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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 . S c , U n i v e r s i t y of 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 the Department of Chemistry-• We accept t h i s t h e s i s as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA May, 1969 @ John M. McAndless 1969 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e a n d S t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d b y t h e Head o f my D e p a r t m e n t o r b y h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n -D e p a r t m e n t o f Chemistry The U n i v e r s i t y o f B r i t i s h C o l u m b i a V a n c o u v e r 8, C a n a d a D a t e A p r i l 15, 196Q i ABSTRACT Supervisor: P r o f e s s o r R. Stewart The p o t e n t i a l use of 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 dihydro d e r i v a t i v e as a redox couple f o r the p t e r i d i n e and f l a v i n systems i s i n v e s t i g a t e d . The syntheses of the above compounds and r e l a t e d lumazine d e r i v a t i v e s are described and t h e i r spectroscopic and chromatographic p r o p e r t i e s are ta b u l a t e d . While examining the p.m.r. spectr a o f 6,7,8-trimethyllumazine (TML) and 7 , 8 - d i h y d r o - 6 , 7 , 8 - t r i m e t h y l -lumazine (DHTML) i n deuterium oxide media, the hydrogens of the C-7 methyl group and of the C -6 methyl group i n the former and l a t t e r compounds r e s p e c t i v e l y were observed to undergo exchange. In the pH range -0 .4 to 8.0, the exchange of TML Is subject to general acid-base c a t a l y s i s . The pseudo f i r s t -order exchange ra t e constant (k-^) was found to be the sum of s e v e r a l c a t a l y t i c terms. Prom measurements of the exchange r a t e - b u f f e r dependency at various pH values, the f o l l o w i n g values of the s p e c i f i c c a t a l y t i c constants ( 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 . 1 4 , ^ ^ - 2 = 5 2 . 9 The mechanism o f the a c i d - and base-eatalyzed 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 i i i s explained i n terms o f the formation of n e u t r a l and i o n i c intermediates which have long conjugated bonding arrangements. Pseudo f i r s t - o r d e r r a t e constants have been tabulated f o r the general a c i d - c a t a l y z e d exchange o f DHTML i n the pH range - 0 . 4 to 8 . 2 l e a d i n g to an unusual pH-rate p r o f i l e . The marked decrease i n exchange ra t e s and anomalous ra t e p l o t s i n the more a c i d i c pH regions i s a t t r i b u t e d to a c i d - c a t a l y z e d covalent h y d r a t i o n across the C (6)-N (5) double bond and e q u i l i b r a t i o n o f hydrated and unhydrated c a t i o n s . Evidence f o r h y d r a t i o n i s presented and the exchange mechanism i s disc u s s e d . 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 the pH range 5 to 1 2 . 5 by spectrophotometry and pot e n t i o m e t r i c techniques. The second-order r e a c t i o n proceeds i n a complicated sequence of steps, some of which are dependent upon the i o n i z a t i o n o f the r e a c t i o n intermediate, t r i m e t h y l -lumazine. Beyond pH 1 1 , the 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 c o n c e n t r a t i o n . At pH 1 2 , the absence of a primary isotope e f f e c t and the i n h i b i t o r y e f f e c t of added ferrocyanide i n d i c a t e that the 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 one-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 i c form of DHTML. The r e s u l t i n g mesoroeric r a d i c a l can 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 i o n . A very negative A value was obtained f o r the r e a c t i o n at 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 ions o f i i i the same charge. A study of the e f f e c t s o f potassium 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 r e a c t i o n . A l i m i t e d study of 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. The hydroxylated anion of TML r e a c t s v i a a h i g h l y coloured intermediate to produce 7-oxo - 6 , 8-dimethyllumazine. Further i n v e s t i g a t i o n i n t o the r e a c t i o n i s r e q u i r e d . i v TABLE OF CONTENTS Page INTRODUCTION . . . . . . . . 1 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 . 1 B. P h y s i c a l and Chemical P r o p e r t i e s of 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 .. .. ... 3 , 2 . Ring cleavage r e a c t i o n s . 4 3 . Covalent h y d r a t i o n 6 4 . Reduction and o x i d a t i o n .. ... 11 C. Methods o f P r e p a r a t i o n .. .. ... 19 1. Isay r e a c t i o n 19 2 . C y c l i z a t i o n o f pyrimidines 21 3 . Synthesis from pyrazine intermediates 22 D. B i o l o g i c a l Importance o f P t e r i d i n e s 24 E. Redox Reactions o f the F l a v i n s • ... 30 OBJECTS OF THE PRESENT RESEARCH .. 35 EXPERIMENTAL . . 37 A. C h a r a c t e r i z a t i o n o f the Prepared 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 the Lumazines ... 39 P a r t I : STABILITY OF THE LUMAZINE DERIVATIVES IN SOLUTION A. I n t r o d u c t i o n .. . 5 9 B. Experimental 59 C. Results 6 l D. D i s c u s s i o n .. 72 1. 5-Amino - 4-methylamino - 2 ,6-dihydroxypyrimidine. 72 2 . H y d r o l y s i s o f the lumazines 74 P a r t I I : HYDROGEN-DEUTERIUM EXCHANGE A. I n t r o d u c t i o n ... 85 B. Experimental .. 86 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 . 86 2 . Treatment o f the data In exchange experiments. 88 C. Res u l t s 90 1 . Trimethyllumazine r . a. Assignment o f the exchanging group .. . 9 0 b. pH-rate p r o f i l e 92 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 98 V Page 2 . 7 , 8 - D i h y d r o - 6 , 7 , 8 - t r i m e t h y l l u r n a z i n e a. Assignment o f the exchanging group .. ... 107 b. pH-rate p r o f i l e 109 c. E f f e c t o f b u f f e r on the exchange ra t e .. . 114 d. C a t a l y s i s by metal c a t i o n s 116 D. D i s c u s s i o n 121 1. Mechanism o f the exchange process a. Trimethyllumazine ... 121 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 .. ... 125 2 . Hydration 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 . B i o l o g i c a l i m p l i c a t i o n s .. ... 138 P a r t I I I : OXIDATION OF TRIMETHYLLUMAZINE AND DIHYDRO-TRIMETHYLLUMAZINE A. I n t r o d u c t i o n . l 4 0 B. Experimental .. l 4 l 1 . P u r i f i c a t i o n of s o l v e n t s and m a t e r i a l s l 4 l 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 .. . 142 3 . Techniques employed i n k i n e t i c measurements .. . l 4 5 i . Spectroscopy l 4 5 i i . Potentiometry l48 4 . Treatment of the k i n e t i c data 151 C. Re s u l t s . . .. 153 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. Products o f the r e a c t i o n and s t o i c h i o m e t r y . 153 b. E f f e c t o f hydroxide i o n 154 c. A c t i v a t i o n parameters l 6 3 d. E f f e c t o f added ferro c y a n i d e 166 e. E f f e c t of s p e c i f i c anions l 6 8 f. E f f e c t o f potassium i o n 170 g. K i n e t i c isotope e f f e c t .. 172 2 . Potentiometry ... 175 3 . Reaction o f 6 , 7 , 8 - t r i m e t h y l l u m a z i n e w i t h f e r r i c y a n i d e 178 a. Spectrophotometry r e s u l t s .. .. 178 b. Potentiometry 179 4 . Miscellaneous r e a c t i o n s . 180 a. D i s p r o p o r t i o n a t i o n experiment .. .. .. . l 8 0 b. Permanganate o x i d a t i o n . l 8 0 . v i Page D. D i s c u s s i o n .. l 8 l SUGGESTIONS FOR FURTHER WORK .. 190 BIBLIOGRAPHY . . . . .. ' 193 v i i LIST OF TABLES Page Ia Spectroscopic data f o r prepared compounds . . ... 55 l b I o n i z a t i o n constants o f lumazine d e r i v a t i v e s 56 I I P.m.r. s p e c t r a l data f o r lumazine d e r i v a t i v e s 57 I I I Chromatography o f prepared compounds . 58 IV FL. values and fluorescence c h a r a c t e r i s t i c s or various compounds 70 V Rates of exchange o f 6 , 7 , 8 - t r i m e t h y l -lumazine i n D p 0 i n the pH range - 0 . 4 to 8 . 0 95 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 100 V I I Rates o f exchange o f 7 , 8 - d i h y d r o - 6 , 7 , 8 -triroethy1lumazine i n D 2 0 i n the pH range - 0 . 4 to 8 . 2 . . 113 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 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 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 , 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/ferricyanlde = 0 . 5 158 X E f f e c t o f pH on 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 , various i n i t i a l r e a c t a n t concentrations .. .. ... 158-9 XI A c t i v a t i o n parameters, DHTML-ferricyanide r e a c t i o n at pH 1 2 . 0 164 X I I E f f e c t o f added fer r o c y a n i d e on the o x i d a t i o n r e a c t i o n 167 X I I I E f f e c t o f anions on the DHTML-f e r r i c y a n i d e o x i d a t i o n r e a c t i o n ... 168 XIV E f f e c t o f potassium i o n on the DHTML-f e r r i c y a n i d e o x i d a t i o n r e a c t i o n . ... 171 v i i i XV K i n e t i c i sotope e f f e c t . 175 XVI O x i d a t i o n r e a c t i o n ; r a t e constants determined by potentiometry 176 -8 XVII TML-ferricyanide r e a c t i o n ; r e a c t i o n r a t e s determined by potentiometry 179 i x LIST OF FIGURES Page 1 5-Amino - 4-methylamino - 2 , 6-dihydroxypyrimidine i n 0.5N sodium hydroxide .. .. . 6 3 2 7 , 8-Dihydro - 6 , 7 , 8-trimethyllumazine i n 0.5N sodium hydroxide, anaerobic c o n d i t i o n s ... 64 3 7 , 8-Dihydro - 6 , 7 , 8-trimethyllumazine i n 0.050M KgHPO^ at pH f» anaerobic c o n d i t i o n s .. ... 65 4 6 , 7-Diphenyl - 8-methyllumazine i n 0.050M KpHPOh at pH 12 66 5 6 , 7 , 8-Trimethylluroazine i n 0.5N sodium hydroxide 67 6 7-Oxo -6 ,8-dimethyllumazine i n 0.050M K2l^0u a t pH 12 . . . . . 68 7 7 , 8-Dihydro - 6 , 7 , 8-trimethyllumazine i n 0.050M K^HPO^ at pH 9, anaerobic c o n d i t i o n s 69 8 T y p i c a l exchange p a t t e r n o f 6 , 7 * 8-trimethyl-lumazine . . . . .. ... 91 9 TML exchange k i n e t i c s , t y p i c a l r a t e p l o t ... 94 10 TML exchange k i n e t i c s , r e l a t i o n between r a t e constant and pH 96 11 TML exchange k i n e t i c s , pH-rate p r o f i l e .. .. . 9 7 12 TML exchange k i n e t i c s , r e l a t i o n between r a t e constant 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 . constant and b u f f e r c o n c e n t r a t i o n at 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 constant and b u f f e r c o n c e n t r a t i o n at pH 2.0 . 103 15 T y p i c a l exchange p a t t e r n o f 7>8-dihydro-6 , 7 ^ 8-trimethyllumazine " . . . 108 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 112 18 DHTML exchange k i n e t i c s , pH-rate p r o f i l e .. . 115 X 19 DHTML exchange k i n e t i c s , e f f e c t of 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 .. .. . I l 8 20 O x i d a t i o n k i n e t i c s , t y p i c a l r a t e p l o t , s pectrophotometry method .. .. 156 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 va 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 r a t e .. .. .. . 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 hydroxide i o n c o n c e n t r a t i o n l 6 l 23 O x i d a t i o n k i n e t i c s , f i r s t - o r d e r r e l a t i o n s h i p between hydroxide 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 r a t e " 162 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 fe 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 O x i d a t i o n k i n e t i c s , e f f e c t of potassium Ion on the o x i d a t i o n r a t e , 173 27 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 potassium Ion c o n c e n t r a t i o n and o x i d a t i o n r a t e 174 28 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 x i . 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 Pr o f e s s o r Ross Stewart f o r h i s many h e l p f u l suggestions and guidance 1 d u r i n g the course o f t h i s work. I would a l s o l i k e to thank the N a t i o n a l Research 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 Is extended to Mr. Roland W. Burton 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 running o f nuclear 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 of t h e i r b i o l o g i c a l importance and extensive d i s t r i b u t i o n i n nature, have a t t r a c t e d more a t t e n t i o n than any other f a m i l y o f t e t r a -azanaphthalenes. However, the amounts of 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 d e r i v a t i v e s commonly i s o l a b l e are, i n ge n e r a l , exceedingly s m a l l . That f a c t , coupled w i t h the r a t h e r inconvenient p r o p e r t i e s o f the p t e r i d i n e s - - f o r example; d i f f i c u l t y of 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 the study o f t h e i r chemistry an exp e r i m e n t a l l y d i f f i c u l t f i e l d . Only more r e c e n t l y w i t h the advent o f modern a n a l y t i c a l techniques has exact 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 nucleus ( i ) , c o n s i s t i n g o f fused pyrazine and pyri m i d i n e r i n g s , i s numbered according to the Ring Index 1; t h i s numbering system i s widely adopted i n recent p u b l i c a t i o n s . According to t h i s assignment the most recent systematic 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 or 1,3,5,3 - tetra-azanaphthalene. A numbering system, shown In ( l l ) r e l a t e d to that o f the purines has been used e x t e n s i v e l y i n p u b l i c a t i o n s from European l a b o r a t o r i e s . Because o f the frequent reference made to them throughout t h i s work, the 2 numbering systems used f o r pyr i m i d i n e s ( i l l ) and the i s o a l l o x a z i n e s (IV) are a l s o i n c l u d e d . 5 ( 1 ) 4 -G 5 2 3 ( i n ) 8 9 P I 2 5 10 . 4 n (iv) 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 nucleus has been the subject o f s e v e r a l c a l c u l a t i o n s ' ^ 3 1 2 The d i s t r i b u t i o n o f " i f-electron charges, although d i f f e r i n g ' s l i g h t l y i n value depending, on the method o f c a l c u l a t i o n , c a n be considered r e p r e s e n t a t i v e as shown i n ( V ) 2 . As i n d i c a t e d , the carbon atom at p o s i t i o n 7 i n the nucleus enjoys l e s s Tf-e l e c t r o n charge than does the one at the 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 o f p t e r i d i n e d e r i v a t i v e s . 1.142 1.204 0.877r" , " V > 6 . 8 8 l 0.9 0 ? 70 1.111 0.822 (V) 3 A l b e r t has pointed out t h a t , because o f the l o c a l i z a t i o n o f the ten TT - e l e c t r o n s a v a i l a b l e f o r aromatic s t a b i l i z a t i o n on the e l e c t r o n - a t t r a c t i n g hetero 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 p t e r i d i n e s 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 displacement r e a c t i o n s r a t h e r 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 . The chemistry and b i o l o g y of p t e r i d i n e s have been 2-8 treated i n c o n s i d e r a b l e d e t a i l i n s e v e r a l reviews. A b r i e f summary of some of t h e i r more important p r o p e r t i e s , methods o f s y n t h e s i s and b i o l o g i c a l s i g n i f i c a n c e p e r t i n e n t to the present I n v e s t i g a t i o n w i l l be presented. B. P h y s i c a l and Chemical 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 The extreme i n s o l u b i l i t y and high and i n d e f i n i t e m e l t i n g p o i n t s of the m a j o r i t y of p t e r i d i n e d e r i v a t i v e s render the usual 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 alue. The p u r i t y o f p t e r i d i n e s 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. S u i t a b l e s o l v e n t s 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 3$ aqueous ammonium c h l o r i d e and butan-l-ol-5M a c e t i c a c i d (7 :3) . The m a j o r i t y o f p t e r i d i n e s , and the intermediates used i n t h e i r p r e p a r a t i o n can be detected by i r r a d i a t i o n w i t h u l t r a -v i o l e t l i g h t at 254 mu, s i n c e they e i t h e r absorb (appearing as b l a c k spots on the white paper) or f l u o r e s c e i n c h a r a c t e r i s t i c c o l ours at that wave-length. 4 U l t r a v i o l e t spectroscopy a l s o 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 constant 2 4 l 4 15 16 spectrum. Extensive t a b u l a t i o n s ' ' J 'of the s p e c t r a , as w e l l as the i o n i z a t i o n constants i n the form of pKa value§, of 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 st 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 s p e c t r a o f p o t e n t i a l l y tautomeric hydroxy- and aminopteridine 2 13 d e r i v a t i v e s , i t has been concluded ' that 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 - h e t e r o c y c l i c amines e x i s t i n the true amino form. 2. Ring cleavage r e a c t i o n s This subject has been reviewed e x t e n s i v e l y by 17 T a y l o r who pointed out that probably every known p t e r i d i n e may be cleaved 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 which i s opened but the ease w i t h which t h i s occurs depends upon the s u b s t i t u e n t s present. This tendency of the p t e r i d i n e nucleus to l o s e the p y r i m i d i n e ( r a t h e r than the pyrazine) r i n g on a c i d and a l k a l i n e h y d r o l y s i s i s consonant wi t h the e l e c t r o n 2 d e n s i t y diagrams which show the p y r i m i d i n e moiety to be more p o l a r and hence more r e a d i l y attacked. P t e r i d i n e i t s e l f i s cleaved i n one hour by b o i l i n g normal potassium hydroxide, 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, alkylamino or mercapto groups i n p o s i t i o n 2 are destroyed i n one minute when b o i l e d i n normal sodium l 8 a hydroxide. On the other hand, l e u c o p t e r i n ( 2 - a m i n o - 4 , 6 , 7 -5 t r i h y d r o x y p t e r l d l n e ) r e s i s t s a l k a l i and o n l y under such vigorous a c i d treatment as to cause d i s r u p t i o n o f the pyrazine r i n g does t h i s compound succumb. The vigorous 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 lumazine 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-aminopyrazine and 19 s u b s t i t u t e d p y razine amino a c i d s . The l a t t e r can be decarboxylated by heating 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 aminopyrazines, important as intermediates i n the s y n t h e s i s o f s u l f a drugs. When the pyrazine r i n g of a p t e r i d i n e c a r r i e s a hydroxyl group, b a s i c h y d r o l y s i s r e s u l t s In cleavage o f t h i s r i n g to subsequently 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-hydroxy- as w e l l as 6-hydroxypteridine give 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 conjugated lactams 8-methyl - 7-pteridinone and 6 , 8-dimethyl-7 - p t e r i d i n o n e (VI; R = H and R = CH^ r e s p e c t i v e l y ) are hydrolyzed i n normal sodium hydroxide at 1 0 0 ° f o r two hours 21 to 5-amino - 4-methylaminopyrimidine ( V I I ) , as determined by 20 paper chromatography. i ^ OH" " V ^ l j H 2 , (VII) D i s r u p t i o n o f the pyrazine r i n g i s a l s o the case 6 f o r 5 j 6 - d i h y d r o p t e r i d i n e s which undergo f a c i l e h y d r o l y s i s to p y r i m i d i n e d e r i v a t i v e s . For i n s t a n c e , 5 , 6-dihydro - 7-hydroxy-p t e r i d i n e i s i n s t a n t l y converted i n t o 4-amino -5-carboxymethyl-aminopyrimidine i n b o i l i n g normal sodium h y d r o x i d e . 1 ^ 3 However, 5 > 6 , 7 j 8-tetrahydropterIdInes, which are merely s u b s t i t u t e d 4 , 5-diaminopyrimidines, are s t a b l e to both a c i d and base h y d r o l y s i s . P t e r i d i n e s which are cleaved by a l k a l i are a l s o 21 22 s u s c e p t i b l e to cleavage by amines. ' Extensive l i s t s o f the products o f h y d r o l y s i s and aminolysis 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 of c o n d i t i o n s have been 4 , 8 p u b l i s h e d . 3 . Covalent h y d r a t i o n R e v e r s i b l e covalent h y d r a t i o n across C=N bonds occurs i n a number of 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 the c a t i o n ) , 1 , 4 , 6 - t r i a z a -naphthalene (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 . This phenomenon has been reviewed In 7 , 2 3 , 2 4 , 2 5 s e v e r a l a r t i c l e s . At present, two hypotheses 26 apparently cover the known examples o f covalent h y d r a t i o n . Hydration may be expected at a double 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 centers are present so that the double bond no longer p a r t i c i p a t e s g r e a t l y i n aromatic conjugation and (b) i f formation o f the water adduct i s s t r o n g l y s t a b i l i z e d by resonance. P o s t u l a t e (a) i s f u l f i l l e d by those h e t e r o c y c l e s c o n t a i n i n g a C=N l i n k a g e and those covered by p o s t u l a t e (b) embrace the usual types o f reso n a t i n g systems + H E -NH-C=NH > -N=C-NH R + such as the amidinium, guanidinium and urea systems. Representative o f these systems are the q u i n a z o l i n e c a t i o n s , ^ the 2-aminopteridine cation^S and the 2-hydroxpteridine c a t i o n . ^ Covalent h y d r a t i o n can u s u a l l y be recognized i n h e t e r o c y c l i c systems by the presence 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 . The usual anomalies i n i o n i z a t i o n constants caused by covalent h y d r a t i o n are the strengthening o f bases and weakening o f a c i d s , as expected f o r the d e l e t i o n of a double 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 , or one thousand-fold, from the norm.30 In. spectroscopy, a l a r g e s h i f t o f the ab 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 observed. Normally, the long-wavelength a b s o r p t i o n o f p t e r i d i n e does not s h i f t more than lOmu when an i o n i s formed from the n e u t r a l s p e c i e s . Moreover, i n the absence o f hy d r a t i o n the spectrum of the anion of a hydroxy pteridine should "be i d e n t i c a l to that of the n e u t r a l species o f the corresponding primary amine.3,31 By the 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 s p e c t r o s c o p i c techniques, q u a n t i t a t i v e data has been o b t a i n e d 3 2 f o r the k i n e t i c s o f the covalent 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 or base and i s r e v e r s i b l e . The hyd 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 predominantly i n the lactam . form, may be used as an example. The r e a c t i o n f o l l o w s f i r s t -order k i n e t i c s , and over the pH range 4 .5 to 12.4 at 2 0 ° , the times f o r h a l f - c o m p l e t i o n range from 0 .5 to 375 seconds. •20 The mechanism-7 f o r the a c i d - and base-catalyzed r e a c t i o n i s p o s t u l a t e d to i n v o l v e the sequences (A) and (B) r e s p e c t i v e l y : (A) anhydrous H (B) O H a c t i v a t e d complex H hydrate H O H ' The presence of a methyl group at 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 extent, 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 of a combination o f s t e r i c and e l e c t r o n i c e f f e c t s . Recently, some examples of transannular covalent l 4 15 34 h y d r a t i o n have been found. ' ' When a methyl or a l k y l group i s present i n the 8 - p o s i t i o n of the p t e r i d i n e , t r a n s -annular h y d r a t i o n can occur across the 1,7 or 3,7 p o s i t i o n s . 9 For example, 6 , 7 , 8 - t r i m e t h y l - 4 - p t e r i d i n o n e ( V I I I ) i n a l k a l i shows the c h a r a c t e r i s t i c hypsochromy corresponding to the formation o f the anion (IX) by 3 , 7-hydration followed by l o s s of a proton. The requirements 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) are adequately s a t i s f i e d by t h i s system. students have c o l l a b o r a t e d i n a study o f the h y d r a t i n g p r o p e r t i e s i n a s e r i e s o f 8 - a l k y l l u m a z i n e s . 