine tween  The were  i f any  t h e two  non-inductive  interactions are operating  b  rings.  slopes of E against log(i b e t v / e e n 7 0 a n d 9 0 mv.  i)/(i)  f o r most  compounds  19'  A for  the  reduction  p o t e n t i a l has  reduction  pound  XXXII  of  was  one  not  ring  of  isolated  been  estimated  XlXb  to  but  was  CONH  dithionite  photometrically  A  a*  was  value  with  the  ring at  pH  7,  for  E°  in  to the  a 1  predicted -287  in  that  2  N  N \CH >i(  assayed  situ  coeffi-  3.6).  reduction  effect  v/hich  the has  =y  The  -280 of  mv.  the  is  in  -280  proximity no  spectro-  extinction  potentiometric  mv.  XlXb  2  the  p o t e n t i a l of  appear and  v/as  approximately  H NOO \ = .  which  inductive  , of  XlXa  and  in Section  be  reduction  i t v/ould  rings  by  i s given  gives  observed  Thus pyridine  1.0  reduction  method  estimated  of  pyridinium potential  (the  determined  potential  prepared  (XXXII)  by  was  dihydropyridine.  CONH,  (XlXb)  cient  work  2e~  CONH,  XlXb  this  CONH,  +  from  a  in  effect  at  pH  7.  unreduced reduction  good  agreement  mv. of on  the the  two  oxidation-  a : n=l o : n=  Z  200  reduction ed  behaviour  for simply  on  the  of  these  basis  of  compounds inductive  that  cannot  effects.  be  account-  201  4.5  Comparison  Before parties  of  the  + MAD  HO  +  of  HAD  effects  (Ia)  and  +  and  i-JMN  of  the  + MHN  with  the  ribosidyl  (II) can  OH  HO  be  Series  I  group  Compounds  on  the  evaluated,  pro-  the  OH  (la)  HO  OH (II)  inductive  effect  Intuitively, by  the  one  presence  of  the  would of  ribose expect  oxygen  group i t to  i n the  must be  ring  first  be  determined and  estimated. primarily  therefore  to  be  202  quite  similar  group.  to the inductive  The p r o p e r t i e s  carbamoylpyridinium XVIII.  However,  o f NAD  chloride  effect  o f the methoxymethyl  , NMN  and 1-methoxymethy1-3 -  (Vllle)  the inductive  a r e summarized  effect  i n Table  of the ribose  group  OH -CH OCH 2  ' HO will  also  I OH  contain  on  C-2' w h i c h  to  that  -CH CHpOH * * p  a contribution  should  3  exert  from  the hydroxyl  an i n d u c t i v e  of the hydroxethyl  group.  effect  In t h i s  group  quite  work  3  a  similar value  'CONH  2  N  CH OCH 2  3  (Vllle) of  0.55 h a s b e e n  0.11  the  gives  ribose  constants with  a predicted  group.  (III)  Table  substituent. substituent  are also  from  by assuming group.  V, p a g e  49  Combining  I t should  these  compared  and two  o f 0.66 f o r  potentials  the correlations a value  substituent  constant  the reduction  a n d NMN  predicted  the r i b o s i d y l  see  Hence  f o r NAD  values  compounds  a  f o r the methoxymethyl  f o r the hydroxyethyl  values  of  used  and  i n Table  rate XVIII  of the Series  I  o f 0.66 f o r t h e o * - v a l u e be n o t e d  that  a  different  203 Table Selected Properties carbamoylpyridinium  XVIII  o f N A D , NMN , chloride. +  +  +  l-methoxymethyl-3-  +  NAD (Ia) Polarographic reduction p o t e n t i a l a t 1 0 ~ M. (mv)  and  +  NMN MMCPy (II) (Vllle)  P r e d i c t e d from 0* = .66 f o r r i b o s e group  -715  •830  -679  •698  Polarographic reduction potential at 2xl0 M. (mv)  •683  -810  -657  •684  Polarographic reduction p o t e n t i a l a t < 5 x l 0 ~ ^ M. (mv)  -6777  -703  -639  •642  2  - 3  Potentiometric standard r e d u c t i o n p o t e n t i a l (mv)  •105 ( t h i s w o r k ) •1 0 4 -95 -97 •108 3 2  •108  3 0  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 flavin (M -sec )  53  .25  .61  2.4  Rate o f a c i d - d e c o m p o s i tion of dihydropyridine by H 0 (M-l-sec )  10.2  7.7  18  15  Rate o f a c i d - d e c o m p o s i tion of dihydropyridine by a c e t i c a c i d (M !sec~l) x 10  .91  7.7  5.9  _ 1  +  - 1  - 1  3  -  3  90  204  -CONH,  "1  (III)  tfsva.iue-  can  CT-value  o f 0.66 r e p o r t e d b y W e l l s In  -be. c a l c u l a t e d , f o r t h e iribos,e_ .group, f r o m 'the  general, the properties  investigated  i n this  work  V6  o f NAD  are i n fair  l-methoxymethyl-3-carbamoylpyridinium with  those  predicted  f o r the methoxymethyl  assuming  +  ( l a ) and  agreement chloride  a a*-value  group.  NMN (II)  with  +  those o f  (Vllle)  o f 0.66 f o r  and the  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 (la)  (II)  (Vllle)  OH  NAD  were  approximately  30 mv. m o r e  negative  l-methoxymethyl-3-carbamoylpyridinium trations dicted  a n d 0 t o 30 mv. m o r e  from  0* e q u a l s  NMN  +  appears  of  the dimeric  0.66.  t o be i n h i b i t e d  have  slopes  than  i s NAD ,  product  greater which  +  polarographic  than  chloride  negative  than  a t a l l concen-  would  The p o l a r o g r a p h i c to a greater  1 0 0 mv.,  extent  see Sections  i s the probable  reduction  cause  p o t e n t i a l s found  N  d 1  be  pre-  reduction of by  (XVI) ( p l o t s o f E a g a i n s t  than  those o f  adsorption  log(i<j -  i)/(i)  3 . 5 . 1 a n d 4.1)  o f the very for this  negative  compound a t  N  H  %  n  (XVI)  1  -3 concentrations the  polarographic  25 mv. m o r e negative reduction chloride ence and  above  M.  reduction  negative  than  10  -4  would  than  At concentrations p o t e n t i a l o f NMN  that  o f NAD  be p r e d i c t e d  from  +  +  i s  and about either  below  5 x 10  approximately 60 mv. m o r e  the polarographic  p o t e n t i a l of l-methoxymethyl-3-carbamoylpyridinium o r from  between  a a*-value  o f 0.66.  the polarographic  the predicted  T h e 0 t o 30 mv.  reduction  p o t e n t i a l s may r e f l e c t  differ-  p o t e n t i a l s o f NAD an e f f e c t  of  the  +  M  206  adenine  (L) m o i e t y  difficult that  to reduce.  i t may n o t b e The  of  toward  NAD  +  The d i f f e r e n c e  i s so s m a l l ,  o f NMN  tion  potential  +  quite  well  agrees  quite  found  for  The agreement  3-carbamoylpyridinium  HO  with  the value  basis  potential  however,  reduction predicted  potential using a  the potentiometric reduction  poten-  well  reduc-  with  the s l i g h t l y  lower  l-methoxymethyl-3-carbamoylpyridinium between  chloride  NMN  +  (II) and 1-methoxymethyl-  (Vllle)  would  be e x p e c t e d  on  OH  (II)  the  more  significant.  o f 0.66, w h e r e a s  tial  chloride.  thepyridinium ring  potentiometric (two-electron)  agrees  0"*-value  making  (Vllle)  of inductive o f NAD  +  effects.  The p o t e n t i o m e t r i c r e d u c t i o n  (la) i sapproximately  1 0 mv. m o r e  negative  207  than  those  chloride, group to  o f NMN which  T  and  1-methoxymethyl-3-carbamoylpyridinium  a g a i n may r e f l e c t  (L) toward  making  an e f f e c t  the pyridinium ring  o f the adenine more  difficult  reduce. The  pyridines that  rate  rates  observed  They  a n d NMN  again  +  than  would tial.  that  would  be e x p e c t e d  In t h i s o f NADH  work,  similar  values  potential  i t has been i s much  found  of  found slower  f o r NMNH  1-methoxymethyl-3-carbamoyl-l,4-dihydropyridine  ions.  of  the  that the than  reduction  rate  the  f o r cyanide  of the rate  of i t s standard  also  study  affinity  of the oxidation  were  effect  to pyridinium  larger  by f l a v i n s  on t h e b a s i s  the oxidation  i n their  on t h e b a s i s  with  Surprisingly,  A similar  addition  h a d a much  dihydro-  agreement  s m a l l e r than  and C o r d e s ^  be e x p e c t e d  of oxidation  reduction  +  compound.  o f 0.66.  of cyanide  NAD  reaction.  Since  a r e i n good  model  a CT-value  by L i n d q u i s t  observed  rate  using  and e q u i l i b r i a  addition  dard  +  of the corresponding  area l l considerably  predicted  rates  ion  o f NAD  of oxidation  o f t h e 1-methoxymethyl  these  was  rates  poten-  and s t a n (LI)  and  (Ve) t h e n t h e  203 slower  rates  are probably  HO  related  i n some  the oxygen  two-fold  rate  atom  on  the a-carbon  enhancement (Ve)  ing  larger  flavin  (X).  presence  (Ve)  dihydropyridine between  to the  OH  (LI)  of  way  the  over  the  1-substituent.  The  of 1-methoxymethyl-3-carbamoyl-1,4 NMNH  ribosidyl  Regardless,  of  ( L I ) may  g r o u p and  the two-fold  R  (XXI)  reflect  rate  the  steric  crowd-  approaching  enhancement  of  209  NADH  (XXV) o v e r  tion  of the transition The  have  very  chloride dihydro slowly or  NMNH  only  state  reaction  similarly  than  by t h e a d e n i n e  i n which  NAD  i n which  NADH  thebasis  from  of the polarographic  reduction  to  b e no s i g n i f i c a n t  effect  molecule  There  pyridinium  ring  negative  from  may b e a v e r y by t h e a d e n i n e  reduction  rate  o f NADH.  between  of  much  more  reduction  the adenine  small  of oxidation there  portion  stabilization  resulting  The f o r m a t i o n  potentials,  properties  p o t e n t i a l s o f NAD  the adenine  0.66.  by f l a v i n s ,  on t h e o x i d a t i o n - r e d u c t i o n  coenzyme.  