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Exchange and oxidation reactions of 6,7,8-trimethyllumazine and its dihydroderivative McAndless, John M. 1969

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THE EXCHANGE AND OXIDATION REACTIONS OP 6,7j8-TRIMETHYLLUMAZINE AND ITS DIHYDRO DERIVATIVE  by JOHN M. McANDLESS B.Sc,  U n i v e r s i t y o f B r i t i s h Columbia,  1965  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY  i n t h e Department of  Chemistry-•  We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e required standard  THE UNIVERSITY OF BRITISH COLUMBIA May, 1969  @  John M. McAndless  1969  In  presenting  an  advanced  the I  Library  further  for  this degree shall  agree  scholarly  by  his  of  this  written  thesis  in p a r t i a l  f u l f i l m e n t of  at  University  of  the  make that  i t freely  permission  purposes  may  representatives. thesis  for  It  financial  available for  of  Chemistry  The U n i v e r s i t y o f B r i t i s h V a n c o u v e r 8, Canada  A p r i l 15,  196Q  by  the  is understood gain  Columbia  for  extensive  granted  permission-  Department  Date  be  British  shall  reference  Head  be  requirements  Columbia,  copying  that  not  the  of  and  of my  I agree  for that  Study.  this  thesis  Department  copying  or  allowed  without  or  publication my  i  ABSTRACT Supervisor:  P r o f e s s o r R. S t e w a r t  The p o t e n t i a l use o f 6 , 7 , 8 - t r i m e t h y l l u m a z i n e ( 6 , 7 , 8 - t r i m e t h y l - 2 , 4 - d i o x o p t e r i d i n e ) and I t s d i h y d r o d e r i v a t i v e as a redox c o u p l e f o r t h e p t e r i d i n e and f l a v i n systems i s investigated.  The s y n t h e s e s o f t h e above compounds and r e l a t e d  lumazine d e r i v a t i v e s a r e d e s c r i b e d and t h e i r s p e c t r o s c o p i c and c h r o m a t o g r a p h i c p r o p e r t i e s a r e t a b u l a t e d . W h i l e examining the p.m.r. s p e c t r a o f 6,7,8t r i m e t h y l l u m a z i n e (TML) and 7 , 8 - d i h y d r o - 6 , 7 , 8 - t r i m e t h y l l u m a z i n e (DHTML) i n d e u t e r i u m o x i d e media, the  the hydrogens o f  C-7 m e t h y l group and o f the C - 6 methyl group i n t h e former  and l a t t e r compounds r e s p e c t i v e l y were o b s e r v e d t o undergo exchange. In t h e pH range - 0 . 4 t o 8.0, t h e exchange o f TML I s subject to general acid-base c a t a l y s i s .  The pseudo  first-  o r d e r exchange r a t e c o n s t a n t (k-^) was found t o be the sum o f s e v e r a l c a t a l y t i c terms.  Prom measurements o f t h e exchange  r a t e - b u f f e r dependency a t v a r i o u s pH v a l u e s , the f o l l o w i n g v a l u e s o f t h e s p e c i f i c c a t a l y t i c c o n s t a n t s ( i n l.mole "''min, ) 1  have been c a l c u l a t e d : V  - 1.2, k  H 3 P 0 4  = 0.41, k  H 2 P Q 4  - = 0.14, ^ ^ - 2  = 52.9  The mechanism o f t h e a c i d - and b a s e - e a t a l y z e d exchange o f TML i s o u t l i n e d .  The r e l a t i v e l y f a c i l e exchange  ii  i s e x p l a i n e d i n terms o f t h e f o r m a t i o n o f n e u t r a l and i o n i c i n t e r m e d i a t e s w h i c h have l o n g c o n j u g a t e d  bonding arrangements.  Pseudo f i r s t - o r d e r r a t e c o n s t a n t s  have been t a b u l a t e d  f o r t h e g e n e r a l a c i d - c a t a l y z e d exchange o f DHTML i n t h e pH range - 0 . 4 t o 8 . 2 l e a d i n g t o an u n u s u a l pH-rate p r o f i l e .  The  marked d e c r e a s e i n exchange r a t e s and anomalous r a t e p l o t s i n the more a c i d i c pH r e g i o n s i s a t t r i b u t e d t o a c i d - c a t a l y z e d c o v a l e n t h y d r a t i o n a c r o s s t h e C ( 6 ) - N ( 5 ) double bond and e q u i l i b r a t i o n o f hydrated f o r h y d r a t i o n i s presented  and unhydrated c a t i o n s .  Evidence  and t h e exchange mechanism i s  discussed. The  f e r r i c y a n i d e o x i d a t i o n o f DHTML was i n v e s t i g a t e d  i n t h e pH range 5 t o 1 2 . 5 by s p e c t r o p h o t o m e t r y and potentiometric techniques. i n a complicated  The s e c o n d - o r d e r r e a c t i o n proceeds  sequence o f s t e p s , some o f w h i c h a r e dependent  upon t h e i o n i z a t i o n o f t h e r e a c t i o n i n t e r m e d i a t e , t r i m e t h y l lumazine.  Beyond pH 1 1 , t h e o x i d a t i o n r a t e i s d i r e c t l y  p r o p o r t i o n a l to the hydroxide  i o n concentration.  At pH 1 2 , t h e absence o f a p r i m a r y  i s o t o p e e f f e c t and  the i n h i b i t o r y e f f e c t o f added f e r r o c y a n i d e i n d i c a t e t h a t t h e o x i d a t i o n proceeds v i a an i n i t i a l r a p i d and r e v e r s i b l e onee l e c t r o n a b s t r a c t i o n from t h e a n i o n i c form o f DHTML. The r e s u l t i n g mesoroeric r a d i c a l c a n undergo f u r t h e r r e a c t i o n s w i t h f e r r i c y a n i d e i o n o r w i t h hydroxide A very negative A  ion.  v a l u e was o b t a i n e d  f o r the  r e a c t i o n a t pH 1 2 , c o n s i s t e n t w i t h a r e a c t i o n between i o n s o f  iii  the same c h a r g e .  A s t u d y o f the e f f e c t s o f p o t a s s i u m i o n  on the o x i d a t i o n r a t e i n d i c a t e s the p a r t i a l p a r t i c i p a t i o n o f the a s s o c i a t e d  species  KFe(CN)g  i n the  reaction.  A l i m i t e d s t u d y o f the r e a c t i o n between t r i m e t h y l lumazine and  f e r r i c y a n i d e was  undertaken.  a n i o n o f TML  reacts v i a a h i g h l y coloured  produce 7 - o x o - 6 , 8 - d i m e t h y l l u m a z i n e . i n t o the r e a c t i o n i s r e q u i r e d .  The  hydroxylated  intermediate  Further  to  investigation  iv  TABLE OF CONTENTS INTRODUCTION  ..  . .  Page 1  ....  A. B.  The P t e r i d i n e s ; I n t r o d u c t i o n and Nomenclature . P h y s i c a l and C h e m i c a l P r o p e r t i e s o f the P t e r i d i n e s 1. C h a r a c t e r i z a t i o n .. .. ... 2. Ring cleavage r e a c t i o n s . 3. Covalent hydration 4 . R e d u c t i o n and o x i d a t i o n .. ...  C.  Methods o f P r e p a r a t i o n .. .. 1. I s a y r e a c t i o n 2. C y c l i z a t i o n o f pyrimidines 3 . S y n t h e s i s from p y r a z i n e i n t e r m e d i a t e s  D.  B i o l o g i c a l Importance o f P t e r i d i n e s  E.  Redox R e a c t i o n s  o f the F l a v i n s •  OBJECTS OF THE PRESENT RESEARCH EXPERIMENTAL  1 3, 4 6 11  . . . 19 19 21 22 24 . . . 30 35  ..  37  ..  A. C h a r a c t e r i z a t i o n o f t h e P r e p a r e d Compounds .. . 3 7 B. P r e p a r a t i o n and P u r i f i c a t i o n o f t h e Lumazines . . . 39 P a r t I : STABILITY OF THE LUMAZINE DERIVATIVES IN SOLUTION A. B. C. D.  Introduction .. . Experimental Results Discussion .. 1. 5 - A m i n o - 4 - m e t h y l a m i n o - 2 , 6 - d i h y d r o x y p y r i m i d i n e . 2. H y d r o l y s i s o f the lumazines  Part I I :  HYDROGEN-DEUTERIUM  59 59 6l 72 72 74  EXCHANGE  A. B.  Introduction ... Experimental .. 1. P r e p a r a t i o n o f samples f o r k i n e t i c s t u d i e s . 2 . Treatment o f t h e d a t a I n exchange e x p e r i m e n t s .  85 86 86 88  C.  Results 1. Trimethyllumazine r . a. Assignment o f t h e exchanging group b. pH-rate p r o f i l e c. E f f e c t o f b u f f e r c o n c e n t r a t i o n on exchange r a t e  90 ..  .90 92 98  V  Page 2. 7,8-Dihydro-6,7,8-trimethyllurnazine a. Assignment o f t h e exchanging group .. b. pH-rate p r o f i l e c. E f f e c t o f b u f f e r on t h e exchange r a t e d. C a t a l y s i s by m e t a l c a t i o n s D.  Discussion 1. Mechanism o f the exchange p r o c e s s a. T r i m e t h y l l u m a z i n e b. 7 , 8 - D i h y d r o - 6 , 7 , 8 - t r i m e t h y l l u m a z i n e  . . . 107 109 .. . 114 116 121  ..  . . . 121 . . . 125  2 . H y d r a t i o n o f 7 , 8 - d i h y d r o - 6 , 7 , 8 - t r i m e t h y l l u m a z i n e . 132 3. Biological implications .. . . . 138 P a r t I I I : OXIDATION OF TRIMETHYLLUMAZINE AND DIHYDROTRIMETHYLLUMAZINE A. B.  Introduction . E x p e r i m e n t a l .. 1. P u r i f i c a t i o n o f s o l v e n t s and m a t e r i a l s 2 . P r e p a r a t i o n o f b u f f e r and sample s o l u t i o n s 3 . Techniques employed i n k i n e t i c measurements i . Spectroscopy i i . Potentiometry  l40 l4l l4l .. . 142 .. . l 4 5 l45 l48  4 . Treatment o f t h e k i n e t i c d a t a C.  151  Results . . .. 1. O x i d a t i o n o f 7 , 8 - d i h y d r o - 6 , 7 , 8 - t r i m e t h y l l u m a z i n e w i t h potassium f e r r i c y a n i d e a. P r o d u c t s o f t h e r e a c t i o n and s t o i c h i o m e t r y . b. E f f e c t o f h y d r o x i d e i o n c. A c t i v a t i o n parameters d. E f f e c t o f added f e r r o c y a n i d e e. E f f e c t o f s p e c i f i c a n i o n s f. E f f e c t o f potassium i o n g. K i n e t i c i s o t o p e e f f e c t .. 2. Potentiometry 3. Reaction o f 6,7,8-trimethyllumazine with ferricyanide a. S p e c t r o p h o t o m e t r y r e s u l t s .. .. b. P o t e n t i o m e t r y 4. Miscellaneous reactions a. D i s p r o p o r t i o n a t i o n experiment b. Permanganate o x i d a t i o n  ..  153 153 154 l63 166 l68 170 172  . . . 175 178 178 179 ..  . 180 .. . l 8 0 . l80.  vi  Page D.  Discussion  ..  SUGGESTIONS FOR FURTHER WORK BIBLIOGRAPHY  ....  ..  l 8 l 190  .. '  193  vii  LIST OF TABLES Ia  Page  Spectroscopic data f o r prepared compounds ..  ...  55  lb  I o n i z a t i o n c o n s t a n t s o f lumazine derivatives  56  II  P.m.r. s p e c t r a l d a t a f o r l u m a z i n e derivatives  57  III IV V  Chromatography o f p r e p a r e d compounds  .  F L . v a l u e s and f l u o r e s c e n c e c h a r a c t e r i s t i c s o r v a r i o u s compounds 70 Rates o f exchange o f 6 , 7 , 8 - t r i m e t h y l l u m a z i n e i n D 0 i n t h e pH range - 0 . 4 to 8 . 0  95  E f f e c t o f b u f f e r c o n c e n t r a t i o n on t h e exchange r a t e o f 6,7,8-trimethyllumazine  100  p  VI VII  58  Rates o f exchange o f 7 , 8 - d i h y d r o - 6 , 7 , 8 t r i r o e t h y 1 l u m a z i n e i n D 0 i n t h e pH range - 0 . 4 to 8.2 .. 113 2  VIII  E f f e c t o f b u f f e r c o n c e n t r a t i o n on t h e exchange r a t e o f 7 , 8 - d i h y d r o - 6 , 7 , 8 trimethyllumazine .  117  IX  E f f e c t o f pH on t h e o x i d a t i o n r e a c t i o n o f DHTML w i t h f e r r i c y a n i d e , i n i t i a l concentration r a t i o DHTML/ferricyanlde = 0 . 5 158  X  E f f e c t o f pH on t h e o x i d a t i o n r e a c t i o n o f DHTML w i t h f e r r i c y a n i d e , v a r i o u s i n i t i a l reactant concentrations .. .. . . . 1 5 8 - 9  XI XII XIII XIV  A c t i v a t i o n parameters, DHTML-ferricyanide r e a c t i o n a t pH 1 2 . 0 164 E f f e c t o f added f e r r o c y a n i d e oxidation reaction  on t h e  E f f e c t o f a n i o n s on t h e DHTMLferricyanide oxidation reaction  167  ...  168  E f f e c t o f p o t a s s i u m i o n on t h e DHTMLferricyanide oxidation reaction . ...  171  viii  XV XVI XVII  Kinetic isotope effect  .  Oxidation reaction; rate constants d e t e r m i n e d by p o t e n t i o m e t r y TML-ferricyanide reaction; reaction rates d e t e r m i n e d by p o t e n t i o m e t r y  175 176-8 179  ix  LIST OF FIGURES 1  Page  5-Amino-4-methylamino-2,6-dihydroxypyrimidine i n 0.5N sodium h y d r o x i d e .. ..  .63  2  7 , 8 - D i h y d r o - 6 , 7 , 8 - t r i m e t h y l l u m a z i n e i n 0.5N sodium h y d r o x i d e , a n a e r o b i c c o n d i t i o n s . . . 64  3  7 , 8 - D i h y d r o - 6 , 7 , 8 - t r i m e t h y l l u m a z i n e i n 0.050M KgHPO^ a t pH f» a n a e r o b i c c o n d i t i o n s .. . . . 65  4  6 , 7 - D i p h e n y l - 8 - m e t h y l l u m a z i n e i n 0.050M KpHPOh a t pH 12  66  5  6 , 7 , 8 - T r i m e t h y l l u r o a z i n e i n 0.5N sodium hydroxide  67  6  7-Oxo-6,8-dimethyllumazine a t pH 12 ..  i n 0.050M  K l^0u 2  ..  . 68  7  7 , 8 - D i h y d r o - 6 , 7 , 8 - t r i m e t h y l l u m a z i n e i n 0.050M K^HPO^ a t pH 9, a n a e r o b i c c o n d i t i o n s  8  T y p i c a l exchange p a t t e r n o f 6 , 7 * 8 - t r i m e t h y l lumazine . . . . .. . . . 91  9  TML exchange k i n e t i c s ,  10  69  . . . 94  t y p i c a l rate plot  TML exchange k i n e t i c s , r e l a t i o n between r a t e c o n s t a n t and pH  96  11  TML exchange k i n e t i c s , p H - r a t e p r o f i l e  12  TML exchange k i n e t i c s , r e l a t i o n between r a t e c o n s t a n t and b u f f e r c o n c e n t r a t i o n .. .. . . . 101  13  TML exchange k i n e t i c s , r e l a t i o n between r a t e . c o n s t a n t and b u f f e r c o n c e n t r a t i o n a t pH 4.5 . 102  14  TML exchange k i n e t i c s , r e l a t i o n between.rate c o n s t a n t and b u f f e r c o n c e n t r a t i o n a t pH 2.0  ..  .97  ..  .  103  15  T y p i c a l exchange p a t t e r n o f 7>8-dihydro6,7^8-trimethyllumazine  16  DHTML exchange k i n e t i c s , t y p i c a l r a t e p l o t f o r the n e u t r a l species .. .. .. .. . . . I l l  17  DHTML exchange k i n e t i c s , t y p i c a l r a t e p l o t f o r the c a t i o n  18  DHTML exchange k i n e t i c s , p H - r a t e p r o f i l e  " . . . 108  112 .. . 115  X  19  DHTML exchange k i n e t i c s , e f f e c t o f b u f f e r c o n c e n t r a t i o n on t h e exchange r a t e .. ..  .Il8  20  Oxidation k i n e t i c s , t y p i c a l rate p l o t , s p e c t r o p h o t o m e t r y method .. ..  21  O x i d a t i o n k i n e t i c s , e f f e c t o f pH and v a r y i n g r e a c t a n t c o n c e n t r a t i o n on the o x i d a t i o n rate .. .. .. . 160  22  O x i d a t i o n k i n e t i c s , r e l a t i o n s h i p between o x i d a t i o n r a t e and h y d r o x i d e i o n concentration  l6l  Oxidation k i n e t i c s , f i r s t - o r d e r relationship between h y d r o x i d e i o n c o n c e n t r a t i o n and oxidation rate "  162  23  156  24  O x i d a t i o n k i n e t i c s , a c t i v a t i o n parameters  . 165  25  O x i d a t i o n k i n e t i c s , e f f e c t o f added f e r r o c y a n i d e on the o x i d a t i o n r a t e ..  . 169  26  Oxidation k i n e t i c s , e f f e c t o f potassium Ion on the o x i d a t i o n r a t e  27  28  ..  ,173  O x i d a t i o n k i n e t i c s , r e l a t i o n s h i p between p o t a s s i u m I o n c o n c e n t r a t i o n and o x i d a t i o n rate  174  O x i d a t i o n k i n e t i c s , p o t e n t i o m e t r i c method  . 177  xi  . ACKNOWLEDGEMENTS I wish to express s i n c e r e a p p r e c i a t i o n to P r o f e s s o r Ross S t e w a r t f o r h i s many h e l p f u l s u g g e s t i o n s and guidance  1  d u r i n g t h e c o u r s e o f t h i s work. I would a l s o l i k e t o thank the N a t i o n a l R e s e a r c h C o u n c i l f o r generous f i n a n c i a l a s s i s t a n c e . A p p r e c i a t i o n I s extended t o Mr. Roland W. B u r t o n f o r h i s t e c h n i c a l a s s i s t a n c e i n the r u n n i n g o f n u c l e a r magnetic resonance s p e c t r a .  1  INTRODUCTION A.  The P t e r i d i n e s ; I n t r o d u c t i o n  and Nomenclature  The p t e r i d i n e s , because o f t h e i r b i o l o g i c a l importance  and e x t e n s i v e d i s t r i b u t i o n i n n a t u r e , have  a t t r a c t e d more a t t e n t i o n than any o t h e r f a m i l y o f t e t r a azanaphthalenes. occurring  However, t h e amounts o f n a t u r a l l y  p t e r i d i n e d e r i v a t i v e s commonly i s o l a b l e a r e , i n  general, exceedingly small. rather  That f a c t , c o u p l e d w i t h t h e  inconvenient properties  example; d i f f i c u l t y  o f the p t e r i d i n e s - - f o r  o f combustion and low s o l u b i l i t y i n most  s o l v e n t s — h a s made t h e s t u d y o f t h e i r c h e m i s t r y an experimentally d i f f i c u l t  field.  Only more r e c e n t l y w i t h t h e  advent o f modern a n a l y t i c a l t e c h n i q u e s has e x a c t knowledge o f these substances  become a v a i l a b l e .  The p t e r i d i n e n u c l e u s  ( i ) , c o n s i s t i n g o f fused  p y r a z i n e and p y r i m i d i n e r i n g s , i s numbered a c c o r d i n g t o t h e R i n g Index ; 1  t h i s numbering system i s widely adopted i n recent  publications.  A c c o r d i n g t o t h i s assignment t h e most r e c e n t  s y s t e m a t i c name f o r p t e r i d i n e i s p y r a z i n o ( 2 , 3 - d ) p y r i m i d i n e o r 1,3,5,3 - t e t r a - a z a n a p h t h a l e n e .  A numbering system, shown I n  ( l l ) r e l a t e d t o t h a t o f t h e p u r i n e s has been used in publications  from European l a b o r a t o r i e s .  f r e q u e n t r e f e r e n c e made t o them throughout  extensively  Because o f t h e t h i s work, t h e  2 numbering systems used f o r p y r i m i d i n e s ( i l l ) isoalloxazines  (IV) are also  and t h e  included.  5 ( 1 ) 4 -  G  2  5  3  8 9P 5  (in)  10 (iv)  I  .4  2  n  The e l e c t r o n d i s t r i b u t i o n about the p t e r i d i n e n u c l e u s has been t h e s u b j e c t  of several  The d i s t r i b u t i o n o f " i f - e l e c t r o n  c a l c u l a t i o n s ' ^  charges, although d i f f e r i n g '  s l i g h t l y i n v a l u e depending, on t h e method o f be c o n s i d e r e d r e p r e s e n t a t i v e  3  calculation,can  as shown i n ( V ) . As 2  indicated,  the c a r b o n atom a t p o s i t i o n 7 i n the n u c l e u s e n j o y s l e s s Tfe l e c t r o n charge than does t h e one a t t h e 6 p o s i t i o n and t h i s may p l a y a v i t a l r o l e i n c e r t a i n r e a c t i o n s derivatives.  1.142  0.877r" " V ,  1.204  >6.88l 70  0.9 0 ? 1.111  0.822  (V)  of pteridine  1  2  3  Albert  has  p o i n t e d out  l o c a l i z a t i o n o f the t e n s t a b i l i z a t i o n on  t h a t , because o f  TT - e l e c t r o n s  a v a i l a b l e f o r aromatic  the e l e c t r o n - a t t r a c t i n g h e t e r o atoms,  p t e r i d i n e i s somewhat e l e c t r o n - d e f i c i e n t .  Hence the  r e a d i l y undergo n u c l e o p h i l i c s u b s t i t u t i o n and reactions  rather The  the  pteridines  displacement  than e l e c t r o p h i l i c s u b s t i t u t i o n .  c h e m i s t r y and  biology  o f p t e r i d i n e s have been 2-8  treated  i n considerable d e t a i l i n several reviews.  b r i e f summary o f some o f t h e i r more i m p o r t a n t methods o f s y n t h e s i s  and  properties,  biological significance  to the p r e s e n t I n v e s t i g a t i o n w i l l be  presented.  B.  o f the  P h y s i c a l and 1.  Chemical P r o p e r t i e s  pertinent  Pteridines  Characterization The  extreme i n s o l u b i l i t y and  melting points  o f the m a j o r i t y  h i g h and  indefinite  of p t e r i d i n e d e r i v a t i v e s  the u s u a l c r i t e r i a o f p u r i t y o f l i t t l e v a l u e . pteridines  A  The  render  purity  of  i s most r e l i a b l y determined by the use of' paper 2  and  t h i n - l a y e r chromatographic techniques.  Suitable  f o r the d i f f e r e n t i a t i o n o f p t e r i d i n e d e r i v a t i v e s are aqueous ammonium c h l o r i d e and The  majority  v i o l e t l i g h t a t 254 as b l a c k s p o t s on  s i n c e t h e y e i t h e r absorb  the w h i t e p a p e r ) o r f l u o r e s c e  c h a r a c t e r i s t i c colours  (7:3).  the i n t e r m e d i a t e s used i n  can be d e t e c t e d by i r r a d i a t i o n w i t h mu,  3$  butan-l-ol-5M acetic acid  o f p t e r i d i n e s , and  t h e i r preparation  solvents  at that wave-length.  ultra-  (appearing in  4  U l t r a v i o l e t spectroscopy also provides  a convenient  means o f c h a r a c t e r i z a t i o n , e.g., by c r y s t a l l i z a t i o n to 2 4 l 4 15 16 spectrum. E x t e n s i v e  tabulations ' '  as w e l l as the i o n i z a t i o n c o n s t a n t s  'of  J  the  constant  spectra,  i n the form o f pKa  value§,  o f p t e r i d i n e d e r i v a t i v e s are a v a i l a b l e . From numerous s t u d i e s o f the u l t r a v i o l e t and i n f r a r e d spectra of p o t e n t i a l l y tautomeric d e r i v a t i v e s , i t has  hydroxy- and 2 13  been c o n c l u d e d '  aminopteridine  t h a t the hydroxy  p t e r i d i n e s p r e f e r the amido tautomer whereas, i n g e n e r a l , N-heterocyclic 2.  amines e x i s t i n the t r u e amino form.  Ring cleavage r e a c t i o n s T h i s s u b j e c t has  been reviewed e x t e n s i v e l y  by  17 Taylor may  who  pointed  be c l e a v e d  out t h a t p r o b a b l y e v e r y known p t e r i d i n e  by b a s i c h y d r o l y s i s under s u f f i c i e n t l y  vigorous  c o n d i t i o n s . In most cases i t i s the p y r i m i d i n e r i n g w h i c h i s opened but the ease w i t h w h i c h t h i s o c c u r s depends upon the substituents present. to l o s e the p y r i m i d i n e a c i d and  T h i s tendency o f the p t e r i d i n e n u c l e u s ( r a t h e r than the p y r a z i n e )  a l k a l i n e h y d r o l y s i s i s consonant w i t h the  r i n g on electron  2  d e n s i t y diagrams more p o l a r and  w h i c h show the p y r i m i d i n e m o i e t y to  be  hence more r e a d i l y a t t a c k e d .  P t e r i d i n e i t s e l f i s cleaved normal p o t a s s i u m h y d r o x i d e ,  i n one  hour by  boiling  whereas those p t e r i d i n e s c a r r y i n g  h y d r o x y l , amino, a l k y l a m i n o o r mercapto groups i n p o s i t i o n 2 a r e d e s t r o y e d i n one minute when b o i l e d i n normal sodium l8a  hydroxide.  On  the o t h e r hand, l e u c o p t e r i n  (2-amino-4,6,7-  5  trihydroxypterldlne)  r e s i s t s a l k a l i and o n l y under such  v i g o r o u s a c i d t r e a t m e n t as to cause d i s r u p t i o n o f the p y r a z i n e r i n g does t h i s compound succumb.  The v i g o r o u s h y d r o l y s i s  o f r e a d i l y a c c e s s i b l e l u m a z i n e and s u b s t i t u t e d  lumazines  ( 2 , 4 - d i h y d r o x y p t e r i d i n e s ) has y i e l d e d 2 - a m i n o p y r a z i n e  and  19 s u b s t i t u t e d p y r a z i n e amino a c i d s .  The l a t t e r can be  d e c a r b o x y l a t e d by h e a t i n g w i t h 80$  s u l f u r i c a c i d to g i v e  s u b s t i t u t e d a m i n o p y r a z i n e s , i m p o r t a n t as i n t e r m e d i a t e s i n the s y n t h e s i s o f s u l f a d r u g s . When the p y r a z i n e r i n g o f a p t e r i d i n e c a r r i e s a h y d r o x y l group, b a s i c h y d r o l y s i s r e s u l t s I n c l e a v a g e o f t h i s r i n g to s u b s e q u e n t l y y i e l d a p y r i m i d i n e d e r i v a t i v e .  For  20  example,  7 - h y d r o x y - as w e l l as 6 - h y d r o x y p t e r i d i n e g i v e  4,5-diaminopyrimidlne i n d i l u t e a l k a l i .  S i m i l a r l y , the  c o n j u g a t e d lactams 8 - m e t h y l - 7 - p t e r i d i n o n e and  6,8-dimethyl-  7 - p t e r i d i n o n e ( V I ; R = H and R = CH^ r e s p e c t i v e l y ) a r e h y d r o l y z e d i n normal sodium h y d r o x i d e a t 1 0 0 ° to 5 - a m i n o - 4 - m e t h y l a m i n o p y r i m i d i n e  f o r two  hours  21  ( V I I ) , as determined  20  paper  chromatography. i  OH"  ^  "V^lj H , 2  (VII)  D i s r u p t i o n o f the p y r a z i n e r i n g i s a l s o the case  by  6  for  5j6-dihydropteridines  w h i c h undergo f a c i l e h y d r o l y s i s t o For instance, 5,6-dihydro-7-hydroxy-  pyrimidine derivatives.  p t e r i d i n e i s i n s t a n t l y converted aminopyrimidine  i n t o 4-amino-5-carboxymethyl-  i n b o i l i n g normal sodium  However, 5 > 6 , 7 j 8 - t e t r a h y d r o p t e r I d I n e s , substituted 4,5-diaminopyrimidines, and  hydroxide. ^ 1  3  which a r e merely a r e s t a b l e to b o t h a c i d  base h y d r o l y s i s . P t e r i d i n e s w h i c h a r e c l e a v e d by a l k a l i a r e a l s o 21  s u s c e p t i b l e to cleavage the p r o d u c t s  by amines.  22  '  Extensive l i s t s o f  o f h y d r o l y s i s and a m i n o l y s i s o f p t e r i d i n e  d e r i v a t i v e s under a wide v a r i e t y o f c o n d i t i o n s have been 4,8  published. 3.  Covalent  hydration  R e v e r s i b l e c o v a l e n t h y d r a t i o n a c r o s s C=N bonds occurs  i n a number o f n i t r o g e n - c o n t a i n i n g h e t e r o c y c l e s ,  i n c l u d i n g q u i n a z o l i n e (as t h e c a t i o n ) ,  1,4,6-triaza-  n a p h t h a l e n e (as the c a t i o n ) and p t e r i d i n e and some o f i t s hydroxy d e r i v a t i v e s .  T h i s phenomenon has been reviewed I n  7,23,24,25  several a r t i c l e s .  At p r e s e n t , two hypotheses 26  a p p a r e n t l y c o v e r t h e known examples o f c o v a l e n t h y d r a t i o n . H y d r a t i o n may be expected  a t a d o u b l e bond (a) i f s u f f i c i e n t  e l e c t r o n - a t t r a c t i n g c e n t e r s a r e p r e s e n t so t h a t the double bond no l o n g e r p a r t i c i p a t e s g r e a t l y i n a r o m a t i c and  conjugation  (b) i f f o r m a t i o n o f the w a t e r adduct i s s t r o n g l y  s t a b i l i z e d by r e s o n a n c e . P o s t u l a t e (a) i s f u l f i l l e d  by those  c o n t a i n i n g a C=N l i n k a g e and those covered  heterocycles  by p o s t u l a t e (b)  embrace t h e u s u a l types o f r e s o n a t i n g systems + -NH-C=NH  HE > -N=C-NH  R  +  such as t h e a m i d i n i u m , g u a n i d i n i u m Representative  and u r e a  systems.  o f these systems a r e t h e q u i n a z o l i n e c a t i o n s , ^  the 2 - a m i n o p t e r i d i n e  c a t i o n ^ S and t h e 2 - h y d r o x p t e r i d i n e  cation.^ Covalent  h y d r a t i o n c a n u s u a l l y be r e c o g n i z e d i n  h e t e r o c y c l i c systems by t h e p r e s e n c e o f anomalous i o n i z a t i o n constants  and u l t r a v i o l e t s p e c t r a .  i o n i z a t i o n constants  The u s u a l a n o m a l i e s i n  caused by c o v a l e n t h y d r a t i o n a r e t h e  s t r e n g t h e n i n g o f bases and weakening o f a c i d s , as expected f o r t h e d e l e t i o n o f a d o u b l e bond.  The anomalous  constant  u s u a l l y d i f f e r s by about 3pK u n i t s , o r one t h o u s a n d - f o l d , the norm.30  In. s p e c t r o s c o p y ,  from  a l a r g e s h i f t o f the a b s o r p t i o n  to s h o r t e r wavelengths ( a hypsochromic s h i f t ) , consonant w i t h the d e l e t i o n o f a double bond, i s u s u a l l y o b s e r v e d .  Normally,  the l o n g - w a v e l e n g t h a b s o r p t i o n o f p t e r i d i n e does n o t s h i f t more than lOmu when an i o n i s formed from t h e n e u t r a l s p e c i e s . Moreover, i n t h e absence o f h y d r a t i o n t h e spectrum o f the anion of a hydroxy p t e r i d i n e should "be i d e n t i c a l to t h a t o f the n e u t r a l species o f the corresponding  p r i m a r y amine.3,31  By t h e a p p l i c a t i o n o f r a p i d - r e a c t i o n p o t e n t i o m e t r i c and  spectroscopic techniques,  obtained32  q u a n t i t a t i v e d a t a has been  f o r the k i n e t i c s o f the c o v a l e n t h y d r a t i o n r e a c t i o n  o f a number o f p t e r i d i n e d e r i v a t i v e s . The r e a c t i o n i s c a t a l y z e d by e i t h e r a c i d o r base and i s r e v e r s i b l e .  The h y d r a t i o n o f  8  2 - h y d r o x y p t e r i d i n e , which e x i s t s p r e d o m i n a n t l y form, may  be used as an example. The  reaction follows  o r d e r k i n e t i c s , and o v e r the pH range 4 . 5 the times  i n the l a c t a m .  to 12.4  f o r h a l f - c o m p l e t i o n range from 0.5  first20°,  at  to 375  seconds.  •20  The  mechanism-  f o r the a c i d - and  7  base-catalyzed  i s p o s t u l a t e d to i n v o l v e the sequences (A) and  reaction (B) r e s p e c t i v e l y :  (A)  anhydrous  activated  complex  hydrate  H  H  H  (B)  O  O  The  H  '  H  p r e s e n c e o f a m e t h y l group a t the s i t e  of„_  h y d r a t i o n u s u a l l y h i n d e r s , to a l a r g e e x t e n t , the h y d r a t i o n r e a c t i o n as a r e s u l t o f a c o m b i n a t i o n  o f s t e r i c and e l e c t r o n i c  effects. R e c e n t l y , some examples o f t r a n s a n n u l a r l4  h y d r a t i o n have been found. group i s p r e s e n t  15  '  covalent  34  '  When a m e t h y l o r  alkyl  i n the 8 - p o s i t i o n o f the p t e r i d i n e , t r a n s -  a n n u l a r h y d r a t i o n can o c c u r a c r o s s the 1,7  o r 3,7 p o s i t i o n s .  9  F o r example,  6,7,8-trimethyl-4-pteridinone (VIII) i n  a l k a l i shows t h e c h a r a c t e r i s t i c hypsochromy c o r r e s p o n d i n g to  the f o r m a t i o n o f the a n i o n ( I X ) by 3 , 7 - h y d r a t i o n  by l o s s o f a p r o t o n . The r e q u i r e m e n t s  followed  o f the h y d r a t i o n  p o s t u l a t e s (a) and (b) a r e a d e q u a t e l y s a t i s f i e d by t h i s system.  (VIII)  (IX)  V/. P f l e i d e r e r and co-workers  and A. A l b e r t and h i s  s t u d e n t s have c o l l a b o r a t e d i n a s t u d y o f the h y d r a t i n g properties i n a series of 8-alkyllumazines. Investigations 1 5  w i t h l u m a z i n e s h a v i n g e i t h e r an i o n i z a b l e hydrogen o r a methyl group a t N-3  showed t h a t the n e u t r a l and c a t i o n i c  s p e c i e s p r e s e n t e d no a n o m a l i e s .  I n a l k a l i however, the a b s o r p t i o n  s p e c t r a o f the l u m a z i n e s were c h a r a c t e r i z e d by v a r i a b l e hypsochromic s h i f t s and were h i g h l y s e n s i t i v e t o s m a l l changes i n the n a t u r e o f the s u b s t i t u e n t s i n the 6 - ,  7-  o r 8-  p o s i t i o n s . These v a r i a t i o n s were t r a c e d to t h e f o r m a t i o n o f e q u i l i b r i u m m i x t u r e s o f two mono-anions  (one o f w h i c h i s  h y d r a t e d a c r o s s the 1 , 7 - p o s i t i o n ) and a d i a n i o n . F o r example, the two mono-anions o f 8-methyllumazine  were found t o e x i s t  10  i n r o u g h l y e q u a l p r o p o r t i o n s a t e q u i l i b r i u m , w i t h each a n i o n s t a b i l i z e d by resonance o f the n e g a t i v e charge i n the p y r i m i d i n e r i n g o f the m o l e c u l e . hydrate  On the o t h e r hand, the p r o p o r t i o n o f  i n the n e u t r a l s p e c i e s o f 8-methyllumazine was found  to be o n l y one p a r t i n 5700.  15  These s t u d i e s have been g r e a t l y f a c i l i t a t e d  by the  14  observation  that o x i d a t i o n of 2-hydroxy-4-methylpteridines  w i t h aqueous permanganate a t room temperature and pH a p p r o p r i a t e to the f o r m a t i o n o f the h y d r a t e .demethylation  results i n  a t the 4 - p o s i t i o n and f o r m a t i o n o f l u m a z i n e s  (2,4-dihydroxypteridines).  T h i s r e a c t i o n has been shown to  be g e n e r a l f o r d i a g n o s i n g the p o s i t i o n o f h y d r a t i o n i n 34 azanaphthalenes and I s r e m i n i s c e n t o f the o x i d a t i o n o f 1 - m e t h y l c y c l o h e x a n o l to cyclohexanone w i t h aqueous p o t a s s i u m 35 permanganate a t room t e m p e r a t u r e .  Thus, those 8 - a l k y l -  p t e r i d i n e s c a r r y i n g a methyl group a t the s i t e o f h y d r o x y l ion  a t t a c k are s u s c e p t i b l e to o x i d a t i v e demethylation  with 34  permanganate as shown i n the f o l l o w i n g r e a c t i o n scheme: pH 4  CH  3  >HC 3  pH 9 -H 0 2  11  S i n c e c o v a l e n t h y d r a t i o n o f p t e r i d i n e s can be regarded  as a n u c l e o p h i l i c a d d i t i o n , g e n e r a l a d d i t i o n o f  n u c l e o p h i l e s t o those p t e r i d i n e s s u s c e p t i b l e t o h y d r a t i o n might be e x p e c t e d .  I t has been s h o w n ^ t h a t  "quinonoid"  37  p t e r i d i n e s such as (X; R = Ch^CHgOH.)  r e a d i l y undergo  n u c l e o p h i l i c a t t a c k a t p o s i t i o n 7 . Thus, on d i s s o l v i n g i n strong a l k a l i ,  (X) g i v e s a s t r o n g hypsochromic s h i f t i n  a b s o r p t i o n i n d i c a t i v e o f f o r m a t i o n o f t h e hydrated  anion  ( X I ; R = CH CH 0H, R' = OH). I n t h e same manner and a t lower 2  2  pH v a l u e s , (x) add  and s i m i l a r y e l l o w  "quinonoid" p t e r i d i n e s  hydrogen c y a n i d e , sodium s u l f i t e , e t c . w i t h complete  d e c o l o u r i z a t i o n to y i e l d compounds ( X I ; R' = CN, SO^ , e t c . ) . -  R  8  HC 3  H  (x)  4.  " HlH e  (XI)  Reduction Reduction  and o x i d a t i o n o f p t e r i d i n e and i t s d e r i v a t i v e s has  been c a r r i e d o u t u s i n g a wide v a r i e t y o f reagents and c o n d i t i o n s . Extensive reviews  on t h i s s u b j e c t have been  2 4 8  published. ' '  Most r e s u l t s i n d i c a t e t h a t t h e p y r a z i n e  ring  12  o f the m o l e c u l e i s more s u s c e p t i b l e to r e d u c t i o n than the pyrimidine ring.  For i n s t a n c e , r e d u c t i o n o f p t e r i d i n e  i t s e l f w i t h l i t h i u m aluminum h y d r i d e g i v e s  5,6,7,8-tetra-  38  hydropteridine  while hydrogenation  of 4-hydroxy-2,o,7-  t r i m e t h y l p t e r i d i n e over n i c k e l y i e l d s i n i t i a l l y a yellow 7 , 8 - d i h y d r o d e r i v a t i v e w h i c h passes on f u r t h e r  hydrogenation 39  to the c o r r e s p o n d i n g 5 , 6 , 7 , 8 - t e t r a h y d r o compound. In 1 9 6 l , the r e d u c t i o n o f 2 - h y d r o x y p t e r i d i n e  was  Re- w i t h a number o f r e a g e n t s and the compound investigated  was  found to r e s i s t c a t a l y t i c r e d u c t i o n i n n e u t r a l o r a c i d i c  s o l u t i o n o v e r Raney n i c k e l o r p l a t i n u m o x i d e . However, i n a l k a l i n e media r e d u c t i o n v/ith p o t a s s i u m b o r o h y d r i d e , d i t h i o n i t e o r c a t a l y t i c a l l y o v e r Raney n i c k e l o r gave 2 - h y d r o x y - 3 , 4 - d i h y d r o p t e r i d i n e  sodium  palladium  as the major p r o d u c t w i t h  t r a c e s o f s u b s t a n c e s r e s u l t i n g from r e d u c t i o n o f the ring.  2-Hydroxy-6-methylpteridine  manner to y i e l d  the c o r r e s p o n d i n g  dihydropteridine.  pyrazine  r e a c t e d i n an analogous 2-hydroxy-6-methyl-3,4-  These r e s u l t s r e p r e s e n t  the o n l y  instance  o f r e d u c t i v e a t t a c k on the p y r i m i d i n e r i n g so f a r r e p o r t e d . 