1 5 I n v e s t i g a t i o n s with lumazines having e i t h e r an i o n i z a b l e hydrogen or a methyl group at N-3 showed that the n e u t r a l and c a t i o n i c species presented no anomalies. In a l k a l i however, the ab s o r p t i o n s p e c t r a of the lumazines 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 to small changes i n the nature o f the s u b s t i t u e n t s i n the 6 - , 7 - or 8-p o s i t i o n s . These v a r i a t i o n s were traced to the formation o f e q u i l i b r i u m mixtures o f two mono-anions (one o f which i s hydrated across the 1 , 7 - p o s i t i o n ) and a d i a n i o n . For example, the two mono-anions o f 8-methyllumazine were found to e x i s t ( V I I I ) (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 10 i n roughly equal proportions at e q u i l i b r i u m , w i t h each anion s t a b i l i z e d by resonance of the negative charge i n the pyrimidine r i n g o f the molecule. On the other hand, the p r o p o r t i o n o f hydrate i n the n e u t r a l species o f 8-methyllumazine was found 15 to be only one part i n 5700. 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 o b s e r v a t i o n that o x i d a t i o n of 2-hydroxy - 4-methylpteridines w i t h aqueous permanganate at room temperature and pH appropriate to the formation of the hydrate r e s u l t s i n .demethylation at the 4 - p o s i t i o n and formation of lumazines ( 2 , 4 - d i h y d r o x y p t e r i d i n e s ) . This r e a c t i o n has been shown to be general f o r diagnosing 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 Is reminiscent of the o x i d a t i o n o f 1-methylcyclohexanol to cyclohexanone w i t h aqueous potassium 35 permanganate at room temperature. 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 at the s i t e of hydroxyl io n 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 w i t h 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 C H 3 >-H 3 C pH 9 -H20 11 Since covalent h y d r a t i o n of 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 , general a d d i t i o n o f nu c l e o p h i l e s to those p t e r i d i n e s s u s c e p t i b l e to hyd r a t i o n might be expected. I t has been shown^ that "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 at 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) gives a strong hypsochromic s h i f t i n abs o r p t i o n i n d i c a t i v e of formation o f the hydrated anion (XI; R = CH 2CH 20H, R' = OH). In the same manner and at lower pH values, (x) and s i m i l a r yellow "quinonoid" p t e r i d i n e s add hydrogen cyanide, 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 (XI; R' = CN, SO^ -, e t c . ) . 8 (x) H R H 3C " e H l H (XI) 4. Reduction and o x i d a t i o n Reduction 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 out using 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 subject have been 2 4 8 p u b l i s h e d . ' ' Most r e s u l t s i n d i c a t e that the pyrazine r i n g 12 o f the molecule 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 p y r i m i d i n e r i n g . 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 hydride gives 5 , 6 , 7 , 8 - t e t r a -38 h y d r o p t e r i d i n e 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-dihydro d e r i v a t i v e which passes on f u r t h e r hydrogenation 39 to the corresponding 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-hydroxypteridine was Re-i n v e s t i g a t e d w i t h a number of reagents and the compound 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 or a c i d i c s o l u t i o n over Raney n i c k e l or platinum oxide. However, i n a l k a l i n e media r e d u c t i o n v/ith potassium borohydride, sodium 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 over Raney n i c k e l o r palladium 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 as the major product w i t h traces o f substances r e s u l t i n g from r e d u c t i o n of the pyrazine r i n g . 2-Hydroxy - 6-methylpteridine reacted i n an analogous manner to y i e l d the corresponding 2-hydroxy - 6-methyl - 3 , 4 -d i h y d r o p t e r i d i n e . These r e s u l t s represent 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 reported. In the expected manner, 2 - 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 , prepared unambiguously., by ...reductive . c y c l i z a t i o n o f- 2-hydroxy 5 - n i t r o - 4 - a c e t o n y l a m l n o p y r i m i d i n e , gives 2 - h y d r o x y - 5 , 6 , 7 , 8 -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 . The unexpected course o f the r e d u c t i o n o f 2-hydroxypteridine has prompted a d e t a i l e d study o f the r e d u c t i o n o f a l l mono- and p o l y -4 l 42 8 h y d r o x y p t e r i d i n e s ' and the r e s u l t s have been summarized. 13 Condensation o f 2,4,5-triamino-6-hydroxypyrimidine w i t h benzoin leads to the formation o f two isomeric 6,7-d 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 the sol v e n t i n which the 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 5j6-dihydro d e r i v a t i v e i s formed i n 25$ aqueous e t h a n o l . This isomer can be converted to the more s t a b l e " j3 " isomer or 7 , 8-dihydro 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 a c i d . 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 the condensation i n a c e t i c a c i d s o l v e n t . 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 reviewed. In the 8-a l k y l p t e r i n s e r i e s ( 8-alkyi - 2-amino-4-hydroxypteridines), r e d u c t i o n of 8 - e t h y l l e u c o p t e r i n (XII) w i t h sodium amalgam gives 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 ) which can be r e o x i d i z e d by oxygen over platinum oxide as p r e v i o u s l y 46,47 described f o r the o x i d a t i o n o f dihyd 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, the only s t a b l e product i s o l a t e d i s 8 - J 3-hydroxyethylisoxanthopterin (XV; R = CH 2CH 20H). C 2 H- 5 Ck (XII) •C,2H5-O-( X I I I ) 14 (XIV) (XV) S i m i l a r r e s u l t s are obtained w i t h the corresponding 2-amino-4 , 7 - d i h y d r o x y - 6 - p t e r i d i n e c a r b o x y l i c a c i d (XIV; R = H) where r e d u c t i o n w i t h sodium amalgam gives a product 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 (to give a 5 , 6-dihydro d e r i v a t i v e ) and to o x i d a t i o n to 2-amino - 4 , 7-dihydroxypteridine (XV; R = H). Analogously, 2 , 4-dlamino - 7-hydroxy - 6-pteridine c a r b o x y l i c a c i d i s reduced by z i n c and a l k a l i or sodium amalgam, followed 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 5 , 6 - d i h y d r o p t e r i d i n e and subsequent r e o x i d a t i o n to 2 , 4-diamino - 7-hydroxy-48 p t e r i d i n e . The o n l y 8-alkyl-dihydrolumazine so f a r r e p o r t e d " " -i s 8-( J3 -hydroxyethyl ) - 6 , 7-dimethyl - 7 , 8-dihydrolumazine (XI; R = CH2CH2OH, R 1 = H ) , 1 5 ' 3 6 ' ^ 9 formed by the c a t a l y t i c r e d u c t i o n of the parent lumazine w i t h platinum oxide c a t a l y s t 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 , e s p e c i a l l y those u n s u b s t i t u t e d i n the pyrazine 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 , are extremely 1 5 s u s c e p t i b l e to dehydrogenation, o f t e n accomplished merely by atmospheric oxygen. For example,5 ° when 2-amino-4-hydroxy-5*6,7*8-tetrahydropteridine s u l f i t e i s exposed to the a i r , three unstable products assigned the 5*6-, 5*8- and 7*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 apparently formed. These can be separated by paper chromatography and 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 . Further o x i d a t i o n o f the Isomeric mixture i n s o l u t i o n gives 2-amino-4-hydroxy-p t e r i d i n e . 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 presence of a n u c l e o p h i l e * a t t a c k by t h i s n u c l e o p h i l e at the 6-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 formation of a 6-substituted p t e r i d i n e . Evidence that the 7*8-dihydropteridines are i n v o l v e d In the formation o f the 6-substituted d e r i v a t i v e s has been provided by an unambiguous synt h e s i s of 2-amino-4-hydroxy-7*8-dihydropteridine and a study of i t s a d d i t i o n 51 r e a c t i o n s . 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 which give the 6-substituted p t e r i d i n e s on mild o x i d a t i o n . The course of the o x i d a t i o n o f 6,7-disubstituted - 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 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 5 2 shown to proceed i n two stages: slow o x i d a t i o n to a 5*6-d i h y d r o p t e r i d i n e followed by r a p i d i s o m e r i z a t i o n o f the l a t t e r compound to a 7*8-dihydropteridine. 7,8-Dihydro-6-hydroxy-, l o > 1 : > 5*6-dihydro-7-hydroxy- 1^ b and 2-amino-7,8-dihydro-4,6-dihydroxypteridine^^ are dehydrogenated i n good y i e l d s by c o l d a l k a l i n e potassium l b 47 permanganate. T h i s appears to be the best g e n e r a l reagent f o r the purpose even though d i h y d r o p t e r i d i n e s with s e v e r a l e l e c t r o n - d o n a t i n g s u b s t i t u e n t s can be o x i d i z e d by m i l d e r reagents such as oxygen i n a l k a l i n e s o l u t i o n ( u n c a t a l y z e d ) , hydrogen p e r o x i d e , a l k a l i n e s i l v e r n i t r a t e , methylene 55 b l u e , sodium hypobromite, benzoquinone, e t c . U n t i l l a t e , l i t t l e i n f o r m a t i o n 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 dihydro p t e r i d i n e s has been r e p o r t e d . Recently,^ 8-monohydro-and 6 , 7 , 8 - t r i -h y d r o p t e r i d i n e r a d i c a l c a t i o n s have been detected by e . s . r . techniques i n the hydrogen peroxide o x i d a t i o n o f N(5)~ m e t h y l - 6 , 7 - d i p h e n y l - 5 , 6 - d i h y d r o - and N ( 5 )-methyl - 6 , 7-diphenyl 5 , 6 , 7 , 8 - t e t r a 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 r e s p e c t i v e l y . In the hydrogen peroxide o r a e r i a l o x i d a t i o n o f the l a t t e r compound (XVI) to the corres p o n d i n g dihydro p t e r i d i n e a 56 57 h y d r o x y l a t e d i n t e r m e d i a t e (XVII) i s proposed. ' Reduction o f (XVII) i n t r i f l u o r o a c e t i c a c i d over platinum oxide a f f o r d e d the c o r r e s p o n d i n g h y d r o x y l a t e d t e t r a h y d r o d e r i v a t i v e ( X V I I l ) . Mass spectrometry, u l t r a v i o l e t and n u c l e a r resonance s p e c t r a , as w e l l as chemical a n a l y s i s c o n f i r m these s t r u c t u r e s . (XVI) H 3 C 9 8 : x v n ) (XVIII) 17 E.s.r. i n v e s t i g a t i o n s 58 have a l s o shown that a one-electron o x i d a t i o n 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 and t e t r a h y d r o f o l i c a c i d occurs w i t h peroxide i n t r i f l u o r o a c e t i c a c i d r e s u l t i n g i n unstable c a t i o n i c r a d i c a l s o f red c o l o u r which can be trapped at l i q u i d n i t r o g e n temperature. 59 In another recent paper, q u a n t i t a t i v e s t u d i e s have shown that organic hydroperoxide formation should be regarded as the primary process 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 f l a v i n s ( 9 - s u b s t i t u t e d i s o a l l o x a z i n e s ) . The sequence of events i n the a u t o x i d a t i o n of a r e p r e s e n t a t i v e tetrahydro-p t e r i d i n e (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 2 > H^PterOOH ( v i a H^Pter*) i i . H^PterOOH + H 20 -H^PterOH + H 20 2, or i i i . H 3PterOOH + Substrate-H — * H^PterOH + Substrate-OH, or the peroxide may a l s o o x i d i z e i t s hydrogenated precursor: i v . H^PterOOH + IfyPter 2H"3PterOH where o o 18 The hydroxylated p t e r i d i n e s (H^PterOH) may then dehydrate to form "quinonoid" dihydro p t e r i d i n e s (H^Pter) which isomerize r a p i d l y ( c f . reference 52) to 7 , 8 - d i h y d r o p t e r i d i n e s , followed by r a t h e r slow conversion to the parent p t e r i d i n e s w i t h the consumption of oxygen and peroxide. The course of the r e a c t i o n was followed 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 measuring the oxygen uptake and amounts of peroxide present at various stages i n the r e a c t i o n . 4-2 +3 The r e a c t i o n s of Cu*" and Fe w i t h t e t r a h y d r o -p t e r i d i n e s under anaerobic c o n d i t i o n s have been I n v e s t i g a t e d and p r e l i m i n a r y r e s u l t s have been published i n a recent communication.^ 0 By the use of polarographic techniques +2 a p p l i c a b l e to p t e r i d i n e s i t was demonstrated that Cu forms 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 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 r e a c t i o n s i n which "quinonoid" and 7 , 8 - d i h y d r o p t e r i d i n e intermediates are formed. The r e a c t i o n s i n v o l v e d , using 2 - amino - 4-hydroxy - 5 , 6 , 7 , 8-tetrahydropteridine ( t e t r a h y d r o -p t e r i n ) as an example, are i l l u s t r a t e d as f o l l o w s : i . O v e r a l l r e a c t i o n ; 4Fe +3 + t e t r a h y d r o p t e r i n — > 4 F e + 2 + o x i d i z e d p t e r i n i o f a s t i i . t e t r a h y d r o p t e r i n + 2Fe" r j > quinonoid-dihydropterin + Fe+2 i i i . q u i n o n o i d - d i h y d r o p t e r i n — > 7 , 8 - d i h y d r o p t e r i n i v . q u i n o n o i d - d i h y d r o p t e r i n + 7,8-dihydropterin »• t e t r a h y d r o p t e r i n + p t e r i n . In 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 converted to 19 q u i n o n o i d - d i h y d r o p t e r i n , 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 support of t h i s process i s the f a c t that the quinonoid form i n the absence o f excess oxidant y i e l d s 7,8-dihydropterin, o x i d i z e d p t e r i n and t e t r a h y d r o p t e r i n . The t r i h y d r o p t e r i n r a d i c a l ^ Is probably an o b l i g a t o r y intermediate i n the r e a c t i o n but i t could not be detected i n t h i s case. C. Methods o f P r e p a r a t i o n 1. Isay r e a c t i o n The p r e p a r a t i v e routes to p t e r i d i n e s have been 2 3 4 8 e x t e n s i v e l y reviewed. •* The c l a s s i c a l and most commonly used s y n t h e t i c route i n v o l v e s condensation of a 4 ,5-diamino-py r i m i d i n e w i t h a 1 , 2-dicarbonyl compound or w i t h an <?C-halo-carbonyl compound and 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 . This r e a c t i o n , f i r s t noted by I s a y , ^ 1 has been 2 employed by a number of i n v e s t i g a t o r s . The general r e a c t i o n scheme may be represented by (XIX—>XXI). M o d i f i c a t i o n s to g i v e 8- or 5 - a l k y l p t e r i d i n e s are p o s s i b l e by employing a pyrimidine p r e c u r s o r - i n which one o f the amino groups i s a l k y l a t e d . (XIX) (XX) (XXI) 20 In t h i s s y n t h e t i c method the f o l l o w i n g s u b s t i t u e h t s can be Incorporated i n t o the p t e r i d i n e product: R± and R 2; H, OH, OR, COOH, COOR, a l k y l , a r y l , e t c . i n various combinations. R^; H, OH, NHg, NHR. R^j H, OH, NH 2, NHR, SH, S R . : No d i f f i c u l t y i s encountered when the 1 , 2-dicarbonyl compound i s symmetrical. When i t i s not, condensation o f t e n leads to a mixture o f isomeric products. However, i t i s f r e q u e n t l y p o s s i b l e to c o n t r o l the r i n g c l o s u r e by s u i t a b l e adjustment P 8 o f the pH of the r e a c t i o n s o l u t i o n . This can 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 the 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 of the Isay r e a c t i o n i s the 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 keto-methylene compound r e a c t s w i t h a 4-amino - 5-nitroso--p y r i m i d i n e d e r i v a t i v e to give a p t e r i d i n e . This method has the advantage over the c l a s s i c a l s y n t h e s i s i n that the o r i e n t a t i o n of the s u b s t i t u e n t s i n the 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 can a l s o be accomplished from <£-substituted carbonyl compounds and 4 , 5-diamino-p y r i m i d i n e s . Thus, aldehydo- and ke 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 to give 7 - a l k y l - 5 , 6 -dihydro - 2-amino - 4-hydroxypteridines which are spontaneously o x i d i z e d 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) give p a i r s o f isomeric 21 43 44 d i h y d r o p t e r i d i n e s ' as p r e v i o u s l y discussed. 2. C y c l i z a t i o n of pyrimidines 4 - C h l o r o - 5 - n i t r o p y r i m i d i n e s r e a c t w i t h amino-acetone to give 5-nitro-4-pyrimidylaminoacetones ( X X I I ) , some of which can be c y c l i z e d to 7 , 8-dihydro - 6-methyl-64 p t e r i d i n e s by hydrogenation over Raney n i c k e l . S i m i l a r l y , 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 the e t h y l e s t e r o f g l y c i n e to give e t h y l - 2 - c h l o r o - 5 - n i t r o - 4 - p y r i m i d y l a m i n o acetate (XXIII) which 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 water to 2-chloro - 7 * 8-dihydro - 6-hydroxy-65 65 p t e r i d i n e . 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,^ r e a c t 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 (XXII) can be replaced w i t h hydroxy groups (by an acetate b u f f e r ) or exchanged f o r primary 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 are e a s i l y 65 removed by hot hyd r o i o d i c a c i d and phosphorus; dehydrogenation to the parent 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 potassium permanganate. O H 3 C - C - C H ^ N v > k ^ a (XXII) O C 2 H 5 ° : C " C H 2 N n ^ N ^ J L L 0 2 N -(XXIII) 22 Because o f the 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 can be used f o r the p r e p a r a t i o n of various 6-hydroxypteridines and f o r determining the o r i e n t a t i o n o f 6- or 7-hydroxypteridines prepared by the Isay r e a b t i o n . 3. Synthesis from pyrazine Intermediates Despite the advantages o f the general p t e r i d i n e syntheses o u t l i n e d above, they s u f f e r from the disadvantage that the requirement o f a 4 , 5-diaminopyrimidine as one component more or l e s s l i m i t s the choice of s u b s t i t u e n t s that can be incorporated i n the pyrim i d i n e r i n g . To overcome t h i s disadvantage, methods have been developed whereby the pyr i m i d i n e r i n g i s constructed from a pyrazine intermediate w i t h r e s u l t a n t g r e a t e r f l e x i b i l i t y . These developments have 67 been summarized by T a y l o r . One s y n t h e t i c route to 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 three steps: 1. p r e p a r a t i o n o f a 4-hydroxy- or 2 , 4 - d i h y d r o x y p t e r i d l n e (lumazine) by conventional methods, i i . cleavage o f the pyrim i d i n e ring, o f the hydroxy:-. 19 p t e r i d i n e , and i i i . r e c o n s t r u c t i o n of the p t e r i d i n e nucleus on the pyrazine l 8 a , 2 1 , 6 8 , 6 9 r i n g to giv e the des i r e d product. U n f o r t u n a t e l y , o b t a i n i n g the r e q u i s i t e pyrazine 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 circumvent t h i s d i f f i c u l t y , an a l t e r n a t e general s y n t h e s i s o f the pyrazine Intermediates 70 has been developed, which permits p r e p a r a t i o n of 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 2 3 3-hydroxy - 4 , 5-diaminopyrazole (XXIV) 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 ) -pyrazine (XXV) which i s subsequently cleaved r e d u c t i v e l y over Raney n i c k e l to give a 2-aminopyrazine - 3-carboxamide ( X X V I ) . E l a b o r a t i o n of the p y r i m i d i n e r i n g i s then accomplished 8 by standard methods. For example, a d d i t i o n o f benzoyl c h l o r i d e to the pyrazine (XXVI) r e s u l t s i n the formation o f a 2 - p h e n y l - 4 - 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 ( X X V I I ) . R "CO H 2 H + OH $i z R (XXIV) (XXV) (xxv) XBX C6HnCOC^. 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 by heating 2~aminopyrazine - 3-carboxamide w i t h e t h y l formate 1 8 a and a c e t i c anhydride under r e f l u x . The syntheses o f monohydroxy-, polyhydroxy-, amino-and aminohydroxypteridines using the various methods 2 , 4 , 8 described have been reviewed e x t e n s i v e l y . 24 D. B i o l o g i c a l Importance o f P t e r i d i n e s 71 The name " f o l i c a c i d " has been coined to designate a substance, present i n leaves and mammalian organs, which i s able to s t i m u l a t e the growth o f c e r t a i n organisms, notably the bacterium Streptococcus f a e c a l i s , and which has been recognized as part o f the vit a m i n B complex. At present, the term i s taken to encompass a f a m i l y o f conjugated p t e r i d i n e s . The s p e c i f i c substance to which the 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 or PGA (XXVIII) and the 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 (XXIX). P t e r o y l g l u t a m i c a c i d proved to be the 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 p t e r i d i n e s w i t h 6-p-carboxyanilinomethyl side chains. 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 a c t i v i t y 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 have been adequately * 2 , 8 , 7 2 reviewed. (XXVIII); ) COOH ;-NH-CH-CH2-CH2-COOH (XXIX); COOH 25 Xanthopterin ( 2 - a m i n o - 4 , 6 - d i h y d r o x y p t e r i d i n e ) , which i s the yellow c o l o u r i n g matter present i n tne bands of wasps, i s not confined o n l y to the i n s e c t world.,There are i n d i c a t i o n s that i t may be the 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 the development o f p r i m i t i v e c e l l s i n t o the 73 t y p i c a l c e l l s o f the kidney. When mammals of various species 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 enlarge to many times t h e i r normal s i z e , although 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 stop, the kidneys s l o w l y r e v e r t to normal s i z e . 2-Amino - 4-hydroxypteridine 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 molecule does not produce kidney enlargement. Many p t e r i d i n e s have been found to i n h i b i t growth by a c t i n g as analogues to other p t e r i d i n e s concerned w i t h normal metabolism. The subject o f f o l i c a c i d antagonists 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 reviewed. Only a few o f the simpler p t e r i d i n e s e x h i b i t growth-retarding 2 p r o p e r t i e s , r e s u l t i n g from competition w i t h the f o l i c a c i d s or t h e i r p r e c u r s o r s . For example, 2 , 4-diamino - 6 , 7-diphenyl-. p t e r i d i n e suppresses the growth o f the 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. gallinaceum, an e f f e c t enhajnced by sulfonamides 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 -glutamic acid. 7 5 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 biochemical 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). In 1956 , 76 77 Masuda ' reported the i s o l a t i o n , along w i t h r i b o f l a v i n 26 (XXX) , o f two deoxyribose d e r i v a t i v e s of lumazine from the mycelium o f E. a s h b y i i . These d e r i v a t i v e s were subsequently shown^' ^*78,79 t Q ^ e 6 ^ 7 _ , j i m e t h y l - 8 - D - r i b i t y l l u m a z i n 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 degradation experiments, u l t r a v i o l e t measurements and syntheses. The i s o l a t i o n o f the same two substances from the 80 mycelium o f A. g o s s y p i i has a l s o been reported. H (XXX) O P H (XXXI) O H H 3 e o (XXXII) R = CH 2(CHOH) 3CH 2OH The c l o s e s i m i l a r i t y between the above r i b i t y l -lumazines and r i b o f l a v i n r a i s e s the question as to whether the lumazines serve as precursors to r i b o f l a v i n . Such a 78 8 l 82 p o s t u l a t e has been suggested by s e v e r a l i n v e s t i g a t o r s ' ' and i s s u b s t a n t i a t e d by s e v e r a l o b s e r v a t i o n s . In an enzyme-c a t a l y z e d r e a c t i o n , two moles of 6 , 7 ~ d i m e t h y l - 8 - - r i b i t y l -27 lumazine reacted to produce one mole of r i b o f l a v i n and other u n i d e n t i f i e d products v i a a route i n v o l v i n g the methyl groups and the 6- and 7-carbon moiety of the lumazine molecule. This 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, was shown to 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 experiments. 