complex  d i d not be-  reacted  p o t e n t i a l s , and r a t e s  dihydropyridines  oxidation  +  o f the corresponding  a a*-value  the corresponding  more  stabiliza-  moiety.  a n d NMN  a n d NMNH  of  +  +  decomposition  be p r e d i c t e d  potentiometric  NAD  a weak  d i d l-methoxymethyl-3-carbamoyl-l,4-dihydropyridine  would On  reflects  to l-methoxymethyl-3-carbamoylpyridinium  was i n t h e a c i d compounds,  than  (LI)probably  appears  of  the  of this of  the  i n the s l i g h t l y and s l i g h t l y  +  o f a charge  and t h e p y r i d i n i u m  faster  transfer  rings  o f NAD  +  88 has of  been this  proposed study  by C i l e n t o  show  oxidation-reduction  that  and S c h r e i e r ,  the effects  properties  o f such  o f NAD  +  but the results a complex  are very  small  on  the  a t best.  210  5.  SUGGESTIONS  In as  FOR F U R T H E R  some  areas,  i thas answered.  RESEARCH  this  work h a s p o s e d  Although  the adenine  a s many moiety  questions o f NAD  +  (Ia)  (Ia)  was  found  tion the of  t o have  properties ribose  ring  oxidation  interest  the  a-carbon  retardation  t o be r e s p o n s i b l e  a n d NMNH  (1) i f t h i s  substituents study,  slower  slow  the r e a c t i o n  occurs  a s was f o u n d  with  t h e 1-methoxymethyl  ring  t o have  and e l s e w h e r e  v/ith  attached  a similar  of the 1-substituent or transition  to  rate inter-  state to  rate.  ( 6 8 ) , v/as f o u n d  work  hetero-atoms  can e f f e c t  the oxygen  reduction  with  be o f  also  two-electron  compared  I t would  rates  rate  the dihydropyridine  +2.8  o f oxygen i n  f o r the slower  by f l a v i n s .  (2) i f a n y o t h e r  a n d (3) how  oxidation-reduc-  the presence  o f the 1-substituent  with  reaction  on the  appears  acts  The  effect  molecule,  t o know  i n this  little  of this  o f NADH  1-alkoxymethyl group  very  reaction 15  of pyridinium a reaction  constants  76 ' f o r three  constant  o f +3.7  analogous  ions  found  shown i n of only i n this  reactions.  211  H H ONHr  CONH, + H  +  -cr  2e  ( 6 8 )  (IV)  The  d i f f e r e n c e was  between  the  a t t r i b u t e d to a r e s o n a n c e  r i n g - n i t r o g e n and  H.  .H  the  interaction  3-carbamoyl.group of  IV,  9 NH.  To  further test  determine  this  hypothesis,  a reaction constant  f o r the  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 1,4-dihydropyridines could  be  i t would  ( L I l ) f o r v/hich the  H  H •Cl  N  I  (LIl)  interesting  two-electron  s u c h as  eliminated.  be  to  reduction  1-substituted-3-chlororesonance  interaction  212  Further pyridinium) enzymatic in  this  studies  compounds  NAD  work  into would  reactions. (XlXa  1,1'-alkylenebis(3-carbamoylbe u s e f u l  The  a n d b)  two  as p o s s i b l e  compounds  exhibited  models  of this  no d i r e c t  type  of used  interactions  :  n= l  :  n =2  (XIX )  between of  t h e two  t h e two  pounds  may  be  rings  which  potentials  have  affected  The  b u t t h i s may  to a l i g n  o f XIX  particularly XXXIl.  rings  very  short  compounds  by d i r e c t  between rates  properly  have  i n these  due two  having  longer  rings  of oxidation  (XXXII)  to an  The  methylene  between  XXXII  bridges  t h e two  and  com-  reduction  of the half-reduced  of both  inability  specific  methylene bridges.  interactions  t h e two  been  XXXIII  rings, form  compounds enzymatic  by  flavins  reactions.  could  also  be  of  interest  as  model  214  6.  EXPERIMENTAL  6.1  Buffers The  a  Manesty  in  water OOBE  used  glass  t h e d r y box were  throughout s t i l l .  boiled  this  work  Solutions  was d i s t i l l e d  which  and f l u s h e d  with  were  t o be  nitrogen  in used  while  cooling. Buffers  were  reagent  grade  Acetate  buffers: The  a  1.0  M.  1.0 M. added  3.4. of  chemicals;  required  solution  perchloric  Acetate  Solutions  t h e l . 0 M .  from  amount  acid  o f sodium into  required  W a t e r was buffers  then  were  o f 0.1 M.  (or of a  0.1  sodium solution  weighed ing  1.0 M.  a flask.  prepared  were  used.  crystals  t o make  The volume o f  the desired  added  buffers  amount  solution  perchlorate  up t o t h e  pH  was  required  a t p H ' s 5 . 6 5 , 4.6 a n d  were  prepared  (Tris)  buffers:  o r sodium  the f l a s k .  perchloric  of Tris  was w e i g h e d  of the proper  into  acetate  obtained  by  dilution  buffers.  required  M.)  methods  to give  Tris(hydroxymethyl)aminomethane The  commerically  the following  was w e i g h e d  by p i p e t t e .  volume.  prepared  ionic  crystals  into  acid  a flask.  chloride strength  The d e s i r e d  t o make The  required  a 1.0 amount  to prepare  ( 1 . 0 o r 0.1) was  pH was  (or hydrochloric  M.  then  obtained  by  acid)  pipette.  by  add-  215  vjater  was  added  ;-.a red. a t pK * s  to  required  amount  obtain  the desired  pH  up t o t h e f i n a l a t pit's  were p r e -  v/as w e i g h e d  v/as a d d e d volume.  o f 7.0  into  hydroxide  by p i p e t t e  a  dihydrochloride flask.  The  required  to  a n d w a t e r v/as  Ethylenediamine  buffers  v/ere  t o make  an  and 10.4.  buffers: The  required  amount  0.1  M . solution  0.1  I ' . o r 1.0 M . s o d i u m  desired  buffers  9.4 .  of ethylonediamine  o r 1.0 ?1. s o d i u m  o f 0.1  Glycine  Tris  Buffers:  volune  prepared  volume.  0.7, 3 . 'J a nd  a 0.05 I ' . s o l u t i o n  make  added  to the f i n a l  7 . 1 , o . 1,  E thylenedian ine The  up  v/as w e i g h e d  pH v/as a d d e d  to  the f i n a l  volume.  of  3.3, 9 . 2 ,  9.5,  of glycine into  hydroxide  by p i p e t t e . Glycine  a  flask. required Water  buffers  9.3, a n d 1 0 . 3 .  crystals  was  were  The volume to obtain then  of the  added  prepared  up  a t pii • s  216  6.2  Oxygen-free The  HE-43-2 Vacuum used and  dry  fitted  box with  supply  of  modified through used  dry  their  a  for oxygen-free  Model  HE-493  Corporation.  the  "oxygen-free"  was  used  Atmospheres  to  cause  inert  found  the  Dri-Train  before  the  so  that  helium  tank  purchased helium  unsuitable  be  inlet gas  was  gas  was  Nitrogen be-  system  box.  the  from  gas  to  the  to  Model  purity  gas  entering  a  (High  incoming  29  was  purity  The  in Figure  connect  High  content).  as  to  work  Dri-Train  atmosphere.  N i t r o g e n were  oxygen  was  forced  Copper inlet  tubing  on  the  box. Materials  in  Work  the  from the  antechamber  the door  During  dry  to  box  between  the  introduced used  to  be and  transferred the  through the  refill  the the  dry  valve A  antechamber  box  the  antechamber  evacuation procedure, to  to  through  antechamber.  (see and  dry  evacuated Figure  the  fresh  box  dry  helium  valve B  to  were and  refilled  29)  twice  box  was  gas  placed  before  opened.  was  replenish  slowly that  Dry DriTrain  From tank  helium  (He  Box  atmosphere)  C  To vacuum pump  Figure  29.  Dry-box  and  accessories  i—  1  218  6.3  Synthesis  of Quaternary  Quaternary responding was  pyridinium  pyridines  prepared  from  and  Pyridinium  Salts  salts  prepared  alkyl  chloroacetic  were  halides. acid  Methyl  from  the  cor-  chloroacetate  and m e t h a n o l  by  the method  89 of  Clinton  and  Laskowski.  prepared  by  propanol  i n place  other out  the method  alkyl  The consisted alkyl  of C l i n t o n  of methanol.  halides  further  Isopropyl  were  noted  general  method  of mixing  for preparing  30 m i l l i m o l e s  were  filtered,  vacuum.  and  2-  and  used  the with-  and  washed  Individual  the p y r i d i n i u m of the pyridine  (usually  refluxing with  salts  f o r one  acetone  variations  acetone  on  and  and  or  day. dried  this  The under  scheme  are  below.  Forty-five 30 m i l l i m o l e s  millimoles  (98%)  of crude  crude  product  filtered.  turned  cloudy.  (m.p.:  and  product was  The  was  product dec.)  (3.66 g r a m s ) ,  mis. of acetone  after  dissolved  Acetone  202-3°  10.0  chloride:  of chloroacetone  of nicotinamide  dimethylformamide,  -15°  each  i n 15 m i s . o f s o l v e n t  l-Acetonyl-3-carbamoylpyridinium  and  commercially  also  by u s i n g  A l l of the p y r i d i n e s  obtained  acetone/dimethylformamide)  aspirator  Laskowski  was  purification.  halide  crystals  and  chloroacetate  refluxing i n methanol  added  (5.0 mis)  8.0  mis. of  yielded  f o r two  6.3  days.  ( 8 mis/gram)  a t 50° u n t i l  the  r e c r y s t a l l i z e d upon  with  grams  The a t 50°  solution  cooling  to  219 Elemental  analysis:  calculated: found:  C:  50.35;  H:  5.13;. N:  13.05  C:50.42;H:5.10;N:13.26.  1-Carborne 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 Thirty-seven  millimoles  g r a m s ) , 30 m i l l i m o l e s of  dimethyIformamide  grams The  crude  50°  and  product  12.0  product  was  filtered. cloudy.  The  (m.p.:  165-6° d e c ) .  mis  was  product  chloroacetate  ( 3 . 6 6 g r a m s ) , 6.0  found:  yielded  6.3  refluxing  f o r one  day.  i n methanol added  (5 m i s . / g r a m )  a t 50° u n t i l  recrysta.llized  upon  C:  C:  46.85;  46.64;  H:  H:  4.77;  Thirty millimoles  of  millimoles  5.06;  N:  solution  cooling,  11.80.  