2-hydroxy-7,8-dihydropteridine,  I n the e x p e c t e d manner, prepared  unambiguously., by ...reductive . c y c l i z a t i o n o f- 2-hydroxy  5-nitro-4-acetonylamlnopyrimidine,  gives  t e t r a h y d r o p t e r i d i n e on f u r t h e r r e d u c t i o n . course  2-hydroxy-5,6,7,8The  o f the r e d u c t i o n o f 2 - h y d r o x y p t e r i d i n e  unexpected has  a d e t a i l e d s t u d y o f the r e d u c t i o n o f a l l mono- and hydroxypteridines  4l  '  42  and  prompted poly-  the r e s u l t s have been summarized.  8  13  Condensation  of  2,4,5-triamino-6-hydroxypyrimidine  w i t h b e n z o i n l e a d s t o t h e f o r m a t i o n o f two i s o m e r i c 6,7d i p h e n y l d i h y d r o p t e r i d i n e s depending upon t h e s o l v e n t i n w h i c h t h e r e a c t i o n was c a r r i e d o u t . ^ ' ^  The "<£" isomer o r  the l a b i l e 5 j 6 - d i h y d r o d e r i v a t i v e i s formed i n 2 5 $ aqueous ethanol.  T h i s isomer c a n be c o n v e r t e d  t o the more s t a b l e  " j3 " isomer o r 7 , 8 - d i h y d r o d e r i v a t i v e by the a c t i o n o f a c e t i c acid.  The l a t t e r isomer can be formed d i r e c t l y by c a r r y i n g  out t h e c o n d e n s a t i o n  i n acetic acid solvent.  The r e d u c t i o n and r e o x i d a t i o n o f s e v e r a l model  45 o - s u b s t i t u t e d p t e r i d i n e s have been r e v i e w e d . alkylpterin series  I n t h e 8-  (8-alkyi-2-amino-4-hydroxypteridines),  r e d u c t i o n o f 8 - e t h y l l e u c o p t e r i n ( X I I ) w i t h sodium amalgam g i v e s 8 - e t h y l - 7 j 8 - d i h y d r o x a n t h o p t e r i n ( X I I I ) w h i c h c a n be r e o x i d i z e d by oxygen o v e r p l a t i n u m o x i d e as p r e v i o u s l y  46,47 d e s c r i b e d f o r the o x i d a t i o n o f d i h y d r o x a n t h o p t e r i n . When 8 - ^ - h y d r o x y e t h y l i s o x a n t h o p t e r i n - 6 - c a r b o x y l i c a c i d (XIV; R = CH2CH2OH) i s reduced  w i t h sodium amalgam, t h e o n l y  isolated i s 8-J3-hydroxyethylisoxanthopterin  s t a b l e product  (XV; R = CH CH 0H). 2  2  C H2  •,2 5C  5  Ck  H  O-  (XII)  (XIII)  14  (XIV)  (XV)  S i m i l a r r e s u l t s a r e o b t a i n e d w i t h the c o r r e s p o n d i n g 2-amino4,7-dihydroxy-6-pteridine  carboxylic acid  (XIV; R = H) where  r e d u c t i o n w i t h sodium amalgam g i v e s a p r o d u c t  very s u s c e p t i b l e  to d e c a r b o x y l a t i o n ( t o g i v e a 5 , 6 - d i h y d r o d e r i v a t i v e ) and t o o x i d a t i o n to 2-amino-4,7-dihydroxypteridine Analogously,  2,4-dlamino-7-hydroxy-6-pteridine  (XV; R = H). carboxylic acid  i s reduced by z i n c and a l k a l i o r sodium amalgam, f o l l o w e d by f a c i l e d e c a r b o x y l a t i o n o f the r e s u l t i n g and  5,6-dihydropteridine  subsequent r e o x i d a t i o n t o 2 , 4 - d i a m i n o - 7 - h y d r o x y 48  pteridine. The o n l y 8 - a l k y l - d i h y d r o l u m a z i n e so f a r r e p o r t e d " " i s 8-( J3 (XI;  -hydroxyethyl)-6,7-dimethyl-7,8-dihydrolumazine  R = CH2CH2OH, R  1  = H),  1 5  '  3 6  r e d u c t i o n o f the p a r e n t lumazine  '^  9  formed by the c a t a l y t i c  with platinum oxide  catalyst  i n water. The 5 , 6 , 7 , 8 - t e t r a h y d r o p t e r i d i n e s ,  especially  those  u n s u b s t i t u t e d i n the p y r a z i n e r i n g and c a r r y i n g e l e c t r o n r e l e a s i n g s u b s t i t u e n t s i n the p y r i m i d i n e r i n g , a r e e x t r e m e l y  -  15  s u s c e p t i b l e to d e h y d r o g e n a t i o n , o f t e n a c c o m p l i s h e d m e r e l y by a t m o s p h e r i c oxygen.  F o r e x a m p l e , 5 ° when 2-amino-4-hydroxy-  5 * 6 , 7 * 8 - t e t r a h y d r o p t e r i d i n e s u l f i t e i s exposed to the a i r , three unstable  products assigned  the 5*6-,  5*8-  d i h y d r o p t e r i d i n e s t r u c t u r e s are a p p a r e n t l y be s e p a r a t e d by paper chromatography and  formed.  pteridine.  These can  Further  oxidation  m i x t u r e i n s o l u t i o n g i v e s 2-amino-4-hydroxy-  I f the o x i d a t i o n i s c a r r i e d out i n the p r e s e n c e  of a nucleophile*  a t t a c k by t h i s n u c l e o p h i l e a t the  p o s i t i o n r e s u l t s i n the u l t i m a t e f o r m a t i o n pteridine.  of a  been p r o v i d e d  o f the 6 - s u b s t i t u t e d  6-  6-substituted  E v i d e n c e t h a t the 7 * 8 - d i h y d r o p t e r i d i n e s  i n v o l v e d I n the f o r m a t i o n has  7*8-  have been  c h a r a c t e r i z e d by t h e i r u l t r a v i o l e t s p e c t r a . o f the I s o m e r i c  and  are  derivatives  by an unambiguous s y n t h e s i s o f 2-amino-  4 - h y d r o x y - 7 * 8 - d i h y d r o p t e r i d i n e and  a study of i t s a d d i t i o n  51 reactions.  The  7*8-dihydro compound undergoes a wide  v a r i e t y o f n u c l e o p h i l i c a d d i t i o n s l e a d i n g to  6-substituted  t e t r a h y d r o p t e r i d i n e s w h i c h g i v e the 6 - s u b s t i t u t e d on m i l d o x i d a t i o n .  The  pteridines  c o u r s e o f the o x i d a t i o n o f  6,7-  d i s u b s t i t u t e d - 2 - a m i n o - 4 - h y d r o x y - 5 * 6,7* 8-tetrahydropteridines by oxygen under p h y s i o l o g i c a l c o n d i t i o n s o f pH has been 52  shown  to proceed i n two  stages:  slow o x i d a t i o n to a  d i h y d r o p t e r i d i n e f o l l o w e d by r a p i d i s o m e r i z a t i o n o f l a t t e r compound to a  the  7*8-dihydropteridine.  7,8-Dihydro-6-hydroxy-, and  5*6-  lo>1:>  5*6-dihydro-7-hydroxy- ^  2 - a m i n o - 7 , 8 - d i h y d r o - 4 , 6 - d i h y d r o x y p t e r i d i n e ^ ^ are  dehydrogenated i n good y i e l d s by c o l d a l k a l i n e p o t a s s i u m  1  b  lb  permanganate. T h i s appears for  t o be t h e b e s t  general  reagent  the purpose even though d i h y d r o p t e r i d i n e s w i t h  electron-donating reagents  such  several  s u b s t i t u e n t s c a n be o x i d i z e d by m i l d e r  as o x y g e n i n a l k a l i n e  hydrogen peroxide,  alkaline  solution  (uncatalyzed),  silver nitrate,  47  methylene  55 blue,  sodium hypobromite, benzoquinone, e t c . Until  late,  little  information concerning  the  mechanism o f o x i d a t i o n o f t e t r a h y d r o and d i h y d r o p t e r i d i n e s has  been r e p o r t e d . R e c e n t l y , ^ 8 - m o n o h y d r o - a n d  hydropteridine techniques  radical  6,7,8-tri-  c a t i o n s have been d e t e c t e d  i n the hydrogen peroxide  oxidation of  by e . s . r . N(5)~  methyl-6,7-diphenyl-5,6-dihydroand N ( 5 ) - m e t h y l - 6 , 7 - d i p h e n y l 5,6,7,8-tetrahydro-2-amino-4-hydroxypteridine respectively. In  the hydrogen p e r o x i d e  compound  or aerial  o x i d a t i o n o f the l a t t e r  (XVI) to the c o r r e s p o n d i n g  dihydro  pteridine 56  hydroxylated of the  (XVII)  intermediate  (XVII)  in trifluoroacetic  corresponding  acid  hydroxylated  Mass s p e c t r o m e t r y , as w e l l as c h e m i c a l  ultraviolet  over  :xvn)  57 '  platinum  Reduction  oxide  tetrahydro derivative  afforded (XVIIl).  and n u c l e a r r e s o n a n c e s p e c t r a ,  analysis confirm  H3C 9 (XVI)  i s proposed.  a  these s t r u c t u r e s .  8 (XVIII)  17  E.s.r. investigations 58  have a l s o shown t h a t a one-electron  oxidation of 5,6,7,8-tetrahydropteridine  t e t r a h y d r o f o l i c a c i d occurs with peroxide acid r e s u l t i n g i n unstable w h i c h can be trapped  and  in trifluoroacetic  c a t i o n i c r a d i c a l s o f red  at l i q u i d nitrogen  colour  temperature.  59  In another recent paper, shown t h a t o r g a n i c  q u a n t i t a t i v e s t u d i e s have  hydroperoxide formation  should  be  regarded  as the p r i m a r y p r o c e s s i n the a u t o x i d a t i o n o f reduced p t e r i d i n e s and  flavins  (9-substituted  i s o a l l o x a z i n e s ) . The  e v e n t s i n the a u t o x i d a t i o n o f a r e p r e s e n t a t i v e pteridine  sequence o f tetrahydro-  (H^Pter), 2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-  t e t r a h y d r o p t e r i d i n e , i s i n d i c a t e d i n the f o l l o w i n g scheme: i.  Hj^Pter  +  0  ii.  H^PterOOH  +  H0  iii.  H PterOOH  +  S u b s t r a t e - H — * H^PterOH  3  o r the p e r o x i d e iv.  may  H^PterOOH  > H^PterOOH  2  2  ( v i a H^Pter*)  -H^PterOH  +  H 0 , or 2  +  a l s o o x i d i z e i t s hydrogenated +  IfyPter  2H"3PterOH  where  o  2  o  Substrate-OH, precursor:  18  The  hydroxylated  p t e r i d i n e s (H^PterOH) may  form " q u i n o n o i d "  d i h y d r o p t e r i d i n e s (H^Pter) w h i c h  r a p i d l y ( c f . r e f e r e n c e 52) by r a t h e r slow c o n v e r s i o n  stages  The  the  c o u r s e o f the r e a c t i o n measuring  amounts o f p e r o x i d e p r e s e n t  at  various  i n the r e a c t i o n . +3  4-2  The  r e a c t i o n s o f Cu*"  p t e r i d i n e s under a n a e r o b i c and  followed  to the p a r e n t p t e r i d i n e s w i t h  f o l l o w e d by r e c o r d i n g u l t r a v i o l e t s p e c t r a and  the oxygen uptake and  isomerize  to 7 , 8 - d i h y d r o p t e r i d i n e s ,  consumption o f oxygen and p e r o x i d e . was  then d e h y d r a t e to  and  Fe  with  c o n d i t i o n s have been I n v e s t i g a t e d  p r e l i m i n a r y r e s u l t s have been p u b l i s h e d  communication.^  in a  By the use o f p o l a r o g r a p h i c  0  tetrahydro-  a p p l i c a b l e to p t e r i d i n e s i t was  recent  techniques +2  demonstrated t h a t Cu  a s t a b l e complex w i t h t e t r a h y d r o p t e r i d i n e s , w h i l e  forms  Fe "^ +  o x i d i z e s t e t r a h y d r o p t e r i d i n e s v i a a complex s e r i e s o f 7,8-dihydropteridine  r e a c t i o n s i n which "quinonoid"  and  intermediates  reactions involved, using  a r e formed.  The  2-amino-4-hydroxy-5,6,7,8-tetrahydropteridine  (tetrahydro-  p t e r i n ) as an example, a r e i l l u s t r a t e d as f o l l o w s : i.  Overall reaction; 4Fe 3 +  ii. iii. iv.  +  tetrahydropterin —>  tetrahydropterin  +  i o  2Fe"  4Fe  + 2  +  oxidized pterin  f a s t  > quinonoid-dihydropterin + Fe+2 quinonoid-dihydropterin—>7,8-dihydropterin quinonoid-dihydropterin + 7,8-dihydropterin »• tetrahydropterin + pterin. I n the l a s t r e a c t i o n the t e t r a h y d r o p t e r i n i s c o n v e r t e d to rj  19  quinonoid-dihydropterin,  the l a t t e r a c t i n g as a c a t a l y s t f o r  the o x i d a t i o n o f 7 , 8 - d i h y d r o p t e r i n by F e 3 .  In s u p p o r t  t h i s process  form i n the  +  i s the f a c t t h a t the q u i n o n o i d  absence o f excess o x i d a n t y i e l d s 7,8-dihydropterin, p t e r i n and  t e t r a h y d r o p t e r i n . The  C.  Methods o f 1.  in this  oxidized  trihydropterin radical^  p r o b a b l y an o b l i g a t o r y i n t e r m e d i a t e c o u l d not be d e t e c t e d  of  Is  i n the r e a c t i o n but i t  case.  Preparation  Isay r e a c t i o n The  p r e p a r a t i v e r o u t e s to p t e r i d i n e s have been 2  3 4  e x t e n s i v e l y reviewed.  8  •*  The  c l a s s i c a l and  used s y n t h e t i c r o u t e i n v o l v e s c o n d e n s a t i o n pyrimidine with a 1,2-dicarbonyl c a r b o n y l compound and  most commonly  of a 4,5-diamino-  compound o r w i t h an  can be c a r r i e d out under a wide v a r i e t y  o f c o n d i t i o n s . T h i s r e a c t i o n , f i r s t noted by I s a y , ^ 2 employed by a number o f i n v e s t i g a t o r s . The scheme may  <?C-halo-  be r e p r e s e n t e d  by  1  has  general r e a c t i o n  ( X I X — > X X I ) . M o d i f i c a t i o n s to  g i v e 8- o r 5 - a l k y l p t e r i d i n e s a r e p o s s i b l e by employing a pyrimidine p r e c u r s o r - i n w h i c h one  o f the amino groups i s  alkylated.  (XIX)  (XX)  been  (XXI)  20  I n t h i s s y n t h e t i c method t h e f o l l o w i n g s u b s t i t u e h t s c a n be I n c o r p o r a t e d i n t o the p t e r i d i n e p r o d u c t : R  ±  and R ; 2  H, OH, OR, COOH, COOR, a l k y l , a r y l , etc. i n various combinations.  R^;  H, OH, NHg, NHR.  R^j  H, OH, NH , NHR, SH, 2  SR.  :  No d i f f i c u l t y i s encountered when t h e 1 , 2 - d i c a r b o n y l compound i s s y m m e t r i c a l . When i t i s n o t , c o n d e n s a t i o n o f t e n l e a d s t o a m i x t u r e o f i s o m e r i c p r o d u c t s . However, i t i s f r e q u e n t l y p o s s i b l e t o c o n t r o l t h e r i n g c l o s u r e by s u i t a b l e adjustment 8  P  o f t h e pH o f t h e r e a c t i o n s o l u t i o n . T h i s c a n be a t t r i b u t e d  3  to s l i g h t d i f f e r e n c e s i n b a s i c i t y between t h e amino groups of the 4,5-diaminopyrimidine. An i n t e r e s t i n g m o d i f i c a t i o n o f t h e I s a y r e a c t i o n i s t h e s o - c a l l e d Timmis r e a c t i o n ^ ' - ' i n which an a c t i v e k e t o - m e t h y l e n e compound r e a c t s w i t h a 4 - a m i n o - 5 - n i t r o s o - p y r i m i d i n e d e r i v a t i v e to g i v e a p t e r i d i n e .  T h i s method has  the advantage o v e r t h e c l a s s i c a l s y n t h e s i s i n t h a t t h e o r i e n t a t i o n o f t h e s u b s t i t u e n t s i n t h e 6- and 7 - p o s i t i o n s o f the p t e r i d i n e i s known. The s y n t h e s i s o f p t e r i d i n e s c a n a l s o be a c c o m p l i s h e d from <£-substituted c a r b o n y l compounds and 4 , 5 - d i a m i n o pyrimidines.  Thus, a l d e h y d o - and k e t o - a l c o h o l s r e a c t w i t h  2,4,5-triamino-6-hydroxypyrimidine dihydro-2-amino-4-hydroxypteridines  to give 7 - a l k y l - 5 , 6 which a r e  spontaneously  oxidized i n a i r to the corresponding 7 - a l k y l p t e r i d i n e s . Aromatic k e t o - a l c o h b l s (e.g. benzoin) g i v e p a i r s o f i s o m e r i c  21  43 44 dihydropteridines 2.  '  as p r e v i o u s l y d i s c u s s e d .  C y c l i z a t i o n of pyrimidines 4-Chloro-5-nitropyrimidines  r e a c t w i t h amino-  acetone to give 5-nitro-4-pyrimidylaminoacetones  (XXII),  some o f which can be c y c l i z e d t o 7 , 8 - d i h y d r o - 6 - m e t h y l -  64 p t e r i d i n e s by h y d r o g e n a t i o n  o v e r Raney n i c k e l .  Similarly,  2 , 4 - d i c h l o r o - 5 - n i t r o p y r i m i d i n e condenses w i t h t h e e t h y l e s t e r o f g l y c i n e to give acetate  ethyl-2-chloro-5-nitro-4-pyrimidylamino  ( X X I I I ) w h i c h may be c a t a l y t i c a l l y reduced and then  c y c l i z e d by b o i l i n g w a t e r to 2 - c h l o r o - 7 * 8 - d i h y d r o - 6 - h y d r o x y 65 65 pteridine. Other 5 - n i t r o - c h l o r o p y r i m i d i n e s , as w e l l as 2-amino-4-chloro-6-hydroxy-5-phenylazopyrimidine,^ react i n a s i m i l a r manner.  C h l o r i n e s u b s t i t u e n t s i n the p y r i m i d y l -  aminoacetones ( X X I I ) can be r e p l a c e d w i t h hydroxy groups (by an a c e t a t e b u f f e r ) o r exchanged f o r p r i m a r y o r s u b s t i t u t e d amino groups.  C h l o r i n e s i n the d i h y d r o p t e r i d i n e s a r e e a s i l y 65 removed by h o t h y d r o i o d i c a c i d and phosphorus; d e h y d r o g e n a t i o n to the p a r e n t p t e r i d i n e s i s r e a d i l y a f f e c t e d 18b by c o l d a l k a l i n e p o t a s s i u m permanganate.  O  O  H3C-C-CH^Nv>k^a  C  2H5°  : C  "  C H  2  N n  ^N^  02N(XXII)  (XXIII)  J L L  22  Because o f t h e f l e x i b i l i t y o f t h i s r e a c t i o n , i t c a n be used for  the p r e p a r a t i o n o f various 6-hydroxypteridines  t h e o r i e n t a t i o n o f 6- o r 7 - h y d r o x y p t e r i d i n e s  determining prepared 3.  and f o r  by t h e I s a y r e a b t i o n . Synthesis Despite  from p y r a z i n e  Intermediates  t h e advantages o f t h e g e n e r a l p t e r i d i n e  s y n t h e s e s o u t l i n e d above, they s u f f e r from t h e d i s a d v a n t a g e t h a t t h e r e q u i r e m e n t o f a 4 , 5 - d i a m i n o p y r i m i d i n e as one component more o r l e s s l i m i t s t h e c h o i c e o f s u b s t i t u e n t s t h a t can be i n c o r p o r a t e d i n t h e p y r i m i d i n e r i n g . To overcome t h i s disadvantage,  methods have been developed whereby t h e  pyrimidine r i n g i s constructed  from a p y r a z i n e  with resultant greater f l e x i b i l i t y . 67  intermediate  These developments have  been summarized by T a y l o r . One  s y n t h e t i c r o u t e t o p t e r i d i n e s by c l o s u r e o f  the p y r i m i d i n e r i n g i n v o l v e s t h r e e  steps:  1. p r e p a r a t i o n o f a 4-hydroxy- o r 2 , 4 - d i h y d r o x y p t e r i d l n e (lumazine)  by c o n v e n t i o n a l methods,  ii.  c l e a v a g e o f t h e p y r i m i d i n e r i n g , o f t h e hydroxy:-. 19 pteridine, and i i i . r e c o n s t r u c t i o n o f t h e p t e r i d i n e n u c l e u s on t h e p y r a z i n e l8a,21,68,69 r i n g to give the desired  product.  U n f o r t u n a t e l y , o b t a i n i n g t h e r e q u i s i t e p y r a z i n e may r e q u i r e a m u l t i s t e p s y n t h e s i s . To c i r c u m v e n t  this difficulty,  a l t e r n a t e general synthesis o f the pyrazine 70 has been d e v e l o p e d ,  an  Intermediates  w h i c h p e r m i t s p r e p a r a t i o n o f 4-hydroxy-  p t e r i d i n e s c a r r y i n g a s u b s t i t u e n t i n the 1 - p o s i t i o n .  A  23  3-hydroxy-4,5-diaminopyrazole  ( X X I V ) i s condensed w i t h a  1 , 2 - d i c a r b o n y l compound to g i v e a l ( H ) - p y r a z o l o - ( 3 , 4 - b ) p y r a z i n e (XXV) which i s s u b s e q u e n t l y c l e a v e d r e d u c t i v e l y o v e r Raney n i c k e l to g i v e a  2-aminopyrazine-3-carboxamide  ( X X V I ) . E l a b o r a t i o n o f the p y r i m i d i n e r i n g i s then  accomplished  8  by s t a n d a r d methods. F o r example,  a d d i t i o n o f benzoyl  c h l o r i d e t o the p y r a z i n e ( X X V I ) r e s u l t s i n the f o r m a t i o n o f a 2-phenyl-4-hydroxypteridine derivative  R "CO  (XXVII).  OH  H H 2  +  $i  R  z  (XXIV)  (xxv)  XBX  (XXV)  C HnCOC^. 6  R'^TKTVJHR (XXVI)  (XXVII)  S i m i l a r l y , 4 - h y d r o x y p t e r i d i n e i s formed i n good y i e l d heating 2~aminopyrazine-3-carboxamide  with ethyl  by  formate  18a  and a c e t i c a n h y d r i d e under r e f l u x . The  s y n t h e s e s o f monohydroxy-, p o l y h y d r o x y - , amino-  and a m i n o h y d r o x y p t e r i d i n e s  u s i n g the v a r i o u s methods 2,4,8  d e s c r i b e d have been r e v i e w e d e x t e n s i v e l y .  24  D.  B i o l o g i c a l Importance The name " f o l i c  o f Pteridines 71 acid"  has been c o i n e d t o d e s i g n a t e  a s u b s t a n c e , p r e s e n t i n l e a v e s and mammalian o r g a n s ,  which  i s a b l e t o s t i m u l a t e t h e growth o f c e r t a i n o r g a n i s m s , n o t a b l y the b a c t e r i u m S t r e p t o c o c c u s f a e c a l i s , and which has been r e c o g n i z e d as p a r t o f t h e v i t a m i n B complex. A t p r e s e n t , t h e term i s t a k e n t o encompass a f a m i l y o f c o n j u g a t e d  pteridines.  The s p e c i f i c s u b s t a n c e t o which t h e name was i n i t i a l l y a p p l i e d i s p t e r o y l - L - g l u t a m i c a c i d o r PGA ( X X V I I I ) and t h e parent a c i d f o r the f a m i l y i s p t e r o i c a c i d P t e r o y l g l u t a m i c a c i d proved  (XXIX).  t o be t h e f i r s t o f a s e r i e s o f  n a t u r a l l y o c c u r r i n g pteridines with 6-p-carboxyanilinomethyl s i d e c h a i n s . The i s o l a t i o n , s y n t h e s i s and b i o l o g i c a l o f p t e r o y l g l u t a m i c a c i d and i t s congeners * 2,8,72 reviewed.  (XXVIII);  (XXIX);  have been a d e q u a t e l y  ) COOH ;-NH-CH-CH-CH-COOH 2  COOH  activity  2  25  Xanthopterin  (2-amino-4,6-dihydroxypteridine),  which i s the yellow c o l o u r i n g matter present  i n tne bands  o f wasps, i s n o t c o n f i n e d o n l y t o t h e i n s e c t w o r l d . , T h e r e a r e i n d i c a t i o n s t h a t i t may be t h e n a t u r a l o r g a n i z e r r e s p o n s i b l e f o r t h e development o f p r i m i t i v e c e l l s i n t o t h e 73  t y p i c a l c e l l s o f the kidney.  When mammals o f v a r i o u s  are i n j e c t e d w i t h x a n t h o p t e r i n the kidneys times  t h e i r normal s i z e , a l t h o u g h  e n l a r g e t o many  they remain normal i n  f u n c t i o n . When the i n j e c t i o n s s t o p , t h e k i d n e y s to  species  slowly revert  normal s i z e . 2 - A m i n o - 4 - h y d r o x y p t e r i d i n e has a s i m i l a r  e f f e c t but f u r t h e r s i m p l i f i c a t i o n o f the xanthopterin. 2  m o l e c u l e does n o t produce k i d n e y  enlargement.  Many p t e r i d i n e s have been found t o i n h i b i t growth by a c t i n g as analogues t o o t h e r p t e r i d i n e s concerned w i t h normal m e t a b o l i s m . The s u b j e c t o f f o l i c a c i d a n t a g o n i s t s 74  as a n t i b a c t e r i a l and a n t i - c a n c e r agents has been r e v i e w e d . Only a few o f the s i m p l e r p t e r i d i n e s e x h i b i t g r o w t h - r e t a r d i n g 2  properties,  r e s u l t i n g from c o m p e t i t i o n w i t h t h e f o l i c  acids  o r t h e i r p r e c u r s o r s . F o r example, 2 , 4 - d i a m i n o - 6 , 7 - d i p h e n y l - . p t e r i d i n e suppresses  t h e growth o f t h e m a l a r i a l p a r a s i t e  i n c h i c k s i n f e c t e d w i t h P. g a l l i n a c e u m , an e f f e c t enhajnced by s u l f o n a m i d e s  and s i g n i f i c a n t l y i n h i b i t e d by p t e r o y l -  acid.75  glutamic  U n t i l r e c e n t l y , a l l the known n a t u r a l l y o c c u r r i n g p t e r i d i n e s and those o f b i o c h e m i c a l s i g n i f i c a n c e were d e r i v a t i v e s o f p t e r i n (2-am.ino-4-hydroxypteridine).  I n 1956,  7 6 77  Masuda  '  reported the i s o l a t i o n , along w i t h  riboflavin  26  (XXX) , o f two d e o x y r i b o s e d e r i v a t i v e s o f lumazine from t h e mycelium o f E. a s h b y i i . These d e r i v a t i v e s were s h o w n ^ ' ^*78,79  t  ^  Q  e  subsequently  6^7_,ji thyl-8-D-ribityllumazine m e  (XXXI) and 6 - m e t h y l - 7 - o x o - 8 - D - r i b i t y l l u m a z i n e  (XXXII) by  d e g r a d a t i o n e x p e r i m e n t s , u l t r a v i o l e t measurements and s y n t h e s e s . The i s o l a t i o n o f t h e same two substances  from t h e  80  mycelium o f A. g o s s y p i i has a l s o been r e p o r t e d .  P H  H (XXX)  O  O H  H e 3  (XXXI)  o  (XXXII)  R = CH (CHOH) CH OH 2  3  2  The c l o s e s i m i l a r i t y between t h e above  ribityl-  l u m a z i n e s and r i b o f l a v i n r a i s e s t h e q u e s t i o n as t o whether the l u m a z i n e s s e r v e as p r e c u r s o r s t o r i b o f l a v i n . Such a 78 8 l 82  postulate  has been suggested  and i s s u b s t a n t i a t e d catalyzed  reaction,  by s e v e r a l i n v e s t i g a t o r s  ' '  by s e v e r a l o b s e r v a t i o n s . I n an enzymetwo moles o f 6 , 7 ~ d i m e t h y l - 8 - - r i b i t y l -  27  l u m a z i n e r e a c t e d t o produce one mole o f r i b o f l a v i n and o t h e r u n i d e n t i f i e d p r o d u c t s v i a a r o u t e i n v o l v i n g the methyl  groups  and the 6- and 7-carbon moiety o f the lumazine m o l e c u l e . T h i s r o u t e , r a t h e r than one i n v o l v i n g the r i b i t y l group, shown t o be c o r r e c t by i s o t o p i c l a b e l l i n g  was  experiments.  6 - M e t h y l - 7 - o x o - 8 - r i b i t y l l u m a z i n e does not appear t o p a r t i c i p a t e i n the b i o s y n t h e s i s o f r i b o f l a v i n ^ but may  arise  directly  from the 6 , 7 - d i m e t h y l - 8 - r i b i t y l l u m a z i n e i t s e l f . 3 ^ 9 6  A s e r i e s o f lumazines w i t h d i f f e r e n t s u b s t i t u e n t s a t p o s i t i o n 8 and carbon atoms 6 and 7 have been s y n t h e s i z e d but o n l y one o f t h e s e s u b s t a n c e s  (6,7-dimethyl-8-(l -D-5 1  1  d e o x y r i b i t y l ) l u m a z i n e ) i s c o n v e r t e d to an analogous  flavin  ( i s o a l l o x a z i n e ) ."^ The c o n v e r s i o n o f the d i m e t h y l r i b i t y l l u m a z i n e  into  r i b o f l a v i n i n v i t r o i n the absence o f any o t h e r source o f  84 carbon has a l s o been r e p o r t e d .  When 6 , 7 - d i m e t h y l - 8 - r i b i t y l -  lumazine i s heated f o r 15 hours i n a phosphate pH 7-3  under n i t r o g e n * r i b o f l a v i n i s formed  buffer at  i n 55$  yield.  A s m a l l amount o f a p y r i m i d o p t e r i d i n e , i d e n t i c a l to t h a t o b t a i n e d from s e l f - c o n d e n s a t i o n o f 5 - a m i n o - 4 - D - r i b i t y l a m i n o 2 , 6 - d i h y d r o x y p y r i m i d i n e , ^ i s o l a t e d from the m o t h e r - l i q u o r s  4Q o f the r e a c t i o n l e n d s s u p p o r t to the suggested mechanism  «4  y >  o f the r e a c t i o n : i.  r i n g - o p e n i n g o f the " q u i n o n o i d " lumazine  initiated  by n u c l e o p h i l i c a t t a c k a t p o s i t i o n 7 ( c f . r e f e r e n c e 36) (XXXI  *• X X X I I I ii.  ^ XXXIV),  a l d o l c o n d e n s a t i o n i n v o l v i n g two m o l e c u l e s o f (XXXIV)  28  to g i v e a d e r i v a t i v e  (XXXV) o f d i m e r i c b i a c e t y l as suggested  86  by B i r c h , iii.  and  c y c l i z a t i o n o f t h i s i n t e r m e d i a t e w i t h the l o s s o f  one d i a m i n o p y r i m l d i n e m o i e t y to g i v e r i b o f l a v i n  (XXX).  R  R  H(xxxr)  5-^3^  (XXXIII)  (XXXIV)  R LH  (XXXIV)-H>  I  3  (XXX)  4-  H  9  N ^  H (XXXV)  The o v e r a l l s i m i l a r i t y between the c h e m i c a l s y n t h e s i s o f r i b o f l a v i n from the lumazine p r e c u r s o r and the c o r r e s p o n d i n g enzymatic r e a c t i o n s u g g e s t s t h a t the l a t t e r may w e l l proceed by a s i m i l a r mechanism. A r e l a t e d  cyclization  employing a d e r i v a t i v e o f d i m e r i c b i a c e t y l has p r e v i o u s l y been r e p o r t e d .  37  -  49 Very r e c e n t l y ,  J  i t has been demonstrated  t h a t the  r e a c t i o n between two m o l e c u l e s o f the 8 - r i b i t y l l u m a z i n e t o g i v e r i b o f l a v i n i s not p e c u l i a r t o t h i s lumazine d e r i v a t i v e .  29  Thus, r e f l u x i n g 2 - i m i n o ~ 4 - h y d r o x y - 8 - ( ^  -hydroxyethyl)-6,7-  d i m e t h y l p t e r i d i n e (XXXVI) i n phosphate b u f f e r a t pH  7.3  f o r 24 hours under n i t r o g e n gave 49$ o f the 2-imino-4hydroxy-6,7-dimethyl-9-(f  -hydroxyethyl)benzo(g)pteridine  ( X X X V I I ) . S i m i l a r l y , 8-( f> - h y d r o x y e t h y l ) - 6 , 7 - d i m e t h y l l u m a z i n e gave the c o r r e s p o n d i n g 9~{f  -hydroxyethyl)-6,7-dimethyl-  i s o a l l o x a z i n e , w i t h the s e l f - c o n d e n s a t i o n p r o d u c t s o f 5amino-4-j*-hydroxyethylamino-2,6-dihydroxypyrimidine as a r e a c t i o n  isolated  by-product.  R  ,NH  H C 3  (XXXVI)  (XXXVII)  CH CHeoH  R=  2  87 I t i s known  t h a t p t e r i d i n e s can be formed  b i o g e n e t i c a l l y from a n o t h e r b i o l o g i c a l l y i m p o r t a n t f a m i l y o f heterocyclesy~the "purineST"Thusy -  "the~p t e r I d i n e s ~ c a n ~ b e  c o n s i d e r e d as the l i n k between the p u r i n e s and the i s o a l l o x a z i n e system ( f l a v i n s ) . The c h e m i s t r y and b i o l o g i c a l 88 QO and the i s o a l l o x a z l n e s 8 ' 8Q f u n c t i o n s o f the p u r i n e s ' y  y  have been r e v i e w e d . The c h e m i c a l c o n v e r s i o n o f p u r i n e d e r i v a t i v e s i n t o p t e r i d i n e s has a l s o been i n v e s t i g a t e d .  87,91,92  30  C o n s i d e r a b l e a t t e n t i o n has been focused on n a t u r a l l y occurring  reduced p t e r i d i n e s because o f an i n c r e a s e d  u n d e r s t a n d i n g o f t h e i r importance i n numerous b i o l o g i c a l 50 93 Q4 95 95 reactions. As one example, f o l i c a c i d c a n be hydrogenated e n z y m a t i c a l l y acid during  the s y n t h e s i s  to dihydroo f leucovorin  and t e t r a h y d r o f o l i c (N(5)-formyl-5,6,7,8-  t e t r a h y d r o f o l i c acid) i n a n i m a l t i s s u e s and m i c r o - o r g a n i s m s . B o t h the d i h y d r o and t e t r a h y d r o the s t a b i l i z i n g f o r m y l  d e r i v a t i v e s not possessing  group a r e v e r y u n s t a b l e t o m i l d  o x i d a t i o n and s o l u t i o n s exposed t o the a i r q u i c k l y decompose w i t h l i b e r a t i o n o f p-aminobenzoyl-glutamic a c i d , and  xanthopterin,  o t h e r p t e r i d i n e s . The b i o l o g i c a l importance o f t h e  f o r m y l t e t r a h y d r o f o l i c a c i d s r e s t s p r i m a r i l y on the f a c t  that  t h e y s e r v e as a s o u r c e o f one c a r b o n fragments i n t h e biosynthesis  o f a host o f p h y s i o l o g i c a l l y important substances 8  such as p u r i n e s , E.  pyrimidines  Redox R e a c t i o n s o f the The  and amino a c i d s . Flavins  8-alkyllumazines  are s t r u c t u r a l l y very s i m i l a r  to t h e f l a v i n s (isoalloxazines)...._. Whether the _.two  systems can..._  be expected t o e x h i b i t s i m i l a r b e h a v i o u r w i t h r e s p e c t t o o x i d a t i o n and r e d u c t i o n  i s n o t so a p p a r e n t , however.  While  the p o s s i b i l i t y o f the p t e r i d i n e s and t h e i r reduced d e r i v a t i v e s s e r v i n g as a redox system has o n l y r e c e n t l y come t o l i g h t , the redox p r o p e r t i e s  o f the f l a v i n s are w e l l known and have 89,104,105,106,107  been the s u b j e c t  o f several  reviews.  F l a v i n s are reduced by sodium d i t h i o n i t e , c a t a l y t i c  31  hydrogenation,  z i n c and h y d r o c h l o r i c a c i d , e t c . The r e s u l t i n g  1,10-dihydro i s o a l l o x a z i n e d e r i v a t i v e s ( l e u c o f l a v i n s ) a r e r a p i d l y a u t o x i d i z e d , thereby r e q u i r i n g anaerobic i n the r e d u c t i o n procedure.  conditions  The f l a v i n s a r e n o r m a l l y  found  in their oxidized state, their oxidation-reduction potentials being c o n s i d e r a b l y negative to the p o t e n t i a l o f atmospheric 89,108  oxygen. On t h e b a s i s o f p o t e n t i o m e t r i c t i t r a t i o n s , magnetic measurements and s p e c t r a l o b s e r v a t i o n s s e v e r a l i n v e s t i g a t o r s , some time ago, concluded  t h a t t h e f l a v i n s a r e o x i d i z e d and  reduced i n two d i s t i n c t o n e - e l e c t r o n s t e p s and t h e r e f o r e 105,109  pass t h r o u g h a t r u e s e m i q u i n o i d The s e m i q u i n o i d  state of oxidation.  i n t e r m e d i a t e s were found t o have a c o n s i d e r a b l e  l i f e t i m e under c e r t a i n c o n d i t i o n s . of f l a v i n s a t the semiquinoid 109 i n d e t a i l by B e i n e r t  The s p e c t r a l c h a r a c t e r i s t i c s  l e v e l have been i n v e s t i g a t e d  who observed two bands a t 570 mu and  900 mu d u r i n g t h e r e d u c t i o n o f f l a v i n mononucleotide (FMN; r i b o f l a v i n c o n t a i n i n g a 5'-phosphate m o i e t y ) . a t 570 mu i s due t o t h e f r e e r a d i c a l ,  The absorbance  110  the presence o f 111  w h i c h has been c o n f i r m e d  by e . s . r . measurements,  while the  broad a b s o r p t i o n band i n t h e 900 mu r e g i o n has been a t t r i b u t e d on t h e b a s i s o f t e m p e r a t u r e - and c o n c e n t r a t i o n - d e p e n d e n c e t o a bimolecular  (or higher molecular)  complex o f t h e semi109  quinones w i t h each o t h e r o r w i t h components o f t h e s o l u t i o n . 110  Gibson,  Massey and A t h e r t o n ,  on t h e o t h e r hand, b e l i e v e i t  to be a c h a r g e - t r a n s f e r a b s o r p t i o n o f a FMN-FMNHg complex..  32  The  k i n e t i c s o f t h e r e d u c t i o n o f r i b o f l a v i n by  d i t h l o n i t e a t pH 9 have been s t u d i e d u s i n g a r a p i d  mixing  The k i n e t i c s o f t h e appearance o f t h e 900 mu  apparatus.  a b s o r p t i o n band suggested t h a t i t was due t o two m o l e c u l e s 112  o f t h e semiquinone  b u t i t i s s t i l l d o u b t f u l whether t h e  semiquinone i s an i n t e r m e d i a t e  i n t h i s r e a c t i o n o r whether  i t i s formed by a d i s p r o p o r t i o n a t i o n r e a c t i o n between o x i d i z e d and reduced The  flavin.  mechanism o f t h e r e d u c t i o n by reduced  nicotinamide-adenine  d i n u c l e o t i d e (NADH) and by d i h y d r o -  l l p o i c a c i d have been s t u d i e d .  I n t h e case o f NADH, t h e  r e a c t i o n i s thought t o proceed by a h y d r i d e - i o n t r a n s f e r . The  evidence i s derived  from t h e e f f e c t s o f s u b s t i t u e n t s  i n b o t h f l a v i n and n i c o t i n a m i d e , s o l v e n t e f f e c t s and t h e ^ 4- * - 4 108,113,114,115 m  deuterium isotope e f f e c t .  u  The a l t e r n a t i v e ,  a f a s t r e v e r s i b l e e l e c t r o n t r a n s f e r f o l l o w e d by a r a t e determining excluded.  hydrogen a b s t r a c t i o n , has n o t been e n t i r e l y A recent r e p o r t  two-electron  1 1  ^ lends  r e d u c t i o n scheme.  f u r t h e r support to the  The r e d u c t i o n w i t h  l i p o i c a c i d i s a l s o b e l i e v e d t o be a t w o - e l e c t r o n  dihydroprocess  on t h e b a s i s o f s i m i l a r s u b s t l t u e n t e f f e c t s , b u t a fastd i s s o c i a t i o n o f one o f t h e t h i o l groups o f t h e l i p o i c a c i d precedes the r e d u c t i o n , ^ 0 8 The FMN),  k i n e t i c s o f t h e a u t o x i d a t i o n o f FMNHg (reduced  as f o l l o w e d by r a p i d - r e a c t i o n t e c h n i q u e s , a r e  c o n s i s t e n t w i t h t h e o c c u r r e n c e o f two s i m u l t a n e o u s mechanisms.  