6-Methyl-7-oxo-8-ribityllumazine does not appear to 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 a r i s e d i r e c t l y 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 6 ^ 9 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 at p o s i t i o n 8 and carbon atoms 6 and 7 have been synthesized but o n l y one of these substances (6,7-dimethyl-8-(l 1-D - 5 1 -d e o x y r i b i t y l ) l u m a z i n e ) i s converted to an analogous f l a v i n ( i s o a l l o x a z i n e ) ."^ The conversion 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 i n t o r i b o f l a v i n i n v i t r o i n the absence of any other source o f 84 carbon has a l s o been reported. 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 b u f f e r at 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 i n 55$ y i e l d . A s m a l l amount of 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 that obtained from s e l f - c o n d e n s a t i o n o f 5-amino-4-D-ribitylamino-2,6-dihydroxypyrimidine, ^ i s o l a t e d from the mother-liquors 4Q «4 o f the r e a c t i o n lends support to the suggested mechanism y > o f the r e a c t i o n : i . ring-opening of the "quinonoid" lumazine i n i t i a t e d by n u c l e o p h i l i c a t t a c k at p o s i t i o n 7 ( c f . reference 36) (XXXI *• XXXIII ^ XXXIV), i i . a l d o l condensation i n v o l v i n g two molecules o f (XXXIV) 28 to g i v e a d e r i v a t i v e (XXXV) o f dimeric b i a c e t y l as suggested 86 by B i r c h , and i i i . c y c l i z a t i o n o f t h i s intermediate w i t h the l o s s of one diaminopyrimldine moiety to gi v e r i b o f l a v i n (XXX). R H-(xxxr) 5-^3^ (XXXIII) R (XXXIV) ( X X X I V ) - H > L H 3 I ( X X X V ) (XXX) R 4-H 9 N ^ H The o v e r a l l s i m i l a r i t y between the chemical s y n t h e s i s of r i b o f l a v i n from the lumazine precursor and the corresponding enzymatic r e a c t i o n suggests that 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 c y c l i z a t i o n employing a d e r i v a t i v e o f dimeric b i a c e t y l has p r e v i o u s l y 37 -been reported. 49 Very r e c e n t l y , J i t has been demonstrated that the r e a c t i o n between two molecules o f the 8 - r i b i t y l l u m a z i n e to giv e r i b o f l a v i n i s not p e c u l i a r to 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-imino~4-hydroxy-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 at pH 7.3 f o r 24 hours under n i t r o g e n gave 49$ of the 2-imino-4-hydroxy-6,7-dimethyl-9-(f -hydroxyethyl)benzo(g)pteridine (XXXVII). S i m i l a r l y , 8-( f> -hydroxyethyl)-6,7-dimethyllumazine gave the corresponding 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 products of 5-amino-4-j*-hydroxyethylamino-2,6-dihydroxypyrimidine i s o l a t e d as a r e a c t i o n by-product. R H 3 C ,NH (XXXVI) (XXXVII) R = C H 2 C H e o H 87 I t i s known that 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 another b i o l o g i c a l l y important f a m i l y o f heterocyclesy~the -"purineST"Thusy "the~p t e r Idines~can~be considered as the l i n k between the purines and the i s o -a l l o x a z i n e system ( f l a v i n s ) . The chemistry and b i o l o g i c a l 88 QO 8 8Q f u n c t i o n s o f the purines ' y and the i s o a l l o x a z l n e s ' y have been reviewed. The chemical conversion of purine 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 . 8 7 , 9 1 , 9 2 30 Considerable a t t e n t i o n has been focused on n a t u r a l l y o c c u r r i n g reduced p t e r i d i n e s because of an increased understanding 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 r e a c t i o n s . As one example, f o l i c a c i d can be hydrogenated e n z y m a t i c a l l y to dihydro- and tetrahydrofolic a c i d during the s y n t h e s i s o f l e u c o v o r i n ( N ( 5 ) - f o r m y l - 5 , 6 , 7 , 8 -t e t r a h y d r o f o l i c acid) i n animal t i s s u e s and micro-organisms. Both the dihydro and tetrahydro d e r i v a t i v e s not possessing the s t a b i l i z i n g formyl group are very unstable to mild o x i d a t i o n and s o l u t i o n s exposed to 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 , x a n t h o p t e r i n , and other p t e r i d i n e s . The b i o l o g i c a l importance o f the 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 they serve as a source o f one carbon fragments i n the bi o s y n t h e s i s 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 pur i n e s , pyrimidines and amino a c i d s . E. Redox Reactions of the F l a v i n s The 8-alkyllumazines are s t r u c t u r a l l y very s i m i l a r to the f l a v i n s (isoalloxazines)...._. Whether the _.two systems can..._ be expected to e x h i b i t s i m i l a r behaviour w i t h respect to o x i d a t i o n and re d u c t i o n i s not so apparent, however. While the p o s s i b i l i t y of 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 only r e c e n t l y come to l i g h t , the redox p r o p e r t i e s of the f l a v i n s are w e l l known and have 89 ,104 ,105 ,106 ,107 been the subject o f s e v e r a l 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 ) are 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 c o n d i t i o n s i n the r e d u c t i o n procedure. The f l a v i n s are normally found i n t h e i r o x i d i z e d s t a t e , t h e i r o x i d a t i o n - r e d u c t i o n p o t e n t i a l s 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 of atmospheric 8 9 , 1 0 8 oxygen. On the b a s i s of po 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 observations s e v e r a l i n v e s t i g a t o r s , some time ago, concluded that the f l a v i n s are o x i d i z e d and reduced i n two d i s t i n c t one-electron steps and therefore 1 0 5 , 1 0 9 pass through a true semiquinoid s t a t e of o x i d a t i o n . The semiquinoid intermediates were found to have a considerable 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 . The s p e c t r a l c h a r a c t e r i s t i c s of f l a v i n s a t the semiquinoid l e v e l have been i n v e s t i g a t e d 109 i n d e t a i l by B e i n e r t who observed two bands at 570 mu and 900 mu during the r e d u c t i o n of 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 moiety). The absorbance 110 at 570 mu i s due to the free r a d i c a l , the presence o f 111 which has been confirmed by e.s.r. measurements, whi l e the broad ab s o r p t i o n band i n the 900 mu regi o n has been a t t r i b u t e d on the basis o f temperature- and concentration-dependence to a bim o l e c u l a r (or higher molecular) complex o f the semi-109 quinones w i t h each other or w i t h components of the s o l u t i o n . 110 Gibson, Massey and Atherton, on the other 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 of a FMN-FMNHg complex.. 32 The k i n e t i c s of the r e d u c t i o n of r i b o f l a v i n by d i t h l o n i t e at pH 9 have been studied using a r a p i d mixing apparatus. The k i n e t i c s o f the appearance o f the 900 mu abso r p t i o n band suggested that i t was due to two molecules 112 o f the semiquinone but i t i s s t i l l d o u b t f u l whether the semiquinone i s an intermediate i n t h i s r e a c t i o n or 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 f l a v i n . The mechanism o f the 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 dihydro-l l p o i c a c i d have been s t u d i e d . In the case of NADH, the r e a c t i o n i s thought to proceed by a hy d r i d e - i o n t r a n s f e r . The evidence i s derived from the e f f e c t s o f s u b s t i t u e n t s i n both f l a v i n and nicoti n a m i d e , s o l v e n t e f f e c t s and the ^ 4- * - 4 - 108 ,113,114 , 115 m u deuterium isotope e f f e c t . 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 followed by a r a t e -determining hydrogen a b s t r a c t i o n , has not been e n t i r e l y excluded. A recent r e p o r t 1 1 ^ lends f u r t h e r support to the two-electron r e d u c t i o n scheme. The red u c t i o n w i t h dihydro-l i p o i c a c i d i s a l s o b e l i e v e d to be a two-electron process on the ba s i s of 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 , but a fast-d i s s o c i a t i o n o f one of the t h i o l groups of the l i p o i c a c i d precedes the r e d u c t i o n , ^ 0 8 The k i n e t i c s o f the a u t o x i d a t i o n o f FMNHg (reduced FMN), as followed by r a p i d - r e a c t i o n techniques, are c o n s i s t e n t w i t h the occurrence of two simultaneous mechanisms. 117 33 In the f i r s t , the semiquinone o f the f l a v i n r e acts w i t h oxygen by a f r e e - r a d i c a l path: PMN + FMNHV 2FMNH' FMNH- + i0 2 > FMN + ^HgOg w h i l e the competitive process i n v o l v e s a two-electron o x i d a t i o n through a f l a v i n peroxide: FMNR_ + 0 2 2 FMN + H 2 0 2 Chemical evidence f o r the hydroperoxide 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 the analogous t e t r a h y d r o p t e r i d i n e s i s that the N-10 s u b s t i t u t e d dihydro-a l l o x a z i n e s are r e s i s t a n t to a u t o x i d a t i o n while the N-9 , ' , 59,118 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 ; are very s e n s i t i v e . The 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 anaerobic "conditions which have been exposed to l i g h t over prolonged periods resembles those obtained by the dark r e d u c t i o n of the f l a v i n by d i t h i o n i t e . This led to the suggestion that the mechanism of photobleaching ( r e a c t i o n i n the absence of ex t e r n a l e l e c t r o n donors) i s re d u c t i o n by w a t e r - s p l i t t i n g , p r o v i d i n g a chemical analogue f o r the w a t e r - s p l i t t i n g r e a c t i o n i n photosynthesis: 119,120 34 FMN + H 0 — — — * - FMNH* + OH* 2 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, Oster, and others ' ' and supporting evidence has been provided. The suggestion i s that during photobleaching an Intra 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 occurs i n which 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 i s o a l l o x a z i n e nucleus i s reduced. The evidence f o r the 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 35 OBJECTS OP THE PRESENT RESEARCH Only r e c e n t l y has the 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 of t h e i r importance i n b i o l o g i c a l systems. The s t u d i e s have been confined so f a r to the o x i d a t i o n o f simple t e t r a h y d r o p t e r i d i n e s to the corresponding dihydro compounds w i t h reagents such as i r o n ( i l l ) , hydrogen peroxide and oxygen i t s e l f . While a wide v a r i e t y o f reagents has been employed to o x i d i z e the d i h y d r o p t e r i d i n e s to the parent p t e r i d i n e s , no s t u d i e s concerning the 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 the o x i d a t i o n of 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 undertaken using common oxidants such as potassium f e r r i c y a n i d e and potassium permanganate f o r the 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 the subject o f i n t e n s i v e study i n recent years and a p a r a l l e l between the o x i d a t i o n and h y d r o x y l a t i n g p r o p e r t i e s o f the 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 the simple t e t r a h y d r o p t e r i d i n e s has been noted. A model compound was chosen f o r the redox s t u d i e s which incorporates the s t r u c t u r a l f eatures o f both the 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 which s a t i s f i e s these requirements i s 6 , 7 , 8 - t r i m e t h y l -lumazine (TML). 36 (TML) During the mec h a n i s t i c s t u d i e s o f the o x i d a t i o n . * o f the di h y d r o d e r i v a t i v e o f the above compound (DHTML) i t was observed t h a t an i n t e r e s t i n g i s o t o p i c exchange o f hydrogens o c c u r r e d i n both TML and DHTML and t h i s r e a c t i o n was i n c l u d e d i n the c u r r e n t i n v e s t i g a t i o n . * The 8- J3~hydroxyethyl analogue of DHTML (see structure XLV, page 4.0) has been designated as a 7 ,3-dihydro lumazine]^ an assignment presumably based on that used for certain reduced pter i d i n e d e r i v a t i v e s . This numbering convention has been used i n the present work although i t i s f e l t that the c l a s s i f i c a t i o n of reduced lumazine derivatives such as DHTML as 1,7-dihydro lumazines i s somewhat l e s s misleading. 37 EXPERIMENTAL A. C h a r a c t e r i z a t i o n o f the Prepared Compounds M e l t i n g points o f the prepared compounds were determined i n c a p i l l a r i e s using a Buchi melting-point apparatus and are uncorrected. A l l s p e c t r o s c o p i c measurements were made i n 1 cm. s i l i c a c e l l s using a Bausch and Lomb Model 5 0 2 r e c o r d i n g spectrophotometer equipped w i t h a 3 0 0 ml. l i q u i d capacity-constant temperature holder. Water from a bath thermostated o at 2 5 . 0 C. was pumped through t h i s holder during the r e c o r d i n g o f spectroscopic data. U l t r a v i o l e t and v i s i b l e absorbance maxima and the corresponding e x t i n c t i o n c o e f f i c i e n t s (£) of the prepared pyrimidines and lumazines are c o l l e c t e d i n Table I , along w i t h l i t e r a t u r e references to the s p e c t r a o f the known compounds. A c i d i c and basic i o n i z a t i o n constants of 6 , 7 , 8 - t r i m e t h y l l u r n a z i n e (TML) and 7 / 8 - d i h y d r o - 6 , 7 , 8 -trimethyllumazine (DHTML) were determined by r e c o r d i n g s p e c t r a o f the compounds i n aqueous phosphate b u f f e r s o l u t i o n — at various pH. The determined a c i d i c i o n i z a t i o n , recorded as the pK a, i s c o r r e c t e d to a thermodynamic value according / % 9 6 to equation ( 1 ) : ( l ) pK (thermodynamic) = pK (determined) + 0 . 5 V 5 j/l+l.6 / j J a. a where )J i s the i o n i c s t r e n g t h at h a l f - n e u t r a l i z a t i o n . 38 For the base i o n i z a t i o n c o n s t a n t , P K B H + ' the i o n i c s t r e n g t h term i s s u b t r a c t e d from the determined v a l u e . With 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 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 o f the b u f f e r determines the magnitude o f the c o r r e c t i o n term. The pK values recorded i n Table I f o r TML and DHTML have been c o r r e c t e d f o r the b u f f e r e f f e c t . I n f r a r e d s p e c t r a were recorded on a Perkin-Elmer Model 21 instrument employing potassium bromide p e l l e t sampling t e c h n i q u e s . Proton magnetic resonance (p.m.r.) s p e c t r a o f the prepared lumazines i n deuterium oxide and t r i f l u o r o a c e t i c a c i d s o l v e n t s were obtained on a V a r i a n A - 6 0 60 megahertz spectrometer u s i n g t e t r a m e t h y l s i l a n e (TMS) as e x t e r n a l r e f e r e n c e . The chemical s h i f t s 8 ( p a r t s per m i l l i o n ) from the r e f e r e n c e and c o u p l i n g constants J ( c y c l e s per second) are recorded i n Table I I . T h i n - l a y e r chromatographic techniques were employed to check the p u r i t y and o b t a i n R f values o f the prepared compounds. Samples were prepared by d i s s o l v i n g approximately 1 mg. 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 ml. o f methanol and s p o t t i n g the s o l u t i o n on t h i n - l a y e r p l a t e s w i t h a 4 m i c r o l i t e r c a p a c i t y d i s p e n s i n g c a p i l l a r y . Aluminum oxide G was used as sorbant on the t h i n - l a y e r p l a t e s . The obtained R f, u s i n g two s o l v e n t systems — 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 ( 7 : 3 ) (B) _ are recorded along w i t h l i t e r a t u r e r e f e r e n c e s i n Table I I I . The ascending 39 97 i technique and u l t r a v i o l e t r a d i a t i o n a t 254 mu. were used . f o r development and d e t e c t i o n o f the compounds r e s p e c t i v e l y . Carbon, hydrogen and n i t r o g e n analyses were performed by P. Borda, M i c r o a n a l y s i s L a b o r a t o r y , U n i v e r s i t y o f B r i t i s h Columbia. 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 the Lumazines The method used f o r the p r e p a r a t i o n o f the lumazine 85 d e r i v a t i v e s i s e s s e n t i a l l y t h at o f C r e s s w e l l and Wood and 7 6 , 8 1 o f Masuda. However, because c e r t a i n m o d i f i c a t i o n s and p u r i f i c a t i o n procedures are employed the p r e p a r a t i v e . methods, as w e l l as the p r o p e r t i e s o f the i n t e r m e d i a t e s , w i l l be d i s c u s s e d ' i n d e t a i l . The o u t l i n e o f the p r e p a r a t i v e scheme i s as f o l l o w s : H Q>>kfP \ > ~ OH (XXXVIII) P 0 C l 3 ¥ (XXXIX) NaOH ^ Ty^n CH 3NH 2 O H (XL) H 3C-N y >1 >OH H3 C"' IN NaN02 . »H (XLI) H (XLII) H H 3c-N > J ^ o H H o N T ^ / ^ OH ( X L I I l ) 40 (XLV) 2 , 4 , 6 - T r i c h l o r o p y r i m i d i n e (XXXIX) 9 8 The l i t e r a t u r e - ^ r e p o r t s a y i e l d of approximately 60$ f o r t h i s compound from b a r b i t u r i c a c i d (XXXVIII) w i t h a r e f l u x time of one hour. A higher y i e l d was obtained by using a longer (three hours) r e f l u x time. A 500 ml. three-necked f l a s k equipped w i t h condenser, a d d i t i o n funnel and mechanical s t i r r e r was charged w i t h 50 g. ( 0 . 3 9 moles) of b a r b i t u r i c a c i d (Eastman Organic Chemicals; used without 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 ml. phosphorus o x y c h l o r i d e and 3 8 . 5 ml. N,N-dimethylaniline was added s l o w l y over a period of twenty minutes. The mixture was then r e f l u x e d and s t i r r e d f o r a period o f three hours. 4 l At the end o f t h i s time, the r e s u l t a n t red s o l u t i o n was cooled, poured s l o w l y onto 800 g. of crushed i c e and allowed to stand overnight at room temperature. The s o l u t i o n was then f i l t e r e d and ext r a c t e d w i t h 50 ml. po r t i o n s o f d i e t h y l ether u n t i l the aqueous phase was no longer t u r b i d . The ether e x t r a c t s were combined, washed w i t h water 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 ether removed by f l a s h evaporation and the r e s i d u a l red o i l d i s t i l l e d under reduced pressure to y i e l d 62 g. (0.34 moles, 87$) of 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 9 8 , 9 9 102-105°(34 Torr) 4-Chloro-2, 6-dihydroxypyrimidine (XL) A one l i t r e two-necked f l a s k equipped w i t h condenser and mechanical 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 hydroxide (42 g.) i n water (420 ml.). 2 , 4 , 6 - T r i c h l o r o -p y r i m i d i n e (48 g; 0 . 26 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 that a f t e r approximately four hours a copious q u a n t i t y o f a white s o l i d was deposited. This s o l i d 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 the f i l t r a t e returned to the r e a c t i o n v e s s e l . At the end o f the r e f l u x p e r i o d , the s o l u t i o n was cooled, a c i d i f i e d w i t h concentrated h y d r o c h l o r i c a c i d and r e f r i g e r a t e d overnight. The white product was c o l l e c t e d , added to the i n i t i a l deposit o f white 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 twice from b o i l i n g water 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 to give 35 g. (0.24 42 moles; 9 2 $ ) of 4 - c h l o r o - 2 , 6 - d i h y d r o x y p y r i r o i d i n e . M.p. 298-300°(decomposition); l i t . ^ 300°(decomposltlon) C a l c u l a t e d f o r C ^ N g O g C l ; N = 1 9 . 1 Found; N = 1 9 . 4 $ . 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 (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 water i n a 2 5 0 ml. three-necked f l a s k equipped w i t h condenser, mechanical s t i r r e r and a d d i t i o n f u n n e l . To t h i s was added 5 0 ml. of 4 0 $ aqueous methylamine and the r e a c t i o n mixture then r e f l u x e d f o r s i x hours. Every two hours, 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 period the r e s u l t a n t yellow s o l u t i o n was cooled, a c i d i f i e d w i t h a c e t i c 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 deposited yellow s o l i d was c o l l e c t e d and r e c r y s t a l l i z e d twice from b o i l i n g water to y i e l d 2 1 . 5 g. ( 0 . 1 5 moles; 7 5 $ ) o f 4-rnethylamino-2,6-dihydroxypyrlmidine as pale yellow t a b l e t s . M.p. 2 9 9 ° (decomposition); l i t . ^ 3 0 2 ° (decomposition) 5 - N l t r o s 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 (XLII) To 1 8 . 5 g. ( 0 . 1 3 moles) of 4 - m e t h y l a m i n o - 2 , 6 -dihydroxypyrimidine i n 2 5 0 ml. o f water, 1 9 g. of sodium n i t r i t e was added. To t h i s mixture 1 0 ml. of 5 0 $ a c e t i c a c i d were added dropwise w i t h s t i r r i n g and the r e s u l t a n t red-coloured suspension heated to a f f e c t s o l u t i o n . On f i l t e r i n g and c o o l i n g a red s o l i d p r e c i p i t a t e d . This m a t e r i a l was c o l l e c t e d and r e c r y s t a l l i z e d twice from 5 0 $ a c e t i c a c i d , 43 washed w i t h water and d r i e d to y i e l d 1 8 . 6 g. (0.11 moles; 85$) o f 5-nItroso-4-methylamino - 2 , 6-dihydroxypyrimIdine as red needles. M.p. above 3 5 0 ° (darkens); l i t . 7 6 ' 8 1 above 3 5 0 ° 5-Amino-4-methylamino - 2 , 6-dihydroxypyrimidine ( X L I I I ) 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 s t i r r i n g to a mixture o f 14 g. 5-nitroso-4-methylamino-2,6-dihydroxypyrimidine i n 300 ml. o f water and 50 ml. o f 10% sodium hydroxide. The yellow s o l u t i o n which r e s u l t e d was heated f o r 10 minutes on the steam bath, f i l t e r e d and cooled. At t h i s stage, most of the s o l u t i o n was used d i r e c t l y i n the next step o f the sy n t h e s i s r a t h e r than to attempt 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 ml.) was c h i l l e d overnight and the b i s u l f i t e s a l t o f ( X L I I I ) was obtained as a pale yellow g r a n u l a r s o l i d . The f r e e amine (yellow needles ) was obtained from the s a l t by r e c r y s t a l l i z i n g from water. M.p. 252-256°(decomposition); l i t . 1 0 0 2 5 0 - 2 6 0 °(decomposition) I t was found that the f r e e amine, on standing 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 resinous brown mass over a period o f weeks. When k e p t , i n a desiccator under vacuum, the amine decomposed more s l o w l y . The b i s u l f i t e s a l t proved s t a b l e as a s o l i d and could be kept f o r long periods of time w i t h no signs o f decomposition. 6 , 7 , 8-Trimethyllumazlne (TML) (XLIV; R-^Rg = CH^) The bulk (approx. 320 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 ami 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 ( 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 the n e u t r a l i z e d s o l u t i o n , 25 ml. o f butane - 2 , 3-dione was added and the 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 at 8 0 ° f o r h a l f an hour. The s o l u t i o n was then cooled, f l a s h evaporated to a volume o f approximately 30 ml. and a p p l i e d d i r e c t l y to a 2x50 cm. column o f a c i d -washed a l u m i n a 1 ^ (Shawnigan Chemicals) made up and treated i n i t i a l l y w i t h 100$ ethanol. The column was eluted w i t h 50$ aqueous ethanol and the l e a d i n g green f l u o r e s c e n t band c o l l e c t e d . The solv e n t s were removed on the r o t a r y evaporator and the s o l i d r e s i due r e c r y s t a l l i z e d twice from b o i l i n g e t h a n o l , to which the minimum amount o f water was added to e f f e c t s o l u t i o n , to y i e l d 6 . 