yielding  product  of nicotinamide  i-propyiehloroacetate  dimethy1formamide,  crude  the  at  N:12.15  1-Carbo-i-propoxymethy1-3-carbamoylpyridinium  day,  mis.  analysis:  calculated:  one  (4.0  of acetone  after  dissolved  Acetone  turned  Elemental  of nicotinamide and  (91%) o f c r u d e  of methyl  chloride:  was  and 7.14  10.0  dissolved  (3.66 g r a m s ) ,  (5.5 g r a m s ) ,  mis. of acetone  grams  chloride :  (92%) o f c r u d e i n methanol  5.0  40  mis. of  were . r e f l u x e d product.  (5 m i s / g r a m )  for  The at  50-  o 60  and  began.  filtered. The  Acetone  was  recrystallization  (m.p. : 2 0 2 - 4 °  dec)  added was  until  completed  recrystallization upon  cooling,  220  Elemental  analysis:  calculated: found:  C:  C:  51.06;  50.89;  H:  H:  5.80;  5.69;  N:  N:  10.88.  l-Carbamoylmethyl-3-acetylpyridinium Thirty and of one  3-acetylpyridine  day.  A  yield The  mis/gram)  lization cool,  crude  began  yielding  Elemental  of  and  each  (3.63  dimethylformamide  obtained. (12  millimoles  and  10.0  5.2  grams  product  fine  mis.  were  mixed  of  acetone  (81%)  was  mis/gram)  chloride:  chloroacetamide  grams)  filtered.  (18  of  of  found:  was  and  solution  needle-like  the  and  when  yield 3.0  30.0  C:  C:  50.35;  50.33;  6.14 of  millimoles  The  crude  and  filtered.  213-4°  of  mis.  chloroacetamide  cloudy.  5.0  and  grams) mis.  refluxed  product  added  crystals.  until was  methanol  crystal-  allowed  (m.p.:  for  was  in refluxing  Acetone  H:  H:  5.13;  5.00;  N:  N:  to  214-214.5°  dec.)  dec.)  crude  dimethylformamide,  15.0  each  of  nicotinamide  grams)  was  were  dissolved  Ethanol  cooling,  chloride:  (.95%) o f  (2.80  product  On  grams  13.05  12.87.  l-Carbamoylmethyl-3-carbamoylpyridinium  ed  with  crude  dissolved  (2.80  analysis:  calculated:  A  10.83  was  white  added  until  mis.  (3.66  refluxed  in:water  product  and  day.  mis/gram)  solution  star-like crystals  obtain-  acetone  grams)  f o r one  (2.5  the  of  was  at  65°  became  formed.  (m.p.:  221  Elemental  analysis:  calculated: found:  C:  C:  44.5;  44.63;  H:  H:  4.6;  4.85;  N:  N:  19.46.  1-Carbamoylraethy1-3-fluoropyridinium Four thirty  3-fluoropyridine sodium  iodide  separated tion The in  was  below yield  was  until  product  each  were  refluxed  crystallized stirring  3.92  grams  ethanol  and  by  the o i l i n t o The  (5 m i s / g r a m )  the s o l u t i o n  a c a t a l y t i c amount  and  found:  turned cloudy cooling.  C:  C:  44.09;  43.80;  H:  H:  Thirty  was  millimoles  chloroacetamide and  yielding  4.17;  each  of  the  solu-  layer. was  dissolved  Acetone  (8 m i s / g r a m ) . 160-2°  was  The dec.).  N:  15 m i s . o f a c e t o n e  3.8  grams  193-6°  dec.)  14.70  14.67.  chloride:  a c a t a l y t i c amount  were  (64%) o f c r u d e 100%  N:  o f n i c o t i n o n i t r i l e (3.04  (2.80 g r a m s ) ,  r e c r y s t a l l i z e d from  (m.p.:  product  (m.p.:  4.20;  1-Carbamoylmethyl-3-cyanopyridinium  iodide,  cooling  filtered.  and  r e d o i l which  the acetone  crude  acetone,  analysis:  calculated:  and  The  s e e d i n g o r by  (68%).  mis. of  (2.80 grams)  f o r three days.  r e c r y s t a l l i z e d upon  Elemental  thirteen  of chloroacetamide  (2.91 g r a m s ) ,  0° a n d  refluxing  added  chloride:  mis. of dimethylformamide,  millimoles  19.5  refluxed  product.  ethanol  (80  f o r two The  crude  mis/gram).  grams)  of  sodium  days, product  222  Elemental  analysis:  calculated: found:  C:  C:  48.61;  48.74;  H:  4.10;  1-Carbamoylmethylpyridinium Thirty mis. one  millimoles  of pyridine, day, y i e l d e d  material  was  and  filtered.  the  solution  (m.p.:  H:  4.05; N:  N:  21.23.  chloride:  of chloroacetamide  a n d 15 m i s . o f a c e t o n e , 4.5  grams  dissolved  allowed  (87%) o f c r u d e  in refluxing  Acetone  was  yielding  5.0  refluxing for  product.  The  crude  (10 m i s / g r a m )  added  slowly  needle-like  and  crystals,  analysis:  calculated: found:  C:  C: 4 8 . 7 0 ; 48.82;  H:  H:  5.22; N:  5 . 0 3 ; N:  Three  millimoles  each  chloride:  of 3-hydroxypyridine  acetamide  i n 15.0 m i s . o f a c e t o n e  were  The  crystals  washed  crude  aspirator  give  white  Elemental  were  vacuum  needles  16.23  15.96.  l-Carbamoylmethyl-3-hydroxypyridinium  to  after  methanol  (30 m i s / g r a m )  to c o o l ,  (2.80 grams),  206-206.5° dec.)  Elemental  under  21.27  filtered,  refluxed  and r e c r y s t a l l i z e d (m.p.:  with from  197-9° dec.)  found:  C:  C: 4 4 . 5 6 ; 44.73,  H:  H:  4.78;  4 . 6 8 ; N:  N:  14.85  14.80.  chloro-  f o r one d a y .  acetone,  analysis:  calculated:  and  100%  dried  ethanol  223  To  confirm  rather this  that  than  at  product  alkylation the  was  occured  3-hydroxyl  compared  at  group,  with  the  the the  ring U.V-  spectra  nitrogen spectrum  of  of  l-methyl-3-  97 hydroxypyridinium The  agreement  chlorxde  does  pH  in acidic  confirm N-alkylation,  one  chloride  crude  289  13  245 322  238 313  on (15  a  millimoles  chloroacetate to  After  product  product  was  steam  react  was  the  mis.  200-3°  and  nicotinamide  of  and  mis. of  of  washed  millimoles  of  chloroacetonitrile  was  then  methanol  l i t . m.p.:  nicotinamide  (3.66  grams)  below  acetone. which  filtered,  recrystallization  dec,  (3.32  with  grams)  iodacetate)  to  (5 m i s / g r a m ) ,  0°, The  was  the crude  heated  ethanol  took  204-6°,  for  90  place 206-8°  upon 91  )  chloride:  dimethylformamide,  30  sodium  mixture  1-Cyanomethyl-3-carbamoylpyridinium Six  (3.66  refluxing  reaction  solution  added  monohydrate:  grams)(or  i n water  This  was  of  (3.50  filtered  bath.  (m.p.:  each  i n 20  cooling  dissolved  mis/gram)  cooling.  compound  283  allowed day.  expected.  1  Thirty  were  as  solutions,  this  1-Carboxymethyl-3-carbamoylpyridinium  sodium  basic  1-methyl-3-hydroxy pyridinium  and  and  12  mis.  grams), yielded  of  and 5.1  acetone, 44  millimoles  grams  (86%)  of  224  crude were  product dissolved  steam  bath  cloudy as  after  a  refluxing  i n water  and  ethanol  (4 m i s / g r a m ) .  fine,  Elemental  yellow,  C:  1, l ' - E t h y l e n e b i s Thirty  The  the  H:  H:  very 300°;  90 (8990).  the s o l u t i o n  (m.p.:  N:  on a  turned  recrystallized 236-8°  dec.)  N:  water,  filtered and  pale 90  A  21.03.  chloride) :  of nicotinamide a catalytic and  5.0  were  The  Thirty of  amount  bottle  filtered,  vacuum.  (3.66 g r a m s ) ,  mis. of  i n a pressure  allowed  beige  crystals  with  product  to c o o l .  (E) =  265  (9090);  millimoles  was  max  dissolved and  from  water l i t . m.p.:  (£) =  ' 265  chloride: (3.66 g r a m s ) ,  (3.22 g r a m s ) ,  and  crystals  dec.)  l i t .A  of nicotinamide  2-chloroethanol  time  297°  acetone  carbon  The  second  (m.p.:  sodium  f o r s i x days.  containing decolourizing and  of  10.0  dimethylformamide  washed  crude  recrystallized a  max  21.3  1-(2'-Hydroxyethyl)-3-carbamoylpyridinium  millimoles  crystals  v/as h e a t e d  the p r o d u c t  4.1;  4.14;  dichloride,  crystals  recovered  96  48.6;  48 .42,  aspirator  solution  285°,  C:  to 90-100°  under  give  until  cooling,  mis. of acetone  refluxing  were  Upon  millimoles  resulting  into  to  15.0  heated  dried  added  ( 3-carbamoylpyridinium  of ethylene  were  was  crude  analysis:  found:  iodide,  The  (5 m i s / g r a m ) , w h i c h  c r y s t a l l i n e powder.  calculated:  mis.  f o r 2 days.  5 mis. of  40  dimethylformamide two  days,  crude  and  yielding  p r o d u c t was  f l u f f y white 93 182° ) .  10.0  2.4  mis. of acetone  grams  (40%) o f c r u d e  recrystallized  crystals  (m.p.:  from  100%  195°; l i t .  1-Methoxymethyl-3-carbamoylpyridinium Thirty dissolved mis.  millimoles  i n 110  of chloromethyl methyl and  refluxed  product. ethanol  m.p.:  to  give  195.5-196°,  the  stirred  The  r e c o v e r e d p r o d u c t needed  were  stirring no  92  chloride:  a t room  ether  for  The  o f n i c o t i n a m i d e (3.66 grams)  mis. of acetone  solution  were  temperature.  added  Three  dropwise  c o n t i n u e d f o r two  further  were  to  hours.  purification.  (m.p.  123-5°) Elemental  analysis:  calculated: found: All to  attempts  C:  C:  47.41;  47.20,  H:  H:  5.43;  5.61;  N:  at recrystallization  N:  13.83  14.00.  resulted  i n decomposition  nicotinamide.  l-Methyl-3-carbamoylpyridinium The  preparation  follows  iodide: the method  used  by  Suelter  20 and  Metzler  millimoles iodide, yielding ial  and  for 1-n-propylnicotinamide iodide. o f n i c o t i n a m i d e (7.80 g r a m s ) , 27 m i s . o f m e t h a n o l  13.7  grams  were  (81%) o f c r u d e  v/as r e c r y s t a l l i z e d  from  100%  4.0  refluxed  product.  ethanol,  Sixty-four  mis. of  methyl  f o r four The  crude  yielding  fine  hours mater  226  feather-like 204°  33  crystals.  (m.p.:  207-210°  dec.; l i t  m.p.:  )  i  1,1-Methylenebis(3-carbamoylpyridinium)chloride Thirty and  millimoles  chloroiodomethane  were  refluxed  filtered, vacuum. give  washed The  bright  Elemental  f o r two with  crude  to  found:  C:  compound  was  when  days.  The  acetone  product  i n 15.0  mis  resulting  and  dried  (3.66 of  acetone  crystals  under  218-21°  were  aspirator  v/as r e c r y s t a l l i z e d f r o m (m.p.:  grams)  water  to  dec.)  analysis:  synthesize  even  of nicotinamide  (5.30 grams)  yellow crystals  calculated:  This  each  iodide:  a  C:  3'7.10;  I :  30.20  37.25;  H:  the only  H:  3.33;  3.26;  N:  product  N:  13.32,  12.99,  C l:  C l : 8.23,  recovered during  1-chloromethyl-3-carbamoylpyridinium  five-fold  excess  8.41;  of chloroiodomethane  I:  29.97.  attempts iodide,  was  used.  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 Thirty mis.  of methylene  iodide, were The  12.0  heated 45%  acetone was  millimoles  and  of nicotinamide  chloride,  and  5.0  i n a pressure bottle of crude  dried  dissolved  under  crystals  mis. of  a t 90-95° were ;  water,  of  treated  10.0  sodium  dimethylformamide  for five  filtered,  a s p i r a t o r -vacuum .  in refluxing  (3.66 grams),  a c a t a l y t i c amount  mis. of acetone  yield  chloride) :  The with  days.  washed crude  with product  decolourizing  227  carbon  and  second  time  Elemental  The  recrystallized. from  water  The  (m.p.:  crystals  192-3°  f o r monohydrate:  found:  44.78;  proton  chloride  C:  resonance v/ith  that  iodide,  H:  4.70;  spectrum of  thus  of  C: N:  this  salt  salt  H:  4.61;  N:  16.13  compound  was  almost  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 ) confirming  the  2  iodide  44.95;  15.66.  structure  Chemical s h i f t l H  dichloride  a  dec.)  H  chloride  recrystallized  analysis:  calculated  identical  were  (no. H  of  4  assigned  (XlXa).  hydrogen) H 5  H  6  7.6(1)  9 ..'85(1)  9.2(1)1. .8.5(1)  9.5(1)  7.7(1)  9.9(1)  9.2(1)  9.6(1)  3.5(1)  228  6.4  Synthesis The  method  of  1,4-dihydropyridines.  1,4-dihydropyridines  of  reduction  of  the 20  with  sodium  used  directly  mixtures  of  purity least  as  (vide  The moles  dithionite.  the  approximately (20  to  pyridine), sodium tion  or  After  yellow were under  were For  methylene  pyridinium  their  pyridinium  preparative  or  (e.g.  was  classical salts  salts  were  reaction  dissolving  mis. this  of  and  begun  dithionite sodium  added  washed  was  or  milliHigh  for  and  (2.61  continued  potassium  are  at  grams,  portionwise  replaced This  maintain  sodium stirred  by  the  dithionite until  with  small  Products  continuous  35  was  carbonate  over  a  five  particularly  millimoles  added  pH  and  the to  occurred.  portions which  of  did  c h l o r i d e e x t r a c t was  not  of reac-  than  base, a  . .  8. the  bright Crystals and  dried  crystallize  methylene  potassium  concentrated  the  water,  extraction with  stored.-over  to  greater  i t turned  crystallization  been  water.  solution  r e a c t i o n was  and  10.0  l-methyl-3-carbamoyl-l,4-dihydrc-,  to  until  had  15.0  hydroxide.  the  by  in  of  d i h y d r o p y r i d i n e s which  needed  mixture  which  The  Sodium  mixed  a s p i r a t o r vacuum.  chloride  the  carbonate  as  recovered  '  through  millimoles)  potassium  filtered,  the  95  from  bubbled  acid  colour  '  salt  before  a d d i t i o n of  reaction  were  15  the  mixture  94  consisted  reaction.  period.  sensitive  method  was  millimoles)  minute  '  pyridinium  minutes  throughout  33  by  ante).  nitrogen ten  prepared  corresponding  recovered  general  the  were  hydroxide.  from  about  200  The mis.  229 to  about  solvent fitted sary  20  n l s . on  was  a  noted  to  of  o i l .  evaporator.  under  aspirator  nitrogen bleed.  in order  evaporation  are  rotary  evaporated  with  yellow  a  recover  the  a  vacuum  remaining in a  nitrogen bleed  crystalline  remaining  Individual  The  The  methylene  variations  on  flask v/as  neces'  product;  withou t i t ,  chloride  left  this  general  only  a  method  below.  1-Acetonyl-3-carbamoyl-1,4-dihydropyridine: Ten  millimoles  carlanoylpyridinium v/ater.  Potassium  sodium  dithionite  added for  minutes,  chloride. a in  rotary vacuo  product  The  grams)  chloride  carbonate (2.08  portionwise.  30  (2.14  then  were (1.66  grams,  The  12  mixture  of  v/as  in  grams,  millimoles)  v/as  stirred  evaporator  and  in a  fitted  with  a  recrystallized  from  ethyl  v/as  12  15  remaining  under  with to  about  s o l v e n t was  nitrogen  bleed.  acetate  found:  C:  C:  60.00  GO. 38;  FI :  ; II: G.67; 6.63;  N :  ;i :  15.20.  and  mixed  and  nitrogen  20  mis.  15.5 J 6  on  evaporated The  (m.p. :  analysis:  calculated:  of  methylene  dec. ) Elemental  .mis.  m i l l i m o l e s ) were  concentrated  the  l-acetonyl-3-  dissolved  extracted overnight  extract  flask  crude  crude  118-21°  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 Ten  millimoles  (2.30  grams)  methyl-3-carbamoylpyridinium mis.  of  moles) were  water. and  sodium  mixed  under  and  added  and  washed  product  needles,  which  128-132°  dec.)  Elemental  was  carbonate (2.08  crude  minutes.  were  (1.66  The  The  1-carbomethoxydissolved  grams,  grams,  portionwise.  v/ith  12  resulting  stable  from  12  was  few  20  milli-  stirred  crystals  water.  benzene  in air for a  in  millimoles)  mixture  small portions of  recrystallized  v/ere  of  chloride  dithionite  n i t r o g e n f o r 15  filtered crude  Potassium  ihydropyridine:  to  were  The give  months.  yellow (m.p.:  analysis:  calculated: found:  C:  C;  55.12;  55.11;  H:  H:  6.12;  6.19;  N:  N:  14.29  14.26.  1-Carbo-i-propoxymethy1-3-carbamoy1-1,4-d i h y d r o p y r i di n e : Ten  millimoles  (2.58  3-carbamoylpyridinium water.  Sodium  chloride  dithionite  potassium  carbonate  and  portionwise.  a  added  red  was  (2.76  o i l separated.  overnight  with  reduced  evaporated nitrogen  150  to to  The  dryness The  20  of  1-carbo-i-propoxymethyl-  dissolved  grams,  grams,  20  was  methylene on  _in v a c u o  a  mis.  millimoles,  f o r about  of  and  v/ere  mixed  five  minutes,  extracted continuously chloride.  rotary  in a  product  15  i n 15  millimoles)  stirring  mixture  mis.  crude  of  were  (2.61  After  mis.  about  bleed.  grams)  flask was  The  extract  evaporator, fitted  with  recrystallized  then a from  231  ethyl  acetate  gen.  The c r y s t a l s  to  (20 m i s / g r a m ) , appear  being  allowed  to require  under  s e v e r a l days  a i r f o r noticeable decomposition.  Elemental  to cool  (m.p.:  of  nitro-  exposure  112-4° dec.)  analysis:  calculated: found:  C:  C:  51.06;  50.89;  H:  H:  5 . 8 0 ; N:  5 . 6 9 ; N:  10.83  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  millimoles  3-acetylpyridinium water.  Sodium  chloride  dithionite  potassium  carbonate  and  added  portionwise.  gen  f o r 20 m i n u t e s  methylene  chloride,  fitted  with  product ethyl give in  acetate fine  air.  Elemental  (2.61 grams,  yellow (m.p.:  15 m i l l i m o l e s ) a n d  was  stirred  days  with  then  to dryness  cooling  which  _i_n v a c u o grams  found:  C:  60.00;  under  are stable  60.31;  H:  H:  6.80;  6.67; N:  20 m i s .  in a of  flask crude  recrystallized  N:  for several  15.56  15.30.  from  nitrogen, to  analysis: C:  was  to about  162-3° dec.)  calculated:  nitro-  150 m i s . o f  1.16  m a t e r i a l was  rals/gram),  needles,  yielding  mixed  under  . The f i l t r a t e  the e x t r a c t evaporated  The c r u d e (160  i n 15 m i s . o f  20 m i l l i m o l e s ) w e r e  filtering.  nitrogen bleed,  (64%) .  1-carbamoylmethyl-  dissolved  f o r three  evaporator, a  of crude  The m i x t u r e  before  continuously  a rotary  were  (2.76 grams,  extracted  on  (2.14 grams)  days  232  1-Carbamoylmethy1-3-carbamoyl-1,4-dihydropyridine: Ten  millimoles  carbamoylpyridinium water.  Sodium  (2.15  chloride  dithionite  potassium  carbonate  and  added  portionwise.  gen  for  washed vacuum,  30  66%  yield  of  1.65  from  of  l-carbamoylmethyl-3-  dissolved  grams,  grams,  20  20  (91%).  and  stirred was  under  crude  mis/gram)  giving  rod-like  crystals,  which  of  a i r at  H:  6.1;  room  nitro-  aspirator  crystals  (30  presence  mixed  filtered,  dried  The  of and  under  crudepproduct  grams  millimoles)  was  the  water,  mis.  were  mixture  cold  i n 20  millimoles)  The  water  i n the  179-182°  Elemental  of  yellow,  indefinitely (m.p.:  mis.  yielding  recrystallized  and  were  (3.48  (2.76  ten minutes with  grams)  an  were  overall  are  stable  temperature,  dec.)  analysis:  calculated: found:  C:  C;  53.0;  52.79;  H:  6.21;  N:  N:  23.2  23.05.  