117  33  In t h e f i r s t ,  t h e semiquinone o f t h e f l a v i n r e a c t s w i t h  oxygen by a f r e e - r a d i c a l PMN  path: +  FMNH-  i0  +  w h i l e the competitive process o x i d a t i o n through a f l a v i n  FMNR_  2  +  2FMNH'  FMNHV  > FMN +  2  ^HgOg  involves a two-electron  peroxide:  0  FMN  2  Chemical evidence  f o r the hydroperoxide  +  H0 2  2  intermediate i n the  r e o x i d a t i o n o f reduced i s o a l l o x a z i n e s and t h e analogous t e t r a h y d r o p t e r i d i n e s i s t h a t t h e N-10 s u b s t i t u t e d d i h y d r o a l l o x a z i n e s a r e r e s i s t a n t t o a u t o x i d a t i o n w h i l e t h e N-9  , '  ,  s u b s t i t u t e d ones ( i s o a l l o x a z i n e s ; a r e v e r y The  sensitive.  59,118  s p e c t r a o f r i b o f l a v i n s o l u t i o n s under a n a e r o b i c  " c o n d i t i o n s which have been exposed t o l i g h t o v e r  prolonged  p e r i o d s resembles those o b t a i n e d by t h e d a r k r e d u c t i o n o f the f l a v i n by d i t h i o n i t e .  This l e d to the suggestion  the mechanism o f p h o t o b l e a c h i n g  that  ( r e a c t i o n i n t h e absence o f  e x t e r n a l e l e c t r o n d o n o r s ) i s r e d u c t i o n by w a t e r - s p l i t t i n g , 119,120 p r o v i d i n g a c h e m i c a l analogue f o r t h e w a t e r - s p l i t t i n g r e a c t i o n in  photosynthesis:  34  FMN  +  H 0 2  ———*-  FMNH*  +  OH*  However, a more s a t i s f a c t o r y mechanism has been suggested by 11*5 121 122 123 Holrnstrom, O s t e r , and o t h e r s ' ' and s u p p o r t i n g e v i d e n c e has been p r o v i d e d .  The s u g g e s t i o n i s t h a t d u r i n g  p h o t o b l e a c h i n g an I n t r a m o l e c u l a r d i s p r o p o r t i o n a t i o n r e a c t i o n o c c u r s i n w h i c h the r i b i t y l s i d e - c h a i n i s o x i d i z e d and the isoalloxazine  n u c l e u s i s reduced.  The e v i d e n c e  8Q s i d e - c h a i n p a r t i c i p a t i o n has been summarized.  7  f o r the  35  OBJECTS OP THE PRESENT RESEARCH  Only r e c e n t l y has t h e o x i d a t i o n o f f u l l y reduced p t e r i d i n e d e r i v a t i v e s been i n v e s t i g a t e d i n a q u a n t i t a t i v e manner i n s p i t e o f t h e i r i m p o r t a n c e i n b i o l o g i c a l The s t u d i e s have been c o n f i n e d simple  tetrahydropteridines  systems.  so f a r t o t h e o x i d a t i o n o f  to the c o r r e s p o n d i n g  dihydro  compounds w i t h r e a g e n t s such as i r o n ( i l l ) , hydrogen p e r o x i d e and  oxygen i t s e l f .  W h i l e a wide v a r i e t y o f r e a g e n t s has  been employed t o o x i d i z e t h e d i h y d r o p t e r i d i n e s  to the  p a r e n t p t e r i d i n e s , no s t u d i e s c o n c e r n i n g t h e mechanism o f the o x i d a t i o n have been p u b l i s h e d .  An i n v e s t i g a t i o n o f the  k i n e t i c s and mechanism o f t h e o x i d a t i o n o f a d i h y d r o p t e r i d i n e has t h e r e f o r e been u n d e r t a k e n u s i n g common o x i d a n t s  such as  p o t a s s i u m f e r r i c y a n i d e and p o t a s s i u m permanganate f o r t h e purpose. The redox r e a c t i o n s o f f l a v i n n u c l e o t i d e s  have  been t h e s u b j e c t o f i n t e n s i v e s t u d y i n r e c e n t y e a r s and a p a r a l l e l between t h e o x i d a t i o n and h y d r o x y l a t i n g  properties  o f t h e d i h y d r o i s o a l l o x a z i n e s ( l e u c o f l a v i n s ) and t h e s i m p l e t e t r a h y d r o p t e r i d i n e s has been n o t e d .  A model compound was  chosen f o r t h e redox s t u d i e s w h i c h i n c o r p o r a t e s  the s t r u c t u r a l  f e a t u r e s o f b o t h t h e p t e r i d i n e and i s o a l l o x a z i n e n u c l e i . A model w h i c h s a t i s f i e s t h e s e r e q u i r e m e n t s i s l u m a z i n e (TML).  6,7,8-trimethyl-  36  (TML)  During  of  the dihydro d e r i v a t i v e  was o b s e r v e d  that  hydrogens o c c u r r e d was  *  the m e c h a n i s t i c  included  studies  o f the .  o f t h e a b o v e compound  an i n t e r e s t i n g  isotopic  oxidation * (DHTML) i t  exchange o f  i n b o t h TML and DHTML and t h i s  i n the c u r r e n t  reaction  investigation.  The 8- J3~hydroxyethyl analogue o f DHTML (see s t r u c t u r e XLV, page 4.0) has been designated as a 7,3-dihydro lumazine]^ an assignment presumably based on that used f o r c e r t a i n reduced pteridine derivatives.  T h i s numbering convention has been  used i n the present work although i t i s f e l t t h a t the c l a s s i f i c a t i o n o f reduced lumazine d e r i v a t i v e s such as DHTML as 1,7-dihydro lumazines i s somewhat l e s s m i s l e a d i n g .  37  EXPERIMENTAL A.  C h a r a c t e r i z a t i o n o f the P r e p a r e d Compounds M e l t i n g p o i n t s o f the p r e p a r e d compounds were  determined i n c a p i l l a r i e s using a Buchi a p p a r a t u s and  are  melting-point  uncorrected.  A l l s p e c t r o s c o p i c measurements were made i n 1 s i l i c a c e l l s u s i n g a Bausch and  Lomb Model 502  cm.  recording  s p e c t r o p h o t o m e t e r equipped w i t h a 3 0 0 ml. l i q u i d capacityconstant  t e m p e r a t u r e h o l d e r . Water from a b a t h  thermostated  o a t 2 5 . 0 C. was  pumped t h r o u g h t h i s h o l d e r d u r i n g  r e c o r d i n g o f s p e c t r o s c o p i c d a t a . U l t r a v i o l e t and absorbance maxima and  the c o r r e s p o n d i n g  (£) o f the p r e p a r e d p y r i m i d i n e s and  of 6,7,8-trimethyllurnazine  trimethyllumazine  (TML)  visible  extinction  lumazines a r e  i n Table I , along w i t h l i t e r a t u r e references o f the known compounds. A c i d i c and  the  coefficients collected  to the  basic i o n i z a t i o n  and  spectra constants  7/8-dihydro-6,7,8-  (DHTML) were determined by  recording  s p e c t r a o f the compounds i n aqueous phosphate b u f f e r a t v a r i o u s pH. The as the pK , to e q u a t i o n a  (l)  pK  determined a c i d i c i o n i z a t i o n ,  i s c o r r e c t e d to a thermodynamic v a l u e / % 96 (1):  recorded according  ( d e t e r m i n e d ) + 0 .5V5j/l+l.6 jJ  (thermodynamic) = pK a.  solution—  /  a  where )J i s the i o n i c s t r e n g t h a t h a l f - n e u t r a l i z a t i o n .  38 For  the base  term  ionization  PKBH+'  constant,  i s s u b t r a c t e d from  the determined  the  ionic  value. With  strength the  c o n c e n t r a t i o n o f the p o l y v a l e n t phosphate b u f f e r g r e a t l y exceeding  the  sample c o n c e n t r a t i o n s i n t h i s  case,  the  ionic  s t r e n g t h c o n t r i b u t i o n o f the b u f f e r determines  the  of  i n Table  the c o r r e c t i o n  f o r TML  and  t e r m . The  s p e c t r a were r e c o r d e d  M o d e l 21  instrument  sampling  techniques. Proton  acid  values recorded  employing  magnetic  lumazines  potassium  resonance  i n deuterium  s o l v e n t s were o b t a i n e d  spectrometer  chemical  t h e r e f e r e n c e and are recorded  coupling constants  obtain R  s p o t t i n g t h e s o l u t i o n on  J  60  the  megahertz  as e x t e r n a l  (cycles per  techniques  v a l u e s o f the  f  by  dissolving  thin-layer  ml.  from  second)  s o r b a n t on  R,  u s i n g two  and  butan-l-ol-5M  the  literature  acetic  acid  3$  (7:3)  references i n Table  employed  prepared approximately  o f methanol 4  Aluminum o x i d e  thin-layer plates.  s o l v e n t systems —  were  plates with a  capacity dispensing capillary.  used  with  pellet  i n Table I I .  was f  (TMS)  o f the a p p r o p r i a t e m a t e r i a l i n 0.1  as  Perkin-Elmer  8 (parts per m i l l i o n )  compounds. Samples were p r e p a r e d  microliter  effect.  trifluoroacetic  a V a r i a n A-60  on  shifts  to c h e c k t h e p u r i t y and  and  a  bromide  o x i d e and  T h i n - l a y e r chromatographic  mg.  on  I  (p.m.r.) s p e c t r a o f  using tetramethyl silane  r e f e r e n c e . The  1  magnitude  DHTML have b e e n c o r r e c t e d f o r t h e b u f f e r  Infrared  prepared  pK  ;  The  obtained  ammonium c h l o r i d e  (A)  (B) _ a r e r e c o r d e d I I I . The  G  ascending  along  39 97  technique  f o r development  h y d r o g e n and n i t r o g e n  b y P. B o r d a , M i c r o a n a l y s i s  of B r i t i s h B.  r a d i a t i o n a t 254 mu. were used .  and d e t e c t i o n o f t h e compounds r e s p e c t i v e l y .  Carbon, performed  i  and u l t r a v i o l e t  analyses  were  Laboratory,  University  Columbia.  Preparation The  and P u r i f i c a t i o n  method used  o f t h e Lumazines  f o r the preparation  o f the lumazine  85 derivatives  i s essentially  that o f Cresswell  and Wood  and  76,81 o f Masuda. purification  However, b e c a u s e  p r o c e d u r e s a r e employed  methods, a s w e l l be d i s c u s s e d ' i n is  c e r t a i n m o d i f i c a t i o n s and  as t h e p r o p e r t i e s detail.  the preparative .  o f the intermediates,  The o u t l i n e o f t h e p r e p a r a t i v e  will scheme  as f o l l o w s :  H  Q>>kfP \ > ~ OH  P 0 C  ¥  l3  (XXXVIII)  ^  NaOH  3  y  >  3  IN NaN02  HoNT^/^ OH  . »H (XLI)  (XLII)  3  H  H c-N > J ^ o  3 "' C  CH NH  (XL)  H H C-N >1 OH  n  OH  (XXXIX)  H  Ty^  (XLIIl)  H  2  40  (XLV)  2,4,6-Trichloropyrimidine  (XXXIX)  98  The 60$  literature-^  f o r t h i s compound from b a r b i t u r i c a c i d  r e f l u x time o f one a longer  addition  hour. A h i g h e r y i e l d was  (three hours) r e f l u x A 500  (0.39  reports a y i e l d of  (XXXVIII) w i t h a o b t a i n e d by  ml. three-necked  moles) o f b a r b i t u r i c a c i d  f l a s k equipped w i t h c o n d e n s e r , charged w i t h 50  (Eastman Organic  38.5  ml.  ml.  N,N-dimethylaniline  was  added s l o w l y o v e r a p e r i o d o f twenty m i n u t e s . The  was  then r e f l u x e d and  stirred  g.  Chemicals;  f u r t h e r p u r i f i c a t i o n ) . A s o l u t i o n o f 156  phosphorus o x y c h l o r i d e and  using  time.  f u n n e l and m e c h a n i c a l s t i r r e r was  used w i t h o u t  approximately  f o r a period o f three  mixture hours.  4l  At t h e end o f t h i s t i m e , t h e r e s u l t a n t r e d s o l u t i o n was c o o l e d , poured s l o w l y onto 800 g. o f crushed to  s t a n d o v e r n i g h t a t room temperature.  i c e and a l l o w e d  The s o l u t i o n was then  f i l t e r e d and e x t r a c t e d w i t h 50 m l . p o r t i o n s o f d i e t h y l  ether  u n t i l t h e aqueous phase was no l o n g e r t u r b i d . The e t h e r e x t r a c t s were combined, washed w i t h w a t e r and d r i e d over anhydrous sodium s u l f a t e . The d r i e d s o l u t i o n was f i l t e r e d , the e t h e r removed by f l a s h e v a p o r a t i o n and t h e r e s i d u a l r e d o i l d i s t i l l e d under reduced p r e s s u r e  t o y i e l d 62 g. (0.34  moles, 87$) o f 2 , 4 , 6 - t r i c h l o r o p y r i m i d i n e as a c o l o u r l e s s o i l . B.p.  88-90°(l8 T o r r ) ; l i t e r a t u r e  4-Chloro-2,6-dihydroxypyrimidine A one l i t r e and  mechanical  hydroxide  9 8 , 9 9  102-105°(34 T o r r )  (XL)  two-necked f l a s k equipped w i t h condenser  s t i r r e r was charged w i t h a s o l u t i o n o f sodium  (42 g.) i n w a t e r (420 m l . ) .  2,4,6-Trichloro-  p y r i m i d i n e (48 g; 0 . 2 6 moles) was then added and the r e a c t i o n s o l u t i o n r e f l u x e d w i t h s t i r r i n g f o r s i x t e e n hours. I t was found t h a t a f t e r a p p r o x i m a t e l y a copious  f o u r hours  q u a n t i t y o f a w h i t e s o l i d was d e p o s i t e d . T h i s  was c o l l e c t e d by s u c t i o n f i l t r a t i o n and t h e f i l t r a t e to  solid  returned  t h e r e a c t i o n v e s s e l . A t t h e end o f the r e f l u x p e r i o d , t h e  s o l u t i o n was c o o l e d , a c i d i f i e d w i t h c o n c e n t r a t e d  hydrochloric  a c i d and r e f r i g e r a t e d o v e r n i g h t . The w h i t e p r o d u c t  was  c o l l e c t e d , added t o t h e i n i t i a l d e p o s i t o f w h i t e m a t e r i a l and the combinate r e c r y s t a l l i z e d t w i c e from b o i l i n g w a t e r made s l i g h t l y a c i d i c w i t h h y d r o c h l o r i c a c i d t o g i v e 35 g. (0.24  42 moles; 9 2 $ ) o f M.p.  4-chloro-2,6-dihydroxypyriroidine.  298-300°(decomposition); l i t . ^  300°(decomposltlon)  Calculated for C^NgOgCl; N = 1 9 . 1 Found; N =  1 9 . 4 $ .  4 - Methylamino-2,6-dihydroxypyrimidine  (XLI)  4 - C h l o r o - 2 , 6 - d i h y d r o x y p y r i m i d i n e ( 3 0 g; 0 . 2 0 moles) was  added to 1 2 0 ml. o f w a t e r i n a 2 5 0 ml.  f l a s k equipped w i t h c o n d e n s e r , m e c h a n i c a l addition  f u n n e l . To t h i s was  methylamine and hours.  Every  the r e a c t i o n  two  three-necked stirrer  and  added 5 0 ml. o f 4 0 $ aqueous mixture  then r e f l u x e d  for six  h o u r s , an a d d i t i o n a l 2 0 ml. o f the aqueous  methylamine were'added. At the end o f the r e f l u x p e r i o d the r e s u l t a n t  y e l l o w s o l u t i o n was  a c e t i c a c i d and s o l i d was  r e f r i g e r a t e d o v e r n i g h t . The  c o l l e c t e d and  w a t e r to y i e l d 2 1 . 5 g. 2,6-dihydroxypyrlmidine M.p.  2 9 9 °  cooled, a c i d i f i e d with deposited  r e c r y s t a l l i z e d t w i c e from b o i l i n g ( 0 . 1 5 moles; 7 5 $ ) o f as p a l e y e l l o w  (decomposition);  l i t . ^  4-rnethylamino-  tablets. 3 0 2 °  (decomposition)  5 - Nltroso-4-methylamino-2,6-dihydroxypyrimidine 1 8 . 5 g.  To  dihydroxypyrimidine n i t r i t e was  ( 0 . 1 3 moles) o f  t h i s mixture  4-methylamino-2,6-  1 0 ml. o f 5 0 $ a c e t i c  were added d r o p w i s e w i t h s t i r r i n g and  and c o o l i n g  (XLII)  i n 2 5 0 ml. o f w a t e r , 1 9 g. o f sodium  added. To  coloured suspension  yellow  the r e s u l t a n t  heated to a f f e c t s o l u t i o n . On  redfiltering  a red s o l i d p r e c i p i t a t e d . T h i s m a t e r i a l  was  r e c r y s t a l l i z e d t w i c e from 5 0 $ a c e t i c  acid,  c o l l e c t e d and  acid  43  18.6  washed w i t h water and d r i e d to y i e l d 85$) red  g. (0.11  moles;  of 5-nItroso-4-methylamino-2,6-dihydroxypyrimIdine  as  needles. M.p.  above 3 5 0 ° ( d a r k e n s ) ; l i t .  7  6  '  8  above 3 5 0 °  1  5-Amino-4-methylamino-2,6-dihydroxypyrimidine Sodium d i t h i o n i t e  (28  g.) was added s l o w l y w i t h 5-nitroso-4-methylamino-2,6-  s t i r r i n g to a m i x t u r e o f 14 g. d i h y d r o x y p y r i m i d i n e i n 300  (XLIII)  ml. o f water and 50 m l . o f  sodium h y d r o x i d e . The y e l l o w s o l u t i o n w h i c h r e s u l t e d  10% was  heated f o r 10 minutes on the steam b a t h , f i l t e r e d and c o o l e d . A t t h i s s t a g e , most o f the s o l u t i o n was  used d i r e c t l y  i n the  n e x t s t e p o f the s y n t h e s i s r a t h e r than to a t t e m p t the i s o l a t i o n o f the p y r i m i d i n e ( X L I I I ) . An a l i q u o t (30 was  c h i l l e d o v e r n i g h t and the b i s u l f i t e s a l t o f  (XLIII)  was o b t a i n e d as a p a l e y e l l o w g r a n u l a r s o l i d . The ( y e l l o w n e e d l e s ) was o b t a i n e d from the s a l t by  ml.)  f r e e amine recrystallizing  from w a t e r . M.p.  252-256°(decomposition); l i t . It  was  1  0  250-260°(decomposition)  0  found t h a t the f r e e amine, on s t a n d i n g as  a s o l i d o r i n s o l u t i o n , s l o w l y formed a r e s i n o u s brown mass o v e r a p e r i o d o f weeks. When k e p t , i n a d e s i c c a t o r  under  vacuum, the amine decomposed more s l o w l y . The b i s u l f i t e  salt  proved s t a b l e as a s o l i d and c o u l d be k e p t f o r l o n g p e r i o d s of  time w i t h no s i g n s o f d e c o m p o s i t i o n .  6,7,8-Trimethyllumazlne  (TML)  (XLIV; R-^Rg  The b u l k (approx. 320  =  CH^)  ml.) o f the f r e s h l y - p r e p a r e d  44 s o l u t i o n c o n t a i n i n g the b i s u l f i t e s a l t o f the amino-alkylaminopyrimidinediol  ( X L I I I ) was c a r e f u l l y n e u t r a l i z e d w i t h  h y d r o c h l o r i c a c i d . To t h e n e u t r a l i z e d s o l u t i o n , 25 m l . o f b u t a n e - 2 , 3 - d i o n e was added and t h e r e s u l t a n t orange s o l u t i o n s t i r r e d and heated a t 8 0 ° f o r h a l f an hour. The s o l u t i o n was then c o o l e d , f l a s h evaporated  t o a volume o f a p p r o x i m a t e l y  30 m l . and a p p l i e d d i r e c t l y t o a 2 x 5 0 cm. column o f a c i d washed a l u m i n a ^ 1  (Shawnigan C h e m i c a l s ) made up and t r e a t e d  i n i t i a l l y w i t h 1 0 0 $ e t h a n o l . The column was e l u t e d w i t h 5 0 $ aqueous e t h a n o l and t h e l e a d i n g g r e e n f l u o r e s c e n t band c o l l e c t e d . The s o l v e n t s were removed on t h e r o t a r y and  evaporator  t h e s o l i d r e s i d u e r e c r y s t a l l i z e d t w i c e from b o i l i n g  e t h a n o l , t o w h i c h the minimum amount o f w a t e r was added t o e f f e c t s o l u t i o n , t o y i e l d 6 . 6 g. o f as b r i g h t y e l l o w n e e d l e s  6,7,8-trimethyllumazine  (green f l u o r e s c e n c e i n aqueous  solution). M.p. 310-311°(decomposition); l i t .  308-310°  3 7  o 8n  300-301 Calculated f o rC H g  „,  The  .  '  1 Q  N 0 ; 4  2  C, 5 2 . 4 2 ;  (decomp.)  H, 4 . 8 9 ; N, 2 7 . 1 7  Found; C, 5 2 . 3 3 ; ' H, 5 . l 6 ; N , 2 7 . 3 2 $ . , 15,37,101  spectroscopic data  and chromatography  37  o f t h i s compound agree f a v o r a b l y t o t h a t p r e v i o u s l y r e p o r t e d . I n a d d i t i o n t o TML, two a d d i t i o n a l l u m a z i n e d e r i v a t i v e s were a l s o s y n t h e s i z e d from a s e p a r a t e  preparation  of 5-amino-4-methylamino-2,6-dihydroxypyrimidine but, i n these l a t t e r c a s e s , t h e p y r i m i d i n e i n t e r m e d i a t e was i s o l a t e d as t h e f r e e amine b e f o r e c o n d e n s a t i o n  w i t h the appropriate .  45  c a r b o n y l compound. 6,8-Dimethyl-7-oxolumazine  (tautomer o f XLIV; R =  OR, R =  ±  2  CH^)  Crude 5 - a m i n o - 4 - m e t h y l a m i n o - 2 , 6 - d i h y d r o x y p y r i m i d i n e ( l g.) was suspended i n a methanol  (75 ml.) and w a t e r (15 ml.)  s o l v e n t p a i r f o l l o w e d by t h e a d d i t i o n o f 0 . 8 g. o f p y r u v i c a c i d . The m i x t u r e was r e f l u x e d w i t h s t i r r i n g f o r one h o u r , f i l t e r e d and r e f r i g e r a t e d . The p r o d u c t was c o l l e c t e d and recrystalllzed  t w i c e from w a t e r , w i t h a c h a r c o a l t r e a t m e n t  i n c l u d e d i n the f i r s t r e c r y s t a l l i z a t i o n ,  to y i e l d 0 . 6 g.  o f 6 , 8 - d i m e t h y l - 7 - o x o l u m a z i n e as p a l e y e l l o w needles.M.p. above 3 5 0 ° ;  lit. ^ 3  358°(decomposition)  C a l c u l a t e d f o r C g H g N ^ ; C, 4 6 . l 6 ; H, 3 . 8 7 ; N, 2 6 . 9 1 Found; C, 46.2.5;  6 , 7 - D i p h e n y l - 8 - m e t h y l l u m a z ' i n e (XLIV; R  1  H, 4 . 0 0 ;  N,  = R  H  = g c  g  5  26.72$  )  A m i x t u r e o f 1.5 g. crude 5-arnino-4-methylamino2 , 6 - d i h y d r o x y p y r i m i d i n e , 2 . 1 g. b e n z i l , 100 ml. w a t e r , 100 m l . e t h a n o l and 10 m l . c o n c e n t r a t e d ammonium h y d r o x i d e was heated w i t h s t i r r i n g a t 8 5 - 9 0 ° f o r two hours and then f i l t e r e d . The y e l l o w f i l t r a t e was f l a s h e v a p o r a t e d t o about  40 m l . and a p p l i e d d i r e c t l y t o a  2x50 cm. acid-washed  alumina  column. The column was e l u t e d w i t h 50$ aqueous e t h a n o l and the  y e l l o w f l u o r e s c e n t band c o l l e c t e d . The s o l v e n t s were  removed on t h e r o t a r y e v a p o r a t o r and the r e s i d u e recrystalllzed  t w i c e from an e t h a n o l - w a t e r s o l v e n t p a i r t o  y i e l d 1.6 g. o f 6 , 7 - d i p h e n y l - 8 - m e t h y l l u m a z i n e as y e l l o w n e e d l e s .  46 M.p. 304-306°(decomposition); C a l c u l a t e d f o r C ^ H - ^ N ^ ; C, Pound; C,  lit.  1  6  288-292°  69.08; H, 4.27; 68.77; H, 4.58;  16.96 N, l6,8l$  N,  7,8-Dlhydro-6,7,8-trimethyllumazine (DHTML) (XLV) 6,7,8-Trimethyllumazine was s u b m i t t e d t o a number o f r e d u c t i o n procedures  i n an attempt t o s y n t h e s i z e a.  d i h y d r o d e r i v a t i v e o f t h i s compound. Only those methods employing a c i d i c c o n d i t i o n s proved p r o d u c t i v e . S i n c e t h e d i h y d r o compounds produced  proved  t o be s u s c e p t i b l e t o  a e r i a l o x i d a t i o n i n s o l u t i o n , a l l s o l v e n t s used i n work-up and r e c r y s t a l l i z a t i o n were t h o r o u g h l y f l u s h e d w i t h h i g h p u r i t y n i t r o g e n b e f o r e use. As s o l i d s , the compounds were s t a b l e as l o n g as p r e c a u t i o n s t o a v o i d prolonged  exposure  to a i r and m o i s t u r e were t a k e n . 1.  Catalytic  hydrogenation  To a s o l u t i o n o f  1.5 g. (0.0073  moles) o f  6,7,8-  t r i m e t h y l l u m a z i n e i n 50 ml. 5M a c e t i c a c i d , p l a t i n u m o x i d e  (0.025 g.)  was added and h y d r o g e n a t i o n commenced a t  a t m o s p h e r i c p r e s s u r e and ambient temperature. A f t e r 25 'ml. more than t h e t h e o r e t i c a l amount o f hydrogen r e q u i r e d t o produce t h e d i h y d r o l u m a z i n e were taken up, the r e a c t i o n v e s s e l was e v a c u a t e d , r e f i l l e d w i t h n i t r o g e n , s t o p p e r e d t i g h t l y and t r a n s f e r r e d t o a d r y box under n i t r o g e n atmosphere. The r e a c t i o n m i x t u r e was n e a r l y n e u t r a l i z e d w i t h c o n c e n t r a t e d sodium h y d r o x i d e , f i l t e r e d , the f i l t r a t e reduced  t o a volume  o f a p p r o x i m a t e l y 10 ml. on t h e r o t a r y e v a p o r a t o r and c h i l l e d  under n i t r o g e n atmosphere  f o r t h r e e days. A y e l l o w s o l i d  was c o l l e c t e d under a stream o f n i t r o g e n and r e c r y s t a l l l z e d t w i c e from e t h a n o l , t o w h i c h t h e minimum amount o f w a t e r had been added t o e f f e c t s o l u t i o n . A t h i r d  recrystallization  from w a t e r a f f o r d e d 750 mg. ( 0 . 0 0 3 6 moles; 4 9 $ ) o f a d i h y d r o l u m a z i n e as p a l e y e l l o w n e e d l e s . T h i s m a t e r i a l proved to be h y g r o s c o p i c and hence was d r i e d  thoroughly  b e f o r e a n a l y s i s and d e t e r m i n a t i o n o f p h y s i c a l d a t a . M.p.  298-300°(decomposition)  C a l c u l a t e d f o r C ^ - ^ N ^ ; C, 5 1 . 9 2 ;  H, 5 . 8 l ; N,  Found; C, 5 2 . 1 1 ; H, 5 - 6 0 ;  26.91  N, 2 6 . 7 2 $  .  That t h i s compound p o s s e s s e d t h e 7 , 8 - and n o t t h e i s o m e r i c 5 , 8 - d i h y d r o s t r u c t u r e was shown by comparison o f s p e c t r o s c o p i c d a t a ( T a b l e I ) w i t h t h e known a n a l o g u e , dihydro-8-( ^-hydroxyethyl)-6,7-dimethyllumazine.  7,8-  1 5 , 3 6 , 2 + 9  As w e l l , t h e o b s e r v e d p.m.r. s p l i t t i n g p a t t e r n ( T a b l e I I ) can be a t t r i b u t e d to a m e t h y l group and hydrogen a t t a c h e d to t h e same s a t u r a t e d c a r b o n c e n t e r ( p o s i t i o n 7 ) . o f t h e t e r t i a r y n i t r o g e n a t p o s i t i o n 8,  Because  the 5,6-dihydro  s t r u c t u r e i s u n l i k e l y s i n c e a charged s p e c i e s must be i n v o k e d to a l l o w f o r t h e bonding and r e q u i r e d number o f hydrogen atoms. Attempted t h i n - l a y e r chromatography o f DHTML r e s u l t e d i n p a r t i a l o x i d a t i o n to 6 , 7 , 8 - t r i m e t h y l l u m a z i n e as i n d i c a t e d by t h e f o r m a t i o n o f two s p o t s , one o f w h i c h possessed the R  f  v a l u e s and f l u o r e s c e n c e under  ultraviolet  l i g h t c h a r a c t e r i s t i c o f t h e l a t t e r compound. The second s p o t ,  48 appearing and  at R  0 . 3 8 and  i n butanol-5M a c e t i c acid ( 7 : 3 )  0.46  3 $ ammonium c h l o r i d e r e s p e c t i v e l y , i s a s s i g n e d  to  the  dihydrotrimethyllumazine. 2.  Zinc  in hydrochloric acid  This  particular  s y s t e m has  been used  previously  38  to r e d u c e a number o f p t e r i d i n e d e r i v a t i v e s . Zinc ( l . O g.) stirred The  was  combined,  then  1 5 ml.  flash  and  two  filtered  o f w a t e r . The  evaporated  pale  f r o m aqueous e t h a n o l this  296-298  m a t e r i a l proved  microanalysis  ash  during  nearly  was  c a r b o n , h y d r o g e n and  It  the  zinc  filtrate  were  was  nitrogen  collected  three  times  of pale yellow  needles.  hygroscopic.  spectroscopic  vitiated  by  by  p.m.r. d a t a  to t h a t o f  catalytic  the  gave v e r y  nitrogen  and  identical  obtained  c o m b u s t i o n and  latter  minutes.  atmosphere o f  deposited  6 0 0 mg.  mixture  (decomposition)  0  t o be  the  the  TML  approximately  recrystallized  somewhat  dihydrotrimethyllumazine  the  w a s h i n g s and  solid  to y i e l d  m a t e r i a l was  and  for thirty  to a volume o f  yellow  A l t h o u g h the  for  ( 2 5 ml.)  r e f r i g e r a t e d a t 2 ° u n d e r an  d a y s . The  M.p.  to a s o l u t i o n o f  u n d e r n i t r o g e n and  u n d e r a s t r e a m o f n i t r o g e n and  Again,  added  under a stream o f n i t r o g e n  washed w i t h  for  ( 2 g.)  i n IN h y d r o c h l o r i c a c i d  m i x t u r e was  1 0 ml.  dust  this  the  hydrogenation,  formation  different  of  of a  values  to t h o s e p r e v i o u s l y  white for  obtained  compound. 2  i s known '  102  that c e r t a i n heterocycles  possessin  4  9  an i o n i z a b l e h y d r o x y l group p e r i t o a t e r t i a r y r i n g (e.g., 4~hydroxypteridine,  nitrogen  8 - h y d r o x y q u i n o l i n e ) form 1:1 and  1:2 complexes w i t h the i o n s o f d i v a l e n t m e t a l s , i n c l u d i n g z i n c . Hence, i t was suspected  t h a t the m a t e r i a l  obtained  v i a t h e z i n c - h y d r o c h l o r i c a c i d r e d u c t i o n method was a 7 , 8 dihydro-6,7,8-trimethyllumazine-zinc  complex, h a v i n g e i t h e r  s t r u c t u r e (XLVI) o r ( X L V I I ) . The m i c r o a n a l y s i s r e s u l t s f a v o r t h e l a t t e r c h o i c e . The w h i t e a s h produced upon c o m b u s t i o n o f the o r g a n i c m a t e r i a l I s presumably an o x i d e o f z i n c but t h i s was not i n v e s t i g a t e d f u r t h e r . Calculated f o r (C H N40 ) Zn-2H 0 g  1 1  2  2  2  (XLVII.2HgO);  C, 41.91j H, 5.08; N, 21.72 Found; C, 4 1 . 4 6 ; H, 6.01; N, 22.02$  (XLVII)  50  The r e d u c t i v e methods employing b a s i c c o n d i t i o n s f o r example, sodium b o r o h y d r i d e , z i n c , o r sodium d i t h i o n i t e i n sodium h y d r o x i d e - gave m i x t u r e s o f p r o d u c t s from l i t t l e o r no d i h y d r o l u m a z i n e c o u l d be o b t a i n e d .  which  T h i s i s not  s u r p r i s i n g i n view o f the d e l e t e r i o u s e f f e c t o f base on d i h y d r o p t e r i d i n e s - ' ^ as w e l l as h y d r o x y p t e r i d i n e d e r i v a t i v e s 2  1  19 and  lumazines.  7,8-Dihydro-6,7,8-trimethyllumazlne-7-d  (XLVIIl)  (XLVIII) The above .compound was  p r e p a r e d by  catalytic  h y d r o g e n a t i o n i n the same manner as the p r o t i o analogue from 1 g. o f TML  u s i n g d e u t e r i u m r a t h e r than hydrogen gas.  Y/ork-up and p u r i f i c a t i o n p r o c e d u r e s were a l s o y i e l d i n g 200 M.p.  mg.  (XLV)  (20$)  302-304°  similar  o f ( X L V I I I ) as p a l e y e l l o w n e e d l e s . (decomposition)  C a l c u l a t e d f o r C q H ^ N ^ D ; C, 5 1 . 6 7 ; H, 6 . 2 6 ; Found; C,  50.98;  H,  N,  26.78  6.O9.; N,  26.20  The p.m.r. spectrum o f t h i s compound i n D 0 2  (Tablell)  c o n f i r m e d the l o c a t i o n o f the d e u t e r i u m atom a t p o s i t i o n  7,  51  as I n d i c a t e d by the l a c k o f the normal C-7 s p l i t t i n g pattern.  methyl-hydrogen  However, a s m a l l amount o f c o u p l i n g  a l o n g w i t h the appearance o f a s m a l l q u a r t e t resonance a t 4.58  6 i n d i c a t e d t h a t deuterium  at p o s i t i o n 7. sample was  The  was  not f u l l y  incorporated  amount o f the p r o t i o i m p u r i t y i n the a t 4$  estimated  from a measurement o f the a r e a o f  the q u a r t e t resonance as compared to t h a t o f the N - 8 - m e t h y l resonance. The  a b s o r p t i o n d a t a f o r t h i s compound p r o v e d  i d e n t i c a l to t h a t o f DHTML. An i n f r a r e d spectrum o f (XLVTII) e x h i b i t e d a weak a b s o r p t i o n a t 2 2 4 0 cm ^ (C-D) c h a r a c t e r i s t i c s present  as w e l l as the o t h e r  absorption  i n the i n f r a r e d spectrum o f  (XLV).  5 , 6 , 7 , 8 - T e t r a h y d ro - 6 , 7 , 8 - t r irne t h y 1 lumaz i n e I n 20 ml. o f 5M a c e t i c a c i d , 0.42  g. o f  6,7,8-  0 . 0 2 9 g. o f p l a t i n u m o x i d e were  t r i m e t h y l l u m a z i n e and  hydrogenated a t a t m o s p h e r i c  p r e s s u r e and ambient  temperature.  A f t e r f o u r h o u r s , hydrogen uptake ceased w i t h the  total  uptake v e r y c l o s e l y a p p r o x i m a t i n g  value  c a l c u l a t e d f o r two employed-.-  the t h e o r e t i c a l  moles o f hydrogen under the c o n d i t i o n s  Work-up- o;f--the—produc-t was - e s s e n t i a l l y - s i m i l a r - t o -  that described f o r  7,8-dihydro-6,7,8-trimethyllumazine  except  r e c r y s t a l l i z a t i o n from hot e t h a n o l , to  t h a t o n l y one  w h i c h the minimum amount o f w a t e r had been added to a f f e c t s o l u t i o n , was  attempted to y i e l d 60 mg.  yellow granular M.p.  solid.  262-265  0  (decomposition)  of a very pale  52  In s p i t e o f p r e c a u t i o n s  taken to exclude a i r , t h i s  compound proved h i g h l y s u s c e p t i b l e to o x i d a t i o n i n s o l u t i o n even i n t h e p r e s e n c e o f t r a c e q u a n t i t i e s o f a i r (oxygen) t o give' 7 , 8 - d i h y d r o - 6 , 7 . > 8 - t r I m e t h y l l u m a z i n e .  Therefore,  further  r e c r y s t a l l i z a t i o n o f the i s o l a t e d m a t e r i a l was n o t a t t e m p t e d . On s t a n d i n g as a s o l i d , even under n i t r o g e n atmosphere, t h e tetrahydro  compound was p a r t i a l l y c o n v e r t e d  o f weeks t o t h e 7 , 8 - d i h y d r o darkening  over a period  compound as i n d i c a t e d by a  o f t h e y e l l o w c o l o u r o f t h e s o l i d and changes i n  absorption  spectra. I n aqueous s o l u t i o n a t pH 6.5,  the m a t e r i a l  i s o l a t e d from the i n i t i a l r e c r y s t a l l i z a t i o n e x h i b i t e d an u l t r a v i o l e t spectrum w i t h absorbance maxima a t 272 and 302 mu. ( a b s o r p t i o n r a t i o = 0.8 r e s p e c t i v e l y ) . The spectrum o f t h i s s o l u t i o n decayed i n a m a t t e r o f hours on exposure t o the a i r t o a spectrum c h a r a c t e r i s t i c o f DHTML, v i a an intermediate  e x h i b i t i n g an absorbance maximum a t 244 mu (and  p o s s i b l y o t h e r s ) . This t r a n s i e n t intermediate h y d r o p e r o x i d e (XLIX) o r t h e h y d r o x y l a t e d to t h e i n t e r m e d i a t e s of  may be t h e  compound ( L ) , s i m i l a r  p r o p o s e d - ^ i n t h e spontaneous  oxidation  2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetrahydropteridine  i n n e u t r a l o r a l k a l i n e aqueous media. However, d i f f i c u l t y i n handling  the tetrahydrolumazine,  p r i m a r i l y due t o i t s  i n s t a b i l i t y t o o x i d a t i o n , made i n v e s t i g a t i o n o f the s t r u c t u r e o f the observed intermediate  as w e l l as the p r o p e r t i e s and  uses o f t h e t e t r a h y d r o p r e c u r s o r u n a t t r a c t i v e .  53  :?» H  | 3 H  V  o  3  (XLIX; R =  0-OH)  (L; R = OH) The assignment o f t h e 5 , 6 , 7 , 8 - t e t r a h y d r o  structure  i s based on t h e s t r u c t u r a l assignment o f o t h e r f u l l y reduced 2  103  pteridines '  and t h e f a c t t h a t , i n g e n e r a l , t h e p y r a z i n e  r i n g i n p t e r i d i n e s i s u s u a l l y hydrogenated i n p r e f e r e n c e t o 2  the p y r i m i d i n e r i n g .  ^8  However, i t i s noted t h a t t h e U.V.  spectrum o f the t e t r a h y d r o l u m a z i n e d i d n o t resemble t h a t o f 5 - a m i n o - 4 - m e t h y l a m i n o - 2 , 6 - d i h y d r o x y p y r i m i d i n e under t h e same conditions.  O r d i n a r i l y , the u l t r a v i o l e t s p e c t r a o f 5 , 6 , 7 , 8 -  t e t r a h y d r o p t e r i d i n e d e r i v a t i v e s resemble the s p e c t r a o f t h e c o r r e s p o n d i n g 4 , 5 - d i a m i n o p y r i m i d i n e s ( s h i f t e d 10 mu o r l e s s to l o n g e r w a v e l e n g t h s ) as would be expected from t h e l i n k i n g 38,40 o f two amino groups by a d i m e t h y l e n e r e s i d u e . 146  I t has been r e p o r t e d  that the f i r s t  intermediate  i n the o x i d a t i o n o f t e t r a h y d r o p t e r i n i s the 5 , 6 - d i h y d r o p t e r i n which i s r a p i d l y c o n v e r t e d t o t h e more s t a b l e 7 , 8 - d i h y d r o d e r i v a t i v e i n phosphate b u f f e r . The spectrum o f t h e former d i h y d r o d e r i v a t i v e a t pH 6 . 8 i s r e m a r k a b l y s i m i l a r t o t h a t o b s e r v e d f o r t h e t e t r a h y d r o l u m a z i n e a t pH 6 . 5 .  However, t h e  54 5 , 6 - i s o m e r i s u n l i k e l y I n the p r e s e n t case due to the t e r t i a r y n i t r o g e n atom a t p o s i t i o n 8. t h a t the i n i t i a l spectrum  The  possibility  i s a r e s u l t of a mixture of a  p a r t i a l l y o x i d i z e d i n t e r m e d i a t e such as (L) o r a 6,7,8-trimethyllumazine be  excluded.  and the t e t r a h y d r o l u m a z i n e  5,8-dihydrocannot  55 T a b l e Ia Spectroscopic  data  f o r prepared  compounds  2,6-Dihydroxypyrimidines Compound 4-chloro4roethylamino5- n i t r o s o - 4 methylamino5>-amino-4methylamino-  Observed V a l u e s \ (mu. ) log A max. ' 10  PH  t  218,282 266 266  3.91,4.01 4.25 4.41  12.0 12.0 1.0  226,256,316 220,311  4.12,3.64,3.92 1.0 4.11 12.0  ref.  37  Lumazines 6,7,8-trimethyl 6,7-diphenyl8-methyl 6,8-dimethyl7-oxo 7,8-dihydro6,7,8-trimethyl  256,276,404 244,(268), 313,362  4.12,3.98,4.01 1.0 4.26,(3.77) 4.28,3.84 12.0  15,37, 101  268,292,426 243,282, 348,(426)  4.19,4.28,4.12 7.0 4.27,4.13, 12.0 4.01,(3-39)  15  283,328 284,346  4.06,4.11 4.05,4.13  79  236,274,352 280,316 230,284,318  4.01,4.17,3.66 1.0 4.19,3.79 5.0 4.33,4.05,3.75 10.0  ) 277,310 231,282,315  4.16,3.82 5.0 4.32,4.09,3.81 10.0  1.0 7.0  f o r comparison; 7,8-dihydro6,7-dimethyl-  a  - d e t e r m i n e d a t 25.0 i n aqueous m e d i a ; v a l u e s f r o m r e f e r e n c e 15 were d e t e r m i n e d a t 20°'.  ()-  shoulder.  15  quoted from  56  Table l b I o n i z a t i o n constants i n water a t 2 5 . 0 ° C . Lumazine  pK Found  6,7,8-trimethyl  9.85+O.O6  a  P%3H  b  Literature 9.90+0.2  Found .  1 5  9.86+0.06 -  6.7- diphenyl8- methyl  70 4.26+0.2'^ 79 13.20+0.1  -  9.69+O.3  7.29+0.03  -  for  Literature  -  0.85+0.1  -  -  -  O.36+O.I  15  2.86+0.05  comparison;  7,8-dihydro6,7-dimethyl-8- . (^-hydroxyethyl)  7-25+0.09  b - r e f e r e n c e 1 5 , 7 9 v a l u e s determined  1 5  7 9  6,8-dimethyl7 - oxb  7.8- dihydro6,7,8-trimethyl  +  1  5  a t 20°C.. i n water  57  Table I I P.m.r. s p e c t r a l d a t a f o r l u m a z i n e d e r i v a t i v e s Lumazine  Protons  o (p.p.m.) from e x t e r n a l TMS D 0 TFAA  J(cps)  2  6,7,8-trimethyl  C -CH (s)  2.6l  C -CH (s)  2.75  N8-CH (s)  4.02  6  3  7  3  3  7,8-dihydro6,7,8-trimethyl  C -CH (d)  1.19  1.36  C -CH (s)  2.01  2.38  N -CH (sj  3.03  3.10  C -H  4.14  4.57  7  6  8  3  3  3  (q)  7  7,8-dihydro6,7,8-trimethyl -7-d  Cy-CH^s)*  1.40  C -CH (s)  2.40  N -CH (s)  3.10  6  8  TFAA  trifluoroacetic  3  3  acid  c - r u n i n t r i f l u o r o a c e t i c acid-D^O s,d,q - s i n g l e t , d o u b l e t ,  (l:2).  quartet.  * - broad s i n g l e t v/ith f i n e s t r u c t u r e present.  6.5+0.2  6.5+0.2  58  Table I I I "' '  cl  Chromatography Compound  (A)  Found  o f p r e p a r e d compounds R~ I n s o l v e n t f e  Reference  Found  (B)  .. R e f e r e n c e  2,6-Dihydroxypyrimidine 4-chloro-  0.69  0.67  4 - methylamino-  0.53  -  0.33  5- n i t r o s o - 4 methylamino-  O.65  -  0.l6  -  0.42  5-amino-4methylamino (bisulfite salt)0.78  3 7  0.67.  0.67  3 7  0.16  3 7  Lumazine 6 , 7 , 8 - t r i m e t h y l 0.72 6,8-dimethyl7 - oxo  0.57  6.7- diphenyl8- methyl  0.57  0.7237  o.22  70  7 . 8 - dihydrof 6 , 7 , 8 - t r i m e t h y l 0.46  0.54  70 0.09  -  0.42  -  O.38  0.19  d - u s i n g aluminum o x i d e G on g l a s s m i c r o s l i d e s . e - r e f e r e n c e 33 used Whatman's No. 1 paper; r e f e r e n c e 79 employed S c h e i d e r and S c h u l l 2043 paper and butanol-5M a c e t i c a c i d (2:1) s o l v e n t system. f - p a r t i a l o x i d a t i o n to 6 , 7 , 8 - t r i m e t h y l l u m a z i n e observed on attempted chromatography. A - 3% ammonium c h l o r i d e B - butan-1-ol-5M a c e t i c a c i d ( 7 : 3 )  Part  I  STABILITY OP THE LUMAZINE DERIVATIVES IN SOLUTION  59  A.  Introduction I n o r d e r to determine t h e i r s t a b i l i t y  i n aqueous  s o l u t i o n some r e p r e s e n t a n t l v e p y r i m i d i n e s and a l l the lumazine d e r i v a t i v e s prepared o f pH c o n d i t i o n s .  The  dihydroxypyrimidine, p y r i m i d i n e and  i n t h i s work were t e s t e d under a v a r i e t y compounds i n c l u d e d 4-methylamino-2,6-  5-amino-4-methylamino-2,6-dihydroxy-  the 6 , 7 , 8 - t r l m e t h y l - , 6,7-diphenyl-8-methyl-,  6,8-dimethyl-7-oxo- and  7,8-dihydro-6,7.,8-trimethyl-  d e r i v a t i v e s of lumazine.  A commercial sample (K and  L a b o r a t o r i e s ) of 5-aminobarbituric p y r i m i d i n e ) was B.  acid  K  (2,4,6-trihydroxy-  also tested.  Experimental For each sample, 15 mg.  o f m a t e r i a l was  dissolved  i n 2 ml. o f the a p p r o p r i a t e b u f f e r system, c o n s i s t i n g o f 0.10M  potassium  12.0,  o r i n 0.5N  hydrogen phosphate a t pH 1.0, sodium h y d r o x i d e , and  thermostated  a t 45.0  s o l u t i o n was  prepared  Two  mg.  7.0,  9-0  or  the s o l u t i o n  - 0.2° i n a water bath.  A t y p i c a l sample  as f o l l o w s :  o f n i t r o g e n - f l u s h e d b u f f e r s o l u t i o n was  i n t r o d u c e d by s y r i n g e i n t o a v i a l equipped w i t h a t i g h t f i t t i n g r u b b e r cap c o n t a i n i n g the weighed sample under n i t r o g e n atmosphere.  The  to p r o t e c t the c o n t e n t s  v i a l was  wrapped i n aluminum  from l i g h t and  then  thermostated.  At v a r i o u s times s m a l l a l i q u o t s were withdrawn w i t h a m i c r o l i t r e s y r i n g e and  foil  50  s p o t t e d on two s e p a r a t e t h i n - l a y e r  60  plates  prepared  were t h e n chloride acid  w i t h aluminum o x i d e G as  immediately s o l u t i o n and  (7:3)  detected  developed,  w i t h 3$  one  The  the s e p a r a t e d  i r r a d i a t i o n with u l t r a v i o l e t  plates  ammonium  the o t h e r w i t h b u t a n - l - o l - 5 M  s o l v e n t s y s t e m , and by  sorbant.  acetic  compounds  light  a t 254  mu.  I n t h e c a s e o f n o n - f l u o r e s c i n g compounds, Wood's l i g h t 356  mu  was  used.  Occasionally, qualitative o  were o b t a i n e d a t 2 5 . 0 recording  spectrophotometer  concentrations with cell  o f the  In t h i s  w i t h a B a u s c h and  t h e same b u f f e r  instrument  using chromatographic A composite  prepared as  by  and  chromatogram  the  larger plate  the c o m p l e t i o n o f the The  reaction  r a n g i n g up  diluted  to s p e c t r o s c o p i c The  sample c o u l d  reference only.  be  spectroscopic techniques. f o r each  (20x20cm) p l a t e  spotting a large  502  Lomb M o d e l  the b u f f e r s o l u t i o n  the s m a l l e r i n d i v i d u a l p l a t e s  developing  periods  contained  absorption spectra  solution.  manner, t h e p r o g r e s s o f e a c h  monitored  time  on a l i q u o t s  at  sample was a t the  same  were p r e p a r e d  i n butanol-5M a c e t i c  also  and  acid  at"  experiment. s o l u t i o n s were u s u a l l y m o n i t o r e d  t o one  month b u t  p e r i o d s were deemed n e c e s s a r y  i n some c a s e s  to ensure  for-  longer  completion  of  the  reaction. To  note  the e f f e c t  of atmospheric  oxygen,  were made up as p r e v i o u s l y d e s c r i b e d u s i n g b u f f e r not  f l u s h e d w i t h n i t r o g e n beforehand  solutions  and  under a i r i n c l o s e d c o n t a i n e r s .  keeping  samples  solutions . the  sample  6l  F o r t h e samples o f 6 , 7 , 8 - t r i m e t h y l l u m a z i n e and i t s dihydro  d e r i v a t i v e , those components e x h i b i t i n g the same  fluorescence  c h a r a c t e r i s t i c s and R  system were s e p a r a t e d  mechanically  l a y e r p l a t e s and combined.  f  values  i n a given  from t h e developed  solvent thin-  Each component was e l u t e d from  the s o r b a n t w i t h 20 ml o f methanol f o r e i g h t hours i n a Soxhlet apparatus.  The e l u a t e was then c o o l e d , reduced t o  a volume o f a p p r o x i m a t e l y  5 nil on t h e r o t a r y  evaporator,  f i l t e r e d and a q u a l i t a t i v e a b s o r p t i o n spectrum t a k e n on t h e filtrate.  Comparison s p e c t r a o f the s t a r t i n g m a t e r i a l s i n  methanol were a l s o r e c o r d e d .  F o l l o w i n g t h i s , the f i l t r a t e  was e v a p o r a t e d t o d r y n e s s and an i n f r a r e d spectrum r u n on the r e s i d u e .  The s a m p l i n g t e c h n i q u e c o n s i s t e d o f a d d i n g  350 mg o f anhydrous p o t a s s i u m bromide t o t h e r e s i d u e f o l l o w e d by 5 ml o f anhydrous methanol t o form a s l u r r y . s o l v e n t was removed on t h e r o t a r y e v a p o r a t o r  The  and t h e s o l i d  r e s i d u e d r i e d a t 57°/l T o r r f o r one hour, f o l l o w e d by p r e s s i n g o f t h e s o l i d i n t o a p e l l e t i n the u s u a l manner. C.  Results With the exception o f 5-amino-4-methylamino-2,6-  dihydroxypyrimidine  and DHTML under a e r o b i c c o n d i t i o n s , a l l  compounds t e s t e d proved t o be r e l a t i v e l y s t a b l e i n b o t h a c i d i c and n e u t r a l phosphate b u f f e r s o l u t i o n s . of 5-aminobarbituric  The samples  a c i d and 4 - m e t h y l a m i n o - 2 , 6 - d i h y d r o x y -  p y r i m i d i h e were s t a b l e under a l l c o n d i t i o n s employed i n the experiment.  However, e x c e p t f o r these l a t t e r two compounds,  62  a l l samples e x h i b i t e d some d e c o m p o s i t i o n on p r o l o n g e d standing i n neutral or a c i d i c s o l u t i o n at 4 5 . 0 ° . In c o n t r a s t , e x a m i n a t i o n o f the a l k a l i n e of  solutions  the l u m a z i n e s and the a m i n o m e t h y l a m i n o p y r i m i d i n e d i o l  i n d i c a t e d e x t e n s i v e d e c o m p o s i t i o n a f t e r o n l y two o r t h r e e days and suggested a h y d r o l y s i s r e a c t i o n a c c e l e r a t e d by  base.  W i t h the e x c e p t i o n o f the 5 - a m i n o - 4 - m e t h y l a m i n o - 2 , 6 - d i h y d r o x y p y r i m i d i n e and DHTML samples,  the d e g r a d a t i o n p a t t e r n o f  each sample p e r s i s t e d throughout the pH range t e s t e d w i t h i n c r e a s i n g r a t e o f d e c o m p o s i t i o n towards regions.  the more a l k a l i n e  T h e r e f o r e , the r e s u l t s o b t a i n e d f o r the a l k a l i n e  d e g r a d a t i o n experiments p r o b a b l y r e p r e s e n t those o b t a i n e d f o r the samples i n n e u t r a l and a c i d i c media as w e l l . the two compounds mentioned  For  above, r e s u l t s were o b t a i n e d  which i n d i c a t e d p r o c e s s e s o t h e r than h y d r o l y s i s were r e s p o n s i b l e f o r some o f the p r o d u c t s  observed.  Composite chromatograms developed w i t h the butanol-5M  a c e t i c a c i d s o l v e n t system i l l u s t r a t i n g the  d e g r a d a t i v e b e h a v i o u r o f the v a r i o u s compounds t e s t e d a r e p r e s e n t e d i n F i g u r e s 1 to 7. F l u o r e s c e n c e c h a r a c t e r i s t i c s under u l t r a v i o l e t l i g h t and  v a l u e s f o r the v a r i o u s compounds which were  t e s t e d and w h i c h were formed presented i n Table IV.  d u r i n g the e x p e r i m e n t s  are  63  FIGURE 1 5 - A m i n o - 4 - m e t h y l a m i n o - 2 , 6 - d i h y d r o x y p y r i m i d i n e i n 0.5N hydroxide at 4 5 . 0 °  sodium  C  0.4-1  0.3  0.21  B  0.1 1  A  0.0-  0  8 TIME  22 IN  120  CD  HOURS  KEY: AjB_, u n i d e n t i f i e d compounds; C, 2,6-dihydroxypyrirnidine -  5-amino-4-methylamino-  64 FIGURE 2 7,8-Dihydro~6,7,8 t r i m e t h y l l u m a z i n e i n 0.5N sodium h y d r o x i d e a t 45.0 , a n a e r o b i c c o n d i t i o n s  E  0.4iss>  cS$  D  0.3 4  Rf 0.2-1  co  cr- o  G>  o  o  o  o>  B  0.1-  t  0.00  3  6  fl!  A  t  25 75 120 260 4 6 0 TIME IN HOURS  °°  KEY: A, u n i d e n t i f i e d compound ( d a r k c e n t e r ) ; B, 6,8-dimethyl7-oxolumazine; C, 6 , 7 j 8 - t r i m e t h y l l u m a z i n e ; D, 7 , 8 - d i h y d r o - 6 , 7 , 8 - t r i m e t h y l l u m a z i n e ; E, 5-amino4-methylamino-2,6-dihydroxypyrimidine  65  FIGURE 3 7 , 8 - D i h y d r o - 6 , 7 , 8 - t r i m e t h y l l u m a z i n e i n 0.050M. KpHPOh a t pH 7.0/45.0°; a n a e r o b i c c o n d i t i o n s  E  0.4-  D  0.3-  0.2-  O  o  o  o  o  o  o  o  0.  B  0.0-  0  "I  5" ~S TIME  N  10  30  60  0 0  DAYS  KEY: A, u n i d e n t i f i e d compound; B, 7 - o x o - 6 , 8 - d i m e t h y l l u m a z i n e ; C, 6 , 7 , 8 - t r i r a e t h y l l u m a z i n e ; D, 7 , 8 - d i h y d r o - 6 , 7 , 8 t r i m e t h y l l u m a z i n e ; E, 5 - a m i n o - 4 - m e t h y l a m i n o - 2 , 6 dihydroxypyrimldine.  66  FIGURE 4 6,7-DiphenyI-8-methyllumazine i n and 4 5 . 0 ° ; anaerobic c o n d i t i o n s  0.050M KgHPO^ a t pH 12.0  <GBJ>  0  0  2 23 120 TIME IN HOURS  —r~ OO  KEY: A, u n i d e n t i f i e d compound; B, u n i d e n t i f i e d compound; C, 6 , 7 - d i p h e n y l - 8 - m e t h y l l u m a z i n e ; D, 5 - a m i n o - 4 methylamino-2,6-dihydroxypyrimidine  67  FIGURE  5  6 , 7 , 8 - T r i m e t h y l l u m a z i n e i n 0.5N sodium h y d r o x i d e a t 4 5 . 0 °  0.4  03  f  02-j  O  CV  O  O  V  0.1-  "5  5  I ®  24  (SJ)  E)  of  100 22 0 3 § 0 556 TIME IN HOURS  I2 j  O  0  O  0.0-  O  el cb  KEY: A, u n i d e n t i f i e d compound; B, 6 , 8 - d i m e t h y l - 7 - o x o l u m a z i n e ; C, u n i d e n t i f i e d compound; D, 6 , 7 , 8 - t r i m e t h y l l u m a z i n e ; E, 5 - a m i n o - 4 - m e t h y l a m i n o - 2 , 6 - d i h y d r o x y p y r i m i d i n e .  68  FIGURE 6 7-0xo-6,8-dim'ethyllumazine  and  45.0°  i n 0.050M KpHPO^'at pH 1 2 . 0  0.44  0.14  <SX2  43  o  24 T I ME' I N  KEY:  72  122  6b  HOURS  A, 7-oxo-6£-dimethyllumazine; B, 5 - a r a i n o - 4 - m e t h y l a m i n o - 2 , 6 - d i h y d r o x y p y r i m i d i n e  69  : FIGURE  7  7 , 8 - D l h y d r o - 6 , 7 , 8 - t r i m e t h y l l u m a z i n e i n 0.050M KgHPO^ a t pH 9.0 and 45.0°; a n a e r o b i c c o n d i t i o n s  G F E  0,4-  0.3-  0.2-  e>  o  o  \  (  o  o  o  o  D  0.1-  C B  o.o-  A,C  "5  3  2'3 4'8 162 I 92 2c39 4-o0 <*> TIME IN H O U R S -  KEY: A, u n i d e n t i f i e d compound; B, 6 , 8 - d i m e t h y l - 7 - o x o l u m a z i n e ; C, d a r k s p o t accompanied by r e d s t r e a k , p r o b a b l y 5 - a m i n o b a r b i t u r i c a c i d ; D, 6 , 7 , 8 - t r i m e t h y l l u m a z i n e ; E, 7 , 8 - d i h y d r o - 6 , 7 , 8 - t r i m e t h y l l u m a z i n e ; p, u n i d e n t i f i e d p a l e b l u e f l u o r e s c e n t compound; G, 5-amino-4-methylamino-2,6-dihydroxypyrimidine  70 T a b l e IV R  v a l u e s arid f l u o r e s c e n c e c h a r a c t e r i s t i c s o f v a r i o u s compunds  on aluminum o x i d e G TLC p l a t e s developed  w i t h 3$ ammonium  c h l o r i d e (A) and butan-l--ol-5M a c e t i c a c i d  Compound  (A)  R  f  (B) s o l v e n t systems  PI uorescence under U.V. l i g h t  (B)  Lumazines 6,7,8-trimethyl  0.72  0.22  6,8-dimethyl7- oxo  0.57  0.09  dark  6.7- d i p h e n y l 8- m e t h y l  0.57  0.42  yellow-green  7.8- d i h y d r o 6,7,8-trimethyl  o.46  green blue  0.38  Pyrimidines 5-amino-4methylamino-2,6dihydroxy  0.78  0.42  blue-violet  4- methylamino2,6-dihydroxy  0.53  0.33  dark  5- a m i n o b a r b i t u r i c acid  0.09  0.030.12  spot  dark spot,blue (red s t r e a k )  Other compounds unidentified unidentified  0.14 0.35,, s  0.27°  0.05  blue  0.16  light  10-methyl-2,4,6,8tetrahydroxy-pyrimido ( 5 , 4 - g ) p t e r i d i n e ( L I ) 0.23  h  (yellow) blue  blue  unidentified  0.40  (Fig.7)  pale blue  unidentified  0.29  (Fig.4)  sky b l u e  g - on Whatman's Mo. 17 paper.  85  h - Whatman's No. 1 paper.  71  Common t o a l l l u m a z i n e s t e s t e d i s the appearance o f 5-amino-4-methylamino-2,6-dihydroxypyrimidine  at various  s t a g e s i n the r e a c t i o n scheme, e s p e c i a l l y i n b a s i c That t h i s was  solutions.  the p y r i m i d i n e was shown by comparison o f R  v a l u e s i n two s o l v e n t systems and f l u o r e s c e n c e c h a r a c t e r i s t i c s w i t h a s y n t h e s i z e d sample ( b i s u l f i t e s a l t ) .  Attempts to  i d e n t i f y t h i s compound by a b s o r p t i o n s p e c t r o s c o p y a f t e r the S o x h l e t e x t r a c t i o n p r o c e d u r e were s i n g u l a r l y u n s u c c e s s f u l , due to s e l f - c o n d e n s a t i o n and d e g r a d a t i o n o f the p y r i m i d i n e i n b o i l i n g methanol o v e r p r o l o n g e d p e r i o d s . chromatography  However, paper  o f the lumazine s o l u t i o n s f o l l o w e d by e l u t i o n  o f the s e p a r a t e d components w i t h water a t ambient temperatures d i d show the spectrum o f the p y r i m i d i n e f o r the e l u a t e o f the h i g h Rf component.The was  5-amino-4-methylamino-2,6-dihydroxypyrirnidine  found to form the same two p r o d u c t s throughout the pH  range t e s t e d , as shown by comparison o f R^, v a l u e s .  These  compounds were formed r e l a t i v e l y f a s t e r i n the more a l k a l i n e s o l u t i o n s w i t h t h e i r f o r m a t i o n b e i n g i n h i b i t e d to some e x t e n t i n those experiments c a r r i e d o u t under n i t r o g e n atmosphere. A  green f l u o r e s c e n t compound (Rj, = 0.21  in  . b u t a n o l - 5 M a c e t i c a c i d ) d e t e c t e d i n e v e r y chromatogram o f 7 j 8 - d i h y d r o ~ 6 , 7 , 8 - t r i m e t h y l l u m a z i n e was s u b s e q u e n t l y n i d e n t i f i e d as 6 , 7 , 8 - t r i m e t h y l l u m a z i n e .  The presence o f the  l a t t e r compound i s p r o b a b l y due, i n p a r t , to o x i d a t i o n o f  72  the d i h y d r o compound d u r i n g development o f the t h i n - l a y e r plates.  T a i l i n g (green f l u o r e s c e n c e ) o f the spot  to the d i h y d r o l u m a z i n e suggestion.  was  observed and  supports  assigned this  I n the aqueous b u f f e r s o l u t i o n s o f DHTML, the  t r i m e t h y l l u m a z i n e i s d e t e c t e d by a b s o r p t i o n  spectroscopy  an hour a f t e r the s t a r t o f the t e s t s under a e r o b i c c o n d i t i o n s and  f i v e to s i x hours subsequent to t h i s under  anaerobic  c o n d i t i o n s i n n e u t r a l and a c i d i c s o l u t i o n s a t 45.0°. I n a l k a l i n e media, the f o r m a t i o n o f the  trimethyllumazine  i s s l i g h t l y f a s t e r , even i n the presence o f t r a c e amounts o f oxygen d i s s o l v e d i n the r e a c t i o n s o l u t i o n . Under the range o f c o n d i t i o n s employed  the  p o s s i b i l i t y o f two moles o f 6 , 7 , 8 - t r i m e t h y l l u m a z i n e  reacting  to form one mole o f l u m i f l a v i n ( 6 , 7 , 9 - t r i m e t h y l i s o a l l o x a z i n e )  49 a c c o r d i n g to the mechanism o f Rowan and Wood  84 '  arises.  However, no m a t e r i a l v/ith p r o p e r t i e s c h a r a c t e r i s t i c o f f l a v i n was D.  d e t e c t e d i n the chromatograms o f  the  TML.  Discussion 1.  5-Amino-4-methylamino-2,6-dihydroxypyrimidine I t has been r e p o r t e d ^ t h a t 8  amino-2,6-dihydroxypyrimidines  5-amino-4-(substituted)-  form s e l f - c o n d e n s a t i o n  p r o d u c t s when s o l u t i o n s o f the p y r i m l d i n e s are kept under non-reducing  conditions.  These compounds have been  85 identified The  as p y r i m i d o ( 5 , 4 - g ) p t e r i d i n e d e r i v a t i v e s ( L l ) .  a l t e r n a t e condensation  product  ( L i l ) was  e l i m i n a t e d on  73  the b a s i s o f an unambiguous s y n t h e s i s o f t h e 1 0 - ^ - h y d r o x y e t h y l compound ( L I ; R = CHgCHgOH) from b a r b i t u r i c a c i d and 4-  j3-hydroxyethylamino-5-nitroso-2,6-dihydroxypyrimidine.  (LI)  (LU)  In the present study, the higher R  f  component  i n t h e chromatograms o f 5 - a m i n o - 4 - m e t h y l a m i n o - 2 , 6 - d i h y d r o x y p y r i m i d i n e e x h i b i t s b l u e f l u o r e s c e n c e under u l t r a v i o l e t l i g h t and R^ = 0.26 (3$ ammonium c h l o r i d e , Whatman No. 17 p a p e r ) , p r o p e r t i e s s i m i l a r t o those r e p o r t e d f o r the 10-methyltetrahydroxypyrimido(5,4-g)pteridine  85  ( L I ; R = CH^).  On t h e b a s i s o f the above e v i d e n c e , the h i g h R^ component i s t e n t a t i v e l y a s s i g n e d t h e above s t r u c t u r e ( L I ; R = CH^). The r e m a i n i n g low R  f  -  component i s as y e t  u n i d e n t i f i e d due, i n p a r t , t o i t s a p p a r e n t low s o l u b i l i t y i n the w a t e r and methanol s o l v e n t s used t o e l u t e t h e chromatographically separated  compounds.  74  2. H y d r o l y s i s o f the Lumazlnes A once s u r p r i s i n g f e a t u r e o f 8 - a l k y l l u m a z i n e s and c e r t a i n 8 - a l k y l p t e r i d i n e s was t h e anomalous v a r i a b l e hypsochromic s h i f t i n the u l t r a v i o l e t  spectra of neutral  s o l u t i o n s made a l k a l i n e , as w e l l as w i d e l y d i f f e r e n t i o n i z a t i o n c o n s t a n t s among 6 , 7 - d i s u b s t i t u t e d homologues. These a n o m a l i e s shown ^' 3  7 9  16,79,124  were s a t i s f a c t o r i l y e x p l a i n e d when i t was  that 8-alkyllumazines w i t h "quinonoid" s t r u c t u r e s  r e a d i l y undergo n u c l e o p h i l i c a t t a c k a t p o s i t i o n 7 . hypsochromic s h i f t observed  i n base was concluded  Thus, t h e t o be a  consequence o f t h e f o r m a t i o n o f t h e " h y d r a t e d " form i n w h i c h t h e h y d r o x y ! group has e n t e r e d  (Llll)  the 7 - p o s i t i o n o f  the compound i n s p i t e o f t h e s t e r i c h i n d r a n c e o f f e r e d by the a l k y l  group.  (LIII)  Q u a n t i t a t i v e s t u d i e s on the a s p e c t s o f c o v a l e n t O\\  hydration of 8-alkylpterldines  T  '  O  R  and 8 - a l k y l l u m a z i n e s  have been p u b l i s h e d and the r e s u l t s o f these s t u d i e s Using 6 , 7 , 8 - t r i m e t h y l l u m a z i n e t h a t on p r o l o n g e d  15 23  reviewed.  as an example, i t was found  exposure t o a l k a l i the 8 - m e t h y l - 6 , 7 -  15  75  d i s u b s t i t u t e d l u m a z i n e s e x i s t p r e d o m i n a n t l y i n the ring-opened form ( L I V ) r a t h e r than as a s t a b l e h y d r o x y l a t e d a n i o n The r i n g - o p e n e d p r o d u c t s were c h a r a c t e r i z e d by t h e i r  (LIII). absorbance  15 a t 360 mu. T h i s r e a c t i o n , the p y r a z i n e r i n g - o p e n i n g o f 8 - a l k y l p t e r i d i n e s and l u m a z i n e s , has been shown to be reversible.  15,49  (LIV)  Prom the above e v i d e n c e , the presence o f 5-amino4-methylamino-2,6-dihydroxypyrimidine  i n the a l k a l i n e  o f TML and 6,7-diphenyl-8-methyllumazine  solutions  can be e x p l a i n e d i n  terms o f i n i t i a l f o r m a t i o n o f a 7-hydroxylumazine  anion  f o l l o w e d by r e v e r s i b l e r i n g - o p e n i n g t o the c o r r e s p o n d i n g p y r i m i d i n e analogue  (LIV).  The a m i n o a l k y l a m i n o p y r i m l d i n e d i o l  i s t h e h y d r o l y s i s p r o d u c t o f t h e ring-opened  compound.  F u r t h e r s u p p o r t i s l e n t t o t h i s scheme by the o b s e r v a t i o n t h a t o n l y r e l a t i v e l y s m a l l amounts o f the p y r i m i d i n e d i o l a r e formed o v e r p r o l o n g e d p e r i o d s i n n e u t r a l and a c i d i c  solutions  o f t h e two l u m a z i n e s where they e x i s t p r e d o m i n a n t l y i n the " q u i n o n o i d " form ( L V ) .  76  R  0 (LV)  The mechanism o f r i n g - o p e n i n g o f the h y d r o x y l a t e d a n i o n o f the 8 - a l k y l l u m a z i n e s has n o t been looked a t I n detail.  I t I s seen from the s t r u c t u r e o f the p r e f e r r e d  a n i o n ( L I I I ) t h a t t h e r e i s no apparent  d r i v i n g f o r c e to  r i n g - o p e n i n g , w h i c h would r e s u l t i n the f o r m a t i o n o f an u n f a v o r a b l e d i a n i o n w i t h one o f the n e g a t i v e charges on the N-8 suggests  n i t r o g e n a t some s t a g e i n the p r o c e s s .  t h a t the r i n g - o p e n i n g may proceed  p r e f e r r e d 7-hydroxy a n i o n such as  CH  3 H  O  (LVI)  (LVT).  residing  This  from a l e s s  77  In t h i s c a s e , the n e g a t i v e charge on the ring-opened following  species  ( L I V ) can then be s t a b i l i z e d i n  initial the  manner;  W.H  CH3  In the case where R = phenyl., f u r t h e r  stabilization  o f the n e g a t i v e charge by d e l o c a l i z a t i o n i n t o the C - 6 p h e n y l group i s p o s s i b l e i n both  ( L I I I ) and  (LIV).  That the 7 - h y d r o x y - 8 - a l k y l l u m a z i n e a n i o n ( L V I ) i s capable o f existence pK  a  i n a l k a l i i s supported  v a l u e s o f s e v e r a l analogues and model  by the known compounds.  i?6  (For example, 7 - h y d r o x y - 6 - m e t h y l p t e r i d i n e 3,4-dihydro-4-hydroxy-3-methylquinazoline P  K  a  =  7.66)  ~ pK = 6 . 9 1 , ( L V I Ia) 127  78  (LVII)  Chromatograms o f the a l k a l i n e trimethyllumazine also (R  = 0.09  Only was  a c i d ) which  as 6 , 8 ~ d i m e t h y l - - 7 - o x o l u m a z i n e  upon p r o l o n g e d this  showed a b l u e f l u o r e s c e n t s p o t  in, butanol-5M a c e t i c  identified  6,7,8-  solutions of  exposure  was  subsequently  (LVIII; R = R  t o n e u t r a l and  1  = CH^) .  acidic buffers  compound d e t e c t e d i n a p p r e c i a b l e q u a n t i t i e s .  (LVIII) 34 Jacobsen p t e r i d i n e s o f the type  has  r e c e n t l y shown t h a t q u l n o n o i d  (LV; R = a l k y l ,  6,7,8-trimethyllumazine,  R  1  = ^3)'  including  undergo o x i d a t i v e d e m e t h y l a t i o n  a pH a p p r o p r i a t e t o t h e f o r m a t i o n o f t h e h y d r a t e  to form  at the  79  c o r r e s p o n d i n g 7-oxo compound when t r e a t e d w i t h aqueous p o t a s s i u m permanganate.  L i k e w i s e , when oxygen i s passed  through a hot a l k a l i n e s o l u t i o n o f 6 , 7 - d i m e t h y l - 8 - D - r i b i t y l l u m a z i n e , t h i s compound i s s l o w l y c o n v e r t e d to 7 ~ o x o - 6 methyl-S-D-ribityllumazine  2 1 9  ( L V I I I ; R - r i b i t y l , R» =  C  H 3  )'  These, and s i m i l a r r e s u l t s from the 8 - a l k y l p t e r i d i n e s e r i e s , suggest the r e a c t i o n i n v o l v e s s u c c e s s i v e h y d r a t i o n , o x i d a t i o n and d e c a r b o x y l a t i o n a t the s i t e o f d e m e t h y l a t i o n . ' In the p r e s e n t c a s e , t h i s i s supported by the f a c t t h a t o n l y s m a l l amounts o f the oxo compound were produced  from 6 , 7 , 8 -  t r i m e t h y l l u m a z i n e i n those aqueous s o l u t i o n s where the l a t t e r compound i s e s s e n t i a l l y a l l i n the anhydrous q u i n o n o i d form (LV; R = R  = CH ).'  1  3  An a l t e r n a t e p o s s i b i l i t y to e x p l a i n the f o r m a t i o n of 6,8-dimethyl-7-oxolumazine  i s a displacement of a  b i a c e t y l u n i t by p y r u v a t e i n a l k a l i n e s o l u t i o n s o f TML. a t r u e d i s p l a c e m e n t o f the b i a c e t y l u n i t can o c c u r 49 shown  by r e f l u x i n g  was  6,7-dimethyl-8~J3-hydroxyethyllurnazine  (LV; R = CH CH 0H, R* = CH" ) 2  That  2  3  with  «C-oxobutyric a c i d under  n i t r o g e n a t pH 13 and I d e n t i f y i n g the r e a c t i o n p r o d u c t as the 6 - e t h y l - 7 - o x o l u m a z i n e  ( L V I I I ; R = CH CH 0H, R» = e t h y l ) . 2  2  T h i s experiment a l s o demonstrates  the r e v e r s i b l e  ring128  opening o f the 8 - a l k y l l u m a z i n e s .  I t has been shown  however, t h a t b i a c e t y l , r a t h e r than b e i n g o x i d i z e d to p y r u v i c a c i d , r a p i d l y undergoes an a l d o l c o n d e n s a t i o n r e a c t i o n i n aqueous a l k a l i to y i e l d a m i x t u r e o f p r o d u c t s . Only one  of  8o  these acid  products,  - a c e t y l - c£,tf - d i h y d r o x y - c £ - m e t h y l v a l e r i c  methylaminopyrimidinediol lumazine case,  i n some manner t o g i v e  b u t t h e r e i s no e v i d e n c e  biochemical  materials  to s u p p o r t  s t u d i e s by P l a u t  8 3  '  1 2  ^  u  s  t h e 7-oxo-  this.  i g  In any  labelled  n  have shown t h a t o x i d a t i v e r e m o v a l o f t h e m e t h y l 7 occurs  group a t p o s i t i o n of  t h e 5~amino~4-  ( L I X ) , c o u l d c o n c e i v a b l y condense w i t h  biacetyl  by p y r u v a t e  i n preference  in  to  displacement  6,7-dimethyl-8-substituted-  lumazlnes.  Ci HoC| Ho 8, 0=C—C-CH -C-C0 H. O CH3 H 3  3  2  2  (LIX)  It  i s r e p o r t e d ^ that  8- j3 - h y d r o x y e t h y l l u m a z i n e alkali  7,8-dihydro-6,7~dimethyl-  i s r a p i d l y converted  to 6 , 7 - d i m e t h y l - 8 - ^ - h y d r o x y e t h y l l u m a z i n e  by p a p e r c h r o m a t o g r a p h y and u l t r a v i o l e t the h i g h s u s c e p t i b i l i t y  to a e r i a l  7,8-dihydro6,7,8-trimethyllumazine properties lumazine  reported  f o r the  a n a l o g u e and s i m i l a r  solutions  as shown  spectroscopy.  Thus,  o x i d a t i o n observed f o r i s c o n s i s t e n t w i t h the  dihydro-8-J3-hydroxyethylreduced  pteridines.  As w e l l as t h e o x i d a t i o n p r o c e s s ,  unusual  i n hot  ->>•_->  the a l k a l i n e  o f t h e d i h y d r o t r i m e t h y l i u m a z i n e e x h i b i t e d some  features with  r e s p e c t to h y d r o l y s i s .  A large  o  quantity o f a blue  f l u o r e s c e n t component w i t h R^  values  81  similar  p y r i m i d i n e was the  t h a t o f the e l u t e d  a b s o r b a n c i e s a t 350  absorbance r a t i o  sodium h y d r o x i d e .  s p e c t r a o f both the f i n a l  o f DHTML and  exhibited  i n 0.5N  detected  ultraviolet  solution  of  5-amino-4~methylamino~2,6-dihydroxy-  to those o f  = 3-5:1  mu  and  the a m i n o a l k y l a m i n o p y r i m i d i n e d i o l .  product  i s , as y e t , n o t w e l l  ring-opened give  species  degraded  alkaline  component  In water  280 mu  respectively),  However,  (approximate  different  from  The n a t u r e o f  established  b u t i t may  the p y r i r n i d i n e d i o l d e r i v a t i v e - w o u l d  this be  (LX) i n w h i c h f u r t h e r h y d r o l y s i s  that  the  to  be e x p e c t e d t o be  difficult.  (LX)  l8b It pteridine  that 5,6-dihydro-7-hydroxy-  i s reported  i s instantly  converted  i n t o 5~arnino-4-carboxy-  me t hy 1 am i n o py r im-i d i n e by-bo -i-l-Ing- no-rma-l—s o d-i-um—hyd-ro x-i-d e-; this  reaction  i s reversed  normal h y d r o c h l o r i c (LX) would  undergo  d i h y d r o l u m a z i n e and  acid.  by one h o u r o f r e f l u x i n g Thus,  reversible have  d i f f e r e n t .to t h o s e o f  with  i t m i g h t be e x p e c t e d t h a t  ring-closure  to the  7,8-  chromatographic c h a r a c t e r i s t i c s  5-amino-4-methylamino-2,6-dihydroxy-  p y r i m i d i n e . T h e s e e x p e c t a t i o n s were n o t  realized.  82  The  p o s s i b i l i t y o f a t t a c k by h y d r o x i d e  i o n on  the f u s e d - r i n g double bond i n DHTML to form a h y d r o x y l a t e d d i a n i o n ( L X I ) , f o l l o w e d by r i n g - o p e n i n g to the S h i f f base ( L X I I ) , w i t h subsequent p r o d u c t i o n o f  5-aminobarbituric  a c i d i s a p p a r e n t l y e l i m i n a t e d by the absence o f the compound i n the s t r o n g l y b a s i c s o l u t i o n s o f the lumazine.  latter  dihydro  However, the d e t e c t i o n o f a s m a l l amount o f a  b l u e - f l u o r e s c i n g component w i t h R  f  v a l u e s s i m i l a r to those  o f the a m i n o b a r b i t u r i c a c i d i n the pH 9 b u f f e r a f t e r p r o l o n g e d s t a n d i n g i n d i c a t e s t h i s pathway may  7,8-dihydrotrimethyllumazine,  r o l e i n the h y d r o l y s i s o f the  CH 3  p l a y a minor  CH-i  CH-  Ayr  r©"T o-  J  (LXII)  (LXI)  The  a m i n o b a r b i t u r i c a c i d was  not d e t e c t e d i n n e u t r a l o r  a c i d i c s o l u t i o n s o f the d i h y d r o t r i m e t h y l l u m a z i n e v S e v e r a l analogues to ( L X I ) a r e known  '  or  131  a r e p o s t u l a t e d to e x i s t . behavior of carbinolamine  A study  o f the h y d r o l y t i c  V - m e t h y l l s o a l l o x a z i n e i n d i c a t e s t h a t the ( L X I I I ) i s the i n i t i a l p r o d u c t o f  hydroxide 59  attack.  S i m i l a r h y d r o x y l a t e d compounds a r e formed  i n the  83  autoxidation of tetrahydropteridines  and  dihydroisoalloxazine  derivatives.  (LXIII)  The p r e s e n c e o f 7 - o x o - 6 , 8 - d i m e t h y l l u m a z i n e . i n the l a t t e r s t a g e s o f the r e a c t i o n s o f DHTML i n base i n a l l p r o b a b i l i t y a r i s e s from the o x i d a t i v e d e m e t h y l a t i o n o f the 6 , 7 , 8 - t r i m e t h y l l u m a z i n e produced by o x i d a t i o n o f the dihydrolumazine.  I n n e u t r a l and a c i d i c media, p r o l o n g e d  r e a c t i o n i s r e q u i r e d b e f o r e s i g n i f i c a n t q u a n t i t i e s o f the oxo compound can be d e t e c t e d . The r e l a t i v e s t a b i l i t y to h y d r o l y s i s  exhibited  by the 7 - o x o - 6 , 8 - d i m e t h y l l u m a z i n e a n i o n (LXIV) i s a r e s u l t o f the well-known amide resonance, w h i c h i n t h i s case s t r e n g t h e n s the C ^ - N Q bond by p a r t i c i p a t i o n o f the N - 8 lonep a i r e l e c t r o n s as I n (LXV).  (LXIV)  (LXV)  84  D e l o c a l i z a t i o n o f the n e g a t i v e charge as i n d i c a t e d a l s o reduces any p r e d i s p o s i t i o n o f the Cg-N^ to n u c l e o p h i l i c a t t a c k .  i n (LXIV)  double bond  The e x t e n t o f t h i s charge  d e l o c a l i z a t i o n i s r e f l e c t e d i n the s t r o n g a c i d i t y ( p K o f the  a  =  4.26)  7-0x0-6,8-dimethy1lumazine. 6,7-Diphenyl-8-methyllumazine  d i d not form the c o r r e s p o n d i n g  i n aqueous media  6-phenyl-7-oxo-8-methyl-  lumazine as shown by the absence o f a component w i t h  spectral  34 p r o p e r t i e s s i m i l a r to those r e p o r t e d ^ f o r an analogue o f the l a t t e r compound, 6 - p h e n y l - 8 - m e t h y l - 2 , 7 - d i o x o p t e r i d l n e . 34  I t has been r e p o r t e d  that oxidation of  6,7-diphenyl-8-  m e t h y l - 2 - o x o p t e r i d i n e i n a c i d and a l k a l i n e s o l u t i o n d i d not y i e l d the c o r r e s p o n d i n g 7-oxo d e r i v a t i v e . As i n d i c a t e d  by the r e s u l t s o f t h i s e x p e r i m e n t ,  aqueous s o l u t i o n s o f the l u m a z i n e s , i n p a r t i c u l a r a l k a l i n e s o l u t i o n s , undergo conditions with  f a i r l y r a p i d d e c o m p o s i t i o n under the  employed.  S l o w e r d e c o m p o s i t i o n o f the compounds,  the e x c e p t i o n o f DHTML, o c c u r s i n n e u t r a l and  a c i d i c media, e s p e c i a l l y when k e p t under n i t r o g e n The d i h y d r o l u m a z i n e shows a p r e d i s p o s i t i o n  I s not w e l l  atmosphere.  to o x i d a t i o n i n  aqueous media as w e l l as a r e a d y tendency to form products.  weakly  hydrolysis  The n a t u r e o f these p a r t i c u l a r h y d r o l y s i s established. \  products  Part I I HYDROGEN-DEUTERIUM EXCHANGE  85  A.  Introduction W h i l e examining the p r o t o n magnetic  (p.m.r.) s p e c t r a o f 6 , 7 , 8 - t r i m e t h y l l u m a z i n e and  resonance 7,8-dihydro-  6 , 7 , 8 - t r i m e t h y l l u m a z i n e the a m p l i t u d e o f the peak a s s i g n e d to the d o w n f i e l d C-methyl  group o f b o t h o f the above  compounds was observed to d e c r e a s e w i t h time i n a c i d i c b a s i c d e u t e r i u m o x i d e media.  