6 g. of 6 , 7 , 8 - t r i m e t h y l l u m a z i n e as b r i g h t yellow needles (green fluorescence i n aqueous s o l u t i o n ) . M.p. 310-311°(decomposition); l i t . 3 0 8 - 3 1 0 ° 3 7 o 8n 300-301 (decomp.) C a l c u l a t e d f o r C g H 1 Q N 4 0 2 ; C, 5 2 . 4 2 ; H, 4 . 8 9 ; N, 2 7 . 1 7 Found; C, 52 .33 ;' H, 5 . l 6 ; N , 2 7 . 3 2 $ „ , . ' . , 1 5 , 3 7 , 1 0 1 37 The s p e c t r o s c o p i c data and chromatography o f t h i s compound agree f a v o r a b l y to that p r e v i o u s l y reported. In a d d i t i o n to TML, two a d d i t i o n a l lumazine d e r i v a t i v e s were a l s o synthesized from a separate p r e p a r a t i o n o f 5-amino - 4-methylamino - 2 , 6-dihydroxypyrimidine but, i n these l a t t e r cases, the py r i m i d i n e intermediate was i s o l a t e d as the f r e e amine before condensation w i t h the app r o p r i a t e . 45 carbonyl compound. 6 , 8-Dimethyl - 7-oxolumazine (tautomer o f XLIV; R±= OR, R2= CH^) Crude 5-amino-4-methylamino - 2 ,6-dihydroxypyrimidine ( l g.) was suspended i n a methanol (75 ml.) and water (15 ml.) solv e n t p a i r followed by the a d d i t i o n o f 0 .8 g. of pyruvic a c i d . The mixture 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 hour, f i l t e r e d and r e f r i g e r a t e d . The product was c o l l e c t e d and r e c r y s t a l l l z e d twice from water, w i t h a charcoal treatment included 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. of 6 , 8-dimethyl - 7-oxolumazine as pale yellow needles.-M.p. above 3 5 0 ° ; l i t . 3 ^ 358°(decomposition) Ca l c u l a t e d f o r C g H g N ^ ; C, 46. l6; H, 3 . 8 7 ; N, 26.91 Found; C, 46.2.5; H, 4 .00 ; N, 26 .72$ 6 ,7-Diphenyl -8-methyllumaz'ine (XLIV; R 1 = R g = c g H 5 ) A mixture o f 1.5 g. crude 5-arnino-4-methylamino-2 , 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. water, 100 ml. ethanol and 10 ml. concentrated ammonium hydroxide was heated w i t h s t i r r i n g at 8 5 - 9 0 ° f o r two hours and then f i l t e r e d . The yellow f i l t r a t e was f l a s h evaporated to about 40 ml. and a p p l i e d d i r e c t l y to a 2x50 cm. acid-washed alumina column. The column was eluted w i t h 50$ aqueous ethanol and the yellow f l u o r e s c e n t band c o l l e c t e d . The solv e n t s were removed on the r o t a r y evaporator and the residue r e c r y s t a l l l z e d twice from an ethanol-water so l v e n t p a i r to y i e l d 1.6 g. of 6 , 7-diphenyl - 8-methyllumazine as yellow needles. 46 M.p. 304-306°(decomposition); l i t . 1 6 288-292° C a l c u l a t e d f o r C ^H-^N^; C, 69.08; H, 4.27; N, 16.96 Pound; C, 68.77; H, 4.58; N, l6,8l$ 7,8-Dlhydro-6,7,8-trimethyllumazine (DHTML) (XLV) 6,7,8-Trimethyllumazine was submitted to a number of r e d u c t i o n procedures i n an attempt to synthesize a. dihydro 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 . Since the dihydro compounds produced proved to be s u s c e p t i b l e to 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 solvents used i n work-up and r e c r y s t a l l i z a t i o n were thoroughly flushed w i t h h i g h -p u r i t y n i t r o g e n before use. As s o l i d s , the compounds were s t a b l e as long as precautions to avoid prolonged exposure to a i r and moisture were taken. 1. C a t a l y t i c 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 , platinum oxide (0.025 g.) was added and hydrogenation commenced at atmospheric pressure and ambient temperature. A f t e r 25 'ml. more than the t h e o r e t i c a l amount o f hydrogen required to produce the dihydrolumazine were taken up, the r e a c t i o n v e s s e l was evacuated, r e f i l l e d w i t h n i t r o g e n , stoppered t i g h t l y and t r a n s f e r r e d to a dry box under n i t r o g e n atmosphere. The r e a c t i o n mixture was n e a r l y n e u t r a l i z e d w i t h concentrated sodium hydroxide, f i l t e r e d , the f i l t r a t e reduced to a volume o f approximately 10 ml. on the r o t a r y evaporator and c h i l l e d under n i t r o g e n atmosphere f o r three days. A yellow 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 twice from ethanol, to which the minimum amount o f water had been added to e f f e c t s o l u t i o n . A t h i r d r e c r y s t a l l i z a t i o n from water afforded 750 mg. (0 . 0 0 3 6 moles; 49$) of a dihydrolumazine as pale yellow needles. This m a t e r i a l proved to be hygroscopic and hence was d r i e d thoroughly before a n a l y s i s and determination o f p h y s i c a l data. 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, 2 6 . 9 1 Found; C, 5 2 . 1 1 ; H, 5 - 6 0 ; N, 2 6 . 7 2 $ . That t h i s compound possessed the 7 , 8 - and not the isomeric 5 , 8-dihydro s t r u c t u r e was shown by comparison o f sp e c t r o s c o p i c data (Table I ) w i t h the known analogue, 7 , 8 -d i h y d r o - 8 - ( ^ - 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 . 1 5 , 3 6 , 2 + 9 As w e l l , the observed p.m.r. s p l i t t i n g p a t t e r n (Table I I ) can be a t t r i b u t e d to a methyl group and hydrogen attached to the same saturated carbon center ( p o s i t i o n 7 ) . Because o f the 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, 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 species must be invoked to a l l o w f o r the 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 the formation o f two spots, one o f which possessed the R f values and fluorescence under u l t r a v i o l e t l i g h t c h a r a c t e r i s t i c o f the l a t t e r compound. The second spot, 48 appearing a t R 0 . 3 8 and 0.46 i n butanol - 5 M a c e t i c a c i d ( 7 : 3 ) and 3 $ ammonium c h l o r i d e r e s p e c t i v e l y , i s assigned to 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 . 2 . Z i n c i n h y d r o c h l o r i c a c i d T h i s p a r t i c u l a r system has been used p r e v i o u s l y 3 8 to reduce 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 dust ( 2 g.) was added to a s o l u t i o n o f TML ( l . O g.) i n IN h y d r o c h l o r i c a c i d ( 2 5 ml.) and the mixture s t i r r e d under a stream o f n i t r o g e n f o r t h i r t y minutes. The mixture was then f i l t e r e d under n i t r o g e n and the z i n c washed wit h 1 5 ml. o f water. The washings and f i l t r a t e were combined, f l a s h evaporated to a volume o f approximately 1 0 ml. and r e f r i g e r a t e d a t 2 ° under an atmosphere o f n i t r o g e n f o r two days. The p a l e yellow s o l i d d e p o s i t e 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 i z e d three times from aqueous e t h a n o l to y i e l d 6 0 0 mg. o f p a l e yellow n e e d l e s . Again, t h i s m a t e r i a l was somewhat h y g r o s c o p i c . M.p. 2 9 6 - 2 9 8 0 (decomposition) Although the s p e c t r o s c o p i c and p.m.r. data o f t h i s m a t e r i a l proved to be n e a r l y i d e n t i c a l to that 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 obtained by c a t a l y t i c hydrogenation, the m i c r o a n a l y s i s was v i t i a t e d by the formation o f a white ash d u r i n g combustion and gave very d i f f e r e n t values f o r carbon, hydrogen and n i t r o g e n to those p r e v i o u s l y obtained f o r the l a t t e r compound. 2 1 0 2 I t i s known ' t h a t c e r t a i n h e t e r o c y c l e s p o s s e s s i n 4 9 an i o n i z a b l e hydroxyl group p e r i to a t e r t i a r y r i n g n i t r o g e n (e.g., 4 ~ h y d r o x y p t e r i d i n e , 8-hydroxyquinoline) form 1:1 and 1:2 complexes w i t h the ions o f d i v a l e n t metals, i n c l u d i n g z i n c . Hence, i t was suspected that the m a t e r i a l obtained v i a the 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 -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 - z i n c complex, having e i t h e r s t r u c t u r e (XLVI) or (XLVII). The m i c r o a n a l y s i s r e s u l t s favor the l a t t e r c h o i ce. The white ash produced upon combustion of the organic m a t e r i a l Is presumably an oxide of 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 . C a l c u l a t e d f o r (C gH 1 1 N 4 0 2) 2Zn-2H 2 0 (XLVII.2HgO); C, 41.91j H, 5.08; N, 21.72 Found; C, 41.46; 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 borohydride, z i n c , or sodium d i t h i o n i t e i n sodium hydroxide- gave mixtures of products from which l i t t l e or no dihydrolumazine could be obtained. This 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 2 - ' 1 ^ as w e l l as hydroxypteridine d e r i v a t i v e s 19 and lumazines. 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 l n e - 7 - d ( X L V I I l ) (XLVIII) The above .compound was prepared by c a t a l y t i c hydrogenation i n the same manner as the p r o t i o analogue (XLV) from 1 g. of TML using deuterium 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 procedures were al s o s i m i l a r y i e l d i n g 200 mg. (20$) of (XLVIII) as pale yellow needles. M.p. 3 0 2 - 3 0 4 ° (decomposition) C a l c u l a t e d f o r C q H ^ N ^ D ; C, 5 1 . 6 7 ; H, 6 . 2 6 ; N, 2 6 . 7 8 Found; C, 5 0 . 9 8 ; H, 6.O9.; N, 2 6 . 2 0 The p.m.r. spectrum o f t h i s compound i n D 2 0 ( T a b l e l l ) confirmed the l o c a t i o n of the deuterium atom at p o s i t i o n 7 , 51 as I n d i c a t e d by the l a c k o f the normal C-7 methyl-hydrogen s p l i t t i n g p a t t e r n . However, a small amount of c o u p l i n g along w i t h the appearance of a small quartet resonance at 4 . 5 8 6 i n d i c a t e d that deuterium was not f u l l y incorporated at p o s i t i o n 7 . The amount of the p r o t i o i m p u r i t y i n the sample was estimated at 4$ from a measurement of the area o f the quartet resonance as compared to that o f the N -8-methyl resonance. The a b s o r p t i o n data f o r t h i s compound proved i d e n t i c a l to that 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 abs o r p t i o n at 2240 cm ^ (C-D) as w e l l as the other absorption c h a r a c t e r i s t i c s present i n the i n f r a r e d spectrum of (XLV). 5 , 6 , 7 , 8-Te trahyd ro - 6 , 7 , 8 - t r irne thy 1 lumaz ine In 20 ml. of 5M a c e t i c a c i d , 0 . 4 2 g. o f 6 , 7 , 8 -t r i m e t h y l l u m a z i n e and 0 . 0 2 9 g. of platinum oxide were hydrogenated at atmospheric pressure and ambient temperature. A f t e r f o u r hours, hydrogen uptake ceased with the t o t a l uptake very c l o s e l y approximating the t h e o r e t i c a l value c a l c u l a t e d f o r two moles of hydrogen under the c o n d i t i o n s employed-.- 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 - 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 except that o n l y one 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 which the minimum amount of water 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. of a very pale yellow g r a n u l a r s o l i d . M.p. 2 6 2 - 2 6 5 0 (decomposition) 52 In s p i t e of precautions 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 the presence o f trace q u a n t i t i e s o f a i r (oxygen) to give' 7 , 8-dihydro - 6 , 7 . > 8-trImethyllumazine. Therefore, f u r t h e r 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 not attempted. On standing as a s o l i d , even under n i t r o g e n atmosphere, the tetrahydro compound was p a r t i a l l y converted over a period o f weeks to the 7 , 8-dihydro compound as i n d i c a t e d by a darkening o f the yellow c o l o u r o f the s o l i d and changes i n abso r p t i o n s p e c t r a . In aqueous s o l u t i o n at 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 at 272 and 302 mu. (absorption 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 matter o f hours on exposure to the a i r to 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 at 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 may be the hydroperoxide (XLIX) or the hydroxylated compound ( L ) , s i m i l a r to the intermediates proposed-^ i n the spontaneous o x i d a t i o n o f 2-amino-4-hydroxy - 6 , 7-dimethyl - 5 , 6 , 7 , 8 -tetrahydropteridine i n n e u t r a l or 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 to i t s i n s t a b i l i t y to 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 the tetrahydro precursor u n a t t r a c t i v e . 53 H | H 3 V :?» 3 o (XLIX; R = 0-OH) (L; R = OH) The assignment o f the 5 , 6 , 7 , 8 - t e t r a h y d r o s t r u c t u r e i s based on the s t r u c t u r a l assignment of other f u l l y reduced 2 103 p t e r i d i n e s ' and the f a c t t h a t , i n gener a l , the pyrazine 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 preference to 2 ^8 the p y r i m i d i n e r i n g . However, i t i s noted that the U.V. spectrum of the tetrahydrolumazine d i d not resemble that o f 5-amino - 4-methylamino - 2 , 6-dihydroxypyrimidine under the same c o n d i t i o n s . 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 of 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 sp e c t r a of the corresponding 4 , 5-diaminopyrimidines ( s h i f t e d 10 mu or l e s s to longer wavelengths) as would be expected from the l i n k i n g 38,40 of two amino groups by a dimethylene r e s i d u e . 146 I t has been reported that the f i r s t intermediate i n the o x i d a t i o n of 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 converted to the more s t a b l e 7 , 8-dihydro d e r i v a t i v e i n phosphate b u f f e r . The spectrum of the former dihydro d e r i v a t i v e at pH 6 . 8 i s remarkably s i m i l a r to that observed f o r the tetrahydrolumazine at pH 6 . 5 . However, the 54 5 ,6-isomer i s u n l i k e l y In the present case due to the t e r t i a r y n i t r o g e n atom at p o s i t i o n 8 . The p o s s i b i l i t y t hat the i n i t i a l spectrum i s a r e s u l t of a mixture o f a p a r t i a l l y o x i d i z e d intermediate such as (L) or a 5 , 8-dihydro-6 , 7 , 8 - t r i m e t h y l l u m a z i n e and the tetrahydrolumazine cannot be excluded. 55 Table Ia S p e c t r o s c o p i c data f o r prepared compounds 2 , 6-Dihydroxypyrimidines Compound 4-chloro-4- roethylamino-5- n i t r o s o - 4 -methylamino-5>-amino-4-methylamino-6 , 7 , 8 - t r i m e t h y l 6 , 7-diphenyl-8-methyl 6 , 8-dimethyl-7-oxo 7 , 8-dihydro-6 , 7 , 8 - t r i m e t h y l f o r comparison; 7 , 8-dihydro-6 , 7-dimethyl-Observed \ (mu. A max. Values ) l o g t ' 10 PH r e f . 218,282 266 266 3.91,4.01 4.25 4 .41 12.0 12.0 1.0 37 226,256,316 220,311 4.12,3.64,3.92 4.11 1.0 12.0 Lumazines 256,276,404 244,(268), 313,362 4.12,3.98,4.01 4.26,(3.77) 4.28,3.84 1.0 12.0 15,37, 101 268,292,426 243,282, 348,(426) 4.19,4.28,4.12 4.27,4.13, 4.01,(3-39) 7.0 12.0 15 283,328 284,346 4 .06,4 .11 4.05,4.13 1.0 7.0 79 236,274,352 280,316 230,284,318 4.01,4.17,3.66 4.19,3.79 4.33,4.05,3.75 1.0 5.0 10.0 ) 277,310 231,282,315 4.16,3.82 4.32,4.09,3.81 5.0 10.0 15 a - determined a t 25.0 i n aqueous media; values quoted from from r e f e r e n c e 15 were determined a t 20°'. ( ) - s h o u l d e r . 56 Table l b I o n i z a t i o n constants i n water at 2 5 . 0 ° C . Lumazine pK a b P%3H + Found L i t e r a t u r e Found . L i t e r a t u r e 6 , 7 , 8 - t r i m e t h y l 9.85+O.O6 9 . 9 0 + 0 . 2 1 5 - 0 . 8 5 + 0 . 1 1 5 9 . 8 6 + 0 . 0 6 7 9 6,8-dimethyl- 70 7 - oxb - 4 . 2 6 + 0 . 2 ' ^ - -79 1 3 . 2 0 + 0 . 1 6 . 7 - d i p h e n y l - 15 8 - methyl - 9.69+O .3 - O.36+O.I 7 . 8 - dihydro-6 , 7 , 8 - t r i m e t h y l 7 . 29+0 .03 - 2 . 8 6 + 0 . 0 5 f o r comparison; 7 , 8-dihydro-6 , 7 - d i m e t h y l - 8 - . 1 5 (^-hydroxyethyl) 7 -25+0 .09 b - reference 1 5 , 7 9 values determined at 2 0 ° C . . i n water 57 Table I I P.m.r. s p e c t r a l data f o r lumazine d e r i v a t i v e s Lumazine 6 , 7 , 8 - t r i m e t h y l Protons C 6-CH 3(s) C 7-CH 3(s) N8-CH 3(s) 7 , 8-dihydro-6 , 7 , 8 - t r i m e t h y l C 7-CH 3(d) C 6-CH 3(s) N 8-CH 3(sj C 7-H (q) Cy-CH^s)* C 6-CH 3(s) N 8-CH 3(s) 7 , 8 - d i h y d r o -6 , 7 , 8 - t r i m e t h y l -7-d o (p.p.m.) from e x t e r n a l TMS D 2 0 2 . 6 l 2 . 7 5 4 . 0 2 1 .19 2 . 0 1 3.03 4 . 1 4 TFAA 1.36 2.38 3.10 4.57 1.40 2.40 3.10 J(cps) 6.5+0.2 6.5+0.2 TFAA t r i f l u o r o a c e t i c a c i d c - run i n t r i f l u o r o a c e t i c acid-D^O ( l : 2 ) . s,d,q - s i n g l e t , doublet, quartet. * - broad s i n g l e t v/ith fine structure present. 58 Table I I I -cl " ' ' Chromatography o f prepared compounds Compound R~ In sol v e n t (A) f e (B) Found Reference Found .. Reference 2,6-Dihydroxypyrimidine 4 - c h l o r o - 0 . 6 9 0 . 6 7 3 7 0 . 6 7 . 0 . 6 7 3 7 4 - methylamino- 0 . 5 3 - 0 . 3 3 5 - n i t r o s o - 4 -methylamino- O .65 - 0 . l 6 5-amino -4 -methylamino ( b i s u l f i t e s a l t ) 0 . 7 8 - 0 . 4 2 Lumazine 6 , 7 , 8 - t r i m e t h y l 0 . 7 2 0 .7237 o.22 0 . 1 6 3 7 6 , 8 - d i m e t h y l - 7 0 7 0 7 - oxo 0 . 57 0 . 5 4 0 . 0 9 0 . 1 9 6 . 7 - d i p h e n y l -8- methyl 0 . 5 7 - 0 . 4 2 7 . 8 - dihydro- f 6 , 7 , 8 - t r i m e t h y l 0.46 - O .38 d -using aluminum oxide G on g l a s s m i c r o s l i d e s . e -reference 33 used Whatman's No. 1 paper; reference 79 employed Scheider and S c h u l l 2043 paper and butanol-5M a c e t i c a c i d (2:1) sol 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 chloride B - butan-1-ol-5M acetic acid (7 :3) P a r t I STABILITY OP THE LUMAZINE DERIVATIVES IN SOLUTION 59 A. I n t r o d u c t i o n In order 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 re p r e s e n t a n t l v e pyrimidines and a l l the lumazine d e r i v a t i v e s prepared i n t h i s work were tested under a v a r i e t y of pH c o n d i t i o n s . The compounds included 4-methylamino-2,6-dihydrox y p y r i m i d i n e , 5-amino-4-methylamino-2,6-dihydroxy-p y r i m i d i n e and the 6,7,8-trlmethyl-, 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 K L a b o r a t o r i e s ) of 5-aminobarbituric a c i d (2,4,6-trihydroxy-p y r i m i d i n e ) was a l s o t e s t e d . B. Experimental For each sample, 15 mg. of m a t e r i a l was d i s s o l v e d i n 2 ml. o f the appropriate b u f f e r system, c o n s i s t i n g o f 0.10M potassium hydrogen phosphate at pH 1.0, 7.0, 9-0 or 12.0, or i n 0.5N sodium hydroxide, and the s o l u t i o n thermostated at 45.0 - 0.2° i n a water bath. A t y p i c a l sample s o l u t i o n was prepared as f o l l o w s : Two mg. of 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 introduced by syringe 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 rubber 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 v i a l was wrapped i n aluminum f o i l to p r o t e c t the contents from l i g h t and then thermostated. At various times small a l i q u o t s were withdrawn w i t h a 50 m i c r o l i t r e s y ringe and spotted on two separate t h i n - l a y e r 60 p l a t e s prepared w i t h aluminum oxide G as sorbant. The p l a t e s were then immediately developed, one w i t h 3$ ammonium c h l o r i d e s o l u t i o n and the o t h e r w i t h b u t a n - l - o l - 5 M a c e t i c a c i d (7:3) s o l v e n t system, and the separated compounds de t e c t e d by i r r a d i a t i o n w i t h u l t r a v i o l e t l i g h t a t 254 mu. In the case o f n o n - f l u o r e s c i n g compounds, Wood's l i g h t a t 356 mu was used. O c c a s i o n a l l y , q u a l i t a t i v e a b s o r p t i o n s p e c t r a o were obtained a t 25 .0 w i t h a Bausch and Lomb Model 502 r e c o r d i n g spectrophotometer on a l i q u o t s d i l u t e d to s p e c t r o s c o p i c c o n c e n t r a t i o n s w i t h the same b u f f e r s o l u t i o n . The r e f e r e n c e c e l l o f the instrument c o n t a i n e d the b u f f e r s o l u t i o n o n l y . In t h i s manner, the progress o f each sample c o u l d be monitored u s i n g chromatographic and s p e c t r o s c o p i c techniques. A composite chromatogram f o r each sample was a l s o prepared by s p o t t i n g a l a r g e (20x20cm) p l a t e a t the same time as the s m a l l e r i n d i v i d u a l p l a t e s were prepared and d e v e l o p i n g the l a r g e r p l a t e i n butanol-5M a c e t i c a c i d at" the completion o f the experiment. The r e a c t i o n s o l u t i o n s were u s u a l l y monitored f o r -p e r i o d s r a n g i n g up to one month but i n some cases l o n g e r p e r i o d s were deemed nec e s s a r y to ensure completion o f the r e a c t i o n . To note the e f f e c t o f atmospheric oxygen, samples 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 s o l u t i o n s . not f l u s h e d w i t h n i t r o g e n beforehand and keeping the sample s o l u t i o n s under a i r i n c l o s e d c o n t a i n e r s . 6 l For the samples of 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 f values i n a given solvent system were separated mechanically from the developed t h i n -l a y e r p l a t e s and combined. Each component was eluted from the sorbant w i t h 20 ml o f methanol f o r e i g h t hours i n a Soxhlet apparatus. The elu a t e was then cooled, reduced to a volume o f approximately 5 nil on the 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 taken on the f i l t r a t e . Comparison s p e c t r a of 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 recorded. F o l l o w i n g t h i s , the f i l t r a t e was evaporated to dryness and an i n f r a r e d spectrum run on the r e s i d u e . The sampling technique c o n s i s t e d of adding 350 mg of anhydrous potassium bromide to the residue followed by 5 ml of anhydrous methanol to form a s l u r r y . The sol v e n t was removed on the r o t a r y evaporator and the s o l i d r e s i d ue d r i e d at 57°/l Torr f o r one hour, followed by p r e s s i n g of the s o l i d i n t o a p e l l e t i n the usual manner. C. Results With the exception o f 5-amino - 4-methylamino - 2 , 6 -dihydroxypyrimidine and DHTML under aerobic c o n d i t i o n s , a l l compounds tested proved to be r e l a t i v e l y s t a b l e i n both 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 . The samples of 5-aminobarbituric a c i d and 4-methylamino - 2 , 6-dihydroxy-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, except 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 decomposition on prolonged standing i n n e u t r a l 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 , examination of the a l k a l i n e s o l u t i o n s of the lumazines and the aminomethylaminopyrimidinediol i n d i c a t e d extensive decomposition a f t e r only two or three 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. With the exception o f the 5-amino - 4-methylamino - 2 , 6-dihydroxy-p y r i m i d i n e and DHTML samples, the degradation p a t t e r n of each sample p e r s i s t e d throughout the pH range tested w i t h i n c r e a s i n g r a t e o f decomposition towards the more a l k a l i n e r e g i o n s . Therefore, the r e s u l t s obtained f o r the a l k a l i n e degradation experiments probably represent those obtained 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 . For the two compounds mentioned above, r e s u l t s were obtained which i n d i c a t e d processes other 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 of the products 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 degradative behaviour of the various compounds tested are presented i n Figures 1 to 7. Fluorescence 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 values f o r the various compounds which were test e d and which were formed during the experiments are presented i n Table IV. 63 FIGURE 1 5-Amino - 4-methylamino-2,6-dihydroxypyrimidine i n 0.5N sodium hydroxide at 4 5 . 0 ° 0.4-1 0 . 