1-Carbamoylmethyl-3-cyano-1,4-dihydropyridine: Five  millimoles  3-cyanopyridinium and  the  solution  dithionite powders  were  solution. the  final  under  (2.0  chloride was  Crystals addition in a  grams) were  flushed  grams)  mixed  nitrogen  (1.0  and  and  of  with  1-carbamoylmethyl-  i n 7 mis.  nitrogen  gas.  carbonate  p o r t i o n w i s e to  within  reagents.  cold  crude  dissolved  potassium  added  formed  of  water  about The  bath  two  mixture f o r one  of  water  Sodium (1.38  the  grams)  stirred  minutes  after  v/as  stirred  hour.  The  233 crystals  were  and  dried  and  washings  The  methylene  a  rotary  the  f i l t e r e d ,  overnight were  snail  portions  vacuum.  o v e r n i g h t wi th  extract The  was  uatsr  f i l t r a t e chloride.  M e t h y l e n e  evaporated  to dryness  on with  were  conbinc.i  nroduc t and r e c r y s t a l l i zed  from  benzene  were  fornor  The  of  crystals  A yield  product  with  aspirator  extracted  evaporator.  nl$/graa). pure  under  chloride  c;: t r a c t i o n  vmsJied  o f 70 milligrams  obtained.  (approximately  (n.p. =  (100  10%) o f  139-130.5°C)  analysis:  ;;ioriental  calculated: found:  C : 5 8 . d0; i< : 5 5.13;  C:  II ;  5.72;  5.52; J :  :  25.77  25.45.  1-Car b a n o y i n e t h y 1 - 3 - f 1 u o r o - I , 4 -dihy:i r o p y r i d i n e : Five  r.illinolos  ( 0 . 9 5 grans)  f luoropyr id iniura  chloride  The  flushed  solution  (1.3 in  gran:;)  and  alternate  ten.  tion ous  extraction  yielded  than  giving  a  hydroxide  a  to acid  product 10% y i e l d  maintained  couple  they  added  greater and  redissolved.  than  were in  solu-  Continu-  chloride  t h e d i h y d r o p y r i d i n e i s so  decomposition even  product.  that  the product  a t a pll a s h i g h  was r e c r y s t a l l i z e d o f pure  were  stirred  with Methylene  Presumably,  immediately  were  water.  dithionite  o f minutes  I f the crystals  catalysed  Sodium  ( 0 . 9 grams)  t h e pi! b e i n g within  i n 7 m i s . of  ni troqen.  of the f i l t r a t e  be r e c o v e r e d crude  with  t e n minutes,  no p r o d u c t .  susceptible must  formed  immediately.  f o r more  vra s d i s s o l v e d  potassium  portions,  Crystals;  f i l t e r e d  The  was  o f 1-carbanoylrac thyl-3 -  from  benzene  ast e n . (40  siils/gran)  (m.p.: 1 1 6 . 5 - 1 1 8 ° d e c . )  234  Elemental  analysis:  calculated: found:  C:  C:  53.85;  53.88;  H:  H:  5.77;  5.80;  N:  N:  17.95  17.94.  1-Cyanomethy1-3-carbamoyl-1,4-dihydropyridine: One  gram  pyridinium  chloride  sium  carbonate  nite  (1.74  wise. duct  (5 m i l l i m o l e s )  (1.38  grams,  The  to  washed v/ere  with  dissolved  Petroleum solution yellow  of  cooled  under  (m.p.:  were  mis.  then  and  minutes  crystals  were  water..  methylene  water.  30  127-130°  pro-  minutes  filtered  The  yield  portion-  b e f o r e the  chloride  to  dithio-  added  crude and  crystallization  nitrogen  Potas-  sodium  for another  ;  cold  until  of  and  mixed  f o r 30  The  portions  v/as a d d e d  crystals.  Elemental  begun.  in refluxing  ether was  and  i n 12  millimoles)  v/as s t i r r e d  had  small  10  millimoles)  crystallize  crystallization  l-cyanomethyl-3-carbamoyl-  dissolved  grams,  10  mixture  began  was  of  after  and crystals  filtered.  began.  fluffy,  The  pale  dec.)  analysis:  calculated: found:  C:  C:  58.89;  58.72;  H:  H:  5.52;  5.68;  N:  N:  25.77  25.75.  1-Methoxymethyl-3-carbamoy1-1,4-dihydropyridine: Ten  millimoles  carbamoylpyridinium water.  Sodium  chloride  dithionite  potassium  carbonate  and  portionwise.  added  (2.02  (2.76  grams)  l-methoxymethyl-3-  v/ere d i s s o l v e d  (2.61 grams,  The  of  grams, 20  mixture  15  i n 15  millimoles)  millimoles) was  mis.  stirred  were under  of and  mixed  235  nitrogen night  for five  with  150  evaporated maining  to about  s o l v e n t was with  yellow  crystals.  be  from  a  evaporated  with  The  crude  acetate. a glass  The  are stable  Elemental  vacuum  for only  1.16  found:  in a  a  flask (70%) o f  flask  pale  must  the s o l u t i o n  not occur.  few d a y s  The r e -  recrystallized  sealing  will  was  The  under  pure  i n the presence  of  dec.)  C:  C:  57.14;  57.40;  H:  H:  7.14;  7.07;  N:  N:  16.67  16.66.  *-Hydroxyethyl)-3-carbamoyl-l,4-dihydropyridine: Ten  millimoies  (2.02 grams)  of crude  ethyl)-3-carbamoylpyridinium  chloride  of  (2.61 grams,  water.  added  Sodium  alternately.  five  minutes,  mis.  of methylene  about tered,  then  20 m i s  on  dried,  transferred is  extract  grams  were  over-  analysis:  calculated:  1-(2  The  recrystallization  rod before  crystals  106-7°  under  crystals  or r e c r y s t a l l i z a t i o n  (m.p.:  continuously  chloride.  to y i e l d  nitrogen  air.  extracted  20 m i s . o n a r o t a r y e v a p o r a t o r .  nitrogen bleed  ethyl  scraped  then  mis. of methylene  fitted  twice  minutes,  dithionite The  mixture  chloride.  stored  under  to cause  The  under  than  with  evaporated  crystals they  a day's  noticeable discoloration  were  nitrogen for  overnight  nitrogen until  Less  i n 15 m i s .  15 m i l l i m o l e s )  e x t r a c t was  evaporator.  to the d r y box.  sufficient  The  dissolved  stirred  extracted continuously  a rotary  and  was  were  1-(2'-hydroxy-  were  150 to f i l -  were  exposure of the  to a i r crystals.  236  (m.p.:  133-133.5°  Elemental  dec.;  lit.  m.p.:  57.14;  H:  119-21°  1 0 1  )  analysis:  calculated: found:  C:  C:  56.97;  H:  7.14;  N:  N:  16.53.  6.96;  16.67  l-Methyl-3-carbamoyl-l,4-d ihydropyr idine: Ten  millimoles  pyridinium solution grams,  iodide  flushed  15  maintained  The  in  8.  with  in a  and  crystals  dec;  l i t  m.p.:  The  to  remaining  yellow  78°  84°,  (e)  max  =  33  355  recrystallizd  of  a  crude  was  in  water,  wher'e t h i s  compound  decomposes  of  any  buffer.  The  was  recorded  sition few  at  pH  reaction,  percent  an  9.8  but  hour  in  even  this  20  at  this  i s ^observable.  pH  the  was  pH  bright  chloride.  mis.  the  to  on  a  evaporated  obtained  A  yield  (m.p.: (e)  =  of  73.5-  360  were u n s u c c e s s f u l .  literature even  spectrum  study  35  methylene  product  coefficient  as  in  grams,  a  nitrogen bleed.  extinction  acid  turned  98 8 5 . 3>- 8 6 . 8 ° ); X max 98 (6630) ). the  the  (2.61  (1.9  s o l v e n t was  lower  added  had  approximately  with  bright  dithionite  and  extracted continuously  mis.  fitted  of  Attempts  the  water  portions while  i t was  to  of  hydroxide  solution  200  l-methyl-3-carbamoyl-  mis.  Sodium  flask  grams  l i tA  hour),  evaporated  0.4  (7820);  the  crude  i n 15  potassium  approximately  was  of  in alternate  After  one-half  evaporator  vacuo  dissolved nitrogen.  added  above  extract  rotary  with  were  (about  overnight  were  m i l l i m o l e s ) and  millimoles)  yellow  (2..64 g r a m s )  of  slow  in  the  this down  v/as  recorded  absence  compound the  a decomposition  decompoof  a  6.5  Kinetics Kinetic  runs  tion  spectroscopy.  than  five  meter. cell  Whenever  was  which  minutes  Reactions were  from  had h a l f - l i v e s  Bausch  A  continuous  a n d Lomb  wavelength  6.5.1  followed  followed  the path less  than  Spectronic  recording  and times  was  rate  five  were  505 r e c o r d i n g  along  absorbance  i s first  trations  n o t be k n o w n .  0.1 M. 5.65,  need  Tris 4.6  a t pH  order  Buffer  a n d 0.1 M.  solutions  were  one-half  hour  1.0 M.  b u f f e r s were  prepared  by d i s s o l v i n g  0.1  placed  cm.  absorbance  cell  i n the thermostated  photometer .  initial  The b u f f e r s used  for at least  An  recording.  of dihydropyridine  and, t h e r f o r e ,  i n the buffer  single  1,4-dihydropyridines  brated  dihydropyridine  at a  300 a n d 390 n a n o m e t e r s .  7.1 a n d 1.0 M.  a n d 3.4.  on  of 1,4-dihydropyridines i n  between  decomposition  followed  spectrophotometer.  this  by f o l l o w i n g t h e l o s s  i n the region  t h e sample Reactions  of the absorbance  marked  greater  read,  beam.  minutes  absorp-  spectrophoto-  of the l i g h t  of decomposition  was m e a s u r e d  16  not being  Acid-catalyzed Decompositionsof The  or u.v.  had h a l f - l i v e s  on a Cary  was made  were  by v i s i b l e  which  the absorbance  removed  a  acid  were  before  solution.  was cell  filled  were  The  concen1.0 M.. a n d  acetate  at pH s  thermally  equili-  use.  Solutions i n  crystals  The t i m e r  with  compartment  this  1  of the was  started.  solution  and  of the spectro-  238  Solutions the  two  1.0  M.  to  following solution,  50  mis.  with  bance  cell  was  in  a  i n 0.1  second  1.0  than  less  M.  sodium  were  added,  mis.  with  cell  was  the  1.0  as  above,  were  this  M.  i n 1.0  minutes, of  cell  M.  the  the  compartment  mis.  1.0  cm.  Cary  buffer  which  M.  mis. the  a  absor-  16  spectro-  had  a  cm.  solution  and  placed  the  predissolved  buffer  made  0.1  half-  was  M.  An  of  a  placed  1.0  solution  of  and  solution  of  of  diluted  d i h y d r o p y r i d i n e were  and  sample  A  i n the  perchlorate.  