and  That the decrease i n peak  a m p l i t u d e was due to exchange o f the methyl group p r o t o n s was c o n f i r m e d by the a d d i t i o n o f H 0 2  compounds w i t h subsequent of  the exchanged  o f water added.  to D 0 2  s o l u t i o n s o f the  r e g e n e r a t i o n o f the peak a m p l i t u d e  methyl group i n p r o p o r t i o n to the amount Only a s i n g l e methyl group I n each compound  underwent exchange and p r e l i m i n a r y experiments showed a marked e f f e c t o f pH on the r a t e s o f these r e a c t i o n s .  I t was  then deemed o f i n t e r e s t to i n v e s t i g a t e the exchange p r o c e s s more f u l l y . In o r d e r to be c o n s i s t e n t i n the comparison o f exchange r e s u l t s o b t a i n e d i n heavy water w i t h measurements made i n o r d i n a r y aqueous media,  the s t r u c t u r e s o f a l l  compounds a r e drawn i n c o r p o r a t i n g the l i g h t i s o t o p e o f hydrogen. F o r l a b i l e hydrogens, such as the one a t t a c h e d to N-3  i n t r i m e t h y l l u m a z i n e o r the ones p r e s e n t i n the  phosphate  b u f f e r s p e c i e s H^POjj,  H2PO4-,  e t c . , i t may  be  assumed t h a t t h e y a r e r e p l a c e a b l e w i t h d e u t e r i u m where  86  e x p e r i m e n t s have been c a r r i e d o u t i n D^O.  S i m i l a r l y , the  term " p r o t o n a t i o n " r e f e r s t o t h e a t t a c k o f the o r g a n i c s u b s t r a t e by e i t h e r hydronium ion  (D^O"*") depending  i o n (h^O*) o r deuteronium  upon t h e i s o t o p i c c h a r a c t e r o f t h e  aqueous medium. To comply w i t h t h e t e r m i n o l o g y c u r r e n t l y i n use i n the l i t e r a t u r e i n t h e f i e l d "proton" w i l l  o f p.m.r. s p e c t r o s c o p y , t h e term  i n c l u d e hydrogen  atoms bonded c o v a l e n t l y  ( u s u a l l y to carbon). B.  Experimental 1.  P r e p a r a t i o n o f samples f o r k i n e t i c  studies  P.m.r. s p e c t r a and i n t e g r a l s were o b t a i n e d w i t h a V a r i a n HA-100 100 megahertz s p e c t r o m e t e r . The d e u t e r i u m o x i d e s o l u t i o n s used I n t h e exchange s t u d y were t h o r o u g h l y f l u s h e d w i t h h i g h - p u r i t y n i t r o g e n b e f o r e use.  Care was t a k e n to p u r i f y b o t h t h e DgO and sodium  phosphate  b u f f e r i n o r d e r to remove t r a c e s o f heavy m e t a l  impurities.  The Dv>0 was d i s t i l l e d  permanganate and then r e d i s t i l l e d . phosphate  hydrogen  f i r s t from p o t a s s i u m Commercial  disodlum  was d r i e d a t 110° and then r e c r y s t a l l i z e d once from"  a s a t u r a t e d s o l u t i o n i n heavy w a t e r which p r o v i d e d f o r exchange of  the b u f f e r protons.  the DgO and phosphate  The absence o f heavy m e t a l c a t i o n s i n s o l u t i o n s was c o n f i r m e d by t h e 132  Eriochrome B l a c k T i n d i c a t o r  test.  87  The  exchange r e a c t i o n s were c a r r i e d out on  a p p r o x i m a t e l y 0.1M DgO s o l u t i o n s o f TML and phosphate b u f f e r a d j u s t e d The  to the d e s i r e d pH w i t h 10$  pD  D 2 S O 4 .  pH o f the prepared s o l u t i o n s was measured on a Radiometer  Model 2 6 instrument w i t h a g l a s s - c a l o m e l The  0.10M  DHTML i n  measured pH was not c o r r e c t e d values.  Solvent  systems a t H  prepared from the a p p r o p r i a t e t r i f l u o r o a c e t i c acid' ' 1  33  electrode  to g i v e the = -0.37  0  train.  corresponding  and pH 8 . 2 were  mixture o f D20-anhydrous  and D 0 - p y r i d i n e 2  r e s p e c t i v e l y . In  most cases the i o n i c s t r e n g t h o f the b u f f e r was a d j u s t e d g i v e n value  (0.42)  by the a d d i t i o n o f the a p p r o p r i a t e  to a  quantity  o f sodium c h l o r i d e . For each k i n e t i c run, 0 . 3 5 - 0 . 4 5 ml. o f the d e s i r e d pH-adjusted n i t r o g e n - f l u s h e d i n t o a rubber-capped v i a l  b u f f e r s o l u t i o n was i n t r o d u c e d  c o n t a i n i n g 7 - 9 mg. o f sample under  n i t r o g e n atmosphere by means o f 1 ml. for  syringe.  After  approximately 2 0 seconds to a f f e c t s o l u t i o n , the  o f the v i a l were taken up w i t h  contents  the same s y r i n g e and i n j e c t e d  i n t o a rubber-capped N.M.R. tube which had f i l l e d w i t h n i t r o g e n gas.  shaking  In some cases,  p r e v i o u s l y been e s p e c i a l l y i n the  more b a s i c s o l v e n t systems, the sample d i d not d i s s o l v e completely.  In these i n s t a n c e s , s m a l l amounts o f u n d i s s o l v e d  sample were removed by f i l t e r i n g  the contents  o f the  vial  under a stream o f n i t r o g e n i n a d e v i c e c o n s i s t i n g o f a 0 . 2 5 ml. syringe b a r r e l f i t t e d  w i t h a s y r i n g e needle which  the rubber cap on the N.M.R. tube. contained  a s m a l l pad  penetrated  The b a r r e l o f the  o f DgO-washed g l a s s wool.  syringe-  The capped  88  tube c o n t a i n i n g t h e f i l t e r e d for testing.  sample s o l u t i o n v/as then removed  D u r i n g p r e p a r a t i o n p r o c e d u r e s , t h e sample  s o l u t i o n s were t h e r m o s t a t e d a t 3 0 . 0 - 0 . 2 ° i n a c o n s t a n t temperature  bath.  P.m.r. s p e c t r a and peak i n t e g r a l s o f b o t h l u m a z i n e s were r e c o r d e d a t r e g u l a r time i n t e r v a l s .  With the p a r t i c u l a r  s p e c t r o m e t e r employed, t h e DgO s o l v e n t peak (HOD) was used as the i n t e r n a l r e f e r e n c e and t h e i n s t r u m e n t l o c k s i g n a l .  The  temperature o f t h e s p e c t r o m e t e r probe i n t o which the sample tubes were p l a c e d was 3 1 . 4 ° ,  as measured w i t h a Honeywell  Model  134 No. 2732 p o t e n t i o m e t e r and Chromel-Alumel ( r e f e r e n c e j u n c t i o n a t 0°C.).  thermocouple  The thermocouple  had p r e v i o u s l y  been c a l i b r a t e d a g a i n s t a mercury thermometer u s i n g a c o n s t a n t temperature  bath.  A f t e r each measurement d u r i n g a k i n e t i c r u n , t h e d i h y d r o t r i m e t h y l l u m a z i n e sample tubes were t h e r m o s t a t e d a t 3 0 . 0 - 0 . 2 ° i n a water bath.  The exchange o f t h e t r i m e t h y l -  lumazine o c c u r r e d a t a r a t e such t h a t t h e r e a c t i o n c o u l d be m o n i t o r e d i n i t s e n t i r e t y , w i t h o u t removing from t h e probe o f t h e s p e c t r o m e t e r .  t h e sample tube  T h e r e f o r e , t h e exchange  r a t e d a t a r e p o r t e d f o r t h i s compound i s a t 31.4°.. 2.  Treatment o f the d a t a i n exchange  experiments  F o r b o t h TML and DHTML, t h e exchange r a t e s were determined by comparing  t h e r a t i o o f peak areas o f t h e  89  exchanging  C-methyl group and non-exchanging methyl group  w i t h time.  The measurement o f peak a r e a was a c c o m p l i s h e d  .by t h e u s u a l p.m.r. i n t e g r a t i o n t e c h n i q u e and was u s u a l l y done i n d u p l i c a t e .  F o r the f a s t e r r e a c t i o n s o f TML, t h e  i n t e g r a l s o f t h e non-exchanging peaks were determined  once  i n i t i a l l y and s u b s e q u e n t l y o n l y t h e i n t e g r a l o f t h e exchanging peak measured a t r e g u l a r  time i n t e r v a l s ,  Pseudo f i r s t - o r d e r  r a t e c o n s t a n t s (k^) and h a l f - l i v e s  (t±)- were o b t a i n e d 135 g r a p h i c a l r e p r e s e n t a t i o n o f the e q u a t i o n ( 2 ) : 2.3031og lm - k j t  (2)  1 0  where  Im =  [(M/R)^  -  (M/R)  (M/R)^  -  (M/R).  from  Q  M = average a r e a o f exchanging  peak,  R = average a r e a o f r e f e r e n c e (non-exchanging) peak(s), and  t h e s u b s c r i p t s r e f e r t o the r e a c t i o n t i m e . Usually ( M / R )  Q  = 1.0  and ( M / R ^ - ^ O t o 0 . 0 3 w i t h i n  the l i m i t s o f a c c u r a c y a f f o r d e d by n u c l e a r magnetic measurements. determined  resonance  The h a l f - l i f e o f a g i v e n exchange p r o c e s s i s  from t h e measured r a t e c o n s t a n t a c c o r d i n g t o  equation (3): t  A  2  v  = 2.3031og 2 1 0  kj  (3)  1  I n p l o t t i n g o f t h e exchange d a t a t h e method o f  l e a s t - s q u a r e s was employed t o o b t a i n t h e b e s t l i n e .  The  90 e q u a t i o n s used f o r t h i s purpose a r e those g i v e n by  Jaffe!^  F o r b o t h lumazine d e r i v a t i v e s , the N - 8 - C H 3 peak i s used  f o r the r e f e r e n c e as w e l l as the u p - f i e l d C-methyl peak  i n the case o f 6 , 7 , 8 - t r i t n e t h y l l u m a z i n e . The  s l i g h t loss of  c o u p l i n g and peak a r e a o f the u p - f i e l d d o u b l e t i n the  latter  s t a g e s o f the exchange i n the case o f the d i h y d r o t r i m e t h y l lumazine p r e c l u d e s the use o f t h i s peak as a r e f e r e n c e . The  f i r s t - o r d e r , t r e a t m e n t o f the exchange d a t a  proved a p p l i c a b l e to a t l e a s t 75$ b o t h compounds.  The  to 85$  o f the r e a c t i o n f o r  " i n f i n i t y " measurement was  usually  r e p e a t e d s i x to e i g h t times to ensure a c c u r a c y i n the ( M / R ) Q Q term o f e q u a t i o n  (2).  C.  Results  1.  Trimethyllumazine a.  (TML)  Assignment o f the exchanging I t was  observed  group  t h a t the a m p l i t u d e o f the down-  f i e l d C-methyl peak i n the p.m.r. s p e c t r a o f t r i m e t h y l l u m a z i n e i n b u f f e r e d DgO  s o l u t i o n s decreased w i t h time due to exchange  o f the methyl group p r o t o n s w i t h the s o l v e n t .  An example of.  the t y p i c a l exchange p a t t e r n o f t h i s compound i s i l l u s t r a t e d i n Figure  8. The assignment  C-7  o f the exchangeable  c e n t e r to the  methyl group i s s u p p o r t a b l e on the f o l l o w i n g i.  no c o m p l e t e l y c o n j u g a t e d o r d e l o c a l i z e d s t r u c t u r e  be i n v o k e d to account methyl ii.  grounds:  f o r an exchange o f p r o t o n s a t the  can  C-6  group. i n t r i f l u o r o a c e t i c acid with external tetramethyl-  91 FIGURE 8 N -CH 8  ?V  3  C -CH6  . '  HjC^Nv^lvk^O  • H  1  T  c \ \  3  1  .  C -CH 7  ll,  3  1 N  H  ll  0  r  t  i  1  1  1  1  1  b)  c)  J  d)  450  1  i  -v—J 4CO  • : . i . ;—. i . r .  .*.•'.,'."'  350  -  300  c.p.s.  250  "  a) 0.1M TML i n u n b u f f e r e d D 0 (HOD s o l v e n t peak a t S 4.68 ) 2  b ) a f t e r 2.5 min. i n pH 2.0 0.10M Na DP04 a t 3 1 . 4 ° 2  c) a f t e r 13 m i n . d) a f t e r 27 m i n . • e; a f t e r 75 m i n .  92  s i l a n e as r e f e r e n c e , t h e d o w n - f i e l d C-methyi peak i n t h e p^m.r. spectra of a series of closely related pteridine Including  derivatives,  6 , 7 , 8 - t r i m e t h y l - 2 - a m i n o - / f - p t e r i d i n o n e , has been  c o n s i s t e n t l y a s s i g n e d to t h e C-7 m e t h y l g r o u p . r j h  e  r e  i tive a  p o s i t i o n s o f t h e C-metbyl s i g n a l s a r e as expected  after  c o n s i d e r a t i o n o f t h e p.m.r. spectrum o f p t e r i d i n e  itself ^  and  1  t h e known p o l a r i z a t i o n o f bonds i n s i m p l e p t e r i d i n e  derivatives. 7,8-dihydro-6-d3-7,8-trimethyllumazine,  iii.  formed by  the exchange o f t h e C - 6 methyl p r o t o n s o f DHTML ( s e e l a t e r R e s u l t s ) c a n be o x i d i z e d by a i r o r D 0 2  6-d3~7,8-trimethyllumazine. of  2  i n unbuffered D 0 to 2  Comparison o f t h e p.m.r. spectrum  t h i s l a t t e r compound w i t h t h a t o f t r i m e t h y l l u m a z i n e exchanged  i n t h e normal way c o n f i r m s t h e assignment the exchanging group.  o f t h e C-7 methyl as  As e x p e c t e d , t h e C-7 methyl group o f  the 6 - d 3 - t r i m e t h y l l u m a z i n e b e g i n s t o exchange i t s p r o t o n s as w e l l upon f o r m a t i o n from t h e d e u t e r a t e d d i h y d r o p r e c u r s o r . b.  pH-rate  profile  F o r TML, t h e exchange o f t h e C-7 m e t h y l p r o t o n s becomes more r a p i d w i t h i n c r e a s i n g b a s i c i t y and a c i d i t y o f t h e b u f f e r e d D 0 media w i t h t h e s l o w e s t exchange r a t e s o b s e r v e d 2  i n t h e r e g i o n o f pH 4.  The exchange r a t e o f t h e l u m a z i n e i n  (pH 6 . 5 )  was e x t r e m e l y s l o w , w i t h a h a l f - l i f e  unbuffered D 0 2  o f a p p r o x i m a t e l y a week and a h a l f . The exchange p r o c e s s was s t u d i e d i n t h e pH range -0.4 t o 8.0.  Except f o r the very s t r o n g l y a c i d i c  solutions  93  the t r i m e t h y l l u m a z i n e s u b s t r a t e e x i s t s p r e d o m i n a n t l y n e u t r a l species i n t h i s region.  9.  f o r the exchange r e a c t i o n a t v a r i o u s pH i n  NagDPOj^ a t 3 1 . 4 °  i n T a b l e V.  i n Figure  e x p e r i m e n t a l l y determined pseudo f i r s t - o r d e r  rate constants 0.10M  the  A p l o t o f the d a t a f o r a  t y p i c a l exchange experiment i s i l l u s t r a t e d The  as  The  and  i o n i c s t r e n g t h 0.42  are  collected  pH v a l u e s quoted have not been c o r r e c t e d  to the c o r r e s p o n d i n g  pD v a l u e s . • C o r r e l a t i o n c o e f f i c i e n t s  have been i n c l u d e d to i n d i c a t e the degree o f l i n e a r i t y  of  the p l o t t e d d a t a from w h i c h the r a t e c o n s t a n t s were o b t a i n e d . E r r o r s i n the r a t e c o n s t a n t s were found to range from 1 to as e s t i m a t e d  by c o m p a r i s o n o f i d e n t i c a l runs and  depending  upon the number o f d a t a p o i n t s a v a i l a b l e i n d e t e r m i n i n g s l o p e o f the r a t e p l o t  the  line.  A p l o t o f the pseudo f i r s t - o r d e r r a t e ( k l ) v e r s u s pH produces the c u r v e  constants  i l l u s t r a t e d i n Figure  A p l o t o f the l o g a r i t h m s o f the r a t e c o n s t a n t s a g a i n s t g i v e s the c o r r e s p o n d i n g  pH-rate p r o f i l e  (Figure 11).  c o - o r d i n a t e s o f t h i s l a t t e r g r a p h a r e arranged  10. pH  The  to i n d i c a t e  the s l o w e s t r a t e s o c c u r r i n g a t the minimum o f the The  5$  curve.  h a l f - c l o s e d c i r c l e i n c l u d e d i n the above p l o t s r e p r e s e n t s  the t r i f l u o r o a c e t i c a c i d - D 0 s o l v e n t system a t H 2  The  Q  = -0.37.  p H - r a t e p r o f i l e observed i s t y p i c a l o f r e a c t i o n s  w h i c h a r e c a t a l y z e d b o t h by hydronium i o n s ( H 0 ) and +  3  i o n s and  i n w h i c h the u n c a t a l y z e d  hydroxide  reaction i s relatively  s l o w . H o w e v e r , the k i n e t i c measurements were c a r r i e d  out  FIGURE  9  TML EXCHANGE KINETICS  TI ME (MINUTES)  95  Table V H-D exchange o f 6 , 7 , 8 - t r i m e t h y l l u m a z i n e range -0.4 to 8.0, 0.10M lO ^  PH  3  i n DgO i n the pH  Na HP04, u = 0 . 4 2 , T = 31.4°C. 2  (min. )  ti  -1  (min.)  r  2  -0.4  i  350.8  2.0  0.992  1.0  127.5  5.4  0.996  2.0  4l.9  16.5  0.999  3.0  20.4  33.9  0.979  3.5  14.1  49.2  0.998  4.0  17.4  39.9  0.997  4.5  25.7  27.0  0.992  5.0  64.9  10.7  0.981  6.0  375.2  1.8  0.992  7.0  fast  -  -  8.0  very  -  - -  1  -trifluoroacetic  fast  acid-D 0 at H p  0  = -0.37  96  FIGURE 10  TML EXCHANGE KINETICS  FIGURE 11  98 on  the  a c i d s i d e o f n e u t r a l i t y where the hydroxide i o n  concentration that  the  Is n e g l i g i b l e .  T h e r e f o r e , the  exchange r e a c t i o n i s s u b j e c t  to c a t a l y s i s by  o t h e r than o r as w e l l as hydronium and l a c k o f a u n i t slope  results indicate species  hydroxide i o n s .  The  f o r e i t h e r s t r a i g h t l i n e portion of  the  pH-rate p r o f i l e a l s o speaks a g a i n s t  s p e c i f i c c a t a l y s i s by  e i t h e r o f these i o n s .  exchange r e a c t i o n i s  subject  That i s , the  to g e n e r a l a c i d and  base c a t a l y s i s by  the  likely  phosphate  b u f f e r species . The  observed f i r s t - o r d e r r a t e constant at a g i v e n  the  range examined i s p r o b a b l y a composite  pH  within  as  i n d i c a t e d by  k  equation ( 4 ) :  l =o + V k  where  k  Q  HA  +  k  _  2  A  A"2 "A" " 2  +  K  _  constant,  A  - 2  = HPO^" . 2  e f f e c t of buffer concentration  at two  buffer concentration 0 . 1 2 5 M w i t h the  on  —  undertaken to t e s t t h i s scheme. on exchange r a t e  exchange o f the C - T methyl protons i n  solutions buffered  the  A"  _  E f f e c t of buffer concentration The  value by  + k_  HA  = H^POjp A" = H P 0 4 " and  exchange r a t e was c.  HA  , e t c . are s p e c i f i c r a t e c o n s t a n t s r e f e r r i n g to s p e c i e s i n d i c a t e d by the s u b s c r i p t s , and  A study o f the the  K  = u n c a t a l y z e d exchange r a t e  k +,  value  was  pH  values was  v a r i e d over the  i o n i c strength  examined.  DgO The  range 0.015M to  maintained a t a c o n s t a n t  a d d i t i o n o f the a p p r o p r i a t e  amount o f sodium  .  99  chloride. lumazine  Above 0.125M b u f f e r c o n c e n t r a t i o n t h e t r i m e t h y l tended t o p r e c i p i t a t e d u r i n g t h e course o f  measurements w h i c h t h e r e b y v i t i a t e d t h e r e s u l t s .  The r e s u l t s  o f t h i s s e r i e s o f e x p e r i m e n t s a r e c o l l e c t e d i n Table V I . A l i n e a r r e l a t i o n s h i p between t h e pseudo  first-  o r d e r r a t e c o n s t a n t s and b u f f e r c o n c e n t r a t i o n a t pH 2.0 and pH 4 . 5 i s observed  (Figure 12).  The e x t r a p o l a t i o n to zero  b u f f e r c o n c e n t r a t i o n o f t h e p l o t t e d l i n e s i n t e r c e p t s the o r d i n a t e somewhat above the v a l u e e s t i m a t e d  f o r the  u n c a t a l y z e d r a t e c o n s t a n t , e s p e c i a l l y a t pH 2.0, i n d i c a t i n g the c o n c e n t r a t i o n o f hydronium i o n i s a f a c t o r i n d e t e r m i n i n g the magnitude o f the r a t e c o n s t a n t i n t h e a c i d i c Bv,0 media. At pH 4 . 5 , where t h e c a t a l y t i c e f f e c t o f t h e hydronium i o n on t h e exchange i s l i k e l y s m a l l i n comparison to t h e phosphate s p e c i e s , a p l o t o f the n e g a t i v e o f the observed  logarithms  r a t e c o n s t a n t s and t o t a l b u f f e r c o n c e n t r a t i o n  y i e l d s a s t r a i g h t l i n e o f s l o p e 1.01 ( F i g u r e 1 3 ) .  A similar  p l o t a t pH 2.0 g i v e s a s t r a i g h t l i n e b u t o f s l o p e 0 . 8 2 , as i l l u s t r a t e d i n Figure l 4 .  The l e s s - t h a n - u n i t s l o p e i s n o t  s a t i s f a c t o r i l y e x p l a i n e d as y e t .  I f l e s s weight i s p l a c e d on  the v a l u e o f t h e d a t a p o i n t f o r 0.025M b u f f e r c o n c e n t r a t i o n ( t h i s p o i n t was c o r r e c t e d f o r a s m a l l pH change) a l i n e approaching  slope  0 . 9 can be a t t a i n e d .  The are estimated  s p e c i f i c r a t e c o n s t a n t s k-^+, k ^ , k^_ and k _ 2 A  from the r e s u l t s o f t h e exchange r a t e - b u f f e r  c o n c e n t r a t i o n dependency a t t h e two pH v a l u e s and r a t e s a t  100  Table VI E f f e c t o f b u f f e r c o n c e n t r a t i o n on the exchange r a t e o f 6,7,8-trimethyllumazine  at T = 3 1 . 4 ° C ,  10 k;j_ (min.-l)  C o n c e n t r a t i o n NagHPO^ (moles/litre)  J  pH 2 . 0 0.015.  u = 0.42  pH 4 . 5 3.5  -  0.025  16.8  0.050  24.0  13.9  0.075  32.7  18.4  0.100  4.1.9  25.7  0.125  46.3  28.6  J  -  j - c o r r e c t e d f o r s m a l l pH change observed when sample added to t h e b u f f e r s o l u t i o n  101  102  FIGURE  13  TML EXCHANGE KINETICS  3.04  RELATION BETWEEN RATE CONSTANT AND BUFFER CONCENTRATION AT pH 4.5  2.7 4  2.4  •LOG K 10  SLOPE = 1 . 0 1  2.1  1.2  0.8  ~l  i.2  1  r  ~LOG  2.0 10  [ BUFFER ][  103  FIGURE 14  TML EXCHANGE KINETICS RELATION BETWEEN RATE CONSTANT AND CONCENTRATION OF CATALYTIC SPECIES AT pH 2.0  0  -LOG [A] 1 0  A =  H 0  H P0  +  3  3  -  —  •  -  4  +  H P0 " " 2  _  4  104  i n d i v i d u a l pH.  I n the e n s u i n g c a l c u l a t i o n s the v a l u e o f t h e  u n c a t a l y z e d r a t e c o n s t a n t , k , i s assumed t o b e s m a l l i n 1  Q  comparison t o t h e o t h e r c o n s t a n t s .  This I s a reasonable  a s s u m p t i o n i n view o f t h e v e r y slow r a t e o f exchange observed f o r TML i n u n b u f f e r e d D 0 a t pH 6 . 5 . g  The v a l u e o f ky+ may be o b t a i n e d by e x t r a p o l a t i o n o f t h e l i n e f o r t h e b u f f e r c o n c e n t r a t i o n dependency a t pH 2.0 ( F i g u r e 1 2 ) t o zero b u f f e r c o n c e n t r a t i o n .  This operation i s  equivalent to reducing equation (4) t o k  l=V[ 3 H  0 +  ]  (5)  The c o n c e n t r a t i o n s o f t h e v a r i o u s b u f f e r s p e c i e s a t a g i v e n pH c a n be c a l c u l a t e d from the known d i s s o c i a t i o n l4l  constants o f phosphoric a c i d . Although the c o n c e n t r a t i o n o f HgPO^" reaches a.maximum v a l u e (99-3$) o f t h e t o t a l b u f f e r c o n c e n t r a t i o n a t pH 4 . 5 , t h e e f f e c t o f t h e s m a l l amount o f -2  HPO4.  p r e s e n t may n o t be i n s i g n i f i c a n t .  The v a l u e s o f k^-  and k ^ - 2 a r e o b t a i n e d t h e r e f o r e by a p p l i c a t i o n o f t h e f o l l o w i n g equations: (6)  =  V[  %(4.5)  =  k - [A-]  k l  A  A  "]6  + 4 # 5  +  V [ 2  A  " ]6  < >.  2  k -2^A- J  6  2  A  4 < 5  (7)  where t h e s u b s c r i p t s r e f e r t o t h e pH a t w h i c h t h e c o n c e n t r a t i o n o f t h e a p p r o p r i a t e b u f f e r s p e c i e s was c a l c u l a t e d .  The r a t e  c o n s t a n t k-j_ i s t h a t v a l u e a t a g i v e n t o t a l b u f f e r c o n c e n t r a t i o n .  105  The  remaining constant  can be o b t a i n e d by-  s u b s t i t u t i o n o f t h e v a l u e s o f the o t h e r c o n s t a n t s  determined  a c c o r d i n g t o t h e above methods i n t o e q u a t i o n (4) a t a g i v e n pH.  The v a l u e s c a l c u l a t e d f o r the s p e c i f i c  catalytic  c o n s t a n t s a r e as f o l l o w s : k + k  HA  1.2  =  H  k -2 A  ,  °"''  =  k^_  1 .mole~- -min. 41  -1  l' °le~ min.~ m  1  1  =  0 . l 4 l.mole ''"min. ^  =  52.9  l.mole" min." 1  1  A p H - r a t e p r o f i l e may be c o n s t r u c t e d w h i c h c l o s e l y approximates  t h e e x p e r i m e n t a l l y determined  profile  illustrated  i n F i g u r e 10 by u s i n g t h e above v a l u e s o f t h e s p e c i f i c r a t e c o n s t a n t s and a p p l y i n g e q u a t i o n  (4).  Since acid-base c a t a l y s i s i n v o l v e s the t r a n s f e r o f a p r o t o n t o o r from the c a t a l y s t m o l e c u l e , some r e l a t i o n s h i p between t h e e f f e c t i v e n e s s o f t h e c a t a l y s t and i t s s t r e n g t h as an a c i d o r base i s t o be e x p e c t e d .  Bro'nsted suggests  that  a s p e c i f i c c a t a l y t i c c o n s t a n t , k , s h o u l d be r e l a t e d t o t h e d i s s o c i a t i o n constant K  a  a o f an a c i d f o r a g i v e n r e a c t i o n l42  c a t a l y z e d by a s e r i e s o f a c i d s a c c o r d i n g t o (8); k  a  or l o g k where C, G than  a  unity.  a  =  a<  G  = dClog K  +  C  andcCare c o n s t a n t s , the l a t t e r u s u a l l y b e i n g  (8) less  106  S i m i l a r l y , the s p e c i f i c base c a t a l y t i c c o n s t a n t , kft, I s r e l a t e d to the d i s s o c i a t i o n c o n s t a n t K  where G , b  k  b  =  or log k  b  =piog K  D  by ( 9 ) ;  G Kf b  b  +  C»  (9)  C' and j 3 a r e c o n s t a n t s , the l a t t e r u s u a l l y b e i n g  l e s s than u n i t y . I n the p r e s e n t c a s e , a l a c k o-f s u f f i c i e n t d a t a p r e c l u d e s a q u a n t i t a t i v e t r e a t m e n t , i n terms o f the Brc/nsted relations.  A l s o , i t i s not c e r t a i n whether the HgPOi). s p e c i e s -  i s behaving as an a c i d o r base o r whether i t i s a m p h o t e r i c . A comparison  o f the v a l u e s c a l c u l a t e d f o r the s p e c i f i c r a t e  constants indicates that is,  J3is  p r o b a b l y l a r g e r than c£ .  the exchange r e a c t i o n o f TML  i s more e f f e c t i v e l y c a t a l y z e d  by the b a s i c s p e c i e s p r e s e n t i n s o l u t i o n . t h a t the l i n e i n F i g u r e 11  That  The o b s e r v a t i o n  f o r the c a t a l y s i s i n more b a s i c  r e g i o n s i s s t e e p e r than the c o r r e s p o n d i n g l i n e i n the a c i d regions supports  this.  I n s o l u t i o n s more a c i d i c than H b a s i c than pH 6 i n 0.10M  Q  = -0.4  and more  phosphate b u f f e r the exchange rate..  became too r a p i d to measure w i t h the a v a i l a b l e t e c h n i q u e s . I n any c a s e , the i n c r e a s i n g i n s o l u b i l i t y and  decomposition  o f the samples i n the more a l k a l i n e s o l u t i o n s make t h i s a d i f f i c u l t r e g i o n i n which to work. The examined was  exchange o f t r i m e t h y l l u m a z i n e i n the pH range not a c c e l e r a t e d by the a d d i t i o n o f s m a l l amounts  107  of C u  + 2  or Zn  + 2  to the b u f f e r s o l u t i o n s .  I t i s known t h a t  n e u t r a l " q u i n o n o i d " p t e r i d i n e s and l u m a z i n e s , as w e l l as the I s o a l l o x a z i n e s ,  d o  n  o  t  complex a v i d l y with the c a t i o n s o f  heavy m e t a l s . ^  -  1  2.  .  7 , 8 - D i h y d r o - 6 , 7 , 8 - t r i m e t h y l l u m a z i n e (DHTML) a.  Assignment o f t h e e x c h a n g i n g group The a m p l i t u d e d e c r e a s e i n t h e s i n g l e t resonance o f  the d o w n - f i e l d C-methyl group i n the p.m.r. s p e c t r a o f DHTML i s a t t r i b u t e d t o exchange o f t h e C - 6 m e t h y l group p r o t o n s with solvent.  The assignment t o t h i s p a r t i c u l a r group i s  based on t h e f o l l o w i n g i.  grounds;  on c h e m i c a l grounds, no p l a u s i b l e s t r u c t u r e c a n be  Invoked t o a c c o u n t f o r exchange o c c u r r i n g a t t h e C - 7 and N-8 m e t h y l g r o u p s , and ii.  the p.m.r spectrum o f DHTML e x h i b i t s a non-exchanging  doublet resonance. C-7  T h i s resonance has been a s s i g n e d t o t h e  m e t h y l group on s t r u c t u r a l  grounds.  The exchange p a t t e r n o f t h e d i h y d r o l u m a z i n e derivative Is illustrated  i n F i g u r e 15.  O b s e r v a t i o n s o f up t o two weeks d u r a t i o n  indicate  t h a t the p r o t o n a t p o s i t i o n 7 may a l s o be e x c h a n g i n g v e r y s l o w l y as i n d i c a t e d by s m a l l changes I n t h e C - 7 m e t h y l hydrogen c o u p l i n g p a t t e r n and d e c r e a s e i n t h e q u a r t e t peak amplitude.  The l a t t e r d e c r e a s e c a n o n l y be o b s e r v e d i n t h e  t r i f l u o r o a c e t i c a c i d - D 0 s o l v e n t system as t h e q u a r t e t 2  resonance i s u s u a l l y masked by the p r o p i n q u i t y o f t h e HOD  .  108  FIGURE•15 "CHj  -  H p  c -- C H 6  S  0-  N  Cr-CH  o)  Cf-  H  —I—:  . JL .  1  . J ^  1  JL  I  .  L  .  J  —i  1  —i  1  .  I  1  1  i >5 3  c.p.s.  , 1  1  1, l  1  v  J 1  1 L  5CO  40O  300  20O  IOO  a)  0.10M D H T M L i n t r i f l u o r o a c e t i c acid-DpO ( H Q = - 0 . 3 7 ) T = 30.0°, 7 minutes a f t e r s t a r t o f exchange .  b)  a f t e r 4 hours  c)  a f t e r 22 hours  d ) a f t e r 54 hours e)  a f t e r 80 hours  109  s o l v e n t peak I n the b u f f e r e d  DgO systems.  On p r o l o n g e d  I n D 0 s o l u t i o n , some o x i d a t i o n and d e g r a d a t i o n o f  standing  2  the samples i s a l s o o b s e r v e d . however, t h e exchange  I n s p i t e o f these d i f f i c u l t i e s  r e a c t i o n c a n u s u a l l y be f o l l o w e d t o  c o m p l e t i o n b e f o r e much d e c o m p o s i t i o n o c c u r s . b.  pH-rate p r o f i l e The exchange  - 0 . 4 to 8 . 2 .  r e a c t i o n was s t u d i e d  The dependence o f the exchange  i n the pH range r a t e on t h e  a c i d i t y o f the DgO media e x h i b i t e d some u n u s u a l e f f e c t s , e s p e c i a l l y i n the more a c i d i c s o l u t i o n s .  The i n d i v i d u a l r a t e  p l o t s a l s o showed anomalous b e h a v i o u r i n t h i s r e g i o n .  For.  example, t h e d a t a i n t h e pH range - 0 . 4 t o 3 . 0 was c h a r a c t e r i z e d by an i n i t i a l r a t e o f undetermined o r d e r o v e r a p p r o x i m a t e l y 15$ o f the r e a c t i o n f o l l o w e d the r e m a i n d e r . t h i s pH r e g i o n  by a pseudo f i r s t - o r d e r r a t e f o r  The p.m.r. s p e c t r a o f t h e DHTML samples i n (except f o r H  Q  = - 0 . 3 7 ) were c h a r a c t e r i z e d by  a marked b r o a d e n i n g o f t h e C - 6 m e t h y l resonance which tended to become l e s s broad as t h e exchange  proceeded.  As w e l l as  the b r o a d e n i n g o f t h e C - 6 methyl r e s o n a n c e , which a l s o downfield  shifts  w i t h i n c r e a s i n g a c i d i t y o f the b u f f e r , the  appearance o f a s m a l l peak l o c a t e d v e r y c l o s e t o t h e p o s i t i o n o f t h e C - 6 m e t h y l resonance was n o t e d .  The p o s i t i o n o f t h i s  l a t t e r peak s h i f t s o n l y s l i g h t l y w i t h the a c i d i t y o f t h e medium and appears t o d e c r e a s e o n l y v e r y s l i g h t l y i n a m p l i t u d e during  t h e time used t o m o n i t o r t h e exchange  reaction.  This  110  peak ( s i n g l e t ) I s n o t d e t e c t e d i n those b u f f e r sytems i n the pH range 3 . 5 t o 7 . 0 .  That t h i s peak was n o t the r e s u l t o f  an i m p u r i t y i n t h e b u f f e r s o l u t i o n s was c o n f i r m e d by r u n n i n g s p e c t r a o f these s o l u t i o n s b e f o r e t h e i r use i n the p r e p a r a t i o n of  sample s o l u t i o n s . The exchange experiments  i n the pH range 3 - 5 t o  7 . 0 e x h i b i t e d f i r s t - o r d e r k i n e t i c d a t a f o r the e n t i r e and  reaction  t h e p.m.r. s p e c t r a o f the samples d i d n o t e x h i b i t an  i n i t i a l b r o a d e n i n g o f the C-6 methyl  resonance.  The two types o f r a t e p l o t s a t r e p r e s e n t a n t i v e pH a r e i l l u s t r a t e d i n F i g u r e s l 6 and 1 7 . The exchange r a t e o f DHTML i n u n b u f f e r e d DgO (pH 6 . 5 ) was found t o be e x t r e m e l y slow w i t h a h a l f - l i f e o f a p p r o x i m a t e l y two weeks. pH 8 . 2  R e s u l t s f o r t h e exchange i n the pH range above  c o u l d n o t be o b t a i n e d owing t o the i n c r e a s i n g  insolubility  and a c c e l e r a t e d d e c o m p o s i t i o n e x h i b i t e d by the samples. The e x p e r i m e n t a l l y determined r a t e c o n s t a n t s ( k j ) and h a l f - l i v e s  pseudo f i r s t - o r d e r  ( t i ) f o r the C-6  methyl  group hydrogen-deuterium  exchange a t v a r i o u s pH v a l u e s a r e  c o l l e c t e d i n T a b l e VTI.  The e r r o r s i n these r e p o r t e d v a l u e s  range from about 2 t o 8 p e r c e n t as i n d i c a t e d by of  i d e n t i c a l runs.  comparison  I n g e n e r a l , the d a t a y i e l d somewhat l e s s  s a t i s f a c t o r y f i r s t - o r d e r r a t e p l o t s than i n the case o f TML, e s p e c i a l l y i n the i n i t i a l s t a g e s o f the exchange r e a c t i o n . F o r those experiments of  undetermined  Table.  c h a r a c t e r i z e d by an i n i t i a l exchange r a t e  o r d e r , two r a t e c o n s t a n t s a r e r e c o r d e d i n the  The r a t e c o n s t a n t k j and h a l f - l i f e  t i apply to-that  FIGURE 16 DHTML EXCHANGE KINETICS . TYPICAL RATE PLOT (NEUTRAL SPECIES)  TI ME (M I NUTES)  FIGURE 17  4000 TIME  (.Ml  NUTES)  113  Table V I I H-D exchange o f 7 , 8 - d i h y d r o - 6 , 7 , 8 - t r i m e t h y l l u m a z i n e  i n the  r e g i o n pH -0.4 t o 8.2, 0.10M Na HP04, u = 0.42 and T = 3l.4°C. 2  PH  IOVIN  (min. ) - 1  lO ^ 2  (min. ) - 1  ti(min.)  r  2  (40)  8.9  777  0.999  0.5  (73)  19.1  363  0.993  1.0  (54)  31.6  219  0.988  1.5  43.8  158  O.989  2.0  37.6  184  0.996  2.5  29.8  233  0.990  16.1  431  0.999  4.5  10.9  639  0.995  4.9  9-8  708  0.991  5.5  9.9  702  0.992  6.0  10.2  677  0.990  7.0  9.8  708  0.992  8.0  3.4  2040  0.^96  3.9  1777  0.997  -0.37  3.5  8.2  k  •  1  k -trifluoroacetic 1 - pyridine-Dp0  acid-DgO  1  114  p a r t o f the d a t a which e x h i b i t s f i r s t - o r d e r k i n e t i c s .  Where  I t c o u l d be e s t i m a t e d ,  from  k j ^ r e f e r s to the v a l u e d e r i v e d  the b e s t s t r a i g h t l i n e drawn t h r o u g h the c u r v e e x h i b i t e d by the i n i t i a l r a t e d a t a when p l o t t e d i n a f i r s t - o r d e r manner. These v a l u e s a r e p l a c e d i n b r a c k e t s estimates  only.  to i n d i c a t e t h a t they  Correlation coefficients  are  ( r ) have a l s o been  i n c l u d e d to i n d i c a t e the degree o f l i n e a r i t y o f the exchange data. A p l o t o f the pseudo f i r s t - o r d e r r a t e  constants  v e r s u s pH r e s u l t s i n the u n u s u a l p H - r a t e p r o f i l e i n Figure l 8 . estimated  The  t r i a n g l e s i n the F i g u r e r e p r e s e n t  k j ^ w h i l e the c i r c l e s r e p r e s e n t  r a t e . c o n s t a n t s k^.  The  two  the  0  = -0.37  and  pH =. 8.2  the  first-order  h a l f - c l o s e d c i r c l e s represent  s o l v e n t systems t r i f l u o r o a c e t i c acid-DgO and H  illustrated  respectively.  pyridine-D20  the at  For d i s c u s s i o n purposes,  the p r o f i l e has been d i v i d e d i n t o t h r e e s e c t i o n s , I n each o f w h i c h one obtained  form o f DHTML i s p r e d o m i n a n t .  From the pK  f o r t h i s compound (Table l b ) , one  can  values  identify  r e g i o n A as the pH range where the compound e x i s t s m a i n l y  as  the c a t i o n , r e g i o n B where the n e u t r a l s p e c i e s p r e d o m i n a t e s , and  r e g i o n C where the monoanion p r e d o m i n a t e s . c_.  E f f e c t o f b u f f e r on the exchange r a t e The  was  exchange o f  examined a t pH 4 . 