3 0.21 0.1 1 0.0-0 8 2 2 1 2 0 T I M E I N H O U R S CD C B A KEY: AjB_, u n i d e n t i f i e d compounds; C, 5-amino-4-methylamino-2, 6-dihydroxypyrirnidine -64 FIGURE 2 7,8-Dihydro~6,7,8 trimethyllumazine i n 0.5N sodium hydroxide at 45.0 , anaerobic c o n d i t i o n s 0.4-R 0.3 4 f 0.2-1 0.1-0.0-0 iss> cS$ co cr- o G> o o o o> 3 t fl! t 6 25 75 120 260 460 °° TIME IN HOURS E D B A KEY: A, u n i d e n t i f i e d compound (dark c e n t e r ) ; B, 6,8-dimethyl-7-oxolumazine; C, 6,7j8-trimethyllumazine; D, 7,8-dihydro-6,7,8-trimethyllumazine; E, 5-amino-4-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 at pH 7.0/45.0°; anaerobic c o n d i t i o n s 0.4-0.3-0.2-0. 0.0-0 O o o o o o o o "I 5" T I M E ~S 10 3 0 N DAYS 60 0 0 E D B KEY: A, u n i d e n t i f i e d compound; B, 7-oxo - 6 , 8-dimethyllumazine; 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-amino -4-methylamino -2 ,6-d i h y d r o x y p y r i m l d i n e . 6 6 FIGURE 4 6 ,7-DiphenyI -8-methyllumazine i n 0.050M KgHPO^ at pH 12.0 and 4 5 .0°; anaerobic conditions <GBJ> 0 0 —r~ OO 2 2 3 120 TIME IN HOURS KEY: A, unidentified compound; B, unidentified compound; C, 6 , 7-diphenyl - 8-methyllumazine; D, 5-amino -4 -methylamino-2,6-dihydroxypyrimidine 67 FIGURE 5 6 , 7 , 8-Trimethyllumazine i n 0.5N sodium hydroxide at 4 5 . 0 ° 0.4 03 f 02-j 0.1-0.0-O C V O O O O V 0 O I ® (SJ) E) of el "5 5 Ij2 24 100 22 0 3 § 0 556 cb TIME IN HOURS KEY: A, u n i d e n t i f i e d compound; B, 6 , 8-dimethyl - 7-oxolumazine; 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-amino - 4-methylamino-2,6-dihydroxypyrimidine. 68 FIGURE 6 7 -0xo-6 ,8-dim'ethyllumazine i n 0.050M KpHPO^'at pH 12 .0 and 4 5 . 0 ° 0.44 0.14 <SX2 43 o 2 4 7 2 1 2 2 TI ME' I N H O U R S 6b KEY: A, 7-oxo-6£-dimethyllumazine; B, 5-araino - 4-methylamino - 2 , 6-dihydroxypyrimidine 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^ at pH 9.0 and 45.0°; anaerobic c o n d i t i o n s 0,4-0.3-0.2-0.1-o.o-\ ( e> o o o o o o "5 3 2'3 4'8 162 I 92 2c39 4-o0 <*> TIME IN HOURS-G F E D C B A , C KEY: A, u n i d e n t i f i e d compound; B, 6 , 8-dimethyl - 7-oxolumazine; C, dark spot accompanied by red s t r e a k , probably 5-aminobarbituric 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 pale blue f l u o r e s c e n t compound; G, 5-amino - 4-methylamino - 2 , 6-dihydroxypyrimidine 70 Table IV R values arid fluorescence c h a r a c t e r i s t i c s of various compunds on aluminum oxide 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 (B) solvent systems Compound 6 ,7 ,8-trimethyl 6,8-dimethyl-7- oxo 6.7- d i p h e n y l -8- methyl 7.8- dihydro-6 ,7 ,8-trimethyl 5-amino-4-methylamino-2,6-dihydroxy 4- methylamino-2 , 6-dihydroxy 5- a m i n o b a r b i t u r i c a c i d u n i d e n t i f i e d u n i d e n t i f i e d (A) R f (B) Lumazines 0.72 0.22 0.57 0.57 0.09 0.42 o.46 0.38 Pyrimidines 0.78 0.53 0.09 0.42 0.33 0.03-0.12 Other compounds 0.05 0.14 s 0.35,, 0.27° 10-methyl-2,4,6,8-tetrahydroxy-pyrimido h (5 ,4-g)pteridine (LI) 0.23 u n i d e n t i f i e d u n i d e n t i f i e d g - on Whatman's Mo. 17 paper. 0.16 PI uorescence under U.V. l i g h t green dark blue yellow-green 0.40 (Fig.7) 0.29 (Fig.4) b l u e - v i o l e t dark spot dark spot,blue (red s t r e a k ) blue (yellow) l i g h t blue blue pale blue sky blue h - Whatman's No. 1 paper. 85 71 Common to a l l lumazines tested i s the appearance of 5-amino -4-methylamino -2 , 6-dihydroxypyrimidine at various stages i n the r e a c t i o n scheme, e s p e c i a l l y i n basic s o l u t i o n s . That t h i s was the py r i m i d i n e was shown by comparison of R values i n two so l v e n t systems and fluorescence c h a r a c t e r i s t i c s w i t h a synthesized 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 ab s o r p t i o n spectroscopy a f t e r the Soxhlet e x t r a c t i o n procedure 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 degradation o f the pyrimidine i n b o i l i n g methanol over prolonged p e r i o d s . However, paper chromatography of the lumazine s o l u t i o n s followed by e l u t i o n o f the separated components wi t h water at ambient temperatures d i d show the spectrum of the py r i m i d i n e f o r the eluate o f the high Rf component.-The 5-amino -4-methylamino -2 , 6-dihydroxypyrirnidine was found to form the same two products throughout the pH range t e s t e d , as shown by comparison of R^ , values. 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 formation being i n h i b i t e d to some extent i n those experiments c a r r i e d out 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 i n .butanol - 5 M a c e t i c a c i d ) detected i n every chromatogram of 7 j 8-dihydro~ 6 , 7 , 8-trimethyllumazine was subsequently n i d e n t i f i e d as 6 , 7 , 8-trimethyllumazine. The presence o f the l a t t e r compound i s probably due, i n p a r t , to o x i d a t i o n o f 72 the dihydro compound during development of the t h i n - l a y e r p l a t e s . T a i l i n g (green fluorescence) of the spot assigned to the dihydrolumazine was observed and supports t h i s suggestion. In the aqueous b u f f e r s o l u t i o n s o f DHTML, the trimethyllumazine i s detected by absorption spectroscopy an hour a f t e r the s t a r t of the t e s t s under aerobic 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 at 45.0°. In a l k a l i n e media, the formation 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 of trace amounts of 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 of 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 of 6,7,8-trimethyllumazine r e a c t i n g to form one mole of 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 84 according to the mechanism of Rowan and Wood ' a r i s e s . 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 the f l a v i n was detected i n the chromatograms of TML. D. D i s c u s s i o n 1. 5-Amino-4-methylamino-2,6-dihydroxypyrimidine I t has been r e p o r t e d 8 ^ that 5-amino-4-(substituted)-amino-2,6-dihydroxypyrimidines form s e l f - c o n d e n s a t i o n products when s o l u t i o n s o f the pyrimldines are kept under non-reducing c o n d i t i o n s . These compounds have been 85 i d e n t i f i e d as pyrimido(5,4-g)pteridine d e r i v a t i v e s ( L l ) . The 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 synthesis of the 1 0 -^-hydroxy-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- j 3-hydroxyethylamino - 5-nitroso - 2 , 6-dihydroxypyrimidine. (LI) (LU) In the present study, the higher R f component i n the chromatograms of 5-amino -4-methylamino -2 ,6-dihydroxy-py r i m i d i n e e x h i b i t s blue fluorescence 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 paper), p r o p e r t i e s s i m i l a r to those reported f o r the 1 0-methyltetrahydroxypyrimido ( 5 , 4-g)pteridine ( L I ; R = CH^). 85 On the b a s i s of the above evidence, the high R^ component i s t e n t a t i v e l y assigned the above s t r u c t u r e ( L I ; R = CH^).-The remaining low R f component i s as yet u n i d e n t i f i e d due, i n p a r t , to i t s apparent low s o l u b i l i t y i n the water and methanol sol v e n t s used to e l u t e the chromatographically separated compounds. 74 2. H y d r o l y s i s of the Lumazlnes A once s u r p r i s i n g feature of 8-alkyllumazines 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 the 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 s p e c t r a of n e u t r a l s o l u t i o n s made a l k a l i n e , as w e l l as widely d i f f e r e n t i o n i z a t i o n constants among 6 , 7 - d i s u b s t i t u t e d homologues. 1 6 , 7 9 , 1 2 4 These anomalies were s a t i s f a c t o r i l y explained when i t was shown 3^' 7 9 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 at p o s i t i o n 7 . Thus, the hypsochromic s h i f t observed i n base was concluded to be a consequence o f the formation of the "hydrated" form ( L l l l ) i n which the hydroxy! group has entered the 7 - p o s i t i o n of the compound i n s p i t e of the s t e r i c hindrance o f f e r e d by the a l k y l group. ( L I I I ) Q u a n t i t a t i v e s t u d i e s on the aspects o f covalent O \ \ T O R 15 h y d r a t i o n o f 8 - a l k y l p t e r l d i n e s ' and 8-alkyllumazines have been published and the r e s u l t s o f these s t u d i e s reviewed. 15 Using 6 , 7 , 8 - t r i m e t h y l l u m a z i n e as an example, i t was found that on prolonged exposure to a l k a l i the 8-methyl - 6 , 7 -23 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 predominantly i n the ring-opened form (LIV) r a t h e r than as a s t a b l e hydroxylated anion ( L I I I ) . The ring-opened products were c h a r a c t e r i z e d by t h e i r absorbance 15 at 360 mu. This r e a c t i o n , the pyrazine ring-opening o f 8 - a l k y l p t e r i d i n e s and lumazines, has been shown to be 15,49 r e v e r s i b l e . (LIV) Prom the above evidence, the presence o f 5-amino-4-methylamino-2,6-dihydroxypyrimidine i n the a l k a l i n e s o l u t i o n s of TML and 6,7-diphenyl-8-methyllumazine can be explained i n terms of i n i t i a l formation of a 7-hydroxylumazine anion followed by r e v e r s i b l e ring-opening to the corresponding p y r i m i d i n e analogue ( L I V ) . The aminoalkylaminopyrimldinediol i s the h y d r o l y s i s product o f the ring-opened compound. Fu r t h e r support i s l e n t to t h i s scheme by the ob s e r v a t i o n that o n l y r e l a t i v e l y small amounts o f the p y r i m i d i n e d i o l are formed over prolonged periods 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 o f the two lumazines where they e x i s t predominantly i n the "quinonoid" form (LV). 76 R 0 (LV) The mechanism o f ring-opening o f the hydroxylated anion o f the 8-alkyllumazines has not been looked at In d e t a i l . I t Is seen from the s t r u c t u r e o f the p r e f e r r e d anion ( L I I I ) that there i s no apparent d r i v i n g force to ring-opening, which would r e s u l t i n the formation of an unfavorable d i a n i o n w i t h one o f the negative charges r e s i d i n g on the N-8 n i t r o g e n at some stage i n the process. This suggests that the ring-opening may proceed from a l e s s p r e f e r r e d 7-hydroxy anion such as (LVT). CH 3 H O (LVI) 77 In t h i s case , the nega t ive charge on the i n i t i a l r ing-opened spec ies (LIV) can then be s t a b i l i z e d i n the f o l l o w i n g manner; W . H C H 3 In the case where R = phenyl., f u r t h e r s t a b i l i z a t i o n o f the nega t ive charge by d e l o c a l i z a t i o n i n t o the C -6 phenyl group i s p o s s i b l e i n both ( L I I I ) and ( L I V ) . That the 7 - h y d r o x y - 8 - a l k y l l u m a z i n e an ion (LVI) i s capable o f ex i s t ence i n a l k a l i i s supported by the known p K a values o f s e v e r a l analogues and model 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 ~ pK = 6 . 9 1 , a 3,4 -dihydro-4 -hydroxy-3 -methylquinazol ine (LVI I ) 127 P K a = 7 .66) 78 ( L V I I ) Chromatograms o f the a l k a l i n e s o l u t i o n s o f 6 , 7 , 8 -t r i m e t h y l l u m a z i n e a l s o showed a b l u e f l u o r e s c e n t s p o t (R = 0 .09 i n , butanol-5M a c e t i c a c i d ) w h i c h was s u b s e q u e n t l y i d e n t i f i e d as 6 , 8 ~ d i m e t h y l - - 7 - o x o l u m a z i n e ( L V I I I ; R = R 1 = CH^) . Only upon p r o l o n g e d exposure to n e u t r a l and a c i d i c b u f f e r s was t h i s 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 . ( L V I I I ) 34 Jacobsen has r e c e n t l y shown t h a t q u l n o n o i d p t e r i d i n e s o f the type (LV; R = a l k y l , R 1 = ^ 3 ) ' i n c l u d i n g 6 , 7 , 8 - t r i m e t h y l l u m a z i n e , undergo o x i d a t i v e d e m e t h y l a t i o n a t a 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 to form the 79 corresponding 7-oxo compound when treated w i t h aqueous potassium 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 -lumazine, t h i s compound i s s l o w l y converted to 7~oxo -6 -m e t h y l - S - D - r i b i t y l l u m a z i n e 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 successive 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 at the s i t e of demethylation. ' In the present case, t h i s i s supported by the f a c t that o n l y small amounts of the oxo compound were produced from 6 , 7 , 8 -trimethyllumazine 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 quinonoid form (LV; R = R1 = CH 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 formation 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 pyruvate i n a l k a l i n e s o l u t i o n s o f TML. That a true displacement of the b i a c e t y l u n i t can occur was 49 shown by r e f l u x i n g 6 , 7-dimethyl - 8~J3-hydroxyethyllurnazine (LV; R = CH 2CH 20H, R* = CH"3) w i t h «C-oxobutyric a c i d under n i t r o g e n at pH 13 and I d e n t i f y i n g the r e a c t i o n product as the 6-ethyl - 7-oxolumazine ( L V I I I ; R = CH 2CH 20H, R» = e t h y l ) . This experiment a l s o demonstrates the r e v e r s i b l e r i n g -128 opening of the 8 - a l k y l l u m a z i n e s . I t has been shown however, that b i a c e t y l , r a t h e r than being 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 condensation r e a c t i o n i n aqueous a l k a l i to y i e l d a mixture of products. Only one of 8o these p r o d u c t s , - a c e t y l - c£,tf -dihydroxy-c£-methylvaleric a c i d ( L I X ) , c o u l d c o n c e i v a b l y condense with the 5~amino~4-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 some manner to g i v e the 7-oxo-lumazine but there i s no evidence to support t h i s . In any case, b i o c h e m i c a l s t u d i e s by P l a u t 8 3 ' 1 2 ^ u s i n g l a b e l l e d m a t e r i a l s have shown that o x i d a t i v e removal o f the methyl group a t p o s i t i o n 7 occurs i n p r e f e r e n c e to displacement o f b i a c e t y l by pyruvate i n 6 , 7 - d i m e t h y l - 8 - s u b s t i t u t e d -lumazlnes. C H 3 C H 3 8 i o | o , 0 = C — C - C H 2 - C - C 0 2 H . O CH H (LIX) 3 I t i s r e p o r t e d ^ that 7 , 8-dihydro - 6 , 7~dimethyl-8- j3 -hydroxyethyllumazine i s r a p i d l y converted i n hot a l k a l i to 6 , 7-dimethyl - 8 - ^ - h y d r o x y e t h y l l u m a z i n e as shown by paper chromatography and u l t r a v i o l e t s p ectroscopy. Thus, 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 o x i d a t i o n observed f o r 7 , 8 - d i h y d r o 6 , 7 , 8-trimethyllumazine i s c o n s i s t e n t w i t h the p r o p e r t i e s r e p o r t e d f o r the dihydro - 8 - J 3-hydroxyethyl-lumazine analogue and s i m i l a r reduced p t e r i d i n e s . ->>•_-> As w e l l as the o x i d a t i o n process, the a l k a l i n e 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 i u m a z i n e e x h i b i t e d some unusual f e a t u r e s w i t h r e s p e c t to h y d r o l y s i s . A l a r g e o q u a n t i t y o f a blue f l u o r e s c e n t component with R^ values 81 s i m i l a r to those o f 5-amino -4~methylamino~2,6-dihydroxy-p y r i m i d i n e was de t e c t e d i n 0 . 5N sodium hydroxide. However, the u l t r a v i o l e t s p e c t r a o f both the f i n a l degraded a l k a l i n e s o l u t i o n o f DHTML and t h a t o f the e l u t e d component In water e x h i b i t e d absorbancies a t 350 mu and 280 mu (approximate absorbance r a t i o = 3-5:1 r e s p e c t i v e l y ) , d i f f e r e n t from t h a t o f 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 . The nature o f t h i s product i s , as y e t , not w e l l e s t a b l i s h e d but i t may be the ring-opened s p e c i e s (LX) i n which f u r t h e r h y d r o l y s i s to g i v e 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 be expected to be d i f f i c u l t . ( L X ) l 8 b I t i s r e p o r t e d t h a t 5 , 6-dihydro - 7-hydroxy-p t e r i d i n e i s i n s t a n t l y converted i n t o 5~arnino -4-carboxy-me t hy 1 am ino py r im-i d ine by-bo -i-l-Ing- no-rma-l—s o d-i-um—hyd-ro x-i-d e-; t h i s r e a c t i o n i s r e v e r s e d by one hour o f r e f l u x i n g w i t h normal h y d r o c h l o r i c a c i d . Thus, i t might be expected that (LX) would undergo r e v e r s i b l e r i n g - c l o s u r e to the 7 , 8 -dihydro lumazine and have chromatographic c h a r a c t e r i s t i c s d i f f e r e n t .to those o f 5-amino - 4-methylamino - 2 ,6-dihydroxy-p y r i m i d i n e . These e x p e c t a t i o n s were not r e a l i z e d . 82 The p o s s i b i l i t y of a t t a c k by hydroxide i o n on the f u s e d - r i n g double bond i n DHTML to form a hydroxylated d i a n i o n ( L X I ) , followed by ring-opening to the S h i f f base ( L X I I ) , w i t h subsequent production of 5-aminobarbituric a c i d i s apparently e l i m i n a t e d by the absence of the l a t t e r 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 dihydro lumazine. However, the d e t e c t i o n of a small amount of a b l u e - f l u o r e s c i n g component w i t h R f values s i m i l a r to those of 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 prolonged standing i n d i c a t e s t h i s pathway may play a minor r o l e i n the h y d r o l y s i s o f the 7 , 8-dihydrotrimethyllumazine, CH 3 CH-(LXI) CH-i Ayr r©"T J o-(LXII) The a m i n o b a r b i t u r i c a c i d was not detected i n n e u t r a l or a c i d i c s o l u t i o n s of the dihydrotrimethyllumazinev Several analogues to (LXI) are known ' or 131 are p o s t u l a t e d to e x i s t . A study o f the h y d r o l y t i c behavior of 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 that the carbinolamine ( L X I I I ) i s the i n i t i a l product of hydroxide 59 a t t a c k . S i m i l a r hydroxylated compounds are formed i n the 83 a u t o x i d a t i o n of t e t r a h y d r o p t e r i d i n e s and d i h y d r o 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 X I I I ) The presence o f 7-oxo - 6 , 8-dimethyllumazine.in the l a t t e r stages 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 demethylation 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. In n e u t r a l and a c i d i c media, prolonged r e a c t i o n i s required before s i g n i f i c a n t q u a n t i t i e s of the oxo compound can be detected. 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 e x h i b i t e d by the 7-oxo - 6 , 8-dimethyllumazine anion (LXIV) i s a r e s u l t of the well-known amide resonance, which i n t h i s case strengthens the C ^ - N Q bond by p a r t i c i p a t i o n of the N - 8 lone-p a i r e l e c t r o n s as In (LXV). (LXIV) (LXV) 84 D e l o c a l i z a t i o n o f the negative charge as i n d i c a t e d i n (LXIV) 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^ double bond to n u c l e o p h i l i c a t t a c k . The extent 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 strong a c i d i t y (pK a = 4.26) of the 7-0x0 - 6 , 8-dimethy1lumazine. 6 , 7-Diphenyl - 8-methyllumazine i n aqueous media did not form the corresponding 6-phenyl -7-oxo -8-methyl-lumazine as shown by the absence of a component wit h s p e c t r a l 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-phenyl - 8-methyl - 2 , 7-dioxopteridlne. 34 I t has been reported that o x i d a t i o n of 6 , 7-diphenyl - 8 -methyl - 2-oxopteridine 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 corresponding 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 experiment, aqueous s o l u t i o n s o f the lumazines, 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 f a i r l y r a p i d decomposition under the c o n d i t i o n s employed. Slower decomposition of the compounds, w i t h the exception of DHTML, occurs i n n e u t r a l and weakly a c i d i c media, e s p e c i a l l y when kept under n i t r o g e n atmosphere. The dihydrolumazine shows a p r e d i s p o s i t i o n to o x i d a t i o n i n aqueous media as w e l l as a ready tendency to form h y d r o l y s i s products. The nature o f these p a r t i c u l a r h y d r o l y s i s products Is not w e l l e s t a b l i s h e d . \ P a r t I I HYDROGEN-DEUTERIUM EXCHANGE 85 A. I n t r o d u c t i o n While examining the proton magnetic resonance (p.m.r.) spect r a of 6,7,8-trimethyllumazine and 7,8-dihydro-6,7,8-trimethyllumazine the amplitude o f the peak assigned to the downfield C-methyl group o f both of the above compounds was observed to decrease w i t h time i n a c i d i c and b a s i c deuterium oxide media. That the decrease i n peak amplitude was due to exchange of the methyl group protons was confirmed by the a d d i t i o n o f H 20 to D 20 s o l u t i o n s o f the compounds w i t h subsequent regeneration of the peak amplitude of the exchanged methyl group i n p r o p o r t i o n to the amount of water added. Only a s i n g l e methyl group In 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 rates o f these r e a c t i o n s . I t was then deemed of i n t e r e s t to i n v e s t i g a t e the exchange process more f u l l y . In order 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 obtained 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 are drawn i n c o r p o r a t i n g the l i g h t isotope o f hydrogen. For l a b i l e hydrogens, such as the one attached to N-3 i n trimethyllumazine or the ones present i n the phosphate b u f f e r species H^POjj, H 2 P O 4 - , e t c . , i t may be assumed that they are r e p l a c e a b l e w i t h deuterium where 86 experiments have been c a r r i e d out 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 to the a t t a c k of the organic s u b s t r a t e by e i t h e r hydronium i o n (h^O*) or deuteronium i o n (D^ O"*") depending upon the i s o t o p i c c h a r a c t e r of the aqueous medium. To comply w i t h the terminology 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 the f i e l d o f p.m.r. spectroscopy, the term "proton" w i l l 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 s t u d i e s P.m.r. spect r a and i n t e g r a l s were obtained w i t h a Var i a n HA-100 100 megahertz spectrometer. The deuterium oxide s o l u t i o n s used In the exchange study were thoroughly flushed w i t h h i g h - p u r i t y n i t r o g e n before use. Care was taken to p u r i f y both the DgO and sodium hydrogen phosphate b u f f e r i n order to remove traces of heavy metal i m p u r i t i e s . The Dv>0 was d i s t i l l e d f i r s t from potassium permanganate and then r e d i s t i l l e d . Commercial disodlum phosphate was d r i e d at 110° and then r e c r y s t a l l i z e d once from" a saturated s o l u t i o n i n heavy water which provided f o r exchange of the b u f f e r protons. The absence o f heavy metal c a t i o n s i n the DgO and phosphate s o l u t i o n s was confirmed by the 132 Eriochrome B l a c k T i n d i c a t o r t e s t . 87 The exchange reactions were car r i e d out on approximately 0.1M DgO solutions of TML and DHTML i n 0.10M phosphate buffer adjusted to the desired pH with 10$ D 2 S O 4 . The pH of the prepared solutions was measured on a Radiometer Model 26 instrument with a glass-calomel electrode t r a i n . The measured pH was not corrected to give the corresponding pD values. Solvent systems at H 0 = - 0 . 3 7 and pH 8 . 2 were prepared from the appropriate mixture of D20-anhydrous t r i f l u o r o a c e t i c acid' 1' 3 3 and D 2 0-pyridine respectively. In most cases the i o n i c strength of the buffer was adjusted to a given value ( 0 . 4 2 ) by the addition of the appropriate quantity of sodium ch l o r i d e . For each k i n e t i c run, 0 . 3 5 - 0 . 4 5 ml. of the desired pH-adjusted nitrogen-flushed buffer solution was introduced into a rubber-capped v i a l containing 7 - 9 mg. of sample under nitrogen atmosphere by means of 1 ml. syringe. After shaking for approximately 20 seconds to a f f e c t solution, the contents of the v i a l were taken up with the same syringe and injected into a rubber-capped N.M.R. tube which had previously been f i l l e d with nitrogen gas. In some cases, e s p e c i a l l y i n the more basic solvent systems, the sample did not dissolve completely. In these instances, small amounts of undissolved sample were removed by f i l t e r i n g the contents of the v i a l under a stream of nitrogen i n a device consisting of a 0 . 