the  5.0  solution  0.1  Five  started,  sodium with  diluted  in either  immediately  perchlorate.  perchlorate. timer  filled  thermostated  reactions,  compartment  Crystals  prepared  slower  with  five  were  For  reactions  directly.  1.0  buffers  sodium  thermostated  life  in  M.  filled  For  pared  ways. prepared  photometer. of  M.  up  to  50  absorbance  appropriate  in  the  spectropho-  tometer. The after  the  Radiometer The to  mixtures  decomposition 26  pH  Meter  concentration of a  The  titration  tion.  was  to  were begun.  acetic  acid  10.5  The  with  was  with  c o n c e n t r a t i o n of pH  analyzed  standardized at  .phenolphthaTeinri e n d p o i n t  solution. by  reaction  a  a  within  pH pH  was 4  and  determined standard  protonated standard  a  sodium  hours  measured 7  or  by  sodium  Tris  few  was  7  on and  a 10.  titration hydroxide determined  hydroxide  solu-  239  6.5.2  R e a c t i o n Between The  rate  of  mononucleotide following and  470  and  the  of  of  cell  box.  sealed  of  removed  from  ment  the  of  absorbance  mixed  reactions  and  with  to  least  dry  Cary  16.  th  volume cell  with  silicone  i n the  was  rubber  inserted  cell.  6OK  The  the  prepared  greater  and  by 430 The  A  stock  thermally  mixed  than  The  in  of  to  was  s  1.0  the  cm.  reaction and  a  to  keep  the  The  cell  was  thermostated were  f i l l  than  method  flavin  the  the  five  dry  stopper  of  minutes,  taken  compartuntil  the  a..t'. fea's t d p h e - h a l f e'h^our v\  i n the  into top  hours. in a  a  stopper  necessary  two  five  timer  possible,  for this  less  required  sealed  needle  box  half-lives  flavin.  readings  required  dry  of  rubber  placed  unchanged  absorbance  a  and  was  between  box.  was  Absorbance  time  having the  box  region  i n a l l cases.  as  silicone  for at  the  determined  overflowing with  oxygen  i n the  solution  quickly  seal  placed  syringe  dry  double  i t from  reactions,  a  flavin  ways.  i n the as  or  r e a c t a n t s were  half-lives  filled  remained  remove  with  The  The  , h a l f ' hoToo/'much and  following  was  septum.  free  i n excess  dry  cell  and  reduction  i n the  immediately  mixture rubber  present  reactions  absorbance  the  r e a c t a n t s was  r e a c t a n t s were  started  to  the  two  riboflavin  i n the  of  the  For  1,4-Dihydropyridines  between  absorbance  due  was  each  equilibrated  the  of  nanometers  solution  reaction  and  1 , 4 - d i h y d r o p y r i d i n e s was  loss  dihydropyridine  either  the  Flavins  absorbance  cell  to  with  be  used  minutes.  For  stock box  solution  and  and  rubber  space  over  this  needle  ••'  the  these was  cell  septum. the  was  flavin  then  was A  240  sealed  into  allowed  for  was  when  as i n F i g u r e  t o be  cell  into  the c e l l  was  required  was m e a s u r e d  into  a  of  dihydropyridine  to  a c t as a back-up  rubber  septum  with  syringe  being  supply.  The t e s t  inserted  The  removed  t h e d r y box  oxygen  were  mixed . The  from  from  entering  syringe  the box.  stated then  from  the syringe  and a b s o r b a n c e  The a b s o r b a n c e  compartment  evident  way  to  pre-  the s o l u t i o n s  apparatus placed  was  removed  i n the thermo-  spectrophotometer,  continued  of a t least  by oxygen  an hour  in this  mixed.  was  within  the septum.  were  was  leaking  of mixing  until  no  15 m i n u t e s .  into  a  the  contents  of the absorbance  able  with  as the c e l l  recording  caused  sealed  tube  the  The  flavin,  test  containing  before  volume  into  was  of  in a  through  a n d Lomb  small  injected  the timer  a period  was  solution  s o l u t i o n was  and  over  started  cell  cell  of the Bausch  the dihydropyridine  stock  was  of the syringe  time  injected.  placed tube  avenue  nitrogen  i n t h e d r y box and a  s o l u t i o n was  was  method, t h e  longer  high-purity  s o l u t i o n was  vent  the needle  this  of dihydropyridine  and t h e n e e d l e  was  dihydropyridine  for a  dihydropyridine syringe  arrangement  n o t a c t as an  Using  s o l u t i o n was  volume  the  i t could  of oxygen  flushed  stock  This  and, s i n c e  the c e l l .  free  the dihydropyridine The  the c e l l  into  remained  30.  r e l i e v e d when  to the atmosphere,  to d i f f u s e  absorbance  while  tube  injected  exposed  oxygen  than  test  the pressure  solution never  a  the c e l l ,  the s o l u t i o n s  cell  change  Reoxidation was  but this  notice-  was  241  J  I empty t e s t tube  I  I I  ii i i  11 11  syring containing dihydropyridine solution  t e s t tube c o n t a i n i n g a s m a l l amount of dihydropyridine solution Flavin solution  absorbance  Figure  30.  cell  Syringe and a b s o r b a n c e o x y g e n - f r e e work  cell  apparatus f o r  242  longer in  than  this  the reaction  time  f o r any o f t h e s o l u t i o n s  prepared  way. Stock  nucleotide^  solutions were  of riboflavin  prepared  (Xa) and f l a v i n  mono-  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 OR 2  a : R=H b:  milligrams coloured box.  o f sample  Kimax  ring  to completely  solution  dissolve.  solutions  by  dissolving  in  25 m i s . o f b u f f e r  between  three  one-half  amount  that  and t h i r t y  wayelenghths,  were  The amount  minutes.  stir-  by m e a s u r i n g t h e  of dihydropyridine  the reactions  of  of the stock  a t a; minimum,'of ' t h r e e  i n the d r y box. su.ch  i n the dry hour  The c o n c e n t r a t i o n as p o s s i b l e  in ared  flask  of 1,4-dihydropyridines  the required  was c h o s e n  volumetric  at least  as soon  of the s o l u t i o n  Stock  lives  glass  required  was d e t e r m i n e d  absorbance  pyridine  i n 75 t o 1 0 0 m i s . o f b u f f e r  "Ray-sorb"  The r i b o f l a v i n  R=PO.  would  The  of  prepared crystals dihydro-  have  half-  concentration  243  of  the  bance  stock of  initial from  the  the  solution  was  solution  at  concentrations concentrations  appropriate  dilutions.  determined at  of of  least  the the  by  three  reactants stock  measuring  the  wavelengths. were  then  solutions  absorThe  calculated  using  the  244  6.6  Polarography All  polarograms  record.  The  Metrohm  drop  electrode. mercury drop was  of  used.  silver  time  v/as  controller The  flow  time  drop  v/ere  flow  rate 2.5  The  recorded  attached  of  3.25  seconds.  A  potentials  a  regulated at  constants  (m)  on  to  of  0.166  the  the  E  261  seconds  dropping  capillary  scan  rate  of  potassium  0.1  by  were:  a  a  a  natural  volts/minute  against a  chloride  Pola-  mercury  m i l l i g r a m s / s e c o n d and  v/ere m e a s u r e d  chloride-saturated  Metrohm  silver-  reference  elec-  99 trode,  f o r which  observed  to  value  of  222  mv.  polarographic potentials  electrode. current  a  The  Polarecord E  compensation  obtain a  base  which  line  261  v/as u s e d to  the  v / h i c h was  as  refer  standard  is fitted  v/as a d j u s t e d  to  with  hydrogen  a  charging  f o r each  close  as  the  polarogram  possible  to  hor i z o n t a 1 . The sodium  acetate  adjusted was  buffer  the  to  0.1  a  pH  with  preferred  chloride, reduced  at  f o r most  formed  of  compounds 5.65.  sodium  salt  pyridinium salts.  more  to  products Lov/er  with  pH  the  Test known  weight  reaction  solutions of  of  were  these  pyridinium salt  some  or  was  salts,  of  (pH  chloride)  a  such  more  used =  (pH  with  either  diluting  the  were  chloride  by  acid-  Sodium p e r c h l o r a t e  compounds  prepared  acetic  strength  buffers  bis(3-carbamoy1-pyridinium  prevent  ionic  M.  nucleophilic  l-carbamoylmethyl-3-cyanopyridinium methylene  0.1  perchlorate.  since  addition  The  v/as  easily  for  4.6) =  "and  3.4)  hydroxide  dissolving stock  as  ion. a  solution  245  of  pyridinium  jacketed  were  then  through  nitrogen the  in a  polarography  solutions trogen  salt  flow  the was  polarogram  pipetted  vessel  deoxygenated  diverted  of  thermostated  solution for  v/as  volume  to  by at  at  over  recorded,  a  period  of  carry  a  complete  a  water-  These  high  ten the  in  25°.  bubbling least  pass  buffer  purity ni-  minutes. solution  five  test  to  The v/hile  seven  minu t e s. In  order  polarographic potentials  to  waves,  along  to  determine  i , . d  If  the  waves  that  Figure more any as  wave both  31a,  given the  lines  the  wave  measured  volts region A  of  the  more  as  the  the  not  be  for  point  the  wave  plateau  the  at  several  methods  limiting from were  were  current  extrapolated the  were  current,  a l l other linear,  as  extrapolated  to  97%  appeared as  in to  the  of  within  to  more  current  two  presence  the  waves. of  the  31b  plateau the  polaro-  Currents limiting  about the  At  measured  and  betv/een  line.  in Figure  limiting  The  base  ( i ^ ) was base  distance  extrapolated the  two  the  portions  of  measured  isolated  and  3%  wave,  betv/een  before.  