5 ,  7,8-dihydro-6,7,8~trimethyllumazine  i o n i c s t r e n g t h 0.42  30.0° w i t h v a r y i n g b u f f e r c o n c e n t r a t i o n . o f the phosphate b u f f e r was  and The  temperature concentration  v a r i e d i n the range 0.050M to  FIGURE 18  116  0.150M w i t h t h e i o n i c s t r e n g t h m a i n t a i n e d v a l u e by t h e a d d i t i o n o f sodium c h l o r i d e . below  0.050M  Table V I I I .  Buffer  concentrations  were unable t o keep t h e pH o f t h e system  upon s o l u t i o n o f t h e o r g a n i c The  a t the c o n s t a n t  constant  substrate.  r e s u l t s o f t h i s experiment a r e c o l l e c t e d i n Included  i n the Table 1  f o r comparison a r e t h e  r e s u l t s from s i n g l e e x p e r i m e n t s u s i n g a t pH 0 . 5 , 2.0 and 6.0.  0.050M  phosphate b u f f e r  I t was noted t h a t w h i l e t h e amount  o f t h e i n i t i a l r a t e o f undetermined o r d e r was comparable f o r the two b u f f e r c o n c e n t r a t i o n s a t pH 2.0, a marked i n c r e a s e in  t h e e x t e n t o f t h e i n i t i a l r a t e p o r t i o n o f the exchange  d a t a was observed i n d e c r e a s i n g  the c o n c e n t r a t i o n o f the b u f f e r  from 0.10M t o 0.050M a t pH 0.5. A p l o t o f t h e d a t a i n T a b l e V I I I a t pH 4 . 5 r e s u l t s in  the curved  reasonable points.  r e l a t i o n s h i p shown i n F i g u r e 19, a l t h o u g h a  straight line  ( r = O.986) can be drawn through t h e  A l i n e a r r e l a t i o n s h i p would i n d i c a t e t h a t t h e  exchange r e a c t i o n i s s u b j e c t t o c a t a l y s i s by b u f f e r s p e c i e s a t t h i s pH.  T h i s i s a l s o i n d i c a t e d by the r e s u l t s o f the  i n d i v i d u a l e x p e r i m e n t s a t t h e o t h e r pH v a l u e s . d_.  C a t a l y s i s by m e t a l c a t i o n s The  reduction o f 6,7,8-trimethyllumazine  in hydrochloric acid yields  with zinc  7,8-dihydro-6,7,8-trimethyl-  lumazine as t h e z i n c complex ( X L V I I ) .  I t has been r e p o r t e d ' ^  t h a t 8 - a l k y l p t e r i d i n e s and l u m a z i n e s w i t h s t r u c t u r e s s i m i l a r to  DHTML have an i n h e r e n t a f f i n i t y f o r m e t a l s .  3 6  1  117  Table V I I I E f f e c t o f b u f f e r c o n c e n t r a t i o n on the exchange r a t e o f DHTML a t u.=-0.42, T = 3 0 . 0 ° C .  10 k]_ a t pH 4 . 5 (min. )  C o n c e n t r a t i o n Na hT04 (moles/l.) . 2  0.050  4.7  0.075  8.4  0.100  10.9  0.125  12.8  0.150  14.4  f o r comparison: PH  4 10 k  -1 (min. ) 1 ' 0.050M Na HP04 0.100M Na HP04 2  0.5 2.0  7.6 20.9  2  19.1 37.6  4.5  4.7  10.9  6.0  6.0  10.2  118  FIGURE 19 DHTML EXCHANGE KINETICS  /  /  /  /  /  0  2.5  75  5.0  ' ,10.0  12.5  i 0 [ N a H P 0 ] (M.-) 1  a  a  4  I s!o  120  I t was d e c i d e d to t e s t f o r the p o s s i b l e e f f e c t o f heavy m e t a l c a t i o n s on the exchange adding C u C l pH 5 . 0 .  2  catalytic  r e a c t i o n by  to s o l u t i o n s o f the d i h y d r o l u m a z i n e i n DgO  at  The i n s o l u b i l i t y o f c u p r i c phosphate I n w a t e r  p r e c l u d e d the use o f sodium phosphate as the b u f f e r i n g agent. T h e r e f o r e , b u f f e r systems c o n t a i n i n g sodium formate (0.10M) a d j u s t e d t o pH 5 . 0 and c o n t a i n i n g amounts o f c u p r i c r a n g i n g i n c o n c e n t r a t i o n from 0.0M  chloride  to 0.012M were p r e p a r e d .  The i o n i c s t r e n g t h was m a i n t a i n e d a t a c o n s t a n t v a l u e o f 0.15. the  The samples o f DHTML were p r e p a r e d and t h e r m o s t a t e d i n p r e v i o u s l y d e s c r i b e d manner. For  those s o l u t i o n s c o n t a i n i n g C u C l , a n o t a b l e 2  i n i t i a l b r o a d e n i n g o f the C-6 methyl resonance a l o n g w i t h the c h a n g i n g o f the s o l u t i o n c o l o u r from p a l e y e l l o w to orange i n d i c a t e d complex  f o r m a t i o n . The b r o a d e n i n g o f t h i s m e t h y l  resonance p e r s i s t e d ^ d u r l n g the k i n e t i c measurements.  The  o t h e r resonances o f the d l h y d r o . t r i m e t h y l l u m a z i n e were a l s o significantly Due  broadened. to the e x t e n s i v e b r o a d e n i n g o f the C-6  group resonance o n l y the exchange  r a t e i n 0.10M  methyl  sodium  formate a t pH 5 . 0 c o n t a i n i n g 0.006M CuClg c o u l d be measured w i t h any degree o f a c c u r a c y . system (k-^  The exchange  12 x 10"^ m i n . " ) compared to the r a t e i n the 1  same system w i t h no added C u C l catalysis  rate i n this  by Cvr  does o c c u r .  ( 2 . 6 x 10""^ m i n . ) i n d i c a t e s - 1  2  This i s l i k e l y a  consequence  o f the d r i f t o f e l e c t r o n s i n the v i c i n i t y o f the exchanging  121  m e t h y l group towards  the p o s i t i v e metal c a t i o n w i t h the  f o r m a t i o n o f the complex ( L X V I ) .  (LXVI)  D. D i s c u s s i o n 1. Mechanism o f the exchange p r o c e s s a. • T r i m e t h y l l u m a z i n e The s e l e c t i v e decrease i n peak a m p l i t u d e o f the d o w n f i e l d C-methyl group, i n 6 , 7 , 8 - t r i m e t h y l l u m a z i n e _ i s a t t r i b u t e d to a g e n e r a l a c i d - b a s e c a t a l y z e d exchange o f the C-7  methyl p r o t o n s w i t h s o l v e n t , through the i n t e r r n e d i a c y  o f ( L X V ) and ( L X V ) . a  b  The s u b s c r i p t s r e f e r to the i n t e r m e d i a t e  i n a c i d i c media (a) o r the one found i n more b a s i c media (b) respectively.  A s i m i l a r i n t e r m e d i a t e (LXVI) to (LXV^) has 140  been proposed  f o r the exchange o f the C-7  methyl  protons  o f r i b o f l a v i n - 5 ' - p h o s p h a t e (PMN) i n DgO b u f f e r e d a t pH to  6.9  6.8  122  (LXVI)  The r e l a t i v e l y f a c i l e exchange o b s e r v e d f o r the C-7  m e t h y l group i n TML can be e x p l a i n e d i n terms o f t h e  l o n g c o n j u g a t e d s t r u c t u r e and e x t e n s i v e charge d e l o c a l i z a t i o n a v a i l a b l e i n the i n t e r m e d i a t e s ( L X V ) and (LXV ) compensating a  fe  f o r t h e f o r m a t i o n o f the exo methylene s t r u c t u r e . The mechanism o f the exchange i n the more s t r o n g l y a c i d i c d e u t e r i u m o x i d e media i s I l l u s t r a t e d i n the f o l l o w i n g r e v e r s i b l e pathway (Scheme I ) :  123  Scheme I A c i d - c a t a l y z e d Exchange o f TML  CH 3 J.  UH  HC  DA(D 0 ) +  +  3  3  6  ^ 3 H  O-D O-  I  I  Si  I  o  CHH  H  CH-  3 ^Sr^°c  3  H  D  3  3  ^  ^ M  H C 3  6  C  r  v  >  D  *3  I I  o H  H  C  D 2  H °  O  HA ( H D 0 ) +  2  ^ 3 C ^ ^ > ^ p 3  C  W O  IjH  124  A t r i v i a l v a r i a t i o n o f t h i s mechanism i s p r o t o n a t i o n o f t h e 4 - p o s i t i o n oxygen r a t h e r than the 2 - p o s i t I o n oxygen as i n d i c a t e d i n Scheme I . P r o t o n a t i o n a t t h e l a t t e r p o s i t i o n does however a l l o w f o r somewhat more d e r e a l i z a t i o n o f t h e p o s i t i v e charge i n t r o d u c e d i n t o the m o l e c u l e . pKg + o f 6 , 7 j 8 - t r i m e t h y l l u m a z i n e H  As w e l l , t h e  a g r e e s more c l o s e l y t o the  pKgpj+ o f 6 , 7 , 8 - t r i m e t h y l - 2 - p t e r i d i n o n e r a t h e r than t o t h e value o f the isomeric t r i m e t h y l - 4 - p t e r i d i n o n e . Towards more b a s i c r e g i o n s base-catalyzed  127  (above pH 4 ) t h e g e n e r a l  exchange becomes more i m p o r t a n t .  The mechanism  o f t h e exchange i n t h e more b a s i c pH range i s i l l u s t r a t e d by the f o l l o w i n g r e v e r s i b l e pathway (Scheme I I ) : Scheme I I Base-catalyzed  exchange o f TML  125  The o b s e r v a t i o n t h a t no resonance c h a r a c t e r i s t i c of exo methylene protons could be detected i n d i c a t e s the e q u i l i b r i u m shown below l i e s w e l l to the l e f t .  f H  3  H-,C'^ -\ ^ O :  H  <  b.  7,8-Dihydro-6,7,8-trimethyllumazine The d e c r e a s e i n peak a m p l i t u d e o f the d o w n - f i e l d  C-methyl s i n g l e t I n the case o f DHTML i s a t t r i b u t e d to an exchange o f the C-6  m e t h y l p r o t o n s w i t h s o l v e n t t h r o u g h the  intermediacy of (LXVII).  CH H C.  3 H  3  H/V^^  H  H o  (LXVIIj T h i s s t r u c t u r e i s analogous t o the e n o l .tautomer o f an a l i p h a t i c ketone such.as a c e t o n e .  126  I n g e n e r a l , t h e much s l o w e r exchange r a t e s observed f o r t h e d i h y d r o compound i n comparison  with trimethyllumazine  i s a consequence o f t h e l a c k o f a l o n g c o n j u g a t e d f a c i l i t a t i n g charge d e l o c a l i z a t i o n i n t h e former  system case.  I n o r d e r t o e x p l a i n t h e exchange mechanism and the u n u s u a l pH-rate p r o f i l e o f 7 , 8 - d i h y d r o - 6 , 7 , 8 - t r i m e t h y l l u m a z i n e , r e c o u r s e must be made t o an e x a m i n a t i o n o f t h e v a r i o u s i o n i c s p e c i e s t h e m o l e c u l e c a n form. A p o s s i b l e s t r u c t u r e o f t h e mono-cation o f DHTML i.s i n d i c a t e d by t h e r e s o n a n c e - s t a b i l i z e d f o r m u l a  CHj  9^3 H  3 S ^ ^ S - 0  H  H  hi  3  C  H  o+ H  H C 3  etc  r> H  H  -  H  3 & ^ r ^ O  H  (LXVIII).  3  C  ^ ^ ^ 3  Y  OH H  or  H C 3  O H (LXVIII)'  A comparison  o f t h e p K + o f DHTML and s e v e r a l model B H  compounds s u p p o r t s t h i s s t r u c t u r e f o r t h e c a t i o n . v a l u e s a r e g i v e n as f o l l o w s :  These  127  Compound  pK + m  DHTML  (H3O, 2 5 ° )  Reference  2.86  H R (CHo) N-C=C-C=0 2  (LXIX)  H .  T h i s work  (R = H) 3 . 0 5 (R = C H )  144  3.15  145  2.34  6  (  (LXX)  5  X44 145 E v i d e n c e has been p r e s e n t e d  '  t o show t h a t  O-protonation p r e v a i l s i n the formation o f the c a t i o n s o f (LXIX) and ( L X X ) .  The p r o t o n a t e d form o f (LXX), f o r  example, c a n be r e p r e s e n t e d as f o l l o w s :  T h i s s t r u c t u r e i s analogous o f t h e proposed  t o one o f t h e resonance  forms  cation of 7,8-dihydro-6,7,8-trimethyllumazine.  The pKgjj+ o f 7 , 8 - d i h y d r o - 2 - a m i n o - 4 - h y d r o x y - 6 m e t h y l p t e r i d i n e has a r e p o r t e d v a l u e o f 3 . 2 , c o r r e s p o n d i n g t o p r o t o n a t i o n o f t h e N-8 p o s i t i o n . * " ^ 1  however, suggests  An e a r l i e r r e f e r e n c e } ^ •  t h a t t h e c a t i o n o f t h i s compound, as w e l l  as t h e c a t i o n o f t h e 6 , 8 - d i m e t h y l  d e r i v a t i v e , possesses the  8  128  structure  (LXXI).  R  H  O  (LXXI; R = H, CH3) l4g In a r e c e n t p a p e r ,  s u p p o r t f o r N-5 p r o t o n a t i o n f o r t h e s e  compounds and s i m i l a r p t e r i d i n e d e r i v a t i v e s i s adduced  from  the c o r r e l a t i o n o b s e r v e d between t h e p e r t u r b a t i o n e f f e c t on the t r a n s i t i o n energy and t h e s p e c t r o s c o p i c d a t a . I t i s e v i d e n t from d i s a g r e e m e n t s i n t h e l i t e r a t u r e about t h e s t r u c t u r e o f c a t i o n s o f reduced p t e r i d i n e  systems  that a s i n g l e s t r u c t u r e f o r the c a t i o n o f 7 , 8 - d i h y d r o - 6 , 7 , 8 t r i m e t h y l l u m a z i n e cannot be e s t a b l i s h e d w i t h complete c e r t a i n t y at present.  Indeed, t h e r e p o r t e d pK H+ f o r DHTML may B  r e p r e s e n t an e q u i l i b r i u m m i x t u r e o f two ( o r more) c a t i o n s e x i s t i n g together i n solution. The s h i f t i n g o f t h e C - 6 m e t h y l resonance t o l o w e r f i e l d w i t h i n c r e a s i n g a c i d i t y o f t h e aqueous media i s c o n s i s t e n t w i t h t h e e x p e c t e d d e s h i e l d i n g o f t h e methyl group p r o t o n s by t h e f u l l p o s i t i v e charge l o c a t e d on t h e n e i g h b o r i n g n i t r o g e n atom i n t h e N-5 p r o t o n a t e d form o f DHTML ( L X X I I ) .  129  C  H  1  u  | 3  H  b  O  (LXXII) W h i l e i t i s e v i d e n t t h a t i n c r e a s i n g amounts o f the N-5 p r o t o n a t e d form o f DHTML ( L X X I I ) c o u l d account f o r observed i n c r e a s e i n exchange r a t e i n the pH range 3.5 t o 1.0 (Region B, F i g u r e l S ) i t i s n o t c l e a r how t h e o t h e r p o s s i b l e c a t i o n ( L X V I I I ) enhances t h e exchange p r o c e s s , i f i t does so a t a l l .  A r a t e enhancement by a coulombic  effect  might be expected from t h i s l a t t e r I o n . I f a c i d c a t a l y s i s o f t h e exchange o c c u r s by means o f p r o t o n a t i o n a t t h e N-5 p o s i t i o n o f DHTML then t h e r a t e s h o u l d c o n t i n u e t o i n c r e a s e u n t i l such time as the s u b s t r a t e i s f u l l y protonated.  A t t h i s s t a g e , the exchange s h o u l d  proceed a t a c o n s t a n t r a t e as i n d i c a t e d by a l e v e l l i n g o f the pH-rate p r o f i l e i n t h e r e g i o n where DHTML e x i s t s p r e d o m i n a n t l y as t h e c a t i o n (Region A, F i g u r e 18).  In fact,  marked decrease i n the exchange r a t e was observed i n t h i s region.  D i s c u s s i o n o f t h i s f e a t u r e o f the exchange p r o c e s s  i s p r e s e n t e d l a t e r i n t h i s work. F o r m a t i o n o f s i g n i f i c a n t amounts o f t h e mono-anion (LXXIII) o f 7,8-dihydro-6,7,8-trimethyllumazine  above pH 7  130  would be expected to i n h i b i t t h e exchange r e a c t i o n s i n c e the i n t e r m e d i a t e unfavorable  i n t h e exchange p r o c e s s now r e q u i r e s t h e  s i t u a t i o n o f two n e g a t i v e  charges i n c l o s e  p r o x i m i t y I n the same m o l e c u l e .  CH;  (LXXIII)  That i n h i b i t i o n o f t h e exchange does o c c u r i n t h e r e g i o n where t h e s u b s t r a t e  i s a n i o n i c I s i n d i c a t e d by t h e d e c r e a s e  i n exchange r a t e i n the pH r e g i o n s pK  a  around and beyond t h e  ( 7 - 2 9 ) o f DHTML (Region C, F i g u r e 1 8 ) .  ,  The s t u d y o f t h e dependency o f t h e exchange r a t e upon b u f f e r c o n c e n t r a t i o n  i n d i c a t e s t h a t t h e exchange o f "  DHTML i s s u b j e c t t o g e n e r a l  catalysis.  exchange mechanism i s i l l u s t r a t e d  The a c i d - c a t a l y z e d  i n Scheme I I I .  131  Scheme I I I A c i d - c a t a l y z e d exchange o f DHTML  Base c a t a l y s i s o f the exchange p r o c e s s i s n o t i n d i c a t e d i n the p H - r a t e p r o f i l e .  132  2.  Hydration o f 7j8-dihydro-6,7,8-trimethyllumazine The p o s s i b i l i t y a r i s e s t h a t a c i d - c a t a l y z e d  addition  o f t h e elements o f w a t e r t o form a " h y d r a t e d " c a t i o n o f DHTML (LXXIV) i s r e s p o n s i b l e f o r t h e slow exchange r a t e s o b s e r v e d i n t h e pH range 1 . 0 t o - 0 . 4 (Region A, F i g u r e l 8 ) . T h i s p o s s i b i l i t y i s p l a u s i b l e on t h e f o l l o w i n g i.  grounds:  A d d i t i o n o f w a t e r t o a C=N bond c o n v e r t s t h e c a r b o n  atom from an u n s a t u r a t e d t o a s a t u r a t e d s t a t e .  Consequently,  the s i g n a l o f a group bonded d i r e c t l y t o t h i s c a r b o n undergoes  a c o n s i d e r a b l e s h i f t u p f i e l d i n t h e p.m.r. spectrum  o f t h e compound i n s p i t e o f t h e p r e s e n c e o f t h e h y d r o x y l group. ^  However, i f t h e " h y d r a t e " (LXXIV) i s u n s t a b l e w i t h  2  r e s p e c t t o t h e r i n g - o p e n e d form (LXXV) then t h e c r i t e r i o n o f u p f i e l d s h i f t upon h y d r a t i o n no l o n g e r a p p l i e s . because  This i s  t h e r e s u l t i n g c a r b o n y l f u n c t i o n c a n be e x p e c t e d t o  have s i m i l a r d e s h i e l d i n g e f f e c t s on t h e C - 6 m e t h y l group as the i m i n e (C=N) f u n c t i o n does i n t h e unhydrated n e u t r a l form o f DHTML.  H (LXXIV)  (LXXV)  133  The f a c t t h a t t h e m e t h y l resonance o f a c e t o n e d i s s o l v e d i n 0.050M phosphate b u f f e r i n DgO a t pH 1.0 l i e s  slightly  u p f i e l d from t h e C - 6 m e t h y l resonance o f DHTML d i s s o l v e d i n the same b u f f e r c o n f i r m s t h i s e x p e c t a t i o n .  The s m a l l peak  w h i c h appears i m m e d i a t e l y a d j a c e n t to t h e broad C - 6 m e t h y l resonance i n s o l u t i o n s o f DHTML I n t h e pH range 0 . 5 t o 2 . 5 may be t h e c o r r e s p o n d i n g C-methyl resonance o f t h e r i n g opened form (LXXV). In support o f the ring-opening r e a c t i o n i s the 150 observation  that, i n acid solution, pteridine  rapidly  adds a m o l e c u l e o f w a t e r t o form "hydrated p t e r i d i n e " , 3 , 4 dihydro~4-hydroxypteridine,(as the c a t i o n ) .  T h i s compound  s l o w l y undergoes f i s s i o n o f t h e d i h y d r o p y r i m i d i n e r i n g t o form t h e c a t i o n o f 2 - a m i n o m e t h y l e n e a m i n o - 3 ~ f o r m y l p y r a z i n e . S i m i l a r b e h a v i o u r i s shown by 2-, 4 - and 7 - m e t h y l p t e r i d i n e . The r i n g - o p e n i n g r e a c t i o n i s a c i d - c a t a l y z e d w i t h t h e r a t e 151 v a r y i n g l i n e a r l y w i t h hydrogen i o n a c t i v i t y . ii.  ,  H y d r a t i o n i n t h e p t e r i d i n e s e r i e s I s u s u a l l y marked  by s p e c t r a l s h i f t s towards s h o r t e r w a v e l e n g t h s , consonant w i t h t h e r e d u c t i o n o f t h e c o n j u g a t e d pathway i n t h e molecule. ' -^ 2 3  2  I n t h e case o f 7 , 8 - d i h y d r o ~ 6 , 7 , 8 - t r i m e t h y l -  lumazine, a bathochromic s h i f t  ( a s h i f t to longer wavelengths)  o f 34 mu was o b s e r v e d i n g o i n g from t h e n e u t r a l s p e c i e s t o t h e cation.  The change from p t e r i d i n e t o i t s p r o t o n a t e d h y d r a t e  I s s i m i l a r l y accompanied  by a bathochromic s h i f t o f about  150 20 mu,  e x p l a i n e d i n terms o f an i n c r e a s e i n t h e ease w i t h  134  w h i c h e l e c t r o n s on N-3 o f t h e h y d r a t e d p t e r i d i n e s p e c i e s can be e x c i t e d i n t o an o r b i t a l i n w h i c h t h e r e i s an e l e c t r o n t r a n s f e r toward N - 8 .  Anomalous s h i f t s i n t h e s p e c t r a o f  p t e r i d i n e , as d e s c r i b e d i n going  above, and i n q u i n a z o l i n e s o l u t i o n s  from t h e n e u t r a l s p e c i e s  t o the c a t i o n was one o f  the main r e a s o n s f o r t h e s u g g e s t i o n hydrated. ^' ' 1  iii.  2 3  t h a t t h e c a t i o n s were  2 5  Water, b e i n g a weak n u c l e o p h i l e , i s not l i k e l y t o  be s t r o n g l y bonded a c r o s s force operates.  t h e C=N bond u n l e s s some o t h e r  Resonance has been found t o s u p p l y t h e  e x t r a i n f l u e n c e t h a t makes t h e h y d r a t e d form s t a b l e . the p r e s e n t  In  c a s e , resonance s t a b i l i z a t i o n i s o p e r a t i v e i n  b o t h t h e h y d r a t e d c a t i o n (LXXIV) and the c o r r e s p o n d i n g  ring-  1  opened form (LXXV). There i s a d e a r t h o f i n f o r m a t i o n i n t h e l i t e r a t u r e concerning systems.  t h e h y d r a t i n g p r o p e r t i e s o f reduced p t e r i d i n e The 7 , 8 - h y d r a t e d form o f 6 , 7 - d i m e t h y l - 5 , 6 - d i h y d r o -  2 - a m i n o - 4 - h y d r o x y p t e r i d i n e has been p o s t u l a t e d " " ^ as a 1  possible intermediate  i n t h e r e d u c t i o n o f the.. 5 , 6 - d i h y d r o - _._  p t e r i d i n e w i t h reduced t r i p h o s p h o p y r i d i n e  nucleotide  (TPNH).  On t h e b a s i s o f t h e a v a i l a b l e e v i d e n c e , t h e r a t e decrease i n Region A o f the pH-rate p r o f i l e i s explained i n terms o f a c i d - c a t a l y z e d h y d r a t e f o r m a t i o n  d e c r e a s i n g the  c o n c e n t r a t i o n o f the exchanging s p e c i e s .  Establishment  of  an e q u i l i b r i u m m i x t u r e o f h y d r a t e d and unhydrated c a t i o n s may a l s o e x p l a i n t h e anomalous i n i t i a l r a t e o f undetermined  135  o r d e r observed  i n the f i r s t - o r d e r r a t e p l o t s f o r the exchange  process In t h i s region of a c i d i t y . The h y d r a t i o n and exchange scheme i n s t o n g l y a c i d i c media can be r e p r e s e n t e d i n terms o f the f o l l o w i n g e q u i l i b r i a : Scheme I V H y d r a t i o n o f DHTML i n a c i d i c  media  I t i s seen t h a t u n l e s s t h e r e i s a d r i v i n g  force  to s h i f t the e q u i l i b r i a i n the above Scheme towards the ring-opened  form, the p r o p o r t i o n o f h y d r a t e d  c a t i o n s h o u l d be pH-independent.  exchanging  I f t h i s were the c a s e ,  a l e v e l l i n g - o f f r a t h e r than a decrease observed  to  i n r a t e s would  i n the r e g i o n s where the s u b s t r a t e i s f u l l y  be  136  protonated.  The o p e r a t i v e  towards t h e r i n g - o p e n e d  force i n shifting  form i s p r o b a b l y  p r i m a r y amino g r o u p i n t h i s pyrimidine, at  25  ' so t h a t p r o t o n a t i o n  Region A o f the pH-rate p r o f i l e .  the coulombic  the  pyrimidine  effect  present  The b a s i c i t y o f t h i s (decrease  moiety o f the molecule.  o f protonation  o f the primary  i n pKgpj+  value)  o f the p o s i t i v e charge present i n  exchange r a t e which i n i t i a l l y result  o f 4 . 6 0 and 2 . 5 1  under t h e c o n d i t i o n s o f a c i d i t y  amino g r o u p i s u n d o u b t e d l y weakened by  o f the  A n i l i n e and 5-amino-  compound. H  amino g r o u p i s l i k e l y in  protonation  f o r e x a m p l e , have p K g + v a l u e s  respectively  the e q u i l i b r i a  occurs  The d r o p i n t h e  n e a r pH 2 i s l i k e l y  a  o f t h e amino g r o u p i n t h e r i n g - o p e n e d  c a t i o n o f the s u b s t r a t e . Support following .1)  i s lent  to the hydration  scheme o n t h e '  grounds: The c o v a l e n t  hydration  reactions o f pteridines are  1S2 usually  catalyzed  2)  The a r e a  of  by hydrogen i o n s  and h y d r o x i d e  o f the peak assigned  the ring-opened  ions.  y  ,  t o the C-methyl group  c a t i o n o f DHTML i n c r e a s e s w i t h  increasing  a c i d i t y o f the phosphate b u f f e r . 3)  Where e s t i m a t i o n  rate constants in  Figure  18)  o f the magnitude o f the i n i t i a l  ( k j ^ ) c a n be made, t h e i r l i e approximately  along  values  a line  extrapolated  from  the a c i d - c a t a l y z e d p o r t i o n o f the pH-rate  This  indicates that  normal f e a t u r e s  (triangles  t h e e x c h a n g e o f DHTML would  profile. e x h i b i t the  o f a n a c i d - c a t a l y z e d r e a c t i o n were t h e  137  concurrent hydration  r e a c t i o n not also a f a c t o r i n determining  the magnitude o f t h e exchange r a t e .  The o b s e r v a t i o n  that  t h e s e i n i t i a l r a t e s i n a c i d i c b u f f e r s change to s l o w e r  first-  o r d e r r a t e s w i t h time i s i n d i c a t i v e o f the e q u i l i b r a t i o n o f the h y d r a t e d and unhydrated c a t i o n s o f the d i h y d r o 4)  lumazine.  The b r o a d e n i n g and s h i f t i n g o f the C-6 m e t h y l  resonance o f DHTML i n a c i d i c media i s c o n s i s t e n t w i t h  this  scheme. 5)  I t was o b s e r v e d t h a t the s m a l l peak a s s i g n e d  to the  C-methyl group o f the ring-opened c a t i o n decreased o n l y  very  s l i g h t l y i n a r e a d u r i n g t h e c o u r s e o f the exchange r e a c t i o n . T h i s i s i n agreement w i t h t h e e x p e c t a t i o n  t h a t the C-methyl  resonance o f the r i n g - o p e n e d c a t i o n would exchange s l o w l y 153 in  the a c i d i c DgO s o l u t i o n s i n l i n e w i t h the r e p o r t e d  slow exchange o f acetone i n DgSO^. 6)  I n comparing the amount o f t h e I n i t i a l r a t e o f  undetermined o r d e r between two exchange e x p e r i m e n t s c a r r i e d out I n 0.050M'and 0.10M phosphate b u f f e r a t pH 0 . 5 , a marked d e c r e a s e was o b s e r v e d i n g o i n g t o the more buffer.  concentrated  This i s consistent with f a s t e r establishment  o f the  a c i d - c a t a l y z e d e q u i l i b r i u m between h y d r a t e d and unhydrated c a t i o n s i n t h e b u f f e r c o n t a i n i n g more o f the a c i d i c H 3 P O 4 species . Attempts t o form a 2 , 4 - d i n i t r o p h e n y l h y d r a z o n e d e r i v a t i v e under c o n d i t i o n s i d e a l f o r the f o r m a t i o n r i n g - o p e n e d c a t i o n have so f a r been  unsuccessful.  o f the  138  3.  Biological The  Implications  a c t i v a t i o n and  exchange o f a s p e c i f i c methyl  group i n b o t h 7 , 8 - d i h y d r o - 6 , 7 , 8 - t r i m e t h y l - and methyllumazine i s i n t e r e s t i n g .  Most o f the  Important n a t u r a l l y - o c c u r r i n g p t e r i d i n e s  '6,7,8-tri-  biologically  - f o r example, the  f o l i c a c i d group - c o n s i s t o f a p t e r i n s u b s t i t u t e d  In  the  6 p o s i t i o n w i t h a s u b s t i t u t e d aminomethylene m o i e t y . o t h e r hand, c e r t a i n p t e r i d i n e pigments such as  On  the  erythropterin  (2-amino-4,6-dihydroxypteridine-7-pyruvic: a c i d ) a r e s u b s t i t u t e d the 7 p o s i t i o n . and  The  i m p l i c a t i o n i s t h a t the f o l i c  s i m i l a r l y s u b s t i t u t e d p t e r i d i n e s might a r i s e  in  acids  by  c o n d e n s a t i o n o f the a p p r o p r i a t e amine f u n c t i o n w i t h a s i m p l e 7,8-dihydro-6-methyl (or hydroxymethylene)-pteridine d e r i v a t i v e , while condensation with a 7-methylpteridine y i e l d s the p i g m e n t s .  6-position  However, n u c l e o p h i l i c a t t a c k on the  of 7,8-dihydropteridines leading  to 6 - s u b s t i t u t e d  tetrahydroo  p t e r i d i n e s followed  by g e n t l e o x i d a t i o n i s known  the c o r r e s p o n d i n g 6 - s u b s t i t u t e d  to produce  pteridines.  Because t h e y are ov to r i n g n i t r o g e n s a l l m e t h y l groups i n p t e r i d i n e s s h o u l d be a c t i v a t e d and hydrogen-deuterium exchange. o n l y one described  susceptible  to  S u r p r i s i n g l y , the exchange o f  other p t e r i d i n e d e r i v a t i v e aside I n the p r e s e n t work has  from the  two  been r e p o r t e d . ^ - ^  7-methylene p r o t o n o f 7 - a c e t o n y l x a n t h o p t e r i n (LXXVI)  The was  observed to undergo r a p i d d e u t e r i u m exchange i n t r i f l u o r o a c e t i c acid-d  and  d i l u t e sodium d e u t e r o x i d e (NaOD).  139  A l t h o u g h a t t e m p t s to condense v a r i o u s  reagents,  such as b e n z a l d e h y d e , w i t h the methyl group o f 6 - m e t h y l p t e r i d i n e d e r i v a t i v e s have i n the main proved s e v e r a l brominatlons known.^  o f 6 - a l k y l and  unsuccessful,  7 - a l k y l groups a r e  I n g e n e r a l , the y i e l d s o f the mono- and  methylene d e r i v a t i v e s a r e o n l y f a i r .  dibromo-  I n the p r e s e n t  a s i n g l e a t t e m p t to b r o m i n a t e 6 , 7 , 8 - t r i m e t h y l l u m a z i n e  case, in  a c e t i c a c i d produced a s m a l l amount o f a r e d d i s h m a t e r i a l w h i c h d i d not m e l t up to 3 5 0 ° and ( )v  v  272,  320  and  400 mu)  trimethyllumazine i n water.  had  an a b s o r p t i o n  spectrum  somewhat s i m i l a r to t h a t o f Not  the  enough m a t e r i a l was a v a i l a b l e  f o r m i c r o a n a l y s i s o r p.m.r. s p e c t r a l a n a l y s i s . An a t t e m p t to condense benzaldehyde w i t h the' t r i m e t h y l l u m a z i n e i n 75$  a c e t i c a c i d c o n t a i n i n g sodium a c e t a t e  l e d to the r e c o v e r y o f the s t a r t i n g m a t e r i a l s . Because o f the s m a l l amount o f m a t e r i a l a v a i l a b l e , no c o n d e n s a t i o n  or bromination  r e a c t i o n s were attempted w i t h  7,8-dihydro-6,7,8-trimethyllumazine.  Part I I I OXIDATION OP TRIMETHYLLUMAZINE AND DIHYDROTRIMETHYLLUMAZINE  i4o  A.  Introduction The  o x i d a t i o n of 7,8-dihydro-6,7,8-trimethyllumazine  i s i n v e s t i g a t e d u s i n g p o t a s s i u m f e r r i c y a n i d e and  potassium  permanganate f o r the p u r p o s e .  reagents  These p a r t i c u l a r  were chosen i n view o f c e r t a i n p r o p e r t i e s which make them a p p l i c a b l e to the p r e s e n t  study.  Ferricyanide i s reported  to r e a c t w i t h c e r t a i n  o r g a n i c compounds by o n e - e l e c t r o n a b s t r a c t i o n  104 155 156 '  '  157 157  and, The  as a n t i c i p a t e d , e l e c t r o n - r i c h compounds are r e a c t i v e . f a c t t h a t f e r r i c y a n i d e absorbs a t a w a v e l e n g t h removed  from the u l t r a v i o l e t r e g i o n where a b s o r p t i o n by  ferrocyanide  and many o r g a n i c s p e c i e s o c c u r s makes k i n e t i c measurements by spectrophotometric  means a t t r a c t i v e .  The  s p e c t r a o f aqueous  s o l u t i o n s o f f e r r i c y a n i d e do not a l t e r a p p r e c i a b l y o v e r a p e r i o d o f days o r weeks i f p r o t e c t e d from l i g h t and under n i t r o g e n atmosphere.  The  f e r r i c y a n i d e - f e r r o c y a n i d e couple  redox p o t e n t i a l o f  kept the  i s e s s e n t i a l l y independent  158  o f pH above pH 5 which p e r m i t s s t u d i e s u s i n g t h i s system o v e r a f a i r l y wide range o f a c i d i t y by p o t e n t i o m e t r i c methods.  1  A c a u t i o n a r y note must be added however.  A recent review  y  o f the o x i d a t i o n - r e d u c t i o n c h e m i s t r y o f the f e r r i c y a n i d e f e r r o c y a n i d e system p o i n t s out t h a t r e a c t i o n s o f c e r t a i n o r g a n i c compounds w i t h f e r r i c y a n i d e y i e l d r e s u l t s which a r e complex and  t h a t e x p l a n a t i o n i n terms o f a s i m p l e  one-electron  141  t r a n s f e r i s inadequate. study  The r e s u l t s o f Speakman and  o f the o x i d a t i o n o f aldehydes, ketones and  alkanes and K o l t h o f f ' s s t u d y ^ 1  1  Waters  1  nitro-  o f the o x i d a t i o n o f  mercapto-  ethanol a t t e s t to t h i s . Potassium permanganate i s a well-known o x i d a n t i n o r g a n i c c h e m i s t r y and i t s r e a c t i o n s w i t h o r g a n i c s u b s t r a t e s have been reviewed.  The use o f aqueous permanganate to  a f f e c t the removal o f c e r t a i n methyl groups In the.lumazine series-^'^9 ^ B.  g  o  f  p a r t i c u l a r i n t e r e s t i n the p r e s e n t c a s e .  Experimental 1. P u r i f i c a t i o n o f s o l v e n t s and m a t e r i a l s Water that was  used i n the p r e p a r a t i o n o f the  b u f f e r systems and s t o c k sample s o l u t i o n s was  distilled,  b o i l e d , and then cooled by f l u s h i n g w i t h h i g h - p u r i t y n i t r o g e n . Commercial  d i b a s i c potassium phosphate was  by slow r e c r y s t a l l i z a t i o n from aqueous methanol  purified  and d r i e d a t  l60° f o r three days. Commercial  potassium f e r r i c y a n i d e ,  potassium  f e r r o c y a n i d e and potassium c h l o r i d e were p u r i f i e d  by  r e c r y s t a l l i z a t i o n from s a t u r a t e d s o l u t i o n s i n water and i n an evacuated  desiccator over ^2^^  f  o  r  o  n  e  week.  Care  dried was  taken to exclude l i g h t from the c r y s t a l l i z i n g s o l u t i o n s o f the h e x a c y a n o f e r r a t e s by wrapping foil.  the c o n t a i n e r s In aluminum  The p u r i t y o f the' potassium f e r r i c y a n i d e was  checked  by running s e v e r a l u l t r a v i o l e t and v i s i b l e s p e c t r a o f aqueous  l42  s o l u t i o n s o f known c o n c e n t r a t i o n s . The o f potassium  average value found f e r r i c y a n i d e a t 420  T h i s value l i e s  f o r the e x t i n c t i o n c o e f f i c i e n t i s E 420 = 1 , 0 3 9  mu  -  12.  i n the range o f values o f t e n quoted T o r  the  e x t i n c t i o n c o e f f i c i e n t o f f e r r i c y a n i d e Ion a t t h i s wavelength i n aqueous media. A n a l y t i c a l grade potassium n i t r a t e , potassium without  bromate and  permanganate,  potassium  potassium  s u l f a t e were used  further purification. All  under vacuum.  s o l i d s when not i n use were kept The  water was  s t o r e d i n a capped  i n a desiccator polyethylene  b o t t l e under n i t r o g e n atmosphere. 2.  potassium  P r e p a r a t i o n o f b u f f e r and  sample s o l u t i o n s  Stock s o l u t i o n s o f d i b a s i c potassium  phosphate and  c h l o r i d e were prepared  0.500M r e s p e c t i v e l y -  a t 0.250M and  by weighing a c c u r a t e l y the a p p r o p r i a t e amount o f m a t e r i a l i n t o • separate  1 - l i t r e volumetric  with d i s t i l l e d were prepared  water.  f l a s k s and making up to the mark;  0.250M s o l u t i o n s o f the o t h e r  salts  i n a s i m i l a r manner In 25 ml v o l u m e t r i c  Prom the known d i s s o c i a t i o n constants o f  flasks.  phosphoric.  r>l4l a c i d i n water a t 25-0  and  a p p l y i n g the r e l a t i o n s which g i v e  the c o n c e n t r a t i o n s o f the v a r i o u s phosphate s p e c i e s from a known phosphate b u f f e r c o n c e n t r a t i o n a t a g i v e n pH, t  l4l  the  —  i o n i c s t r e n g t h c o n t r i b u t i o n to a s o l u t i o n by the phosphate b u f f e r s p e c i e s a t t h i s pH can be c a l c u l a t e d .  Hence, the i o n i c  strength  143  of  a given  by  the  salt.  buffer  addition of  the  A series of  buffers  typical  graduated  value  amount o f  KC1 or  other  were t h e n made up  i n the  following  to g i v e  ionic strength  volume o f 2 5  ml. 22  approximately free potassium  at  hydroxide or  stream o f  nitrogen  indicated  by  the  until  pH  by  was  R a d i o m e t e r M o d e l 26  pH  meter.  accurately  the  transferred atmosphere buffers  f i n a l pH  to  1.0.  to  adjust  the  carbonate-  under  attained, The  of  a as  contents  q u a n t i t a t i v e l y to a 2 5 mark w i t h removed  s o l u t i o n and container  the  to  ml  beaker  measure  the  under  W i t h t h i s means o f  a c i d i t y or  final  remainder  was  nitrogen  storage  b a s i c i t y maintained  even  t h e i r pH  those over  months.  