2 5 ml. syringe b a r r e l f i t t e d with a syringe needle which penetrated the rubber cap on the N.M.R. tube. The ba r r e l of the syringe-contained a small pad of DgO-washed glass wool. The capped 88 tube c o n t a i n i n g the f i l t e r e d sample s o l u t i o n v/as then removed f o r t e s t i n g . During p r e p a r a t i o n procedures, the sample s o l u t i o n s were thermostated at 3 0 . 0 - 0 . 2 ° i n a constant temperature bath. P.m.r. s p e c t r a and peak i n t e g r a l s of both lumazines were recorded at 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 spectrometer employed, the DgO sol v e n t peak (HOD) was used as the i n t e r n a l reference and the instrument l o c k s i g n a l . The temperature o f the spectrometer probe i n t o which the sample tubes were placed was 3 1 . 4 ° , as measured w i t h a Honeywell Model 134 No. 2732 potentiometer and Chromel-Alumel thermocouple (reference j u n c t i o n at 0°C.). 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 using a constant temperature bath. A f t e r each measurement during a k i n e t i c run, the dihy d r o t r i m e t h y l l u m a z i n e sample tubes were thermostated at 3 0 . 0 - 0 . 2 ° i n a water bath. The exchange of the t r i m e t h y l -lumazine occurred at a r a t e such that the r e a c t i o n could be monitored i n i t s e n t i r e t y , without removing the sample tube from the probe o f the spectrometer. Therefore, the exchange r a t e data reported f o r t h i s compound i s at 31.4°.. 2. Treatment o f the data i n exchange experiments For both TML and DHTML, the exchange rates were determined by comparing the r a t i o o f peak areas o f the 89 exchanging C-methyl group and non-exchanging methyl group w i t h time. The measurement o f peak area was accomplished .by the usual p.m.r. i n t e g r a t i o n technique and was u s u a l l y done i n d u p l i c a t e . For the f a s t e r r e a c t i o n s o f TML, the i n t e g r a l s o f the non-exchanging peaks were determined once i n i t i a l l y and subsequently only the i n t e g r a l o f the exchanging peak measured at 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 constants (k^) and h a l f - l i v e s (t±)- were obtained from 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 equation (2 ) : 2 . 3 0 3 1 o g 1 0lm - k j t (2) [(M/R)^ - (M/R) Q where Im = (M/R)^ - (M/R). M = average area o f exchanging peak, R = average area o f reference (non-exchanging) peak(s), and the s u b s c r i p t s r e f e r to the r e a c t i o n time. U s u a l l y ( M / R ) Q = 1 .0 and ( M / R ^ - ^ O to 0 .03 w i t h i n the l i m i t s o f accuracy a f f o r d e d by nuclear magnetic resonance measurements. The h a l f - l i f e o f a given exchange process i s determined from the measured r a t e constant according to equation ( 3 ) : t A = 2 . 3 0 3 1 o g 1 0 2 (3) 2 k j 1 v In p l o t t i n g o f the exchange data the method o f least- s q u a r e s was employed to o b t a i n the best l i n e . The 90 equations used f o r t h i s purpose are those given by J a f f e ! ^ For both 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 reference as w e l l as the u p - f i e l d C-methyl peak i n the case of 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 l o s s o f c o u p l i n g and peak area of the u p - f i e l d doublet i n the l a t t e r stages o f the exchange i n the case of the d i h y d r o t r i m e t h y l -lumazine precludes 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 , treatment o f the exchange data proved a p p l i c a b l e to at l e a s t 75$ to 85$ of the r e a c t i o n f o r both compounds. The " i n f i n i t y " measurement was u s u a l l y repeated s i x to e i g h t times to ensure accuracy i n the ( M / R ) Q Q term o f equation ( 2 ) . C. Results 1. Trimethyllumazine (TML) a. Assignment of the exchanging group I t was observed that the amplitude o f the down-f i e l d C-methyl peak i n the p.m.r. sp e c t r a o f trimethyllumazine i n b u f fered DgO s o l u t i o n s decreased w i t h time due to exchange of the methyl group protons 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 of the exchangeable center to the C-7 methyl group i s supportable on the f o l l o w i n g grounds: i . no completely conjugated or d e l o c a l i z e d s t r u c t u r e can be invoked to account f o r an exchange of protons at the C-6 methyl group. i i . i n t r i f l u o r o a c e t i c a c i d w i t h e x t e r n a l t e t r a m e t h y l -91 FIGURE 8 N 8 - C H 3 ? V . ' H j C ^ N v ^ l v k ^ O C 7 - C H 3 1 T 1 • H 3 c \ \ N H 1 . 0 r t i C 6 - C H - ll, ll b) c) 1 1 1 d) J 1 1 1 - v — J i • : . i . ;—. i . r . 4 5 0 4 C O 3 5 0 - 3 0 0 2 5 0 .*.•'.,'."' c.p.s. " a) 0.1M TML i n unbuffered D20 (HOD solvent peak at S 4.68 ) b) a f t e r 2.5 min. i n pH 2.0 0.10M Na2DP04 at 3 1 . 4 ° c) a f t e r 13 min. d) a f t e r 27 min. • e; a f t e r 75 min. 92 s i l a n e as r e f e r e n c e , the down-field C-methyi peak i n the p^m.r. sp e c t r a o f a s e r i e s o f c l o s e l y r e l a t e d p t e r i d i n e d e r i v a t i v e s , I n c l u d i n g 6 , 7 , 8-trimethyl -2-amino-/f-pteridinone , has been c o n s i s t e n t l y assigned to the C-7 methyl g r o u p . r j h e r e i a t i v e p o s i t i o n s o f the C-metbyl s i g n a l s are as expected a f t e r c o n s i d e r a t i o n of the p.m.r. spectrum o f p t e r i d i n e i t s e l f 1 ^ and the known p o l a r i z a t i o n o f bonds i n simple p t e r i d i n e d e r i v a t i v e s . i i i . 7 , 8 - d i h y d r o - 6 - d 3 - 7 , 8 - t r i m e t h y l l u m a z i n e , formed by the exchange of the C -6 methyl protons of DHTML (see l a t e r R e s u l t s ) can be o x i d i z e d by a i r o r D 20 2 i n unbuffered D20 to 6 - d 3 ~ 7 , 8 - t r i m e t h y l l u m a z i n e . Comparison o f the p.m.r. spectrum of t h i s l a t t e r compound w i t h that o f trimethyllumazine exchanged i n the normal way confirms the assignment o f the C-7 methyl as the exchanging group. As expected, the C-7 methyl group o f the 6 - d 3-trimethyllumazine begins to exchange i t s protons as w e l l upon formation from the deuterated dihydro p r e c u r s o r . b. pH-rate p r o f i l e For TML, the exchange o f the C-7 methyl protons 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 the buff e r e d D20 media w i t h the slowest exchange rates observed i n the r e g i o n of pH 4. The exchange r a t e o f the lumazine i n unbuffered D20 (pH 6 . 5 ) was extremely slow, w i t h a h a l f - l i f e o f approximately a week and a half.-The exchange process was studied i n the pH range -0.4 to 8.0. Except f o r the very s t r o n g l y a c i d i c s o l u t i o n s 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 predominantly as the n e u t r a l species i n t h i s r e g i o n . A p l o t of the data 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 i n Figure 9 . The 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 r a t e constants f o r the exchange r e a c t i o n at various pH i n 0.10M NagDPOj^ at 3 1 . 4 ° and i o n i c s t r e n g t h 0 . 4 2 are c o l l e c t e d i n Table V. The pH values quoted have not been correc t e d to the corresponding 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 included to i n d i c a t e the degree of l i n e a r i t y o f the p l o t t e d data from which the r a t e constants were obtained. E r r o r s i n the r a t e constants were found to range from 1 to 5$ as estimated by comparison of i d e n t i c a l runs and depending upon the number of data p o i n t s a v a i l a b l e i n determining the slope o f the r a t e p l o t l i n e . A p l o t o f the pseudo f i r s t - o r d e r r a t e constants ( k l ) versus pH produces the curve i l l u s t r a t e d i n Figure 10. A p l o t of the logarithms o f the r a t e constants a g a i n s t pH gives the corresponding pH-rate p r o f i l e (Figure 11). The co-ordinates o f t h i s l a t t e r graph are arranged to i n d i c a t e the slowest r a t e s o c c u r r i n g at the minimum of the curve. The 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 represents the t r i f l u o r o a c e t i c acid-D 20 s o l v e n t system at H Q = -0 . 3 7 . The pH-rate 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 which are c a t a l y z e d both by hydronium ions (H 30 +) and hydroxide ions and i n which the uncatalyzed r e a c t i o n i s r e l a t i v e l y 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 of 6 , 7 , 8 -trimethyllumazine i n DgO in the pH range -0.4 to 8.0, 0.10M Na2HP04, u = 0 . 4 2 , T = 31.4°C. PH l O 3 ^ (min. - 1) t i (min.) 2 r - 0 . 4 1 350.8 2.0 0 .992 1.0 127.5 5 .4 0 .996 2.0 4 l.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 fast - - -i - t r i f l u o r o a c e t i c acid-D p 0 at H 0 = - 0 . 3 7 96 FIGURE 10 TML EXCHANGE KINETICS FIGURE 11 98 on the acid side of n e u t r a l i t y where the hydroxide ion concentration Is n e g l i g i b l e . Therefore, the results indicate that the exchange reaction i s subject to c a t a l y s i s by species other than or as well as hydronium and hydroxide ions. The lack of a unit slope for either straight l i n e portion of the pH-rate p r o f i l e also speaks against s p e c i f i c c a t a l y s i s by e i t h e r of these ions. That i s , the exchange reaction i s l i k e l y subject to general acid and base c a t a l y s i s by the phosphate buffer species . The observed f i r s t - o r d e r rate constant at a given pH within the range examined i s probably a composite value as indicated by equation (4): k l = ko + V + KHA HA + kA_ A" + K A " 2 " A " 2 " _ _ _ where k Q = uncatalyzed exchange rate constant, k +, k , etc. are s p e c i f i c rate constants r e f e r r i n g to species indicated by the subscripts, and HA = H^POjp A" = H 2 P04" and A - 2 = HPO^"2. A study of the e f f e c t of buffer concentration on — the exchange rate was undertaken to test this scheme. c. E f f e c t of buffer concentration on exchange rate The exchange of the C - T methyl protons i n DgO solutions buffered at two pH values was examined. The buffer concentration was varied over the range 0.015M to 0 . 1 2 5 M with the ionic strength maintained at a constant value by the addition of the appropriate amount of sodium . 9 9 c h l o r i d e . Above 0.125M b u f f e r c o n c e n t r a t i o n the t r i m e t h y l -lumazine tended to p r e c i p i t a t e during the course o f measurements which thereby v i t i a t e d the 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 experiments are 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 the pseudo f i r s t -order r a t e constants and b u f f e r c o n c e n t r a t i o n at 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 of the 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 value estimated f o r the uncatalyzed r a t e constant, e s p e c i a l l y at 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 determining the magnitude o f the rat e constant i n the a c i d i c Bv,0 media. At pH 4 . 5 , where the c a t a l y t i c e f f e c t of the hydronium i o n on the exchange i s l i k e l y small i n comparison to the phosphate s p e c i e s , a p l o t o f the negative logarithms o f the observed r a t e constants 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 slope 1.01 (Figure 1 3 ) . A s i m i l a r p l o t at pH 2.0 gives a s t r a i g h t l i n e but of slope 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 slope i s not s a t i s f a c t o r i l y explained as y e t . I f l e s s weight i s placed on the value of the data 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 correct e d f o r a small pH change) a l i n e slope approaching 0 . 9 can be a t t a i n e d . The s p e c i f i c r a t e constants k-^ +, k ^ , k^_ and k A_2 are estimated from the r e s u l t s of the 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 at the two pH values and rates at 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 ra t e o f 6 , 7 , 8 - t r i m e t h y l l u m a z i n e at T = 3 1 . 4 ° C , u = 0.42 Concentration NagHPO^ 10Jk;j_ ( m o l e s / l i t r e ) (min.-l) pH 2 . 0 pH 4 . 5 0.015. - 3 . 5 0 . 0 2 5 1 6 . 8 J -0 . 0 5 0 24 . 0 1 3 . 9 0 . 0 7 5 3 2 . 7 18.4 0 . 1 0 0 4.1.9 2 5 . 7 0 . 1 2 5 46 .3 2 8 . 6 j - co r r e c t e d f o r small pH change observed when sample added to the 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 •LOG10K 2.1 SLOPE =1.01 1.2 0.8 ~ l 1 r i.2 ~ L O G 1 0 [ BUFFER ][ 2.0 103 FIGURE 14 TML EXCHANGE KINETICS RELATION BETWEEN RATE CONSTANT AND CONCENTRATION OF CATALYTIC SPECIES AT pH 2.0 0 - L O G 1 0 [ A ] A = H 3 0 + H 3P0 4 + H 2P0 4" " - — • - _ 104 i n d i v i d u a l pH. In the ensuing c a l c u l a t i o n s the value o f the uncatalyzed r a t e constant, k Q, i s assumed to b e 1 s m a l l i n comparison to the other constants. This Is a reasonable assumption i n view o f the very slow r a t e o f exchange observed f o r TML i n unbuffered D g0 at pH 6 . 5 . The value o f ky+ may be obtained by e x t r a p o l a t i o n o f the l i n e f o r the b u f f e r c o n c e n t r a t i o n dependency at pH 2.0 (Figure 12) to zero b u f f e r c o n c e n t r a t i o n . This o p e r a t i o n i s equ i v a l e n t to reducing equation (4) to k l = V [ H 3 0 + ] (5) The concentrations o f the various b u f f e r species at a given pH can 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 l 4 l 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 value (99-3$) o f the t o t a l b u f f e r c o n c e n t r a t i o n at pH 4 . 5 , the e f f e c t o f the small amount o f - 2 HPO4. present may not be i n s i g n i f i c a n t . The values o f k^-and k ^ - 2 are obtained t h e r e f o r e by a p p l i c a t i o n o f the f o l l o w i n g equations: k l ( 6 ) = V [ A " ] 6 + V 2 [ A " 2 ] 6 < 6 > . % ( 4 . 5 ) = k A- [ A - ] 4 # 5 + k A - 2 ^ A - 2 J 4 < 5 (7) where the s u b s c r i p t s r e f e r to the pH at which the co n c e n t r a t i o n o f the appropriate b u f f e r species was c a l c u l a t e d . The r a t e constant k-j_ i s that value at a given 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 obtained by-s u b s t i t u t i o n o f the values of the other constants determined according to the above methods i n t o equation (4) at a given pH. The values c a l c u l a t e d f o r the s p e c i f i c c a t a l y t i c constants are as f o l l o w s : k H+ = 1 .2 1 .mole~-,-min.-1 kHA = °"''41 l' m°le~ 1min.~ 1 k^_ = 0 . l 4 l.mole ''"min. ^ k A - 2 = 5 2 . 9 l.mole" 1min." 1 A pH-rate p r o f i l e may be constructed which c l o s e l y approximates the exp e r i m e n t a l l y determined p r o f i l e i l l u s t r a t e d i n Figure 10 by using the above values of the s p e c i f i c r a t e constants and ap p l y i n g equation ( 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 proton to or from the c a t a l y s t molecule, some r e l a t i o n s h i p between the e f f e c t i v e n e s s o f the 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 to be expected. Bro'nsted suggests that a s p e c i f i c c a t a l y t i c constant, k , should be r e l a t e d to the a d i s s o c i a t i o n constant K a of an a c i d f o r a given r e a c t i o n l 4 2 c a t a l y z e d by a s e r i e s of acid s according to (8); k a = Ga< or l o g k a = dClog K + C (8) where C, G a andcCare constants, the l a t t e r u s u a l l y being l e s s than u n i t y . 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 constant, kft, Is r e l a t e d to the d i s s o c i a t i o n constant K D by (9); k b = G b K f or l o g k b = p i o g K b + C» (9) where G b, C' and j3 are constants, the l a t t e r u s u a l l y being l e s s than u n i t y . In the present case, a l a c k o-f s u f f i c i e n t data precludes a q u a n t i t a t i v e treatment, i n terms of the Brc/nsted r e l a t i o n s . A l s o , i t i s not c e r t a i n whether the HgPOi).- species i s behaving as an a c i d or base or whether i t i s amphoteric. A comparison o f the values 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 i n d i c a t e s that J3is probably l a r g e r than c£ . That i s , 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 species present i n s o l u t i o n . The o b s e r v a t i o n that the l i n e i n Figure 11 f o r the c a t a l y s i s i n more ba s i c regions i s steeper than the corresponding l i n e i n the a c i d regions supports t h i s . In s o l u t i o n s more a c i d i c than H Q = -0 . 4 and more basi c than pH 6 i n 0.10M 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 techniques. In any case, 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 exchange of trimethyllumazine i n the pH range examined was not a c c e l e r a t e d by the a d d i t i o n o f small amounts 107 o f C u + 2 or Z n + 2 to the b u f f e r s o l u t i o n s . I t i s known that n e u t r a l "quinonoid" p t e r i d i n e s and lumazines, 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 avi d l y with the cations of 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 the exchanging group The amplitude decrease i n the s i n g l e t resonance o f the down-field 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 to exchange o f the C -6 methyl group protons w i t h s o l v e n t . The assignment to t h i s p a r t i c u l a r group i s based on the f o l l o w i n g grounds; i . on chemical grounds, no p l a u s i b l e s t r u c t u r e can be Invoked to account f o r exchange o c c u r r i n g a t the C-7 and N-8 methyl groups, and i i . the p.m.r spectrum o f DHTML e x h i b i t s a non-exchanging doublet resonance. This resonance has been assigned to the C-7 methyl group on s t r u c t u r a l grounds. The exchange p a t t e r n o f the dihydro lumazine d e r i v a t i v e Is i l l u s t r a t e d i n Figure 15. Observations o f up to two weeks d u r a t i o n i n d i c a t e that the proton at p o s i t i o n 7 may a l s o be exchanging very s l o w l y as i n d i c a t e d by small changes In the C-7 methyl-hydrogen c o u p l i n g p a t t e r n and decrease i n the quartet peak amplitude. The l a t t e r decrease can on l y be observed i n the t r i f l u o r o a c e t i c acid-D 20 s o l v e n t system as the quartet 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 the HOD 108 . FIGURE•15 H p N o) C f - H " C H j - c6-- C H S 0- i C r - C H 3 I , >5 c . p . s . 1 . JL . . J — I — : L . ^ . J 1 1 1 , 1 1 1 I JL . J — i 1 1 l v — i 1 1  1 L 5 C O 4 0 O 3 0 0 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 .37) T = 30.0°, 7 minutes a f t e r s t a r t of 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 In the buffered DgO systems. On prolonged standing In D 2 0 s o l u t i o n , some o x i d a t i o n and degradation o f the samples i s a l s o observed. In s p i t e o f these d i f f i c u l t i e s however, the exchange r e a c t i o n can u s u a l l y be followed to completion before much decomposition occurs. b. pH-rate p r o f i l e The exchange r e a c t i o n was studied i n the pH range - 0 . 4 to 8 . 2 . The dependence o f the exchange r a t e on the a c i d i t y o f the DgO media e x h i b i t e d some unusual 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 behaviour i n t h i s r e g i o n . For. example, the data i n the pH range - 0 . 4 to 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 order over approximately 15$ of the r e a c t i o n followed by a pseudo f i r s t - o r d e r r a t e f o r the remainder. The p.m.r. spectr a of the DHTML samples i n t h i s pH reg i o 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 broadening o f the C -6 methyl resonance which tended to become l e s s broad as the exchange proceeded. As w e l l as the broadening o f the C -6 methyl resonance, which a l s o s h i f t s downfield w i t h i n c r e a s i n g a c i d i t y of the b u f f e r , the appearance of a small peak located very c l o s e to the p o s i t i o n o f the C -6 methyl resonance was noted. 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 the medium and appears to decrease only very s l i g h t l y i n amplitude during the time used to monitor the exchange r e a c t i o n . This 110 peak ( s i n g l e t ) Is not detected i n those b u f f e r sytems i n the pH range 3 . 5 to 7 . 0 . That t h i s peak was not the r e s u l t of an i m p u r i t y i n the b u f f e r s o l u t i o n s was confirmed by running s p e c t r a o f these s o l u t i o n s before 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 to 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 data f o r the e n t i r e r e a c t i o n and the p.m.r. spect r a of the samples d i d not e x h i b i t an i n i t i a l broadening of the C -6 methyl resonance. The two types of r a t e p l o t s at representantive pH are i l l u s t r a t e d i n Figures l 6 and 1 7 . The exchange r a t e o f DHTML i n unbuffered DgO (pH 6 . 5 ) was found to be extremely slow w i t h a h a l f - l i f e o f approximately two weeks. Results f o r the exchange i n the pH range above pH 8 .2 could not be obtained owing to the i n c r e a s i n g i n s o l u b i l i t y and a c c e l e r a t e d decomposition 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 pseudo f i r s t - o r d e r r a t e constants ( k j ) and h a l f - l i v e s ( t i ) f o r the C -6 methyl group hydrogen-deuterium exchange at various pH values are c o l l e c t e d i n Table VTI. The e r r o r s i n these reported values range from about 2 to 8 percent as i n d i c a t e d by comparison of i d e n t i c a l runs. In g e n e r a l , the data 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 stages of the exchange r e a c t i o n . For those experiments 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 of undetermined order, two r a t e constants are recorded i n the Table. The r a t e constant k j and h a l f - l i f e t i apply t o - t h a t FIGURE 16 DHTML EXCHANGE KINETICS . TYPICAL RATE PLOT (NEUTRAL SPECIES) TI ME (M I NUTES) FIGURE 17 4000 T I M E ( .M l N U T E S ) 113 Table V I I H-D exchange of 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 re g i o n pH -0.4 to 8.2, 0.10M Na2HP04, u = 0.42 and T = 3l.4°C. PH I O V I N ( m i n . - 1 ) l O 2 ^ ( m i n . - 1 ) ti(min.) 2 r - 0 . 3 7 k (40) 8 . 9 777 0 . 9 9 9 0 . 5 (73) 1 9 . 1 363 0 . 9 9 3 1.0 (54) 3 1 . 6 219 0 . 9 8 8 1.5 4 3 . 8 158 O .989 2 . 0 3 7 . 6 184 0 . 9 9 6 2 . 5 2 9 . 8 233 0 . 9 9 0 3 . 5 • 1 6 . 1 431 0 . 9 9 9 4 . 5 1 0 . 9 639 0 . 9 9 5 4 . 9 9 -8 708 0 .991 5 . 5 9 . 9 702 0 .992 6 . 0 1 0 . 2 677 0 . 9 9 0 7 . 0 9 . 8 708 0 .992 8 . 0 3.4 2040 0 . ^ 9 6 8 . 2 1 3 . 9 1777 0 .997 1 k - t r i f l u o r o a c e t i c acid-DgO 1 - pyridine-Dp 0 114 p a r t o f the data 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 could be estimated, k j ^ r e f e r s to the value derived from the best s t r a i g h t l i n e drawn through the curve e x h i b i t e d by the i n i t i a l r a t e data when p l o t t e d i n a f i r s t - o r d e r manner. These values are placed i n brackets to i n d i c a t e that they are estimates o n l y . C o r r e l a t i o n c o e f f i c i e n t s ( r ) have a l s o been included to i n d i c a t e the degree of l i n e a r i t y of 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 versus pH r e s u l t s i n the unusual pH-rate p r o f i l e i l l u s t r a t e d i n Figure l 8 . The t r i a n g l e s i n the Figure represent the estimated k j ^ w h i l e the c i r c l e s represent the f i r s t - o r d e r r a t e . c o n s t a n t s k^. The two h a l f - c l o s e d c i r c l e s represent the 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 pyridine-D20 at H 0 = -0 .37 and pH =. 8 .2 r e s p e c t i v e l y . 