i , and  limiting  ( i ) as  measured  be  following  extrapolated  second  analysis  positive potentials, respectively.  approximately  single value  inflection  line  linear  between  and  a  could  measured  these  current  from  When  current,  base  potential,  and  The  must  sufficiently  the  distance  graphic  the  and  current  wave.  was  then  negative  the  the  used  out  200  were current.  milli-  plateau  positive potentials. was The  measured current  second  wave  at  the  was causes  (b) Figure  31.  Sample  polarograms  247  a  distortion  currents  were  of  the p l a t e a u  only  measured  region up  of  t o 85%  the measured of  wave  the l i m i t i n g  so  current.  243  6.7  Potentiometry Potentiometric  under  a helium  Beckman  Model  periodically each The  atmosphere.  with  (Figure  to a  stant  Rodkey  and  '  was  such  used  purchased  from  Sigma  to  used  each  convert  at  pH  7.5  natant box. were  time.  potentiometry  to  liquid  25°.  assumed' t o h a v e an hour  apart  used  into  study  as a  a  reached  were  con-  The  to measure  The x a n t h i n e suspension activity  determining  +  the potentiometry  was  Sigma  one u n i t  o f NAD ,  oxidase  by  where  amounts  solutions  quoted  cell,  of  suspension  prepared f o r  i s t h e amount r e q u i r e d  to uric  and t h e s o l i d  medi-  ammonium  o f enzyme were  was  and a  M.  units  suspension  same  NAD  t h e volume  of xanthine  NAD  i n 2.3  Three  The  flavoenzyme  between  viologen.  enzymatic  when  discarded  The d e s i r e d weighed  were  models.  one m i c r o m o l e and  electrode  chloride-calomel electrode  as benzyl  Corp.  specific  as a guide  use each  M.  ferricyanide.  a platinum  recorded  i n this  some NAD  The  calibrated  c o n t a i n i n g 0.1  to a c c e l e r a t e equilibrium  with  was  with  has s u c c e s s f u l l y  tried  sulphate.  was  on a  millivolt.  '  mediator  system  recorded  313 2  oxidase  potential  contents  potentials  14  ator  was m e a s u r e d  to w i t h i n one  xanthine  solution  i n t h e d r y box  f e r r o c y a n i d e and p o t a s s i u m  The c e l l  when  were  instrument  saturated potassium  32).  equilibrium  The  an u n b u f f e r e d  potential  referred  w e r e made  Potentials  G pH m e t e r .  of potassium cell  measurements  acid  per minute  c e n t r i f u g e d , the transferred  NADH, cell  super-  to the d r y  and b e n z y l  viologen  a n d 25 m i s . o f  buffer  249  Figure  32.  Potentiometry  cell.  v/ere  added.  The  total  c o n c e n t r a t i o n of  -3 tween  1 x  of  this  oxidase  A  M.,  solution  were  then  transfer xanthine  well  with  NAD.  ten  used  units  of  brium  was  stant  for at  by  mv.  but  gave  benzyl  solution  was  oxidase  least  pyridinium,  one  other  in a  since  and  were  be-  viologen  con-  One  the  results  reduced  into  to  added  to  the  two  v/ith  cell.  with  models t e s t e d , this,  tested  and of  the  scatter.  Methyl  solutions  of  system  NAD  models.  l-methoxymethyl-3-  cells  each.  and  above  three  After  potentials cell  as  equili-  v/ere  potentials  condiffered  l-methoxymethyl-3-carbamoyl-  ions with  mis.  the  cell  changes  and  potentials  in  the  1-  did  not  pyridinium-  ratio.  riboflavin  potentials  way  two  xanthine  prepared  two  (the c e l l  attempts  were  dihydropyridines  despite  erratic  hour),  systematic  Flavins  NAD  was  1-carbamoylmethy1-3-carbamoylpyridinium  to-dihydropyridine  tion  this.  potentiometry  split  methy1-3-carbamoylpyridinium  models  of  benzyl  v i o l o g e n was  apparently reached  In  NADH  oxidase-benzyl viologen mediator  and  xanthine  the  to d i s s o l v e  the  containing oxidized  The  respond  with  percent  i t into  NAD  carbamoylpyridine  50  10 to  solution  for  5 x  five  The worked  and  being  and  and  -3  10  centration  NAD'  and  are  possible  FMN  are  some  measured  are  more  cases  by  also  l-methyl-3-carbamoyl and  readily  as  1-(2  active.  much  as  shov/ed  added  as  for  reduced  positive  potentials  v i o l o g e n was  3-carbamoylpyridinium  mediators  electromotively  flavins in  as  pyridinium,  by  NAD 1,4-  The  reduc-  than  any  of  200  mv.  but  very a  the  the  little  co-mediator  to  1-carboxymethyl-  *-hydroxyethyl)-3-carbamoyl-  251  pyridinium 10  were  within  viologen. and  since  pyridines  tions  were  that  were  added  the c e l l  Identical  prepared.  Benzyl  potential  were  solutions viologen  measured  NAD m o d e l s  general, were  quantities  corresponding polarography pyridinium  salt  concentration  of a  value,  substituted  fitted  with  were  between  10  -3  a n d 10  -2  with  NAD  agreed  and  +  oxidase  for  to within  5 mv.  potentials of the  into  jacket. chosen Two  the  time the  of mixtures.  weighed  were  to the other.  hours  pyridinium  M.  poten-  the xanthine  a f t e r which  were  orn o t .  i n t h e measurement o f  twelve  a water  and d i h y d r o p y r i d i n e  correct  was a d d e d  over  solu-  was p r e s e n t  and x a n t h i n e  by t h e method  dihydropyridine cell  of identical  the oxidation-reduction  determined  using the  containing  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  In  desired  a constant  be u s e d t o  as a c a t a l y s t  giving  system  1,4-dihydro-  1,4-dihydro-  from  was c o m p a r e d  solution required  to reach  by  viologen  t o one s o l u t i o n and r i b o f l a v i n  riboflavin  with still  potentials  mediator  pH  methyl r e d ,  riboflavin  derived  methyl  t h e method  must  viologen  the f l a v i n s  viologen  potentials.  +  benefits  s i m i l a r whether  measurements,  NADH  The  since  directly  of using  near  p o t e n t i a l of methyl  viologen,  riboflavin  o f methyl  any p o s s i b l e  ensure  benzyl  S d i d not react  The n e c e s s i t y  oxidase/benzyl NAD  viologen,  blue  viologen  To tial  of the reduction  the reduction  negate  methyl  potentials of the solutions  and, t h e r e f o r e ,  pyridines. may  50 mv.  Methyl  alizarin  catalyze  the c e l l  salt  The  and t h e  a Metrohm The w e i g h t o f to give  to four  a  total  milligrams  252  of  riboflavin  o r FMN  (and a p p r o x i m a t e l y  methyl  v i o l o g e n , when u s e d )  flavin  was u s e d  less  than  cantly, act  leaving  15  the t o t a l  t o 20  decomposition catalyzed  in solution  mediator  were  chosen  o f NAD , +  determined  to prevent,  and pH  made  NMN ,  while  where  used  to ten percent  added  to the  cell  the s o l u t i o n f o r  the  the acid The  poten-  l-carbamoylmethyl-3-carbamoylpyridinium  +  betv/een  possible,  minimizing  l-acetonyl-3-carbamoylpyr idinium  pH  8 and 10.  i o n ( H i e )and  ion ( H i d ) could  be  Acid-catalyzed decomposition p o t e n t i o m e t r i c measurements  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  below  pH  9.5  or l-methyl-3-carbamoylpyridinium  The o x i d i z e d  pyridinium and  were  l-carboxymethyl-3-carbamoylpyridinium 10.  f o r i tto  was  of the d i h y d r o p y r i d i n e .  the-dihydropyridines prevented  (iiic)  signifi-  c o n c e n t r a t i o n was  by s t i r r i n g  of pyridinium ions  decomposition  (Illi),  being  of five  mis. of buffer  dissolved  s o FMN  Ribo-  A t pH's  riboflavin  l-methoxymethyl-3-carbamoylpyridinium  of  7.5.  of  minutes.  Buffers  ion  were  than  the c e l l .  precipitated  pyridiniunvdihydropyridine Twenty-five  into  milligram  riboflavin  as a p o t e n t i a l  the c r y s t a l s  tials  greater  (A c o n c e n t r a t i o n o f f l a v i n  required.) and  too l i t t l e  effectively  weighed  t h e pH was  7.5, t h e r e d u c e d  instead. of  when  were  one-half  (IIIh),  forms  of  ions  1-carbamoylmethy1-3-cyanopyridinium  rapidly  with  base  below  1-cyanomethyl-3-carbamoyl-  l-carbamoylmethyl-3-acetylpyridinium (IIIj)  sufficiently  (Ilia)  (Illb)  to prevent  their  ions  (Illk) react  potentials  253  III a:  R  l  =  V  C  H  2  C  0  R  °  b:  CH  c:  CH  d:  CH OCH  e:  CH„COCH„ 2 3  f:  CH COOCH  g:  3  -  CONH CONH  3  0  CONH  CH,OH  2  2  CONH  3  2  CONH CONH  2  CH COOCH(CH )  CONH  2  h:  CH CN  CONH  2  i :  CH CONH  CONH  2  j:  CH CONH  COCH  3  k:  CH CONH  1:  CH CONH  m:  CH^CONH  n:  CH CONH  2  3  2  3  2  2  2  2  CN  2  2  2  F  2  H 2  .  OH  254  from  being  measured  respectively.  At  decomposition (Vh) The  and ester  was  the  of  group ion  only  pyridine  of  of  the  (Illf)  stable  (Vf)  i t was  this  two  greater  lower  than  pH's  6.0,  there  7.5,  i s some  was  so  rapidly  in acidic  media  hydrolyzed  where  the  by  underwent  impossible The  rapid to  acid-catalyzed  determine  more  the  sterically  reduction hindered  It  the  stable  at  this  at pH,  pH  7  30%  1-carboxymethyl  to of  assumed  that  only  allow the  compound  d i h y d r o p y r i d i n e m a t e r i a l had was  that  one  potential  ester  sufficiently even  base  decomposition.  