In strength  of  f o r storage..  of  the  a l i q u o t was  to a p o l y e t h y l e n e  of high  a period  A 4 ml  to  concentration  acid  pH  made up  ml.  t o a volume  desired  f l a s k and  water washings.  added  of  volumes  dropwise a d d i t i o n o f  b e a k e r were t h e n t r a n s f e r r e d  volumetric  The  when d i l u t e d t o a  IN hydrochloric  the  volumes  i n t o a 30  nitrogen.  w a t e r was  followed  the  phosphate b u f f e r  a given  Distilled  syringes,  were measured  stream o f  a desired  ml  and  solutions  beaker under a  were c h o s e n  the  fixed at a desired  appropriate  means o f p i p e t t e s  KC1 stock  KgHPO^ and  of  be  manner: By  and  s o l u t i o n may  0.25  t h i s manner, 0.050M p h o s p h a t e b u f f e r s were made up  Buffer the  solutions  w i t h pH using  ionic strength  Stock solutions  values  ranging  at from  d i f f e r e n t potassium  were a l s o p r e p a r e d  of potassium  ionic  salts 12.0.  a t pH  ferricyanide  12.5  and  l44  p o t a s s i u m permanganate were p r e p a r e d  by w e i g h i n g a c c u r a t e l y  the a p p r o p r i a t e amount o f o x i d a n t i n t o a 10 ml  making up to the mark w i t h d i s t i l l e d w a t e r a t 25.0°.  f l a s k and The  volumetric  c o n c e n t r a t i o n o f the o x i d a n t i n the s t o c k s o l u t i o n was  u s u a l l y i n the neighborhood o f 0.1M. f e r r i c y a n i d e s o l u t i o n was  The  potassium  p r o t e c t e d from l i g h t by wrapping  the v o l u m e t r i c f l a s k i n aluminum f o i l .  The  s o l u t i o n s were  a t 25.0° i n a w a t e r b a t h .  thermostated  O r g a n i c s u b s t r a t e s t o c k s o l u t i o n s were p r e p a r e d  by  the a d d i t i o n o f n i t r o g e n - f l u s h e d d i s t i l l e d w a t e r by means o f a s y r i n g e to a known w e i g h t o f d r y m a t e r i a l under n i t r o g e n c  atmosphere i n a v i a l equipped w i t h a t i g h t - f i t t i n g The  volume o f added w a t e r was  reweighing added was  the v i a l and  rubber  cap.  determined a c c u r a t e l y by  contents.  The  amount o f w a t e r to  be  p r e d e t e r m i n e d so as to g i v e a c o n c e n t r a t i o n o f  sample i n a c e r t a i n r a t i o , u s u a l l y s t o i c h i o m e t r i c , to  the  c o n c e n t r a t i o n o f o x i d a n t when a l i q u o t s o f b o t h sample and o x i d a n t s t o c k s o l u t i o n s were added to the b u f f e r system to initiate  the r e a c t i o n .  prepared  a t 25.0° p r i o r to t h e i r  thermostated The  sample s o l u t i o n s were use.  aqueous s t o c k s o l u t i o n s o f 7 , 8 - d i h y d r o - 6 , 7 , 8 -  trimethyllumazine considered  The  tended to decompose s l o w l y and  unacceptable  were  f o r use i n k i n e t i c measurements  a f t e r about s i x hours from the time the s o l u t i o n was prepared.  Therefore,  originally  a l l k i n e t i c measurements o f a r e l a t e d  n a t u r e were c a r r i e d out on one  sample and  then those runs o f  145'  a d i f f e r e n t series investigated with a freshly-prepared s t o c k sample s o l u t i o n , the l a t t e r sample c o n c e n t r a t i o n  being  a d j u s t e d so as t o v e r y c l o s e l y approximate the s t o c k s o l u t i o n c o n c e n t r a t i o n o f the o r i g i n a l sample.  I n t h i s way, v a r i o u s  s e r i e s o f measurements can be compared w i t h a  reasonable  degree o f c o n f i d e n c e . The  o x i d a n t and t r i m e t h y l l u m a z i n e s t o c k s o l u t i o n s  proved t o be more s t a b l e , e s p e c i a l l y i f p r o t e c t e d from l i g h t and  k e p t under n i t r o g e n atmosphere.  f r e s h s o l u t i o n s were prepared of k i n e t i c runs.  before  I n g e n e r a l , however, the s t a r t o f a new s e r i e s  A g a i n , c a r e was taken to d u p l i c a t e as  c l o s e l y as p o s s i b l e the p r e v i o u s  stock s o l u t i o n concentrations.  3. Techniques employed i n k i n e t i c measurements The  oxidation of dihydrotrimethyllumazine  and the  r e a c t i o n s o f t r i m e t h y l l u m a z i n e were s t u d i e d by two methods. In the pH range 10 to 12.5 s p e c t r o s c o p i c t e c h n i q u e s  were  employed w h i l e p o t e n t i o m e t r i c methods proved a p p l i c a b l e a t a l l pH v a l u e s above 5. i.  Spectroscopy The  u s e f u l n e s s o f t h e s p e c t r o s c o p i c method i n the -  more b a s i c r e g i o n s i n the p r e s e n t  case stems from the f a c t  t h a t a t t h e w a v e l e n g t h o f maximum absorbance f o r the f e r r i c y a n i d e I o n t h e r e i s v e r y l i t t l e absorbance c o n t r i b u t i o n from t h e o r g a n i c s p e c i e s o r from f e r r o c y a n i d e i o n .  Thus, t h e  r e a c t i o n s between f e r r i c y a n i d e and t h e lumazine d e r i v a t i v e s may be f o l l o w e d by s i m p l y m o n i t o r i n g  t h e change i n absorbance  146  T h i s v/as done w i t h a Bausch and Lomb Model 502  a t 420 mu.  recording spectrophotometer. to  Below pH 10, o t h e r c o n t r i b u t i o n s 6,7,8-  t h e t o t a l absorbance a t 420 mu, e s p e c i a l l y from  trimethyllumazine  (>\  m  a  = 4o4 mu), become i n c r e a s i n g l y  x  l a r g e and make t r e a t m e n t complex p r o c e d u r e .  o f the k i n e t i c data a r e l a t i v e l y  At a l l pH v a l u e s  the u l t r a v i o l e t s p e c t r a l  r e g i o n o f t h e r e a c t i o n system e x h i b i t s a l a r g e number o f c l o s e l y spaced and o v e r l a p p i n g a b s o r p t i o n s  thus making t h i s  a r e a u n a t t r a c t i v e from t h e s t a n d p o i n t o f a s i m p l e  kinetic  treatment. A t y p i c a l spectrophotometric  r u n was c a r r i e d o u t  as f o l l o w s : The  r e a c t i o n medium was p r e p a r e d  i n 1 cm.  silica  c e l l s f i t t e d w i t h a s i l i c o n e r u b b e r d i s c i n t h e ground g l a s s neck.  The d i s c s , s i m i l a r to t h e type used as i n j e c t i o n  septa  i n gas chromatography, c o u l d be p i e r c e d r e p e a t e d l y w i t h s m a l l gauge s y r i n g e n e e d l e s w i t h o u t aqueous media. c e l l was f i r s t  Before  l e a k i n g and were i n e r t to  i n t r o d u c t i o n o f a b u f f e r s o l u t i o n , the  f i t t e d w i t h a d i s c and then two s y r i n g e n e e d l e s  were i n s e r t e d through t h i s .  H i g h - p u r i t y n i t r o g e n was passed  t h r o u g h one o f the n e e d l e s to remove a i r from t h e c e l l . means o f a 5 ml s y r i n g e equipped w i t h a Chaney a d a p t e r ml o f 0.050M phosphate b u f f e r a t pH 12.0 and i o n i c 0 . 2 5 was i n t r o d u c e d  i n t o the c e l l  By 3-00  strength  f o l l o w e d by 0.0277 ml o f  0.108M aqueous p o t a s s i u m f e r r i c y a n i d e from a s i m i l a r l y equipped 50 m i c r o l i t r e s y r i n g e .  This p a r t i c u l a r stock s o l u t i o n  c o n c e n t r a t i o n o f f e r r i c y a n i d e produces an absorbance r e a d i n g  147  o f c l o s e to 1.0 a t the s t a r t o f the r e a c t i o n .  The c e l l was  t h e n t r a n s f e r r e d t o t h e c e l l compartment o f t h e s p e c t r o photometer and t h e r m o s t a t e d  a t 2 5 . 0 - 0 . 0 2 ° f o r 15 minutes  under a slow stream o f n i t r o g e n .  A blank s o l u t i o n containing  b u f f e r s o l u t i o n o n l y was used i n t h e r e f e r e n c e beam o f t h e spectrophotometer.  The s y r i n g e n e e d l e s were then removed a n d ,  i n some c a s e s , t h e absorbance r e a d i n g o f t h e b u f f e r e d f e r r i c y a n i d e s o l u t i o n was checked a t 420 mu.  The r e a c t i o n was i n i t i a t e d  by  i n t r o d u c i n g i n t o t h e c e l l 0 . 0 6 1 3 ml o f sample s t o c k s o l u t i o n ( u s u a l l y 2 . 4 2 x 10~ M) o f DHTML o r TML from a 100 m i c r o l i t r e 2  syringe f i t t e d  w i t h a Chaney a d a p t e r .  The c e l l was shaken  b r i e f l y t o mix t h e r e a c t a n t s , c a r e b e i n g taken n o t t o a l l o w the s o l u t i o n t o contact  t h e s i l i c o n e r u b b e r d i s c , and t h i s  was f o l l o w e d by t h e r e c o r d i n g o f absorbance r e a d i n g s a t v a r i o u s time i n t e r v a l s .  a t 420 mu  The r e a c t i o n was f o l l o w e d  until  a s t a b l e r e a d i n g a t 420 mu o v e r a p e r i o d o f t e n minutes was attained.  T h i s r e a d i n g was t a k e n as t h e " i n f i n i t y "  absorbance  o f the r e a c t i o n a t that wavelength. These runs were u s u a l l y done i n t r i p l i c a t e  t o ensure  r e l i a b i l i t y o f the r e s u l t s . Due t o t h e c o m p l e x i t y o f t h e u l t r a v i o l e t s p e c t r a l r e g i o n o f t h e system t h e f a t e o f t h e o r g a n i c m a t e r i a l c o u l d n o t be d e t e r m i n e d by r e p e a t e d did  s p e c t r a l scans.  The r e a c t i o n  p r o v e t o be slow i n a c i d i c b u f f e r s however (see R e s u l t s ) .  A quenching p r o c e d u r e was d e v i s e d by w h i c h t h e p r o g r e s s o f the r e a c t i o n w i t h r e s p e c t t o t h e o r g a n i c s u b s t r a t e c o u l d be  148  determined.  The p r o c e d u r e i s based on the f a c t t h a t z i n c  i n n e u t r a l o r a c i d i c s o l u t i o n react with potassium  ions  ferrocyanide 162  to form t h e v e r y s p a r i n g l y 2K4Fe(CN)  6  +  BZnSO^  s o l u b l e potassium zinc >  KgZn-^FeCCNjgJg  ferrocyanide: +  SKgSO^  A t y p i c a l quenching p r o c e d u r e i s d e s c r i b e d as f o l l o w s : Into o f 1.67  each o f a s e r i e s o f 10 ml e r l e n m e y e r s , 2 . 5 ml  x 10 M z i n c s u l f a t e i n 0.01M s u l f u r i c a c i d were  pipetted.  J  B u f f e r and s t o c k s o l u t i o n s o f f e r r i c y a n i d e and  organic substrate  were i n t r o d u c e d t o a t o t a l volume o f 6 ml  i n a s e p a r a t e 10 ml e r l e n m e y e r equipped w i t h a r u b b e r cap and thermostated a t 2 5 . 0 ° .  The i n i t i a l r e a c t a n t c o n c e n t r a t i o n s  were such as t o c l o s e l y a p p r o x i m a t e those c o n c e n t r a t i o n s used i n the spectrophotometric a n a l y s i s .  A f t e r the i n i t i a t i o n o f  the r e a c t i o n , 0 . 5 ml a l i q u o t s were withdrawn by means o f a 1 ml s y r i n g e  a t v a r i o u s times and i n j e c t e d i n t o t h e z i n c  sulfate solutions.  Each s o l u t i o n was f i l t e r e d i n t u r n  into  a 1 cm. a b s o r p t i o n c e l l and a complete spectrum r e c o r d e d on the Bausch and Lomb Model 502 i n s t r u m e n t . the  filtered  The average pH o f  s o l u t i o n s was found t o be 5 . 3 when pH 1 2 . 0  b u f f e r s o l u t i o n s were used f o r t h e r e a c t i o n medium. ii.  Potentiometry The  oxidation  a p p l i c a t i o n o f p o t e n t i o m e t r y t o the s t u d y o f  reactions  i s described i n d e t a i l elsewhere.  the p r e s e n t s t u d y , a m o d i f i e d form o f t h e N e r n s t e q u a t i o n  In (10)  149  is  used.  V  = ox-red  "  E  2  h  A  A  +  6-1983T.log  ([OX]/[RED] )  (10)  where E  E  h'  ox-red  =  =  T  m  p o t e n t i a l (mV.) o f t h e system u s i n g a platinum-calomel electrode t r a i n ,  e  a  s  u  r  e  d  standard cyanide  p o t e n t i a l o f the f e r r i c y a n i d e - f e r r o couple,  = temperature (°K.)  [_0X~]  = instantaneous  c o n c e n t r a t i o n o f f e r r i c y a n i d e , and  [RED^J  = instantaneous  concentration o f ferrocyanide.  Prom t h e measured E ^ , v a l u e s a t known c o n c e n t r a t i o n r a t i o s o f p o t a s s i u m f e r r i c y a n i d e and p o t a s s i u m f e r r o c y a n i d e i n 0 . 0 5 0 M phosphate b u f f e r a t pH 1 2 . 0 , u = 0 . 2 5 and T = 2 5 . 0 ° C , t h e value o f E ° _ x  r e d  was found t o be 0 . 4 3 3  ±  0.002 v o l t s .  This  agrees f a v o r a b l y w i t h those E ° v a l u e s found i n the 158  literature  164  '  f o r t h e f e r r i c y a n i d e - f e r r o c y a n i d e redox  c o u p l e where s i m i l a r c o n d i t i o n s have been employed. The  k i n e t i c s o f the o x i d a t i o n r e a c t i o n were  d e t e r m i n e d by measuring t h e E i v a l u e o f t h e system, w i t h h  time.  From these measurements t h e change i n t h e c o n c e n t r a t i o n o f f e r r i c y a n i d e w i t h time c a n be c a l c u l a t e d from e q u a t i o n  (10),  assuming one mole o f f e r r i c y a n i d e r e a c t s t o g i v e one mole o f f e r r o c y a n i d e i n the o v e r a l l r e a c t i o n scheme. A t y p i c a l p o t e n t i o m e t r i c r u n was c a r r i e d o u t as follows: The  p o t e n t i a l s were measured w i t h a Radiometer Model  150  26 pH m e t e r u s i n g  the m i l l i v o l t recording  s c a l e and a  calomel  train.  i s equipped w i t h a  electrode  This  instrument  platinum-  s p e c i a l compartment c o n t a i n i n g t h e e l e c t r o d e s and has p r o v i s i o n s for  thermostating,  f o r s t i r r i n g , and f o r m a i n t a i n i n g t h e  r e a c t i o n s o l u t i o n under i n e r t atmosphere. of the instrument,  the platinum  and c a l o m e l  T e f l o n s t i r r e r and n i t r o g e n gas i n l e t a 20 m l p o l y e t h y l e n e and  container.  syringes described  I n t h e compartment electrodes,  tube were f i t t e d  Using  into  t h e same s t o c k s o l u t i o n s  i n t h e spectrpp.h6to.metrie-...method,  s o l u t i o n s o f b u f f e r and p o t a s s i u m f e r r i c y a n i d e were i n j e c t e d into in  the polyethylene  container  t h e l i d o f t h e compartment.  ethylene  through t h e gas o u t l e t The c o n t e n t s  c o n t a i n e r were s t i r r e d  briefly  hole  o f the poly-  and a l l o w e d t o  equilibrate  under a slow s t r e a m o f n i t r o g e n a t 25.0° t 0.02°  for  minutes.  fifteen  The r e a c t i o n was i n i t i a t e d  introduction o f the previously described  amount o f a q u e o u s  DHTML o r TML s o l u t i o n f r o m a 100 m i c r o l i t r e r e a c t i o n s o l u t i o n was s t i r r e d p o t e n t i a l readings For  for five  syringe.  reactions, the glass  recorded. compartment  containing the reactants  i n the polyethylene  wrapped i n aluminum f o i l  to protect the contents  Potentiometric occasionally  triplicate  The  s e c o n d s and t h e n  a t r e g u l a r time i n t e r v a l s  the slower  by t h e  c o n t a i n e r was from  light.  r u n s w e r e done i n d u p l i c a t e a n d t o ensure r e l i a b i l i t y o f the r e s u l t s .  151  4. Treatment o f the k i n e t i c  data  F o r a b i m o l e c u l a r r e a c t i o n o f the aA  +  bB  —  >  type  products  which i s f i r s t - o r d e r w i t h r e s p e c t to the r e a c t a n t s A and the r a t e e x p r e s s i o n i s g i v e n dA/dt where  k  by  -k [.A][B] A  = s p e c i f i c r a t e c o n s t a n t f o r the d i s a p p e a r a n c e the s p e c i e s A,  A  a,b [A]  =  B,  = i n t e g r a l c o e f f i c i e n t s o f the balanced e q u a t i o n , and  of  chemical  c o n c e n t r a t i o n s o f the s p e c i e s A and B r e s p e c t i v e l y  ,[B]  I n the p r e s e n t c a s e , A = f e r r i c y a n i d e i o n and B = o r g a n i c substrate. 165  The  mathematical  r e l a t i o n s d e r i v e d by Benson  d e s c r i b e b i m o l e c u l a r r e a c t i o n s a r e a p p l i c a b l e i n the study.  to  present  F o r the r e a c t i o n between s u b s t r a t e and f e r r i c y a n i d e  i o n i n w h i c h the i n i t i a l r e a c t a n t c o n c e n t r a t i o n s a r e  non-  s t o i c h i o m e t r i c , use i s made o f the i n t e g r a t e d r a t e e x p r e s s i o n (11);  i n (A/B)  =  (bA  Q  - aB )/a.k t 0  A  +  In (A /B )0  (ll)  Q  where the u s u a l b r a c k e t s used to denote c o n c e n t r a t i o n have been o m i t t e d to s i m p l i f y the t r e a t m e n t . A p l o t o f the instantaneous  logarithms  r e a c t a n t c o n c e n t r a t i o n s versus time a f f o r d s a  s t r a i g h t l i n e whose s l o p e has the v a l u e  (bA  Q  - aB )/a'k Q  from w h i c h the v a l u e o f the s e c o n d - o r d e r r a t e c o n s t a n t  A  can  152  be c a l c u l a t e d u s i n g t h e known I n i t i a l c o n c e n t r a t i o n s o f the reactants. F o r those r e a c t i o n s i n which f e r r i c y a n i d e and substrate are present i n s t o i c h i o m e t r i c , o r n e a r l y s t o i c h i o m e t r i c amounts - - t h a t i s , aB -^-'bA -- e q u a t i o n ( l l ) Q  0  165  i s no l o n g e r a p p l i c a b l e .  An a l t e r n a t e e x p r e s s i o n  (12) i s  used t o o b t a i n the r a t e c o n s t a n t : 1/C , - 1/C , ° A  A  where  C , A  =  A  bk t/a«(l - A ) 4A'A> O co  =  a B / b - A , and  =  A  Q  ()  2  12  A  Q  +A/2  A p l o t o f l / C , v e r s u s time y i e l d s a curve' w h i c h v e r y c l o s e l y Pi  approximates  a s t r a i g h t l i n e f o r more than h a l f the r e a c t i o n .  The s l o p e o f t h i s l i n e , w h e n d i v i d e d by b/a, y i e l d s the s p e c i f i c second-order  rate constant k . A  Because A ,  the i n i t i a l  d i f f e r e n c e i n s t o i c h i o m e t r i c c o n c e n t r a t i o n s , was n o r m a l l y a v e r y s m a l l f r a c t i o n o f those c o n c e n t r a t i o n s i n curved  brackets  i n e q u a t i o n ( 1 2 ) , t h i s term c o u l d u s u a l l y be I g n o r e d . For e i t h e r k i n e t i c treatment, a least-squares t r e a t m e n t ^ o f t h e d a t a was used t o o b t a i n the b e s t s t r a i g h t 1 3  line.  153  C.  Results  1.  O x i d a t i o n o f 7,8-dihydro-6,7,8-trimethyllumazine w i t h potassium f e r r i c y a n i d e a.  P r o d u c t s o f the r e a c t i o n and  stoichiometry  By means o f s p e c t r o p h o t o m e t r i c  a n a l y s i s i t was  a s c e r t a i n e d t h a t two moles o f f e r r i c y a n i d e r e a c t  completely  w i t h one mole o f the reduced lumazine to produce two o f f e r r o c y a n i d e and  various organic products.  The  moles  type  o r g a n i c p r o d u c t ( s ) produced depended on the i n i t i a l pH the r e a c t i n g system.  I n the pH r e g i o n 5-0  to 8.0,  of of  the o n l y  organic product  d e t e c t e d when s t o i c h i o m e t r i c amounts o f the  dihydrolumazine  and  f e r r i c y a n i d e were used ( i . e .  D H T M L / f e r r i c y a n i d e = 0.5) balanced  was  6,7,8-trimethyllumazine.  The  r e a c t i o n e q u a t i o n i n t h i s pH r e g i o n i s l i k e l y DHTML + 2 F e ( C N ) ~ 6  3  =  TML + 2Fe(CN)g~  Z|  +  2H+  as shown by s p e c t r a l a n a l y s i s . The  r e a c t i o n becomes r e l a t i v e l y c o m p l i c a t e d  more b a s i c r e g i o n s .  In the pH range 10.5  to 12.5,  o r g a n i c p r o d u c t s o f the r e a c t i o n , when the i n i t i a l  the  i n the final  reactant  c o n c e n t r a t i o n s are s t o i c h i o m e t r i c , a r e 7~oxo-6,8-dimethyll u m a z i n e and what appears to be 5-amino-4-methylamino-2,6dihydroxypyrimidine. procedure,  By the p r e v i o u s l y d e s c r i b e d quenching  6 , 7 , 8 - t r i m e t h y l l u m a z i n e was  found to be  i n t e r m e d i a t e i n the r e a c t i o n i n t h i s pH range.  an  From  measurement o f the absorbance o f the 7-oxo-6,8-dimethyl-  154  lumazine produced,  i t was shown t h a t one mole o f DHTML  y i e l d e d o n l y a p p r o x i m a t e l y one h a l f a mole o f t h e 7-oxo compound w i t h t h e remainder b e i n g t h e p y r i m i d i n e d e r i v a t i v e . That t h e p y r i m i d i n e was n o t p r e s e n t i n s i g n i f i c a n t  quantities  i n t h e aqueous s t o c k s o l u t i o n o f t h e 7,8-dihydro-6,7,8t r i m e t h y l l u m a z i n e used t o i n i t i a t e t h e r e a c t i o n was c o n f i r m e d by r e p e a t e d chromatographic  a n a l y s i s o f the stock s o l u t i o n .  In t h e i n t e r m e d i a t e pH.range 8.0 t o 10.5, a m i x t u r e o f t r i m e t h y l l u m a z i n e , 7-oxo-6,8-dimethyllumazine and s m a l l amounts o f 5-amino-4-methylamino-2,6-dihydroxypyrimidine were found i n t h e f i n a l r e a c t i o n s o l u t i o n s . T h i s and t h e above p r o d u c t a n a l y s e s i n d i c a t e a change i n mechanism i n g o i n g from n e u t r a l t o more b a s i c pH. When n o n - s t o i c h i o m e t r i c amounts o f t h e s t a r t i n g m a t e r i a l s were used w i t h t h e o r g a n i c s u b s t r a t e i n e x c e s s , u n r e a c t e d q u a n t i t i e s o f t h e d i h y d r o lumazine were d e t e c t e d i n t h e f i n a l r e a c t i o n s o l u t i o n s o v e r t h e e n t i r e range o f pH  investigated. F o r t h e r e a c t i o n s c a r r i e d o u t i n b a s i c media, a  s m a l l drop i n t h e pH o f t h e i n i t a l r e a c t i o n s o l u t i o n o f the o r d e r o f 0.01 pH u n i t s o c c u r s d u r i n g t h e c o u r s e o f t h e r e a c t i o n . b.  E f f e c t o f hydroxide i o n O x i d a t i o n r a t e s were determined  by s p e c t r o p h o t o m e t r y  methods i n t h e pH range 10.5 t o 12.5 i n 0.050M phosphate b u f f e r i n o r d e r t o examine t h e e f f e c t o f h y d r o x i d e i o n on t h e  155 reaction.  The i o n i c s t r e n g t h o f t h e b u f f e r s o l u t i o n s was o  m a i n t a i n e d a t 0.25  and t h e temperature a t 25.0  maintaining  the i n i t i a l concentration  and  the concentration  varying  reactant  concentrations  . By  o f f e r r i c y a n i d e constant  o f DHTML, t h e r a t i o o f i n i t i a l  was v a r i e d from 0.25  to 2  (DHTML/ferricyanide r e s p e c t i v e l y ) . I n t h i s pH range, a s e c o n d - o r d e r k i n e t i c  treatment  o f t h e d a t a gave r a t e p l o t s which v e r y c l o s e l y approximated a s t r a i g h t l i n e o v e r 65 t o 75 p e r c e n t o f t h e t o t a l r e a c t i o n . Beyond t h i s p o i n t , t h e r e a c t i o n r a t e tended t o slow down b u t i n a few c a s e s , e s p e c i a l l y i n t h e more b a s i c b u f f e r s , i t began to a c c e l e r a t e . measured a t  The " i n f i n i t y " v a l u e o f a g i v e n  420 mu  u s u a l l y l a y somewhat above  solution  (•~10$)  the  v a l u e c a l c u l a t e d f o r r e m a i n i n g f e r r i c y a n i d e and minor absorbance c o n t r i b u t i o n s o f o r g a n i c length.  a t t h i s wave-  I n t h e case where t h e i n i t i a l r e a c t a n t  r a t i o i s 0.25, for  species  the r e a c t i o n followed  concentration  second-order k i n e t i c s  a p p r o x i m a t e l y 40 p e r c e n t o f t h e t o t a l r e a c t i o n w i t h a  subsequent r a t e a c c e l e r a t i o n .  I n t h i s p a r t i c u l a r case only-  one h a l f t h e t o t a l amount o f p o t a s s i u m f e r r i c y a n i d e  initially  p r e s e n t had been consumed. A t y p i c a l r a t e p l o t f o r t h e o x i d a t i o n o f DHTML by f e r r i c y a n i d e i o n when t h e i n i t i a l r e a c t a n t  concentrations  a r e s t o i c h i o m e t r i c i s i l l u s t r a t e d i n F i g u r e 20. S i m i l a r r a t e p l o t s were o b t a i n e d amounts o f r e a c t a n t s  f o r those r e a c t i o n s i n which were i n i t i a l l y present u s i n g  non-stoichiometric the appropriate  FIGURE  20  OXIDATION KINETICS TYPICAL RATE PLOT, SPECTROPHOTOMETRY METHOD  0  50  100  150  Tl M E ^ S E C O N D S )  200  250  1 5 7  k i n e t i c treatment  o f the d a t a .  Concern t h a t p o s s i b l e s i d e r e a c t i o n s o f the o r g a n i c s u b s t r a t e w i t h t h e b a s i c medium might i n t e r f e r w i t h the k i n e t i c treatment  was r e l i e v e d by the f a c t t h a t the o x i d a t i o n proceeded  at a r e l a t i v e l y , f a s t r a t e . Presented  i n T a b l e I X a r e the observed second-  o r d e r r a t e c o n s t a n t s , k^, p e r t a i n i n g t o the d i s a p p e a r a n c e 9  o f f e r r i c y a n i d e f o r the r e a c t i o n s i n v o l v i n g s t o i c h i o m e t r i c i n i t i a l r e a c t a n t c o n c e n t r a t i o n s a t v a r i o u s pH v a l u e s .  The  d a t a f o r those r e a c t i o n s i n v o l v i n g n o n - s t o i c h i o m e t r i c c o n c e n t r a t i o n s o f r e a c t a n t s a r e c o l l e c t e d i n T a b l e X. Included  i n both Tables are c o r r e l a t i o n c o e f f i c i e n t s  ( r ) to  i n d i c a t e t h e degree o f l i n e a r i t y o f the p l o t t e d d a t a . g e n e r a l , the e r r o r s i n the r e p o r t e d r a t e c o n s t a n t s order o f 0 . 2 identical  to 1 . 5 percent  as e s t i m a t e d  In  a r e o f the  by comparison o f  runs. A p l o t o f the p e r t i n e n t d a t a c o n t a i n e d  i n Tables  IX and X v e r s u s pH r e s u l t s i n the r e l a t i o n s h i p s shown i n Figure 2 1 .  The unexpected a p p a r e n t r a t e - r e t a r d i n g e f f e c t o f  i n c r e a s i n g c o n c e n t r a t i o n o f the o r g a n i c s u b s t r a t e r e l a t i v e to the c o n c e n t r a t i o n o f t h e o x i d a n t i n the more b a s i c pH r e g i o n s is indicated i n this Figure. When the observed r a t e c o n s t a n t s against hydroxide are obtained  are p l o t t e d  i o n concentration, linear relationships  (Figure 2 2 )  I n d i c a t i n g the o x i d a t i o n r e a c t i o n  i s l i n e a r l y dependent on t h e h y d r o x i d e  i o n concentration i n  158  Table IX The e f f e c t o f pH on t h e o x i d a t i o n potassium f e r r i c y a n i d e  r e a c t i o n o f DHTML and  0.050M K HPC>4, u = 0 . 2 5 , T = 2  25.0°C.  I n i t i a l c o n c e n t r a t i o n r a t i o DHTML/ferricyanide = 0 . 5 pH " 1  —A ( i •  1 2 . 5 2  m o l e  ~  l s e c  • ) _ 1  ]  1 0 8 . 8 5  0 . 9 9 9 7  1 2 . 2 5  5 5 . 4 1  0 . 9 9 9 9  1 2 . 0 1  3 5 . 5 2  0 . 9 9 9 8  1 1 . 7 2  2 0 . 9 5  0 . 9 9 9 6  1 1 . 4 9  9 - 3 3  0 . 9 9 9 1  1 1 . 0 6  8 . 8 5  0 . 9 9 8 8  1 0 . 5 2  m - i n i t i a l pH o f r e a c t i o n  solution Table X  I n i t i a l c o n c e n t r a t i o n r a t i o DHTML/ferricyanide = 1 pH  _k ( l , m o l e ~ s e c . ) 1  r  _ 1  A  1 2 . 5 2  4 1 . 8 6  0 . 9 9 8 3  1 2 . 0 1  1 7 . 8 7  O.9996  1 1 . 4 9  8 . 4 5  O.9981  1 1 . 0 6  7 . 8 7  0 . 9 9 9 9  1 0 . 5 2  5 . 9 8  ""  '  0 . 9 9 9 4  159  Table X (continued) I n i t i a l concentration  r a t i o DHTML/ferricyanide k  J2S  2  (l.mole" sec. )  r  12.01  9.16  0.9981  11.47  5.29  0.9994  11.02  3.46  0.9994  I n i t i a l concentration  r a t i o DHTML/ferricyanide = 0 . 2 5  12.01  64.65  the pH range 10 t o 1 2 . 5 .  The extreme r a p i d i t y o f t h e  o x i d a t i o n r e a c t i o n i n more b a s i c s o l u t i o n s p r e v e n t e d i n v e s t i g a t i o n o f the o x i d a t i o n p r o c e s s i n s t r o n g aqueous alkali.  E x t r a p o l a t i o n t o zero h y d r o x i d e i o n c o n c e n t r a t i o n  I n F i g u r e 22 y i e l d s a r e l a t i v e l y s m a l l r a t e  constant  c o n s i s t e n t v/ith the t r e n d toward s l o w e r r a t e s f o r t h e o x i d a t i o n r e a c t i o n s c a r r i e d o u t i n more n e u t r a l b u f f e r solutions. A p l o t o f the logarithms order rate constants  o f the o b s e r v e d second-  versus the negative  hydroxide i o n concentration  logarithm o f the  (Figure 23) a f f o r d s a s t r a i g h t  l i n e o f v e r y n e a r l y u n i t s l o p e I n t h e case o f t h o s e . r e a c t i o n s  160  FIGURE 81 OXIDATION KINETICS  16.1  FIGURE 22  OXIDATION KINETICS RELATIONSHIP BETWEEN OXIDATION RATE AND HYDROXIDE ION CONCENTRATION  162  FIGURE  23  1.00 -L0G [0H"] 1 0  163  where s t o i c h i o m e t r i c i n i t i a l r e a c t a n t c o n c e n t r a t i o n s were employed.  F o r the o t h e r r e a c t i o n s , s i m i l a r p l o t s y i e l d e d  s t r a i g h t l i n e s but w i t h s l o p e s i n c r e a s i n g l y l e s s than u n i t y as the i n i t i a l  c o n c e n t r a t i o n o f the o r g a n i c s u b s t r a t e i n c r e a s e s  r e l a t i v e to t h a t o f the f e r r i c y a n i d e i o n . In  the e x p e r i m e n t s w h i c h f o l l o w , the r e a c t i o n s are  c a r r i e d out i n  0.050M  phosphate b u f f e r a t pH  12.0  using  s t o i c h i o m e t r i c i n i t i a l reactant concentrations unless  otherwise  specified. c_. A c t i v a t i o n Parameters The  energy r e q u i r e m e n t s  o f a g i v e n r e a c t i n g system  can o r d i n a r i l y be determined by measuring the r a t e o f r e a c t i o n at  two o r more d i f f e r e n t t e m p e r a t u r e s .  use i s made o f the log  k/T  where  =  I n the u s u a l manner,  equation  10.319 -  1/T(AH*A.574J  + As*/ *-574 2  (13)  k = measured r a t e c o n s t a n t ,  AH  = enthalpy of a c t i v a t i o n ,  As*  = entropy of a c t i v a t i o n ,  and  T = temperature (°K.). A p l o t o f l o g k/T  a g a i n s t l / T a f f o r d s a s t a i g h t l i n e whose  s l o p e can be used to c a l c u l a t e AH*. of A S equation  A more a c c u r a t e  i s u s u a l l y o b t a i n e d by s u b s t i t u t i o n o f A H  value  back i n t o  (.13) r a t h e r than u s i n g the i n t e r c e p t o f the l i n e w i t h  the o r d i n a t e .  164  The energy o f a c t i v a t i o n , E a , i s g i v e n by AH  + RT, o r o v e r t h e range o f temperatures near room  t e m p e r a t u r e , A H ^ t 0.6 k c a l . m o l e . - 1  In the present case, the v a r i a t i o n o f o x i d a t i o n r a t e w i t h temperature was s t u d i e d u s i n g t h e s p e c t r o p h o t o m e t r i c technique.  The temperature o f t h e c e l l compartment o f t h e  s p e c t r o p h o t o m e t e r was determined a c c u r a t e l y w i t h a Chromel( r e f e r e n c e j u n c t i o n a t 0°) and a  Alumel thermocouple  Honeywell Model 2732 p o t e n t i o m e t e r . The r e s u l t s o f t h e a c t i v a t i o n parameter s t u d y a r e presented i n Table X I . Table XI A c t i v a t i o n p a r a m e t e r s ; o x i d a t i o n o f DHTML w i t h p o t a s s i u m ferricyanide pH = 12.0, 0.050M K HP04, u = 0.25, D H T M L / f e r r i c y a n i d e = 0 . 5 2  Temperature T l°C.r^ (°K.)  k„ (1. mo l e s s e e . " ) 1  25.40  298.56.  36.23.  30.17  303.33  50.51  34,94  308.10  63.50  39.70  312.86  75.40  44.18  317.34  91.34  A p l o t o f 1/T (°K.) v e r s u s - l o g k / T y i e l d s t h e A  l i n e a r r e l a t i o n s h i p i l l u s t r a t e d i n F i g u r e 24.  From the s l o p e  165  FIGURE OXIDATION KINETICS  24  166  o f t h i s l i n e and e q u a t i o n (13) the c a l c u l a t e d magnitudes o f the a c t i v a t i o n parameters a r e as  follows:  =  8.0-±  0.4  A s * =  -24.6  ± 1.3  AH*  Ea  =  8.6  kcal.mole"  1  e.u.  kcal.mole  - 1  I n any m e c h a n i s t i c s t u d y i t i s d e s i r a b l e to compare e x p e r i m e n t a l l y d e t e r m i n e d a c t i v a t i o n parameters w i t h v a l u e s found i n the l i t e r a t u r e f o r s i m i l a r r e a c t i o n s . However, t h e r e i s l i t t l e q u a n t i t a t i v e k i n e t i c d a t a a v a i l a b l e on  systems  s i m i l a r to the p r e s e n t one and t h i s i s e s p e c i a l l y t r u e w i t h regard to a c t i v a t i o n  parameters.  d_. E f f e c t o f added f e r r o c y a n i d e Phosphate 12.0  buffer solutions  (0.050M K^HPO^) a t pH  were p r e p a r e d c o n t a i n i n g amounts o f added p o t a s s i u m  f e r r o c y a n i d e r a n g i n g i n c o n c e n t r a t i o n from 0 to 1.0 The  x 10""%.  l a t t e r c o n c e n t r a t i o n i s a 1 0 0 - f o l d excess o v e r the  i n i t i a l c o n c e n t r a t i o n o f f e r r i c y a n i d e used i n the r e a c t i o n . The i o n i c s t r e n g t h o f the system was m a i n t a i n e d a t 0 . 3 0 the a d d i t i o n o f p o t a s s i u m c h l o r i d e .  I n s p i t e o f the added  f e r r o c y a n i d e , the r e a c t i o n c o n t i n u e d to e x h i b i t k i n e t i c s a l t h o u g h t h e r e was  by  second-order  a tendency f o r the r a t e to  a c c e l e r a t e a f t e r 55 to 70 p e r c e n t c o m p l e t i o n o f the r e a c t i o n . A c o n t r o l experiment showed t h a t p o t a s s i u m f e r r o c y a n i d e and 7 , 8 , - d i h y d r o - 6 , 7 , 8 - t r i m e t h y l l u m a z i n e  do not  167  r e a c t under t h e c o n d i t i o n s o f t h i s e x p e r i m e n t . 6,7,8-trimethyllumazine  Similarly,  and p o t a s s i u m f e r r o c y a n i d e do n o t  appear t o r e a c t . The  r e s u l t s o f t h e experiments c a r r i e d o u t i n t h e  "doped" b u f f e r systems a r e c o l l e c t e d i n T a b l e X I I . reported  second-order r a t e constants  50 p e r c e n t  The  r e f e r to the f i r s t  o f the r e a c t i o n . Table X I I  E f f e c t o f added p o t a s s i u m f e r r o c y a n i d e on t h e o x i d a t i o n r e a c t i o n o f DHTML w i t h f e r r i c y a n i d e pH = 1 2 . 0 , 0.050M KgHPO^, u = 0 . 3 0 ,  I n i t i a l added K ^ F e ( C N ) g  k  n  (moles. 1  A  ( l .mole sec . -1  0.0  - 1  )  42.06  9.7 x 1 0 " -4  .  5  37.78 21.23  9 . 7 x 10 9.7 x 1 0 "  T = 25.0°  .  3  n - corrected for d i l u t i o n  3.31  by added r e a c t a n t s o l u t i o n s .  I t i s e v i d e n t from t h e r e s u l t s i n T a b l e X I I t h a t f e r r o c y a n i d e I o n has an i n h i b i t o r y e f f e c t on t h e r a t e o f t h e oxidation reaction. when n e a r i n g  Therefore,  the completion  the r a t e a c c e l e r a t i o n observed  o f t h e r e a c t i o n s does n o t appear  to be r e l a t e d d i r e c t l y t o t h e p r e s e n c e o f t h e hexacyanoferrate(ll') species.  168  A p l o t o f the r e c i p r o c a l s o f t h e s e c o n d - o r d e r  rate  c o n s t a n t s a g a i n s t f e r r o c y a n i d e c o n c e n t r a t i o n y i e l d s the l i n e a r r e l a t i o n s h i p shown i n F i g u r e 2 5 .  This p a r t i c u l a r  r e l a t i o n s h i p r e f l e c t s the i n v e r s e p r o p o r t i o n a l i t y t h a t e x i s t s between t h e o x i d a t i o n r a t e and t h e c o n c e n t r a t i o n o f the hexacyanoferrate(II) species. £• E f f e c t o f s p e c i f i c  anions  A s e r i e s o f b u f f e r s o l u t i o n s a t pH 12.0 were p r e p a r e d i n w h i c h t h e i o n i c s t r e n g t h was a d j u s t e d t o 0 . 