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 three s e c t i o n s , In each o f which one form of DHTML i s predominant. From the pK values obtained f o r t h i s compound (Table l b ) , one can i d e n t i f y r e g i o n A as the pH range where the compound e x i s t s mainly as the c a t i o n , r e g i o n B where the n e u t r a l species predominates, and r e g i o n C where the monoanion predominates. c_. E f f e c t o f b u f f e r on the exchange r a t e The exchange of 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 was examined at pH 4 . 5 , i o n i c s t r e n g t h 0 . 42 and temperature 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 . The c o n c e n t r a t i o n o f the phosphate b u f f e r was v a r i e d i n the range 0.050M to FIGURE 18 116 0.150M w i t h the i o n i c s t r e n g t h maintained at the constant value by the a d d i t i o n of sodium c h l o r i d e . B u f f e r concentrations below 0 . 050M were unable to keep the pH o f the system constant upon s o l u t i o n o f the organic s u b s t r a t e . The r e s u l t s of t h i s experiment are c o l l e c t e d i n Table V I I I . Included i n the 1Table f o r comparison are the r e s u l t s from s i n g l e experiments using 0 . 0 5 0 M phosphate b u f f e r at pH 0 . 5 , 2.0 and 6.0. I t was noted that w h i l e the amount of the i n i t i a l r a t e o f undetermined order was comparable f o r the two b u f f e r concentrations at pH 2.0, a marked increase i n the extent of the i n i t i a l r a t e p o r t i o n o f the exchange data was observed i n decreasing the co n c e n t r a t i o n of the b u f f e r from 0.10M to 0.050M at pH 0.5. A p l o t o f the data i n Table V I I I at pH 4 .5 r e s u l t s i n the curved r e l a t i o n s h i p shown i n Figure 19, although a reasonable s t r a i g h t l i n e ( r = O.986) can be drawn through the p o i n t s . 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 that the exchange r e a c t i o n i s subject to c a t a l y s i s by b u f f e r species at t h i s pH. This 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 experiments at the other pH values . d_. C a t a l y s i s by metal c a t i o n s The r e d u c 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 w i t h z i n c i n h y d r o c h l o r i c a c i d y i e l d s 7 , 8 - d i h y d r o - 6 , 7 , 8 - t r i m e t h y l -lumazine as the z i n c complex (X L V I I ) . I t has been r e p o r t e d 3 6 ' 1 ^ that 8 - a l k y l p t e r i d i n e s and lumazines 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 inherent a f f i n i t y f o r metals. 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 of DHTML a t u.=-0.42, T = 30 .0°C. Concentration Na 2hT04 (moles/l.) . 10 k]_ at pH 4 . 5 (min. ) 0 . 0 5 0 0 . 0 7 5 0 . 1 0 0 0 . 1 2 5 0 . 1 5 0 4 . 7 8 . 4 1 0 . 9 1 2 . 8 1 4 . 4 f o r comparison: PH 4 -1 10 k (min. ) 1 ' 0.050M Na2HP04 0.100M Na2HP04 0 . 5 2 . 0 4 . 5 6 . 0 7 . 6 2 0 . 9 4 . 7 6 . 0 1 9 . 1 3 7 . 6 1 0 . 9 1 0 . 2 118 FIGURE 19 DHTML EXCHANGE KINETICS / / / / / 0 2.5 5.0 7 5 ' ,10 .0 12.5 I s!o i 0 a [Na a HP0 4 ] (M.-1) 120 I t was decided to t e s t f o r the p o s s i b l e c a t a l y t i c e f f e c t o f heavy metal c a t i o n s on the exchange r e a c t i o n by adding C u C l 2 to s o l u t i o n s o f the dihydrolumazine i n DgO at pH 5 . 0 . The i n s o l u b i l i t y o f c u p r i c phosphate In water precluded the use o f sodium phosphate as the b u f f e r i n g agent. Therefore, b u f f e r systems c o n t a i n i n g sodium formate (0.10M) adjusted to pH 5 . 0 and c o n t a i n i n g amounts of c u p r i c c h l o r i d e ranging i n c o n c e n t r a t i o n from 0.0M to 0.012M were prepared. The i o n i c s t r e n g t h was maintained at a constant value o f 0 . 1 5 . The samples o f DHTML were prepared and thermostated i n the p r e v i o u s l y described 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 2 , a notable i n i t i a l broadening o f the C-6 methyl resonance along w i t h the changing of the s o l u t i o n c o l o u r from pale yellow to orange i n d i c a t e d complex formation. The broadening of t h i s methyl 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 other resonances o f the dlhydro.trimethyllumazine were a l s o s i g n i f i c a n t l y broadened. Due to the extensive broadening of the C-6 methyl group resonance onl y the exchange r a t e i n 0.10M sodium formate at pH 5 . 0 c o n t a i n i n g 0.006M CuClg could be measured w i t h any degree of accuracy. The exchange r a t e i n t h i s system (k-^ 12 x 10"^ min." 1) compared to the r a t e i n the same system w i t h no added C u C l 2 (2 . 6 x 10""^ min. - 1) i n d i c a t e s c a t a l y s i s by Cvr does occur. 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 methyl group towards the p o s i t i v e metal c a t i o n w i t h the formation o f the complex (LXVI). (LXVI) D. D i s c u s s i o n 1. Mechanism o f the exchange process a. • Trimethyllumazine The s e l e c t i v e decrease i n peak amplitude o f the downfield 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 general acid-base c a t a l y z e d exchange o f the C-7 methyl protons w i t h s o l v e n t , through the interrnediacy o f (LXV a) and ( L X V b ) . The s u b s c r i p t s r e f e r to the intermediate i n a c i d i c media (a) or the one found i n more ba s i c media (b) r e s p e c t i v e l y . A s i m i l a r intermediate (LXVI) to (LXV^) has 140 been proposed f o r the exchange of the C-7 methyl protons of r i b o f l a v i n - 5 ' - p h o s p h a t e (PMN) i n DgO buffered at pH 6.8 to 6.9 122 (LXVI) The r e l a t i v e l y f a c i l e exchange observed f o r the C-7 methyl group i n TML can be explained i n terms o f the long conjugated s t r u c t u r e and extensive 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 intermediates (LXV a) and (LXV f e) compensating f o r the formation 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 deuterium oxide 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 of TML CH J. 3 H 3 C UH + D A ( D 3 0 + ) 6 ^ H 3 I I o I O- Si O-D CH-H 3 c ^ S r ^ ° - D H 3 6 CH-H 3 C r ^ M v > D * 3 H 3 C I I o H 3 C ^ O D ^ 3 H 2 C ^ ^ > ^ p IjH H 3 C W ° O HA ( H D 2 0 + ) 1 2 4 A t r i v i a l v a r i a t i o n of t h i s mechanism i s pr o t o n a t i o n o f the 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 at the l a t t e r p o s i t i o n does however allo w f o r somewhat more d e r e a l i z a t i o n of the p o s i t i v e charge introduced i n t o the molecule. As w e l l , the pKg H+ o f 6 , 7 j 8 - t r i m e t h y l l u m a z i n e agrees more c l o s e l y to 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 to the 1 2 7 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 basic regions (above pH 4 ) the general base-catalyzed exchange becomes more important. The mechanism of the exchange i n the more bas 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 of 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 indicates the equilibrium shown below l i e s w e l l to the l e f t . f H 3 H - , C ' ^ : - \ < ^ H O b. 7 , 8-Dihydro - 6 , 7 , 8 -trimethyllumazine The decrease i n peak amplitude o f the down-field C-methyl s i n g l e t In 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 methyl protons w i t h s o l v e n t through the intermediacy o f ( L X V I I ) . CH 3 H H 3C. H / V ^ ^ H H o (LXVIIj This s t r u c t u r e i s analogous to the enol .tautomer of an a l i p h a t i c ketone such.as acetone. 126 In g e n e r a l , the much slower exchange rates observed f o r the dihydro compound i n comparison w i t h trimethyllumazine i s a consequence of the l a c k o f a long conjugated system 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 the former case. In order to e x p l a i n the exchange mechanism and the unusual pH-rate p r o f i l e of 7 , 8 - d i h y d r o - 6 , 7 , 8 - t r i m e t h y l -lumazine, recourse must be made to an examination o f the various i o n i c species the molecule can form. A p o s s i b l e s t r u c t u r e of the mono-cation o f DHTML i.s i n d i c a t e d by the r e s o n a n c e - s t a b i l i z e d formula ( L X V I I I ) . 9^3 H H 3 S ^ ^ S - 0 hi H 3 C -H o+ H or C H j H H 3 & ^ r ^ O H 3 C r> H H 3 C ^ ^ Y OH ^ 3 H H 3 C O H (LXVIII)' etc A comparison of the pK B H+ of DHTML and s e v e r a l model compounds supports t h i s s t r u c t u r e f o r the c a t i o n . These values are given as f o l l o w s : 127 Compound p K m + ( H 3 O , 2 5 ° ) Reference DHTML H R (CHo)2N-C=C-C=0 (LXIX) H . ( (LXX) 2 . 8 6 2 . 3 4 (R = H) 3 . 0 5 (R = C 6H 5) 3 . 1 5 This work 144 145 X44 145 Evidence has been presented ' to show that O-protonation p r e v a i l s i n the formation of the c a t i o n s o f (LXIX) and (LXX). The protonated form of (LXX), f o r example, can be represented as f o l l o w s : This s t r u c t u r e i s analogous to one of the resonance forms of the proposed c a t i o n of 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 . The pKgjj+ o f 7 , 8-dihydro - 2-amino - 4-hydroxy - 6 -m e t h y l p t e r i d i n e has a reported value of 3 . 2 , corresponding to pr o t o n a t i o n of the N-8 p o s i t i o n . * 1 " ^ An e a r l i e r r e f e r e n c e } ^ 8 • however, suggests that the c a t i o n o f t h i s compound, as w e l l as the c a t i o n o f the 6 , 8-dimethyl d e r i v a t i v e , possesses the 128 s t r u c t u r e (LXXI). R H O (LXXI; R = H, CH3) l 4 g In a recent paper, support f o r N-5 p r o t o n a t i o n f o r these 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 observed between the 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 the spectroscopic data. I t i s evident from disagreements i n the l i t e r a t u r e about the s t r u c t u r e of 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 of 7 , 8 - d i h y d r o - 6 , 7 , 8 -trimethyllumazine 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, the reported pKBH+ f o r DHTML may represent an e q u i l i b r i u m mixture of two (or more) c a t i o n s e x i s t i n g together i n s o l u t i o n . The s h i f t i n g o f the C -6 methyl resonance to lower 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 the aqueous media i s c o n s i s t e n t w i t h the expected d e s h i e l d i n g of the methyl group protons by the f u l l p o s i t i v e charge located on the neighboring n i t r o g e n atom i n the N-5 protonated form of DHTML (L X X I I ) . 129 C H 1 u | 3 H b O (LXXII) While i t i s evident that i n c r e a s i n g amounts o f the N-5 protonated form of DHTML (LXXII) could 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 to 1.0 (Region B, F i g u r e l S ) i t i s not c l e a r how the other p o s s i b l e c a t i o n (LXVIII) enhances the exchange process, i f i t does so at a l l . A r a t e enhancement by a coulombic e f f e c t might be expected from t h i s l a t t e r Ion. I f a c i d c a t a l y s i s of the exchange occurs by means of p r o t o n a t i o n a t the N-5 p o s i t i o n of DHTML then the ra t e should continue to increase u n t i l such time as the subs t r a t e i s f u l l y protonated. At t h i s stage, the exchange should proceed at a constant 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 of the pH-rate p r o f i l e i n the reg i o n where DHTML e x i s t s predominantly as the c a t i o n (Region A, Figure 18). In f a c t , marked decrease i n the exchange r a t e was observed i n t h i s r e g i o n . D i s c u s s i o n of t h i s f eature o f the exchange process i s presented l a t e r i n t h i s work. Formation o f s i g n i f i c a n t amounts of the 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 the exchange r e a c t i o n since the intermediate i n the exchange process now re q u i r e s the unfavorable s i t u a t i o n of two negative charges i n cl o s e p r o x i m i t y In the same molecule. C H ; (LXXIII) That i n h i b i t i o n of the exchange does occur i n the region where the substrate i s a n i o n i c Is i n d i c a t e d by the decrease i n exchange ra t e i n the pH regions around and beyond the pK a ( 7 - 2 9 ) of DHTML (Region C, Figure 1 8 ) . , The study of the dependency o f the 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 that the exchange of" DHTML i s subject to general c a t a l y s i s . The a c i d - c a t a l y z e d exchange mechanism i s i l l u s t r a t 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 process i s not i n d i c a t e d i n the pH-rate p r o f i l e . 132 2. Hydration 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 The p o s s i b i l i t y a r i s e s that a c i d - c a t a l y z e d a d d i t i o n o f the elements o f water to form a "hydrated" 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 the slow exchange r a t e s observed i n the pH range 1 .0 to - 0 . 4 (Region A, Figure l 8 ) . This p o s s i b i l i t y i s p l a u s i b l e on the f o l l o w i n g grounds: i . A d d i t i o n o f water to a C=N bond converts the carbon atom from an unsaturated to a saturated 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 to t h i s carbon undergoes a co n s i d e r a b l e s h i f t u p f i e l d i n the p.m.r. spectrum of the compound i n s p i t e o f the presence o f the hydroxyl group. 2^ However, i f the "hydrate" (LXXIV) i s unstable w i t h respect to the ring-opened form (LXXV) then the c r i t e r i o n of u p f i e l d s h i f t upon h y d r a t i o n no longer a p p l i e s . This i s because the r e s u l t i n g carbonyl f u n c t i o n can be expected to 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 the C -6 methyl group as the imine (C=N) f u n c t i o n does i n the unhydrated n e u t r a l form of DHTML. H (LXXIV) (LXXV) 133 The f a c t that the methyl resonance of acetone d i s s o l v e d i n 0.050M phosphate b u f f e r i n DgO at pH 1.0 l i e s s l i g h t l y u p f i e l d from the C -6 methyl resonance o f DHTML d i s s o l v e d i n the same b u f f e r confirms t h i s e x p e c t a t i o n . The small peak which appears immediately adjacent to the broad C -6 methyl resonance i n s o l u t i o n s of DHTML In the pH range 0 . 5 to 2 . 5 may be the corresponding C-methyl resonance of the r i n g -opened form (LXXV). In support of the ring-opening r e a c t i o n i s the 150 o b s e r v a t i o n t h a t , i n a c i d s o l u t i o n , p t e r i d i n e r a p i d l y adds a molecule o f water to form "hydrated p t e r i d i n e " , 3 , 4 -dihydro~4-hydroxypteridine,(as the c a t i o n ) . This compound s l o w l y undergoes f i s s i o n o f the dihydropyrimidine r i n g to form the c a t i o n o f 2-aminomethyleneamino - 3~formylpyrazine. S i m i l a r behaviour i s shown by 2-, 4 - and 7 - m e t h y l p t e r i d i n e . The ring-opening 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 the ra 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 . , i i . H ydration i n the p t e r i d i n e s e r i e s Is 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 wavelengths, consonant w i t h the r e d u c t i o n o f the conjugated pathway i n the molecul e . 2 3 ' 2 - ^ In the 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) of 34 mu was observed i n going from the n e u t r a l species to the c a t i o n . The change from p t e r i d i n e to i t s protonated hydrate Is s i m i l a r l y accompanied by a bathochromic s h i f t o f about 150 20 mu, explained i n terms o f an increase i n the ease w i t h 134 which e l e c t r o n s on N-3 o f the hydrated p t e r i d i n e species can be e x c i t e d i n t o an o r b i t a l i n which there 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 the sp e c t r a o f p t e r i d i n e , as described above, and i n q u i n a z o l i n e s o l u t i o n s i n going from the n e u t r a l species to the c a t i o n was one of the main reasons f o r the suggestion that the c a t i o n s were h y d r a t e d . 1 ^ ' 2 3 ' 2 5 i i i . Water, being a weak n u c l e o p h i l e , i s not l i k e l y to be s t r o n g l y bonded across the C=N bond unless some other f o r c e operates. Resonance has been found to supply the e x t r a i n f l u e n c e that makes the hydrated form s t a b l e . In the present case, 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 both the hydrated c a t i o n (LXXIV) and1 the corresponding r i n g -opened form (LXXV). There i s a dearth o f inf o r m a t i o n i n the l i t e r a t u r e -concerning the 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 systems. The 7 , 8-hydrated form o f 6 , 7 - d i m e t h y l - 5 , 6 - d i h y d r o -2-amino - 4-hydroxypteridine has been postulated" 1"^ as a p o s s i b l e intermediate i n the r e d u c t i o n of the.. 5 , 6-dihydro- _._ 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 n u c l e o t i d e (TPNH). On the b a s i s o f the a v a i l a b l e evidence, the 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 hydrate formation decreasing the co 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 mixture of hydrated and unhydrated c a t i o n s may a l s o e x p l a i n the anomalous i n i t i a l r a t e of undetermined 135 order 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 r e g i o n o f 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 represented i n terms of the f o l l o w i n g e q u i l i b r i a : Scheme IV Hydration of DHTML i n a c i d i c media I t i s seen that unless there i s a d r i v i n g f o r c e 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 hydrated to exchanging c a t i o n should be pH-independent. I f t h i s were the case, a l e v e l l i n g - o f f r a t h e r than a decrease i n r a t e s would be observed i n the regions where the substrate i s f u l l y 136 p r o t o n a t e d . The o p e r a t i v e f o r c e i n s h i f t i n g the e q u i l i b r i a towards the ring-opened form i s prob a b l y p r o t o n a t i o n o f the primary amino group i n t h i s compound. A n i l i n e and 5-amino-p y r i m i d i n e , f o r example, have pKg H+ values o f 4 . 6 0 and 2 .51 a t 25 r e s p e c t i v e l y ' so th a t p r o t o n a t i o n o f the primary amino group i s l i k e l y under the c o n d i t i o n s o f a c i d i t y p r e s e n t i n Region A o f the pH-rate p r o f i l e . The b a s i c i t y o f t h i s amino group i s undoubtedly weakened (decrease i n pKgpj+ value) by the coulombic e f f e c t o f the p o s i t i v e charge p r e s e n t i n the p y r i m i d i n e moiety o f the molecule. The drop i n the exchange r a t e which i n i t i a l l y o c curs near pH 2 i s l i k e l y a r e s u l t o f p r o t o n a t i o n o f the amino group i n the ring-opened c a t i o n o f the s u b s t r a t e . Support i s l e n t to the h y d r a t i o n scheme on the' f o l l o w i n g grounds: .1) 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 s o f p t e r i d i n e s are 1S2 u s u a l l y c a t a l y z e d by hydrogen ions and hydroxide i o n s . y , 2) The are a o f the peak assigned to the C-methyl group o f the ring-opened c a t i o n o f DHTML i n c r e a s e s w i t h i n c r e a s i n g 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 o f the magnitude o f the i n i t i a l r a t e c o n s t a n t s ( k j ^ ) can be made, t h e i r values ( t r i a n g l e s i n F i g u r e 18) l i e approximately along a l i n e e x t r a p o l a t e d 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 p r o f i l e . T h i s i n d i c a t e s t h a t the exchange o f DHTML would e x h i b i t the normal f e a t u r e s o f an a c i d - c a t a l y z e d r e a c t i o n were the 1 3 7 concurrent h y d r a t i o n r e a c t i o n not al s o a f a c t o r i n determining the magnitude of the exchange r a t e . The observation that these 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 slower f i r s t -order r a t e s w i t h time i s i n d i c a t i v e of the e q u i l i b r a t i o n o f the hydrated and unhydrated c a t i o n s of the dihydro lumazine. 4) The broadening and s h i f t i n g of the C-6 methyl 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 t h i s scheme. 5) I t was observed that the small peak assigned to the C-methyl group of the ring-opened c a t i o n decreased only very s l i g h t l y i n area during the course of the exchange r e a c t i o n . This i s i n agreement w i t h the exp e c t a t i o n that the C-methyl resonance o f the ring-opened c a t i o n would exchange s l o w l y 153 i n 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 reported slow exchange o f acetone i n DgSO^. 6) In comparing the amount of the I n i t i a l r a t e o f undetermined order between two exchange experiments c a r r i e d out In 0.050M'and 0.10M phosphate b u f f e r at pH 0 . 5 , a marked decrease was observed i n going to the more concentrated b u f f e r . This i s c o n s i s t e n t w i t h 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 hydrated and unhydrated c a t i o n s i n the 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 H3PO4 species . Attempts to form a 2 , 4-dinitrophenylhydrazone 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 formation o f the ring-opened c a t i o n have so f a r been u n s u c c e s s f u l . 138 3. B i o l o g i c a l I m p l i c a t i o n s The 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 both 7 , 8 - d i h y d r o - 6 , 7 , 8 - t r i m e t h y l - and ' 6 , 7 , 8 - t r i -methyllumazine i s i n t e r e s t i n g . Most of the b i o l o g i c a l l y 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 - 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 moiety. On the other hand, c e r t a i n p t e r i d i n e pigments such as e r y t h r o p t e r i n (2-amino -4 ,6-dihydroxypteridine -7-pyruvic: acid)are s u b s t i t u t e d i n the 7 p o s i t i o n . The i m p l i c a t i o n i s that the f o l i c a c i d s and 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 by condensation of the a p propriate amine f u n c t i o n w i t h a simple 7 , 8-dihydro - 6-methyl (or hydroxymethylene)-pteridine d e r i v a t i v e , w h i l e condensation w i t h a 7-methylpteridine y i e l d s the pigments. However, n u c l e o p h i l i c a t t a c k on the 6 - p o s i t i o n o f 7 , 8 - d i h y d r o p t e r i d i n e s l e a d i n g to 6 - s u b s t i t u t e d t e t r a h y d r o -o p t e r i d i n e s followed by gentle o x i d a t i o n i s known to produce the corresponding 6 - s u b s t i t u t e d p t e r i d i n e s . Because they are ov to r i n g nitrogens a l l methyl groups i n p t e r i d i n e s should be a c t i v a t e d and s u s c e p t i b l e to hydrogen-deuterium exchange. S u r p r i s i n g l y , the exchange o f o n l y one other p t e r i d i n e d e r i v a t i v e aside from the two described In the present work has been r e p o r t e d . ^ - ^ The 7-methylene proton o f 7-acetonylxanthopterin (LXXVI) was observed to undergo r a p i d deuterium 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 deuteroxide (NaOD). 139 Although attempts to condense various reagents, such as benzaldehyde, w i t h the methyl group of 6-methyl-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 u n s u c c e s s f u l , s e v e r a l brominatlons o f 6 - a l k y l and 7 - a l k y l groups are known.