was  ations but  (Vk).  corresponding  l-carbo-i-propoxymethyl-3-carbamoylpyridinium  the  unavoidable  1-carbornethoxymethyl-3-carbamoyl-1,4-dihydro-  compound.  to  9.5,  l-carbomethoxymethyl-3-carbamoyl-  the  lyzed  and  l-cyanomethyl-3-carbamoyl-l,4-dihydropyridine  d ihydropyr idine  Thus  pH's  1-carbamoylmethyl-3-cyano-1,4-dihydropyridine  pyridinium it  at  ion  potential  ester  had  (Ilia)  decomposed  (Illg)  determin-  been  and  of  hydro-  20-30%  after  of  eight  d i h y d r o p y r i d i n e compound  hours. was  present,  1-carbo-i-propoxymethy1-3-carbamoy1-1,4-dihydro-  pyridine  (Vg).  approximately compound  and  With 60  the  centrations  of  cent  total  the (Va).  of  the  form  of  The  a  reduction potential  millivolts  between  1-carboxymethyl  the  two  pyridinium  the  of  1-carbo-i-propoxymethyl  compound ions,  difference  and  only  near one  to  equal two  dihydropyridine concentration should  conper-  be  in  l-carboxymethyl-3-carbamoyl-l,4-dihydropyridine lability  of  the  ester  group  was  utilized  for  the  255  preparation since  at  of  high  l-carboxymethyl-3-carbamoyl-l,4-dihydropyridine pH,  the  1,4-dihydropyridine  ester  (Vf)  of  l-carbomethoxymethyl-3-carbamoyl-  i s r a p i d l y hydrolyzed  and  1-carboxymethyl-3-carbamoyl-1,4-dihydropyridine composed  only  very  Equilibrium was  generally  pyridinium to  reach  was  final and  placed  pH.  a  0.1  rubber  less the  of  cm.  as  of  eight  reduction After  analyzed salt  de-  to  potentiometry cell  in  potential Those  potentials  the  and  cell  hours.  cell  the  dihydropyridine • solution  the  tended  potential  determine  dry  i n an  erlenmeyer  possible  after  removal  of  s o l u t i o n was  three  regions  solving  between  include  and  oxidized  the  systems  absorbance  combinations the  on  was  box.  The  flask  sealed  440  between the  and of  data.  v/ere  from the  used  concentration  Cary  the  values  spectroand  300  and  400  nm.  The  reduced  and  16  box,  nm.  absorbance  least  dry  460  maxima  five  were  linear  calequations  different  resulting used  of  concentrations  flavin  simultaneous At  the  and  oxidized.'.f l a v i n s .  absorbance  give  read  wavelengths  the  to  the  constant  placed  containing  averaged  to  pyridinium  wavelengths  dihydropyridine by  were  absorbance  the  two  wavelength  culated  three  a  quickest.  of  IV>^=^ihydropyriain'e: and of  is  septum.  at  minimum  by  negative  solutions  quickly  photometer  These  within  sample  absorbance  a  determined  s o l u t i o n was  As  at  the  A  i n an  remaining  the  with  concentrations  the  with  as  equilibrium  measured,  (Va)  product  slowly.  reached  ions  the  in  values subsequent  calculations.  When  necessitated  by h i g h d i h y d r o p y r i d i n e c o n -3  centrations solutions  (greater  were  than  diluted  5 x 10  v/ith  M.), t h e p o t e n t i o m e t r i c  buffer  before  the absorbance  was  read . The  concentration  etry  solution  line  was c a l c u l a t e d  least  seven  bration  was d e t e r m i n e d by  were  the l i n e s  solution  an  flask.  erlenmeyer minutes  after  polarography to  minimize  pyridine  mined.  The a n a l y s i s  the polarogram,  buffer  never  varied  Pyridinium  salts  have  photometry  and a t t e m p t s  acetonyl-3-carbamoylpyridinium ions  could  was u s e d ,  made  determinations of these  acetylpyridinium  i s being  than  an a b s o r b a n c e were  and  M.  The  v/ithin  contents  be c a r r i e d  by more  cali-4  t h e d r y box s e a l e d i n  a i roxidation  oxidase  The  the o r i g i n .  was r e c o r d e d  must  from a t  a n d 10  into the out quickly  of dihydroassayed.  t h e pH o f t h e s o l u t i o n  v/hen x a n t h i n e  a n d 270 nm.  squares  10  from  i o n which  calibration  between  the erlenmeyer  the f l a v i n - c a t a l y z e d  Except  particular  260  cell.  A  pairs. . -2  The p o l a r o g r a m  pouring  i n the potentiom-  current  through  was r e m o v e d  to the pyridinium  recording  of least  linear  d i d not pass  potentiometry  tv/o  t h e method  generally  salt  by p o i a r o g r a p h y .  concentration-diffusion  lines  although  of pyridinium  v/as d e t e r -  t h e pH  .05 pH maximum  of a  units. between  to use d i r e c t  compounds.  After  Only  spectro1-  1-carbamoylmethyl-3-  be a s s a y e d  i n this  results  f o r these  tv/o c o m p o u n d s  agreed  with  results  to within  ten percent.  The p r o d u c t  way.  The  the polarographic of acid  catalyzed  257  decomposition  of 1,4-dihydropyridines exhibits 1  bance the  maximum  direct  pyridinium  near  as  290 nm.  ' '  spectrophotometric  '  success method  f o r NAD  f o r NAD  models.  +  in  much  more  coverted aqueous  '  with  inspired  +  and t h e c y a n i d e  have  a 15 m i n u t e  pH ' s a t w h i c h carried  out.  successfully,  +  which  both  underwent  period  pyridines  than  NAD  were  to 4-cyano-l,4-dihydropyridines  solutions  NAD  this  1-acetony1-3-  substituted  acetylpyridinium than  adduct  t o 1-methoxy-  ( H i d )and  since  to use  reduction potentials  quantitatively  ions,  the cyanide  attempts  and  over  of other  was l i m i t e d  ions  (Hie)  negative  cyanide  '  cyanomethyl-3-carbamoylpyridinium  potentials  with  31 3 2  Success  carbamoylpyr idinium ions  not  interferes  determinations  o f Rodkey  methyl-3-carbamoylpyridinium  having  which  compounds.  an a s s a y  method  absor-  9 10 71  14 The  a high  adducts  o f 1-  1-carbamoylmethyl-3more  significant  positive  reduction  decomposition  a t pH 5.6 a n d 8.1 r e s p e c t i v e l y , t h e  the potentiometry I n t h e few c a s e s i tconfirmed  of these  i n which  c o m p o u n d s v/as t h e method  was  used  the concentration determined  by  polarography. The ion  held  methods  polarographic determination of the pyridinium  another which  advantage  were  the p y r i d i n i u m r i n g  unable  over  t h e two s p e c t r o p h o t o m e t r i c  to detect decompositions  intact.  left  I f the reduction potentials of  the  original  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  are  sufficiently  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  different,  which  a s v/as o f t e n  the case,  product then the  by p o l a r o g r a p h y .  One  258 commonly  met  3-carbamoyl duct  had a  negative ester  ions as  group  than  and  of  original  greater  due  was  than  betv/een  the h y d r o l y s i s 10.  The  200 a n d  to. t h e 3 - c a r b a m o y l  of the  3-carboxy 3 0 0 mv.  pro-  more  compound.  The  l-carbomethoxymethyl-3-carbamoylpyridinium  l-carbo-i-propoxymethyl-3-carbamoylpyridinium (Illg)  7 and  pyridinium  reaction  potential  t h e v/ave  underwent  low as  a t pH's  half-wave  groups  (Illf)  decomposition  significant  the r e s u l t i n g  ion (Ilia)  esters  base-catalyzed hydrolysis  on  the  was  at a  l-carboxymethyl-3-carbamoyl-  easily  distinguished  polarograms.  from  the  pH  259  6.8  Absorbance  Spectra  Absorbance photometer Spectral  between  of  most  Table of  of A  near  max  A  max  were were  repeated  until  intervals. the e x t i n c t i o n  extinction  i n this recorded  study  are listed  methy1-3-fluoro-1,4-dihydropyridine i n pure  buffer.  Typical  pyridine  a r e shown  33.  spectra  that  CH3  c:  CH^CH  d:  CH  i t i s shown  was  i n pH  3  2  CONH„ 2 OH  C0NH  CONH.,  salt  2  F  and a  dihydro-  of 1-carbamoyl-  so u n l i k e  separately i n Figure  10  R =CONH  2  b:  1-carboxy-  decompose  recorded  The spectrum  methyl-3-fluoro-1,4-dihydropyridine  those  1-carbamoyl-  R^=CH C00~  spectra of a pyridinium i n Figure  (Vc) ,  ( V I ) , which  a:  in  except  ( V a ) , and  v/ater and t h e r e f o r e were  The  (Vb), l-(2'-  h y d r o x y 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 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  3%.  coefficients  i n water,  l-methyl-3-carbamoyl-1,4-dihydropyridine  rapidly  16 s p e c t r o -  reproducible to within  used  The s p e c t r a were  on a Cary  a t 10 nm.  and t h e c o r r e s p o n d i n g  o f t h e compounds  XIX.  recorded  220 a n d 520 nm.  determinations  coefficients values  s p e c t r a were  34.  the other  260  T a b l e XIX A  max  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 this H  CH  COO  CH3  CONH  2  CONH  0  -  H  -  356  7360  -  360  7280  CH,CH OH  CONH,,  265  4340  357  7510  CH OCH  CONH  264  ^4500  338  6490  266  4860  353  6300  266  4750  347  6900  349  6360  2  2  3  CH „,COCH„ 2 3 CH COOCH 2  CH  CONH CONH  3  COOCH(CH  CH CN  )  CH CONH  2  CONH  CH CONH  2  COCH  2  2  CH CH  t  CONH 2  2  CONH „ 2  were  2  -  CONH CONH  2  2  CN F  n o t measured.  2  3  -  265  4900  340  5820  267  4800  351  6800  267 . 5  4450  370  12000  269.5  4670  334  5680  -  -  see  Figure  34  Work  Figure  33. 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