2 5 by the a d d i t i o n o f d i f f e r e n t potassium s a l t s .  I n t h i s manner, t h e  a n i o n o f t h e s a l t i s m a i n t a i n e d a t a c o n s t a n t v a l u e o f 0.050M w h i l e the t o t a l p o t a s s i u m i o n c o n c e n t r a t i o n i s O.167M u n l e s s otherwise noted.  The r e s u l t s o f these e x p e r i m e n t s a r e  p r e s e n t e d i n the f o l l o w i n g T a b l e . Table  XIII  E f f e c t o f a n i o n s on t h e D H T M L - f e r r i c y a n i d e o x i d a t i o n pH = 12.0, 0.050M KgHPOip u = 0 . 2 5 ,  reaction  T = 25.0°  C o n c e n t r a t i o n ' o f a n i o n = 0.050M, t o t a l p o t a s s i u m i o n c o n c e n t r a t i o n = O.167M u n l e s s o t h e r w i s e n o t e d . Potassium s a l t  k^ ( l . m o l e s e c . ""*") _1  35.56  KC1  KN0  34.72  3  KBr0  28.81  3  K S04°  19.66  2  o - t o t a l potassium i o n =  0.151M  FIGURE 25  170  The r e s u l t s i n d i c a t e t h a t the l a r g e r and more h i g h l y charged a n i o n s have an i n h i b i t o r y e f f e c t on the oxidation reaction rate.  The d e c r e a s e observed i n t h e case  o f p o t a s s i u m s u l f a t e may be due, i n p a r t , t o the t o t a l d e c r e a s e d amount o f p o t a s s i u m i o n o f the b u f f e r s o l u t i o n r e l a t i v e t o those b u f f e r s c o n t a i n i n g f.  the o t h e r s a l t s .  E f f e c t o f potassium i o n Many p o l y v a l e n t  cations associate with ferricyanide  i n aqueous s o l u t i o n but o f the u n i v a l e n t c a t i o n s  166  potassium i o n do s o .  This  and tetraalkyammonium i o n s  l 6 ?  only  a r e known t o  i s r a t h e r s u r p r i s i n g i n view o f the l a r g e  a v a i l a b l e coulombic a t t r a c t i o n between the t e r v a l e n t f e r r i c y a n i d e a n i o n and a c a t i o n .  In c o n t r a s t , there i s  e v i d e n c e f o r a s s o c i a t i o n o f a l a r g e number o f c a t i o n s  ..  ferrocyanide.  with  159,168169  In a study  o f the k i n e t i c s o f the e l e c t r o n  exchange between h e x a c y a n o f e r r a t e ( I I )  and - ( I I I ) i o n s , the  r a t e was found t o depend markedly upon the n a t u r e and concentration  o f the cations present.  between p o t a s s i u m f e r r o c y a n i d e  I n the r e a c t i o n  and p o t a s s i u m p e r s u l f a t e the  r e s u l t s s i m i l a r l y c o r r e l a t e the r a t e c o n s t a n t s w i t h p o t a s s i u m  170,171 Ion c o n c e n t r a t i o n . L i t t l e dependence was found on the Ionic strength at constant c a t i o n concentration. In p a r t i c u l a r , the r a t e i n c r e a s e w i t h i n c r e a s i n g p o t a s s i u m i o n c o n c e n t r a t i o n  170 was c o n c l u d e d  t o be a r e s u l t o f r e a c t i o n between the  171  a s s o c i a t e d i o n s , KPe(CN)g~- and 5  r a t h e r than between  KS2O3",  the c o r r e s p o n d i n g u n a s s o c i a t e d s p e c i e s . At the r i s k o f the r e s u l t s b e i n g l e s s  than  q u a n t i t a t i v e because o f the apparent r a t e - d e c r e a s i n g e f f e c t o f the s u l f a t e a n i o n , the dependence o f the o x i d a t i o n on i n c r e a s i n g p o t a s s i u m i o n was  s t u d i e d a t pH 12.0.  reaction A series  o f 0.050M b u f f e r s were p r e p a r e d i n w h i c h v a r y i n g amounts o f KC1 and KgSO^ were added to i n c r e a s e the t o t a l p o t a s s i u m i o n c o n c e n t r a t i o n w h i l e m a i n t a i n i n g the i o n i c s t r e n g t h c o n s t a n t . The r e s u l t s o f o x i d a t i o n experiments c a r r i e d out i n these b u f f e r systems a r e p r e s e n t e d I n T a b l e Table  XIV.  XIV  E f f e c t o f p o t a s s i u m i o n on the D H T M L - f e r r i c y a n i d e reaction pH  12.0,  0.050M KgHPO^, T =  25.0°,  u =  T o t a l potassium i o n c o n c e n t r a t i o n (moles.I"- -) 1  oxidation  0.40 k^ ( l .mole-^sec . ) - 1  0.250  39.24  0.267  42.16  0.290  47.72  0.317  54.90  p - r a t e s have been determined from s i n g l e runs i n t h i s experiment. A p l o t o f the d a t a i n t h i s T a b l e r e s u l t s i n the r e l a t i o n s h i p shown i n F i g u r e 26.  E x t r a p o l a t i o n o f the l i n e  172  to a c o n c e n t r a t i o n o f potassium i o n equal to 0.151M y i e l d s a r a t e approximately o f the o r d e r found f o r the experiment i n which potassium s u l f a t e alone was used to a d j u s t the i o n i c s t r e n g t h (see Table X I I I ) .  T h i s i n d i c a t e s t h a t the lower r a t e  observed i n t h i s l a t t e r experiment was probably a r e s u l t o f the lower potassium i o n c o n c e n t r a t i o n . A p l o t o f the l o g a r i t h m s o f the r a t e constants and potassium i o n c o n c e n t r a t i o n (Figure27) y i e l d s a f a i r l y s t r a i g h t l i n e o f slope I . 3 6 . g_. K i n e t i c i s o t o p e e f f e c t The presence o f 6 , 7 , 8 - t r i m e t h y l l u m a z i n e as an i n t e r m e d i a t e i n the o x i d a t i o n r e a c t i o n o f DHTML i n b a s i c media r e q u i r e s the rupture o f the C-7 carbon-hydrogen the l a t t e r compound a t some stage In the r e a c t i o n . o x i d a t i o n o f 7,8-dihydro-6,7>8-trlmethyllumazine-7-d  bond i n  The was  examined i n o r d e r to e s t a b l i s h whether the bond-rupture occurs i n the r a t e - c o n t r o l l i n g s t e p .  Measurements were made under  c o n d i t i o n s s i m i l a r to those p r e v i o u s l y employed i n the case o f the p r o t l o compound. The r e s u l t s o f three runs at. pH 12.0 u s i n g the deuterated dihydrolumazine are presented i n Table XV. F o r comparison,  the r a t e constants f o r the o x i d a t i o n r e a c t i o n o f  DHTML under i d e n t i c a l c o n d i t i o n s are I n c l u d e d .  173  FIGURE 2 6  OXIDATION KINETICS EFFECT OF POTASSIUM ION ON THE OXIDATION RATE  GO-  DHTML/FERRICYANIDE = 0 . 5 u  =  0.40,  0 . 0 5 0 M  5  5  T  =  (l.mole~ sec. 1  A 0  pH  =  12.0  -  K  5  2 5 . 0 ° ,  K2HPO4  1  )  -  2  SLOPE = 2 1 6 l.mole  45-  4  3  0  -2  -1  sec.  -  5  -  0.24  0  .  2  6  0  K^"  .  2  8  0  (mole.l" ) 1  .  3  0  0  .  3  2  174  FIGURE 27 OXIDATION KINETICS RELATIONSHIP BETWEEN POTASSIUM ION CONCENTRATION AND OXIDATION RATE (same e x p e r i m e n t a l c o n d i t i o n s as i n FIGURE 26)  .72-  168 SLOPE  LOG, K J 0  =1.36  A  1.64-  .60-  060  0.56 -L0G  10  Q52  0.48  175  Table  XV  Kinetic isotope e f f e c t ; oxidation of 7,8-dlhydro-6,7,8trimethyllumazine w i t h potassium f e r r i c y a n i d e pH = 12.0, 0.050M K HP04, u = 0 . 2 5 , 2  DHTML/ferricyanide k  =0.5  (H)  A  T = 25.0°  k  (l.mole" sec." )  A  (D)  k /k H  (l.mole" sec.  D  )  35.82  30.61  1.17  35.26  28.94  1.22  36.13  30.14  1.20 Mean k / k H  D  =  1.2  The kjj/kj-j v a l u e i s much s m a l l e r than those t h a t a r e o r d i n a r i l y considered primary isotope e f f e c t s .  I n the case o f c a r b o n -  bound hydrogen, a k^/k^ v a l u e o f more than 2 . 0 ^ 1  temperature) suggests  2  ( a t room  t h a t the hydrogen ( o r d e u t e r i u m ) atom  i s b e i n g t r a n s f e r r e d i n the r a t e - c o n t r o l l i n g s t e p o f the reaction. In  the p r e s e n t c a s e , the k^/k^. v a l u e l i e s  the range o f e x p e r i m e n t a l e r r o r n o r m a l l y encountered spectrophotometric allowances  a n a l y s i s technique.  outside w i t h the  T h i s i s so even when  a r e made f o r the f a c t t h a t a 4 p e r c e n t i m p u r i t y o f  the p r o t i o compound  i s p r e s e n t i n the samples o f the d e u t e r a t e d  dlhydrolumazine. 2.  Potentiometry The o x i d a t i o n r e a c t i o n o f DHTML w i t h f e r r i c y a n i d e  was s t u d i e d i n the pH range 5 t o 12 u s i n g the p r e v i o u s l y d e s c r i b e d method o f p o t e n t i o m e t r i c a n a l y s i s .  The i n t e n d e d  176  purpose o f t h i s method v/as t o o b t a i n r a t e d a t a i n the pH range where t h e s p e c t r o s c o p i c t e c h n i q u e  was n o t a p p l i c a b l e . '  However, t h e two methods gave s t r i k i n g l y d i f f e r e n t r e s u l t s . T h i s i s c l e a r l y i l l u s t r a t e d i n the r a t e p l o t s shown i n F i g u r e 28.  Following a r e l a t i v e l y f a s t i n i t i a l r e a c t i o n , the  e x t e n t o f w h i c h depends upon t h e pH, a s l o w e r n e a r l y secondo r d e r d i s a p p e a r a n c e o f f e r r i c y a n i d e was observed p o t e n t i o m e t r i c a l l y . Some a c c e l e r a t i o n o f t h e r a t e was.noted near t h e end o f the reactions. I t i s i m m e d i a t e l y e v i d e n t t h a t the expected d i r e c t comparison o f t h e d a t a o b t a i n e d potentiometric  techniques  by s p e c t r o s c o p i c and  i s n o t r e a l i z e d . I t i s noteworthy t h a t  a t pH 5, where the o r g a n i c s u b s t r a t e i s n e u t r a l r a t h e r than the mono-anion as i s t h e case i n more b a s i c r e g i o n s , a marked i n c r e a s e i n the e x t e n t o f the i n i t i a l r a t e i s o b s e r v e d . The s e c o n d - o r d e r r a t e c o n s t a n t s potentiometric  technique,  r e p r e s e n t a t i v e pH.  determined by the  k^,, a r e p r e s e n t e d  These c o n s t a n t s  i n T a b l e XVI a t  have been c a l c u l a t e d from  the s l o p e s o f the s t r a i g h t l i n e p o r t i o n o f t h e r a t e p l o t s . T a b l e XVI Oxidation r e a c t i o n ; rate constants  determined by p o t e n t i o m e t r y  0.050M K2BTO4, u = 0 . 2 5 , T = 25.0°, D H T M L / f e r r i c y a n i d e = 0 . 5 pH  k p  12.0  21.55  11.0  2.82  9.0  A  2.20  (l.mole sec. ) - 1  - 1  FIGURE 28  TIME (SECONDS)  178  T a b l e XVI ( c o n t i n u e d ) pH  k p (l.mole A  7.0  1.20  5.0  0.15  sec." )  By c o m p a r i s o n o f t h e above d a t a w i t h d a t a o b t a i n e d spectroscopic technique 3.  i t i s evident that k - ~ 2 k p . A  Reaction o f 6,7,8-trimethyllumazine a. S p e c t r o p h o t o m e t r y The  was  by t h e A  with ferricyanide-  results  r e a c t i o n between TML and p o t a s s i u m f e r r i c y a n i d e  monitored a t 420 mu under s i m i l a r c o n d i t i o n s t o those  used i n t h e o x i d a t i o n o f DHTML.  A t pH 7 . 0 , v e r y slow changes  i n t h e absorbance a t 420 mu (and 404 mu) i n d i c a t e d t h a t a r e l a t i v e l y slow r e a c t i o n was o c c u r r i n g w i t h these in neutral solution. and  reactants  I n s t r o n g l y b a s i c media, however, TML.  f e r r i c y a n i d e r e a c t e d r a p i d l y t o y i e l d u l t i m a t e l y 7-oxo-  6 , 8 - d i m e t h y l l u m a z i n e and f e r r o c y a n i d e i o n . By s p e c t r o s c o p i c a n a l y s i s i t was a s c e r t a i n e d t h a t two moles o f t h e hexacyanof e r r a t e ( l l l ) i o n r e a c t s w i t h one mole o f TML t o produce one mole o f t h e 7-oxo compound and two moles o f f e r r o c y a n i d e . T h i s i s somewhat s u r p r i s i n g i n view o f t h e f a c t t h a t o f t h e 7 - o x o l u m a z i n e by o x i d a t i v e d e m e t h y l a t i o n  formation  o f t h e C-7  m e t h y l group o f TML would be e x p e c t e d t o r e q u i r e more than two equivalents o f f e r r i c y a n i d e . When e q u i m o l a r amounts o f f e r r i c y a n i d e and TML a r e allowed  t o r e a c t a t pH 1 2 . 0 , an o r a n g e - c o l o u r e d  ( ^ max ^ 2 reaction.  m x  >  £~10^)  intermediate  appears d u r i n g t h e c o u r s e o f t h e  T h i s m a t e r i a l was u n s t a b l e as i n d i c a t e d by a slow  179  change o f t h e spectrum t o t h a t c h a r a c t e r i s t i c o f 7-oxo-6,8dimethyllumazine,  even a f t e r a l l o f t h e f e r r i c y a n i d e was  presumably consumed. an i n i t i a l  T h i s i n t e r m e d i a t e was n o t d e t e c t e d when  excess o f the o x i d a n t was used. The  disappearance  o f t h e i n t e r m e d i a t e i s a c c e l e r a t e d i n t h e p r e s e n c e o f oxygen. When t h e r e a c t i o n was c a r r i e d o u t i n an e . s . r . tube under t h e same c o n d i t i o n s as d e s c r i b e d above, no s i g n a l c h a r a c t e r i s t i c o f a f r e e r a d i c a l c o u l d be d e t e c t e d . b_.  Potentiometry The r e a c t i o n o f TML and f e r r i c y a n i d e was s t u d i e d  under t h e same c o n d i t i o n s employed I n the o x i d a t i o n o f DHTML. The r a t e p l o t s o f t h e T M L - f e r r i c y a n i d e r e a c t i o n e x h i b i t e d the same I n i t i a l c u r v a t u r e c h a r a c t e r i s t i c o f t h e o x i d a t i o n r e a c t i o n s o f DHTML monitored  by p o t e n t i o m e t r y .  The r e s u l t s o f t h i s  e x p e r i m e n t a r e c o l l e c t e d i n Table X V I I .  The r e p o r t e d r a t e  c o n s t a n t s r e f e r t o t h e s t r a i g h t l i n e p o r t i o n o f the r a t e d a t a when p l o t t e d i n a second-order Table  manner. XVII  T M L - f e r r i c y a n i d e r e a c t i o n ; p o t e n t i o m e t r i c method E f f e c t o f pH on t h e r e a c t i o n r a t e , T M L / f e r r i c y a n i d e = 0.5 0.050M KpHPOip u - 0.25, T = 25.0° pH  k p A  (l.mole sec.~ )  12.04  16.76  9.02  1.82  7.00  o.n  _ 1  1  q  q - i n i t i a l r a t e ; remainder o f r e a c t i o n e x t r e m e l y slow one e q u i v a l e n t o f f e r r i c y a n i d e consumed.  after  180  When e q u i m o l a r amounts o f f e r r i c y a n i d e and TML were a l l o w e d t o r e a c t a t pH 1 2 . 0 , t h e p o t e n t i a l o f t h e r e a c t i o n s o l u t i o n dropped  very r a p i d l y .  The appearance  o f t h e orange-  c o l o u r e d i n t e r m e d i a t e was n o t e d . 4.  Miscellaneous reactions a. D i s p r o p o r t i o n a t i o n  experiment  E q u l m o l a r m i x t u r e s o f 6 , 7 , 8 - t r i m e t h y l l u m a z i n e and i t s d i h y d r o d e r i v a t i v e were examined i n phosphate systems  buffer  under n i t r o g e n atmosphere o v e r t h e pH range 1 t o 1 2 .  No a p p a r e n t r e a c t i o n between t h e above l u m a z i n e s was observed as shown by s p e c t r a l scans o f t h e s o l u t i o n s .  I n base, s m a l l  changes i n t h e composite spectrum o f t h e m i x t u r e c o u l d be a t t r i b u t e d t o t h e slow d e c o m p o s i t i o n o f t h e d i h y d r o l u m a z i n e . Thus, a d i s p r o p o r t i o n a t i o n TML analogous  reaction +  DHTML  2HTML*  to the d i s p r o p o r t i o n a t i o n r e a c t i o n f o r the l e u c o 117  f l a v i n - f l a v i n couple b.  e v i d e n t l y does n o t o c c u r h e r e .  Permanganate o x i d a t i o n The o x i d a t i o n o f 7 , 8 - d i h y d r o - 6 , 7 , 8 - t r i m e t h y l l u m a z i n e  w i t h p o t a s s i u m permanganate produced 7 ~ o x o - 6 , 8 - d i m e t h y l l u m a z i n e • but t h e r e a c t i o n proved t o be too r a p i d t o measure w i t h t h e a v a i l a b l e techniques.  6 , 7 , 8 - T r i m e t h y l l u m a z i n e was found t o  be an i n t e r m e d i a t e i n t h i s  reaction.  181  D.  Discussion The  ferrocyanide  oxidation-reduction system has  chemistry  o f the f e r r i c y a n i d e -  r e c e n t l y been r e v i e w e d . 1 5 9  The  r e a c t i o n s o f f e r r i c y a n i d e as an o x i d i z i n g agent f a l l i n t o o r the o t h e r o f two  classes.  The  f i r s t comprises the  one  classic  r e a c t i o n s which i n v o l v e no more than a s i m p l e e l e c t r o n t r a n s f e r and  are o r d i n a r i l y v e r y r a p i d .  The  second group  t h o s e r e a c t i o n s w h i c h are r e l a t i v e l y slow and by c o m p l i c a t e d  kinetics.  are  includes characterized  An e x a m p l e ^ o f the l a t t e r group 1  o f r e a c t i o n s i s the o x i d a t i o n o f a l d e h y d e s , ketones n i t r o a l k a n e s w i t h f e r r i c y a n i d e i n a l k a l i n e media.  and These  compounds appear to be o x i d i z e d t h r o u g h the a n i o n o f the o r a c i - f o r m and stages.  the r e a c t i o n proceeds i n s e v e r a l  Free organic  enol-  consecutive  r a d i c a l s c o u l d not be d e t e c t e d  by  the  160  vinyl polymerization  t e c h n i q u e and  i t was  suggested  that  the f e r r i c y a n i d e i o n forms a complex w i t h the e n o l i c a n i o n o f the s u b s t r a t e , w h i c h i s then a t t a c k e d  by a second f e r r i c y a n i d e  i o n o r a n o t h e r complex. I t i s evident  from the r e s u l t s o f the p r e s e n t s t u d y  t h a t the o x i d a t i o n o f  1,8~dihydro-6,"J,8-trimethyllumazine  w i t h f e r r i c y a n i d e b e l o n g s to the second group o f r e a c t i o n s this  of  oxidant. I n the pH range examined ( w i t h the e x c e p t i o n  e x p e r i m e n t c a r r i e d out a t pH 5)  the  of  the  dihydrotrimethyllumazine  e x i s t s p r e d o m i n a n t l y as the mono-anion.  However, the p r o d u c t  182  a n a l y s i s shows t h a t t h e p a r t i c u l a r p r o d u c t ( s )  formed i n t h e  o x i d a t i o n r e a c t i o n depends on t h e i o n i z a t i o n o f t h e t r i m e t h y l lumazine i n t e r m e d i a t e  r a t h e r than on t h e i o n i z a t i o n o f t h e That i s , above pH 10 where t h e  dihydrolumazine precursor. trimethyllumazine  e x i s t s as t h e h y d r o x y l a t e d  s t r u c t u r e L I I I ; R = CH3),  a n i o n (see  t h e r e a c t i o n proceeds t o g i v e  u l t i m a t e l y 7-oxo-6,8-dimethyllumazine.  I n t h e pH r e g i o n where  the former compound i s n e u t r a l , t h e o x i d a t i o n o f DHTML y i e l d s only the trimethyllumazine The  itself.  small enthalpy  and energy o f a c t i v a t i o n found  i s i n d i c a t i v e o f a r e a c t i o n pathway p o s s e s s i n g energy r e g u i r e m e n t s .  The l a r g e and n e g a t i v e  r e l a t i v e l y low  e n t r o p y term i s  c o n s i s t e n t w i t h t h e coulombic i n t e r a c t i o n s expected o f two ( o r p o s s i b l y more) s i m i l a r l y charged s p e c i e s corning  together  173 In the t r a n s i t i o n s t a t e o f the r e a c t i o n .  Laidler  t h a t f o r i o n i c r e a c t i o n s such as t h e p r e s e n t  deduces  one t h e  e l e c t r o s t a t i c c o n t r i b u t i o n to the entropy o f a c t i v a t i o n , A can be c r u d e l y e s t i m a t e d A  S  from t h e f o l l o w i n g e q u a t i o n ( l 4 ) :  L .  -  -  1 0 Z  A B  where z^, z ^ = e l e c t r o n i c charges p r e s e n t s p e c i e s A and B r e s p e c t i v e l y . A S~j*  = -30 e.u.  ^  Z  on t h e r e a c t i n g  This gives a value o f  f o r t h e case where t h e r e a c t i n g  species  are t h e t e r v a l e n t f e r r i c y a n i d e i o n and the mono-anion o f DHTML However, t h e dependence o f t h e r a t e on p o t a s s i u m i o n -2 c o n c e n t r a t i o n s u g g e s t s t h a t t h e a s s o c i a t e d s p e c i e s , KFe(CN)g ~ may  be t h e r e a c t i v e o x i d a n t  under t h e c o n d i t i o n s employed.  183  T h i s would l e a d t o a v a l u e o f A s „ „ o f -20 e.u., e. s .  not f a r  ±  determined v a l u e o f A S .  from t h e e x p e r i m e n t a l l y The  s m a l l isotope e f f e c t observed f o r the o x i d a t i o n  r e a c t i o n s u g g e s t s t h a t t h e C-7 carbon-hydrogen bond i s n o t b e i n g broken i n t h e r a t e - c o n t r o l l i n g s t e p o f t h e r e a c t i o n i n b a s i c media. The  k i n e t i c s o f the o x i d a t i o n o f  trimethyllumazine  7,8-dihydro-6,7,8-  by f e r r i c y a n i d e a r e s e c o n d - o r d e r ,  order i n oxidant  and f i r s t - o r d e r I n o r g a n i c  first-  substrate.  The  compexity o f the o x i d a t i o n r e a c t i o n prevents f o r m u l a t i o n o f a complete and q u a n t i t a t i v e mechanism a t p r e s e n t .  However,  the f o l l o w i n g p a r t i a l r e a c t i o n sequence c a n be i n f e r r e d from the r e s u l t s : (1) DHTML  v  +  OH"  7.  — DHTML" + J  HpO  •\  -4  (2)  Fe(CN) "  (3) (4|  Fe(CN)g ° + OH" +; X  J  6  very r a p i d  +  DHTML  X  +  Fe(CN)g  -3  X  .  * TML — > X'  +  reversible -4  Fe(CN)g  A l t h o u g h n o t i n d i c a t e d i n t h e above sequence o f r e a c t i o n s , i t i s expected t h a t some o f t h e f e r r i c y a n i d e w i l l r e a c t as t h e -2 associated  species The  KFe(CN)g  n a t u r e o f X and X' have n o t been e s t a b l i s h e d  with c e r t a i n t y .  I t i s noted t h a t e l e c t r o n a b s t r a c t i o n from  the a n i o n o f DHTML w i l l g e n e r a t e t h e mesomeric r a d i c a l  (LXXVII).  184  etc  <—>  (LXXVII)  (e.g. pH 5)  In s o l u t i o n s where the d i h y d r o l u m a z i n e i s n e u t r a l  an e l e c t r o n a b s t r a c t i o n from the N^-H bond w i l l y i e l d a r a d i c a l c a t i o n w h i c h , upon l o s s o f a p r o t o n , g i v e s radical  (LXXVII).  Further  can l e a d t o t h e p r o d u c t i o n  t h e same  e l e c t r o n a b s t r a c t i o n from (LXXVII) o f 6,7,8-trircethyIlumazine,  as  T h i s c o r r e s p o n d s t o s t e p (3) o f t h e r e a c t i o n  I n d i c a t e d below.  sequence and i s l i k e l y t h e r a t e - c o n t r o l l i n g s t e p f o r t h e o x i d a t i o n r e a c t i o n i n t h e n e u t r a l pH r e g i o n s .  This implies  the e q u i l i b r i u m (2) i s s h i f t e d t o t h e r i g h t - h a n d t h a t t h e r e a c t i o n be f i r s t - o r d e r w i t h r e s p e c t  side i n order  to f e r r i c y a n i d e  ion.  CH  CH-  CH-z  I  °  H ^ ( ^ K > k / Q iH  H C  k  3  8  8  o  that  0  185  A concurrent reaction o f X with hydroxide i o n ( s t e p 4) may be o c c u r r i n g w h i c h a f f e c t s t h e r a t e o f f e r r i c y a n i d e consumption i n t h e more b a s i c s o l u t i o n s by s h i f t i n g the e q u i l i b r i u m i n (2) t o t h e r i g h t .  T h i s does n o t deny t h e  p o s s i b i l i t y o f d i r e c t hydroxide Ion p a r t i c i p a t i o n i n the e q u i l i b r i u m step  itself.  The p o s s i b i l i t y o f d i m e r i z a t i o n o f (LXXVIIJ t o P r o t o n removal from the 7- o r  produce (LXXVTII) e x i s t s . 7'-position  o f t h i s species  o f DHTML a n i o n and  by base c a n l e a d t o the f o r m a t i o n  trimethyllumazine.  CH  3  H C 3  (LXXVIIIj  Dimerization  o f r a d i c a l intermediates i n the  r e a c t i o n s o f c e r t a i n o r g a n i c compounds v / i t h f e r r i c y a n i d e has  159,1-60,175 p r e v i o u s l y been f o r m u l a t e d . Potentiometric Indicates  a n a l y s i s o f the o x i d a t i o n  reaction  t h a t f e r r i c y a n i d e i s consumed r a p i d l y I n t h e i n i t i a l  s t a g e s o f t h e r e a c t i o n o v e r t h e range o f pH i n v e s t i g a t e d . The r a p i d consumption o f f e r r i c y a n i d e a t pH 5, where t h e  186  dihydrotrirnethyllumazine species,  i s l i k e l y due  e x i s t s p r e d o m i n a n t l y as the  neutral  to the decreased coulombic b a r r i e r to  r e a c t i o n i n step (2).  The  r a p i d uptake o f o x i d a n t i s  i n d i c a t e d by s p e c t r o p h o t o m e t r y a n a l y s i s , however.  not  It is  i n f e r r e d t h a t the p r o d u c t i o n o f X causes a c o n t r i b u t i o n the absorbance a t 420  mu  uptake o f f e r r i c y a n i d e .  to  i n a manner which masks the  Initial  I f X p o s s e s s e s the r a d i c a l  structure  (LXXVII) then the masking e f f e c t Is made more p l a u s i b l e . I t i s not c l e a r how substrate  an excess o f the  organic  w i l l cause the observed r e t a r d i n g e f f e c t on  reaction rate.  A s i m i l a r e f f e c t , also unexplained,  encountered b y ' S t e i n and  Tendeloo ^^ i n a study of  The  and  was the  1  r e a c t i o n between p h l o r o g l u c i n o l  ferricyanide.  o x i d a t i o n o f 6 , 7 , 8 - t r i m e t h y l l u m a z i n e v/ith  f e r r i c y a n i d e produces 7 - o x o - 6 , 8 - d i m e t h y l l u m a z i n e In media v i a a h i g h l y - c o l o u r e d ,  A p p a r e n t l y , o n l y two  agent are r e q u i r e d  basic  unstable, intermediate.  l a t t e r s p e c i e s i s l i k e l y a r a d i c a l but confirmed.  the  t h i s could  equivalents  This not  o f the  to complete the r e a c t i o n .  oxidizing  This i s less  than what would be expected f o r the removal o f the C-7 group o f TML  by o x i d a t i o n  acid followed information  to the c o r r e s p o n d i n g  by d e c a r b o x y l a t i o n .  to e l u c i d a t e  be  methyl  carboxylic  Although s u f f i c i e n t  the r e a c t i o n mechanism f u l l y i s  l a c k i n g , the f o l l o w i n g i n i t i a l sequence i s i n f e r r e d :  still  187  CH (5)  TML  +  OH"  CH  3  (6)  H  j^TMLOH"  q H f < k > K  P  H C  +  Pe(CN)  3 fast  -3  X"  6  +  Fe(CN)  -4  3  He  H  11  3  O  X" may be the mesomerlc r a d i c a l (LXXIX) formed by e l e c t r o n a b s t r a c t i o n from t h e h y d r o x y l a t e d lumazine.  anion o f trimethyl-  T h i s would a c c o u n t f o r t h e orange c o l o u r o f t h e  intermediate  observed i n the r e a c t i o n .  •O^HNx^^o  a H , N k / ^ 0  .  Further  (XXXIX)  1  >  etc  o  r e a c t i o n s o f (LXXIX) a r e p o s s i b l e .  For  example; I.  O n e - e l e c t r o n a b s t r a c t i o n from (LXXIX) by a second mole  o f f e r r i c y a n i d e can l e a d t o t h e c a t i o n (LXXX).  Ring-closure,  rearrangement o f t h e C-7 m e t h y l group and subsequent a t t a c k by hydroxide i o n leads  to 7-oxo-6,8-dimethyllumazine w i t h the  188  e l i m i n a t i o n o f methanol.  A s i m i l a r rearrangement o f a c a t i o n  o c c u r s i n the chromic a c i d o x i d a t i o n o f c e r t a i n  tertiary  179  alcohols.  (LXXX) The c a t i o n (LXXX) i s p r o b a b l y n e v e r f u l l y formed, the m e t h y l group rearrangement o c c u r r i n g i n the t r a n s i t i o n s t a t e  leading  to t h i s i o n . ii.  I n t r a m o l e c u l a r hydrogen a b s t r a c t i o n from  l e a d s to ( L X X X I ) .  (LXXIX)  R e a c t i o n o f t h i s l a t t e r r a d i c a l w i t h any  oxygen p r e s e n t i n s o l u t i o n g i v e s the c o r r e s p o n d i n g p e r o x i d e r a d i c a l w h i c h , upon r e a c t i o n w i t h w a t e r , y i e l d s the p e r o x i d e i t s e l f and hydrogen p e r o x i d e .  E l i m i n a t i o n o f formaldehyde and  w a t e r from the o r g a n i c p e r o x i d e produces lumazine.  CH  O (LXXXI)  7-oxo-6,8-dimethyl-  189  The f a s t e r r a t e o f d i s a p p e a r a n c e o f the c o l o u r e d r e a c t i o n i n t e r m e d i a t e i n the p r e s e n c e o f oxygen I s c o n s i s t e n t w i t h the p r e c e d i n g r e a c t i o n sequence. Attempts to d e t e c t formaldehyde and methanol i n the f i n a l s o l u t i o n s o f the T M L - f e r r i c y a n i d e r e a c t i o n by gas chromatography,  as w e l l as a t t e m p t s to d e t e c t C 0  onto A s c a r i t e , have not been s u c c e s s f u l .  2  by a d s o r p t i o n  T h i s i s not a l t o g e t h e r  s u r p r i s i n g i n view o f the s m a l l q u a n t i t i e s o f r e a c t a n t s used. That TML and f e r r i c y a n i d e r e a c t o n l y s l o w l y under c o n d i t i o n s where the o r g a n i c compound i s n e u t r a l i n d i c a t e s t h a t i n c r e a s e d , a c t i v a t i o n o f the C-7 m e t h y l group as w e l l as f o r m a t i o n o f the h y d r o x y l a t e d a n i o n a r e n e c e s s a r y r e q u i r e m e n t s f o r the r e a c t i o n . I t i s evident that there i s l i t t l e  similarity  between the k i n e t i c s o f the o x i d a t i o n o f the p r e s e n t model 177 system and those o f the l e u c o f l a v i n - f l a v i n system. The l a t t e r system i s c h a r a c t e r i z e d by the a b i l i t y to undergo a 117 d i s p r o p o r t i o n a t i o n r e a c t i o n to form a r a d i c a l as w e l l as to undergo o x i d a t i o n and r e d u c t i o n r e a c t i o n s which p r o c e e d 105,109  t h r o u g h a r e l a t i v e l y s t a b l e semiquinone  intermediate.  E x t r a s t a b i l i z a t i o n o f the r a d i c a l i n t e r m e d i a t e i s p r o v i d e d 104  by the benzene r i n g i n the f l a v i n system.  The l a c k o f  t h i s e x t r a s t a b i l i z a t i o n may p l a y a v i t a l r o l e w i t h r e g a r d to the i n s t a b i l i t y o f r a d i c a l i n t e r m e d i a t e s i n the d i h y d r o l u m a z i n e - l u m a z i n e system.  199  • SUGGESTIONS FOR FURTHER WORK  oxidation of 7,8-dihydro-6,7,8-trimethyllumazine  The  w i t h f e r r i c y a n i d e i n v o l v e s a complex s e r i e s o f r e a c t i o n s and more work i n s p e c i f i c a r e a s i s r e q u i r e d t o e l u c i d a t e t h e r e a c t i o n mechanism f u l l y . o f added f e r r o c y a n i d e  For i n s t a n c e , a study o f the e f f e c t s  i o n on t h e r e a c t i o n r a t e and measurement  o f t h e i s o t o p e e f f e c t i n t h e n e u t r a l pH r e g i o n s  i s necessary.  I t would be j u d i c i o u s t o s t u d y t h e o x i d a t i o n r e a c t i o n w i t h another one-electron  r e a g e n t i n o r d e r t h a t a comparison o f  r e s u l t s u s i n g t h e two o x i d a n t s  c a n be made.  w h i c h i s known t o r e a c t by o n e - e l e c t r o n  Manganic  transfer,  acetate, seems a  s u i t a b l e reagent f o r t h i s purpose. In t h e case o f t h e t r i m e t h y l l u m a z i n e - f e r r i c y a n i d e r e a c t i o n i n b a s i c media, a s t u d y o f t h e e f f e c t o f added f e r r o c y a n i d e on t h e r e a c t i o n r a t e i s r e q u i r e d i n o r d e r t o e s t a b l i s h whether t h e i n i t i a l s t e p reversible. coloured  Confirmation  intermediate  polymerize  (6) o f t h e r e a c t i o n i s  o f t h e r a d i c a l n a t u r e o f t h e orange-  might be a c h i e v e d  by a t t e m p t i n g t o  a c r y l o n i t r i l e ^ i n i t s presence. 1  I n c o n j u n c t i o n w i t h t h e exchange s t u d y o f DHTML, a d d i t i o n o f hydrogen p e r o x i d e  to a n e u t r a l s o l u t i o n o f the  compound i n D 0 was employed t o produce 2  A r e l a t i v e l y stable intermediate and  6,7,8~trimethyllumazine.  o f t h i s r e a c t i o n was d e t e c t e d  p r e l i m i n a r y s t u d i e s i n d i c a t e t h a t i t may be the h y d r o -  peroxide  (see s t r u c t u r e L X I ) o r t h e c o r r e s p o n d i n g  hydroxylated  191  compound o f DHTML.  An i n v e s t i g a t i o n o f t h e o x i d a t i o n o f  7,8-dihydro-6,7,8-trimethyllumazine under a wide v a r i e t y o f c o n d i t i o n s  w i t h hydrogen p e r o x i d e seems p o s s i b l e u s i n g  p.m.r. t e c h n i q u e s and s p e c t r o p h o t o m e t r y  both  analysis.  I t would be o f i n t e r e s t t o determine t h e e f f e c t i v e n e s s o f a c i d s and bases on t h e exchange r e a c t i o n s o f both TML and DHTML i n more q u a n t i t a t i v e terms. o f mono-protic  F o r example, use o f a s e r i e s  a c i d s , such as t h e c h l o r o a c e t i c a c i d s ,  than t h e phosphate  species  rather  might a l l o w more a c c u r a t e  c a l c u l a t i o n o f t h e cC parameter 142  i n terms o f t h e B r / n s t e d  relations. An I n t e r e s t i n g companion s t u d y t o t h e t r l m e t h y l lumazine-ferricyanide  r e a c t i o n would be the c o r r e s p o n d i n g  r e a c t i o n between 6 , 7 - d i p h e n y l ~ 8 - m e t h y l l u m a z i n e 34 I t has been shown  J  and f e r r i c y a n i d e .  t h a t o x i d a t i v e removal o f t h e 7-phenyl  group does n o t o c c u r when  6,7-diphenyl-8-methyl-2-oxopteridine  and p o t a s s i u m permanganate a r e p r e s e n t t o g e t h e r i n a c i d and alkaline solution.  Thus, i t may be p o s s i b l e to observe a  eomplexing r e a c t i o n which might o c c u r between t h e 6 , 7 - d i p h e n y l 8-rnethyllumazine and f e r r i c y a n i d e i o n and/or whether e l e c t r o n a b s t r a c t i o n o c c u r s w i t h t h e a n i o n o f t h i s p a r t i c u l a r lumazine derivative. potentiometric  O b s e r v a t i o n s c o u l d be made u s i n g and s p e c t r o s c o p i c  techniques.  An a m b i t i o u s program t o s y n t h e s i z e diphenyl-8-methyllumazine  both  7,8-dihydro-6,7-  d e r i v a t i v e s with various  substituents  i n t h e p h e n y l r i n g s would a l l o w a s t u d y o f the o x i d a t i o n  192  r e a c t i o n i n terms o f s u b s t i t u e n t e f f e c t s .  I t i s expected  t h a t s u b s t i t u e n t s on the 6 - p o s i t i o n r i n g should considerable  have a  e f f e c t on the energy o f the a n i o n o f the  lumazine.  T h i s e f f e c t would be r e f l e c t e d i n the energy r e q u i r e d a b s t r a c t an e l e c t r o n from t h i s a n i o n and requirements of a r a d i c a l intermediate The  to  a l s o i n the energy  analogous to  (LXXVTI).  r e l a t i v e s t a b i l i t y o f d i p h e n y l m e t h y l l u m a z i n e i n aqueous  s o l u t i o n commends i t s use  i n such a s t u d y .  I t would be i n t e r e s t i n g to s t u d y the r e a c t i o n o f DHTML w i t h oxygen, and  f o r that matter other oxidants,  c o n d i t i o n s where the competing h y d r o l y s i s r e a c t i o n o f d i h y d r o l u m a z i n e does not i n t e r f e r w i t h the r e s u l t s . problem may  be c i r c u m v e n t e d by s t u d y i n g the o x i d a t i o n  i n anhydrous medium. dimethylsulfoxide a study.  under the  The  latter  reactions  Preliminary tests indicate that  provides  a s u i t a b l e s o l v e n t system f o r such  193  BIBLIOGRAPHY  1.  A.M. P a t t e r s o n , L.T. C a p e l l and D.F. Walker, "The R i n g Index", Second e d i t i o n , American C h e m i c a l S o c i e t y , i 9 6 0 .  2.  A. A l b e r t , Quart. Revs. Chem. Soc. 6 , 197 ( 1 9 5 2 ) .  3.  M. G a t e s , Chem. Revs. 4 l , 63 ( 1 9 4 7 ) .  4.  "The C h e m i s t r y and B i o l o g y o f P t e r i d i n e s " , C i b a F o u n d a t i o n Symposium, J.A. 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