^ In g e n e r a l , the y i e l d s o f the mono- and dibromo-methylene d e r i v a t i v e s are o n l y f a i r . In the present case, a s i n g l e attempt to brominate 6 , 7 , 8 - t r i m e t h y l l u m a z i n e i n a c e t i c a c i d produced a small amount of a r e d d i s h m a t e r i a l which d i d not melt up to 3 5 0 ° and had an absorption spectrum ( )v v 272, 320 and 400 mu) somewhat s i m i l a r to that o f the t r i m e t h y l l u m a z i n e i n water. Not 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 or p.m.r. s p e c t r a l a n a l y s i s . An attempt 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 acetate l e d to the recovery o f the s t a r t i n g m a t e r i a l s . Because of the small amount of m a t e r i a l a v a i l a b l e , no condensation or bromination r e a c t i o n s were attempted w i t h 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 a r t I I I OXIDATION OP TRIMETHYLLUMAZINE AND DIHYDROTRIMETHYLLUMAZINE i4o A. I n t r o d u c t i o n The o x i d a t i o n o f 7,8-dihydro-6,7,8-trimethyllumazine i s i n v e s t i g a t e d using potassium f e r r i c y a n i d e and potassium permanganate f o r the purpose. These p a r t i c u l a r reagents were chosen i n view of 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 present study. F e r r i c y a n i d e i s reported to r e a c t w i t h c e r t a i n 104 155 156 157 organic compounds by one-electron a b s t r a c t i o n ' ' 157 and, 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 . The f a c t that f e r r i c y a n i d e absorbs at a wavelength removed from the u l t r a v i o l e t r egion where abs o r p t i o n by f e r r o c y a n i d e and many organic species occurs 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 over a p e r i o d o f days or weeks i f protected from l i g h t and kept under n i t r o g e n atmosphere. The redox p o t e n t i a l of 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 i s e s s e n t i a l l y independent 158 of pH above pH 5 which permits s t u d i e s using t h i s system over a f a i r l y wide range of 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 chemistry of 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 that r e a c t i o n s o f c e r t a i n organic 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 are complex and that e x p l a n a t i o n i n terms of a simple one-electron 141 transfer i s inadequate. The r e s u l t s of Speakman and Waters 1 study of the oxidation of aldehydes, ketones and n i t r o -alkanes and Kolthoff's s t u d y 1 ^ 1 of the oxidation of mercapto-ethanol a t t e s t to t h i s . Potassium permanganate i s a well-known oxidant i n organic chemistry and i t s reactions with organic substrates have been reviewed. The use of aqueous permanganate to a f f e c t the removal of c e r t a i n methyl groups In the.lumazine series-^'^9 ^ g o f p a r t i c u l a r i n t e r e s t i n the present case. B. Experimental 1. P u r i f i c a t i o n of solvents and materials Water that was used i n the preparation of the buffer systems and stock sample solutions was d i s t i l l e d , b o i l e d, and then cooled by flushing with high-purity nitrogen. Commercial dibasic potassium phosphate was p u r i f i e d by slow r e c r y s t a l l i z a t i o n from aqueous methanol and dried at l60° f o r three days. Commercial potassium ferricyanide, potassium ferrocyanide and potassium chloride 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 saturated solutions i n water and dried i n an evacuated desiccator over ^2^^ f o r o n e week. Care was taken to exclude l i g h t from the c r y s t a l l i z i n g solutions of the hexacyanoferrates by wrapping the containers In aluminum f o i l . The p u r i t y of the' potassium ferricyanide was checked by running several u l t r a v i o l e t and v i s i b l e spectra of aqueous l42 solutions of known concentrations. The average value found for the e x t i n c t i o n c o e f f i c i e n t of potassium ferricyanide at 420 mu i s E 420 = 1 ,039 - 12. This value l i e s i n the range of values often quoted Tor the ex t i n c t i o n c o e f f i c i e n t of ferricyanide Ion at this wavelength i n aqueous media. A n a l y t i c a l grade potassium permanganate, potassium n i t r a t e , potassium bromate and potassium sulfate were used without further p u r i f i c a t i o n . A l l s o l i d s when not i n use were kept i n a desiccator under vacuum. The water was stored i n a capped polyethylene b o t t l e under nitrogen atmosphere. 2. Preparation of buffer and sample solutions Stock solutions of dibasic potassium phosphate and potassium chloride were prepared at 0.250M and 0.500M respectively -by weighing accurately the appropriate amount of material into• separate 1 - l i t r e volumetric flasks and making up to the mark; with d i s t i l l e d water. 0.250M solutions of the other s a l t s were prepared i n a s i m i l a r manner In 25 ml volumetric f l a s k s . Prom the known d i s s o c i a t i o n constants of phosphoric. r>l4l acid i n water at 25-0 and applying the relations which give the concentrations of the various phosphate species from a l 4 l known phosphate buffer concentration at a given pH, the t — i o n i c strength contribution to a solution by the phosphate buffer species at this pH can be calculated. Hence, the i o n i c strength 143 o f a g i v e n b u f f e r s o l u t i o n may be f i x e d a t a d e s i r e d value by the 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 K C 1 o r o t h e r s a l t . A s e r i e s o f b u f f e r s were then made up i n the f o l l o w i n g t y p i c a l manner: By means o f p i p e t t e s and s y r i n g e s , the volumes o f KgHPO^ and K C 1 s t o c k s o l u t i o n s were measured i n t o a 30 ml. graduated beaker under a stream o f n i t r o g e n . The volumes were chosen to g i v e a d e s i r e d phosphate b u f f e r c o n c e n t r a t i o n and i o n i c s t r e n g t h a t a g i v e n pH when d i l u t e d to a f i n a l volume o f 2 5 ml. D i s t i l l e d water was added to a volume o f a p p r o x imately 2 2 ml f o l l o w e d by dropwise a d d i t i o n o f carbonate-f r e e potassium hydroxide o r IN h y d r o c h l o r i c a c i d under a stream o f n i t r o g e n u n t i l the d e s i r e d pH was a t t a i n e d , as i n d i c a t e d by the Radiometer Model 26 pH meter. The contents o f the beaker were then t r a n s f e r r e d q u a n t i t a t i v e l y to a 2 5 ml v o l u m e t r i c f l a s k and made up to the mark with the beaker water washings. A 4 ml a l i q u o t was removed to measure a c c u r a t e l y the f i n a l pH o f the s o l u t i o n and the remainder was t r a n s f e r r e d to a p o l y e t h y l e n e c o n t a i n e r under n i t r o g e n atmosphere f o r storage.. With t h i s means o f storage even those b u f f e r s o f h i g h a c i d i t y o r b a s i c i t y maintained t h e i r pH over a p e r i o d o f months. In t h i s manner, 0.050M phosphate b u f f e r s a t i o n i c s t r e n g t h 0 . 2 5 were made up w i t h pH values r a n g i n g from 1 2 . 5 to 1 . 0 . B u f f e r s o l u t i o n s u s i n g d i f f e r e n t potassium s a l t s to a d j u s t the i o n i c s t r e n g t h were a l s o prepared a t pH 1 2 . 0 . Stock s o l u t i o n s o f potassium f e r r i c y a n i d e and l44 potassium permanganate were prepared by weighing a c c u r a t e l y the appropriate amount of oxidant i n t o a 10 ml volumetric f l a s k and making up to the mark w i t h d i s t i l l e d water at 25.0°. The c o n c e n t r a t i o n o f the oxidant i n the stock s o l u t i o n was u s u a l l y i n the neighborhood of 0.1M. The potassium f e r r i c y a n i d e s o l u t i o n was protected from l i g h t by wrapping the volumetric f l a s k i n aluminum f o i l . The s o l u t i o n s were thermostated at 25.0° i n a water bath. Organic su b s t r a t e stock s o l u t i o n s were prepared 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 water by means o f a syringe to a known weight of dry 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 rubber cap. The volume of added water was determined a c c u r a t e l y by reweighing the v i a l and contents. The amount of water to be added was predetermined so as to give 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 of oxidant when a l i q u o t s of both sample and oxidant stock s o l u t i o n s were added to the b u f f e r system to i n i t i a t e the r e a c t i o n . The prepared sample s o l u t i o n s were thermostated at 25.0° p r i o r to t h e i r use. The aqueous stock s o l u t i o n s of 7 , 8 - d i h y d r o - 6 , 7 , 8 -trimethyllumazine tended to decompose slow l y and were considered unacceptable 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 o r i g i n a l l y prepared. Therefore, a l l k i n e t i c measurements of a r e l a t e d nature were c a r r i e d out on one sample and then those runs of 145' a d i f f e r e n t s e r i e s i n v e s t i g a t e d w i t h a freshly-prepared stock sample s o l u t i o n , the l a t t e r sample co n c e n t r a t i o n being adjusted so as to very c l o s e l y approximate the stock 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. In t h i s way, various s e r i e s o f measurements can be compared w i t h a reasonable degree o f confidence. The oxidant and trimethyllumazine stock s o l u t i o n s proved to be more s t a b l e , e s p e c i a l l y i f protected from l i g h t and kept under n i t r o g e n atmosphere. In g e n e r a l , however, f r e s h s o l u t i o n s were prepared before the s t a r t o f a new s e r i e s of k i n e t i c runs. Again, care 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 previous stock s o l u t i o n c o n c e n t r a t i o n s . 3. Techniques employed i n k i n e t i c measurements The o x i d a t i o n o f dihydrotrimethyllumazine and the r e a c t i o n s o f trimethyllumazine were studied by two methods. In the pH range 10 to 12.5 spe c t r o s c o p i c techniques were employed wh 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 at a l l pH values above 5. i . Spectroscopy The usefulness o f the spe c t r o s c o p i c method i n the -more b a s i c regions i n the present case stems from the f a c t that at the wavelength o f maximum absorbance f o r the f e r r i c y a n i d e Ion there i s very l i t t l e absorbance c o n t r i b u t i o n from the organic species o r from f e r r o c y a n i d e i o n . Thus, the r e a c t i o n s between f e r r i c y a n i d e and the lumazine d e r i v a t i v e s may be followed by simply monitoring the change i n absorbance 146 at 420 mu. This v/as done w i t h a Bausch and Lomb Model 502 r e c o r d i n g spectrophotometer. Below pH 10, other c o n t r i b u t i o n s to the t o t a l absorbance at 420 mu, e s p e c i a l l y from 6 , 7 , 8 -t r i m e t h y l l u m a z i n e (>\ m a x = 4o4 mu), become i n c r e a s i n g l y l a r g e and make treatment o f the k i n e t i c data a r e l a t i v e l y complex procedure. At a l l pH values 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 the r e a c t i o n system e x h i b i t s a lar g e number o f c l o s e l y spaced and overlapping absorptions thus making t h i s area u n a t t r a c t i v e from the standpoint of a simple k i n e t i c treatment. A t y p i c a l spectrophotometric run was c a r r i e d out as f o l l o w s : The r e a c t i o n medium was prepared i n 1 cm. s i l i c a c e l l s f i t t e d w i t h a s i l i c o n e rubber d i s c i n the ground g l a s s neck. The d i s c s , s i m i l a r to the type used as i n j e c t i o n septa i n gas chromatography, could be pie r c e d repeatedly w i t h small gauge syringe needles without l e a k i n g and were i n e r t to aqueous media. Before i n t r o d u c t i o n of a b u f f e r s o l u t i o n , the c e l l was f i r s t f i t t e d w i t h a d i s c and then two syringe needles 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 through one o f the needles to remove a i r from the c e l l . By means o f a 5 ml syringe equipped w i t h a Chaney adapter 3-00 ml of 0.050M phosphate b u f f e r at pH 12.0 and i o n i c s t r e n g t h 0 .25 was introduced i n t o the c e l l followed by 0.0277 ml o f 0.108M aqueous potassium 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 reading 147 o f c l o s e to 1 .0 at the s t a r t o f the r e a c t i o n . The c e l l was then t r a n s f e r r e d to the c e l l compartment o f the sp e c t r o -photometer and thermostated 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 c o n t a i n i n g b u f f e r s o l u t i o n o n l y was used i n the reference beam o f the spectrophotometer. The syringe needles were then removed and, i n some cases, the absorbance reading o f the buffered f e r r i c y a n i d e s o l u t i o n was checked at 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 the c e l l 0.0613 ml o f sample stock s o l u t i o n ( u s u a l l y 2.42 x 10~ 2M) o f DHTML o r TML from a 100 m i c r o l i t r e s y r i n g e f i t t e d w i t h a Chaney adapter. The c e l l was shaken b r i e f l y to mix the r e a c t a n t s , care being taken not to allow the s o l u t i o n to contact the s i l i c o n e rubber d i s c , and t h i s was followed by the reco r d i n g o f absorbance readings at 420 mu at various time i n t e r v a l s . The r e a c t i o n was followed u n t i l a s t a b l e reading at 420 mu over a pe r i o d o f ten minutes was a t t a i n e d . This reading was taken as the " i n f i n i t y " absorbance o f the r e a c t i o n at 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 to ensure r e l i a b i l i t y o f the r e s u l t s . Due to the complexity o f 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 the system the f a t e o f the organic m a t e r i a l could not be determined by repeated s p e c t r a l scans. The r e a c t i o n d i d prove to 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 procedure was devised by which the progress o f the r e a c t i o n w i t h respect to the organic s u b s t r a t e could be 148 determined. The procedure i s based on the f a c t that z i n c ions 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 r e a c t w i t h potassium ferro c y a n i d e 162 to form the very s p a r i n g l y s o l u b l e potassium z i n c f e r r o c y a n i d e : 2K4Fe(CN) 6 + BZnSO^ > KgZn-^FeCCNjgJg + SKgSO^ A t y p i c a l quenching procedure i s described as f o l l o w s : Into each o f a s e r i e s o f 10 ml erlenmeyers, 2 . 5 ml of 1 .67 x 10 JM 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 p i p e t t e d . B u f f e r and stock 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 s u b s t r a t e were introduced to a t o t a l volume o f 6 ml i n a separate 10 ml erlenmeyer equipped w i t h a rubber cap and thermostated at 2 5 . 0 ° . The i n i t i a l r eactant concentrations were such as to c l o s e l y approximate those concentrations 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 syr i n g e at various times and i n j e c t e d i n t o the z i n c s u l f a t e s o l u t i o n s . Each s o l u t i o n was f i l t e r e d i n turn i n t o a 1 cm. ab s o r p t i o n c e l l and a complete spectrum recorded on the Bausch and Lomb Model 502 instrument. The average pH o f the f i l t e r e d s o l u t i o n s was found to 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 the r e a c t i o n medium. i i . Potentiometry The a p p l i c a t i o n o f potentiometry to the study o f o x i d a t i o n r e a c t i o n s i s described i n d e t a i l elsewhere. In the present study, a modified form o f the Nernst equation (10) 149 i s used. V = E o x - r e d " 2 h A A + 6 - 1 9 8 3 T . l o g ( [ O X ] / [ R E D ] ) (10) where E h ' = m e a s u r e d p o t e n t i a l (mV.) o f the system using a platinum-calomel e l e c t r o d e t r a i n , E o x - r e d = standard 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 -cyanide couple, T = temperature (°K.) [_0X~] = instantaneous c o n c e n t r a t i o n of f e r r i c y a n i d e , and [RED^J = instantaneous c o n c e n t r a t i o n o f f e r r o c y a n i d e . Prom the measured E^ , values at known co n c e n t r a t i o n r a t i o s o f potassium f e r r i c y a n i d e and potassium ferr o c y a n i d e i n 0 .050M phosphate b u f f e r at pH 1 2 . 0 , u = 0 . 2 5 and T = 2 5 . 0 ° C , the value o f E ° x _ r e d was found to 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° values found i n the 158 164 l i t e r a t u r e ' f o r 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 redox couple 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 determined by measuring the E h i value of the system, w i t h time. From these measurements the change i n the co 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 can be c a l c u l a t e d from equation ( 1 0 ) , assuming one mole o f f e r r i c y a n i d e reacts to give one mole of fer 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 run was c a r r i e d out as f o l l o w s : The p o t e n t i a l s were measured w i t h a Radiometer Model 150 26 pH meter u s i n g the m i l l i v o l t r e c o r d i n g s c a l e and a p l a t i n u m -c a l o m e l e l e c t r o d e t r a i n . T h i s i n s t r u m e n t i s equipped w i t h a s p e c i a l compartment c o n t a i n i n g the e l e c t r o d e s and has p r o v i s i o n s f o r t h e r m o s t a t i n g , 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 the r e a c t i o n s o l u t i o n under i n e r t atmosphere. I n the compartment o f the i n s t r u m e n t , the p l a t i n u m and c a l o m e l e l e c t r o d e s , 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 tube were f i t t e d i n t o a 20 ml p o l y e t h y l e n e c o n t a i n e r . U s i n g the same s t o c k s o l u t i o n s and s y r i n g e s d e s c r i b e d i n the 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 i n t o the p o l y e t h y l e n e c o n t a i n e r t h r o u g h the gas o u t l e t h o l e i n the l i d o f the compartment. The c o n t e n t s o f the p o l y -e t h y l e n e c o n t a i n e r were s t i r r e d b r i e f l y and a l l o w e d t o e q u i l i b r a t e 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° f o r f i f t e e n m i n u t e s . The r e a c t i o n was i n i t i a t e d by the i n t r o d u c t i o n o f the p r e v i o u s l y d e s c r i b e d amount o f aqueous DHTML o r TML s o l u t i o n from a 100 m i c r o l i t r e s y r i n g e . The r e a c t i o n s o l u t i o n was s t i r r e d f o r f i v e seconds and the n p o t e n t i a l r e a d i n g s a t r e g u l a r time i n t e r v a l s r e c o r d e d . F o r the s l o w e r r e a c t i o n s , the g l a s s compartment c o n t a i n i n g the r e a c t a n t s i n the p o l y e t h y l e n e c o n t a i n e r was wrapped i n aluminum f o i l t o p r o t e c t t h e c o n t e n t s from l i g h t . P o t e n t i o m e t r i c runs were done i n d u p l i c a t e and o c c a s i o n a l l y 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 . 151 4. Treatment of the k i n e t i c data For a b i m o l e c u l a r r e a c t i o n o f the type aA + bB — > products which i s f i r s t - o r d e r w i t h respect to the reactants A and B, the r a t e expression i s given by dA/dt = - k A [ . A ] [ B ] where k A = s p e c i f i c r a t e constant f o r the disappearance o f the species A, a,b = i n t e g r a l c o e f f i c i e n t s of the balanced chemical equation, and [ A ] , [ B ] concentrations of the species A and B r e s p e c t i v e l y In the present case, A = f e r r i c y a n i d e i o n and B = organic s u b s t r a t e . 165 The mathematical r e l a t i o n s derived by Benson to 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 are a p p l i c a b l e i n the present study. For the r e a c t i o n between sub s t r a t e and f e r r i c y a n i d e i o n i n which the i n i t i a l r e a c t a n t concentrations are non-s t o i c h i o m e t r i c , use i s made of the i n t e g r a t e d r a t e expression (11); i n (A/B) = (bA Q - a B 0 ) / a . k A t + In ( A 0 / B Q ) - ( l l ) where the usual brackets used to denote c o n c e n t r a t i o n have been omitted to s i m p l i f y the treatment. A p l o t of the logarithms instantaneous reactant concentrations versus time a f f o r d s a s t r a i g h t l i n e whose slope has the value (bA Q - a B Q ) / a ' k A from which the value o f the second-order r a t e constant can 1 5 2 be c a l c u l a t e d using the known I n i t i a l concentrations of the r e a c t a n t s . For 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 sub s t r a t e 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 , aBQ-^-'bA0-- equation ( l l ) 165 i s no longer a p p l i c a b l e . An a l t e r n a t e expression (12) i s used to o b t a i n the r a t e constant: 1/C A, - 1/C A, = bkAt/a«(l - A 2 ) (12) ° 4A'A> O co where A = aB Q/b - A Q, and C A, = A +A/2 A p l o t o f l/C , versus time y i e l d s a curve' which very 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 slope o f t h i s line,when 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 r a t e 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 normally a very s m a l l f r a c t i o n of those concentrations i n curved brackets i n equation ( 1 2 ) , t h i s term could u s u a l l y be Ignored. 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 1 3 ^ o f the data was used to o b t a i n the best s t r a i g h t l i n e . 1 5 3 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. Products o f the r e a c t i o n and s t o i c h i o m e t r y By means of spectrophotometric a n a l y s i s i t was a s c e r t a i n e d that two moles of f e r r i c y a n i d e r e a c t completely w i t h one mole of the reduced lumazine to produce two moles o f f e r r o c y a n i d e and various organic products. The type o f organic product(s) produced depended on the i n i t i a l pH o f the r e a c t i n g system. In the pH r e g i o n 5-0 to 8.0, the o n l y organic product detected when s t o i c h i o m e t r i c amounts of the dihydrolumazine and f e r r i c y a n i d e were used ( i . e . DHTML/ferricyanide = 0.5) was 6,7,8-trimethyllumazine. The balanced r e a c t i o n equation 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 complicated i n the more b a s i c r e g i o n s . In the pH range 10.5 to 12.5, the f i n a l organic products o f the r e a c t i o n , when the i n i t i a l r eactant concentrations are s t o i c h i o m e t r i c , are 7~oxo-6,8-dimethyl-lumazine and what appears to be 5-amino-4-methylamino-2,6-d i h y d r o x y p y r i m i d i n e . By the p r e v i o u s l y described quenching procedure, 6,7,8-trimethyllumazine was found to be an intermediate i n the r e a c t i o n i n t h i s pH range. From measurement of the absorbance o f the 7-oxo-6,8-dimethyl-154 lumazine produced, i t was shown that one mole o f DHTML y i e l d e d o n l y approximately one h a l f a mole of the 7-oxo compound w i t h the remainder being the pyri m i d i n e d e r i v a t i v e . That the py r i m i d i n e was not present i n s i g n i f i c a n t q u a n t i t i e s i n the aqueous stock s o l u t i o n o f the 7,8-dihydro-6,7,8-trimethyllumazine used to i n i t i a t e the r e a c t i o n was confirmed by repeated chromatographic a n a l y s i s o f the stock s o l u t i o n . In the intermediate pH.range 8.0 to 10 .5, a mixture of tr i m e t h y l l u m a z i n e , 7-oxo -6,8-dimethyllumazine and small amounts o f 5-amino -4-methylamino-2,6-dihydroxypyrimidine were found i n the f i n a l r e a c t i o n s o l u t i o n s . This and the above product analyses i n d i c a t e a change i n mechanism i n going from n e u t r a l to more ba 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 the s t a r t i n g m a t e r i a l s were used w i t h the organic substrate i n excess, unreacted q u a n t i t i e s o f the dihydro lumazine were detected i n the f i n a l r e a c t i o n s o l u t i o n s over the e n t i r e range o f pH i n v e s t i g a t e d . For the r e a c t i o n s c a r r i e d out i n b a s i c media, a sma l l drop i n the pH o f the i n i t a l r e a c t i o n s o l u t i o n of the order o f 0.01 pH u n i t s occurs during the course o f the r e a c t i o n . b. E f f e c t of hydroxide i o n O x i d a t i o n r a t e s were determined by spectrophotometry methods i n the pH range 10.5 to 12.5 i n 0.050M phosphate b u f f e r i n order to examine the e f f e c t o f hydroxide i o n on the 155 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 b u f f e r s o l u t i o n s was o maintained at 0.25 and the temperature at 25.0 . By maint a i n i n g 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 constant and v a r y i n g the co n c e n t r a t i o n o f DHTML, the r a t i o o f i n i t i a l r e a c t a n t concentrations 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 ) . In t h i s pH range, a second-order k i n e t i c treatment of the data gave r a t e p l o t s which very c l o s e l y approximated a s t r a i g h t l i n e over 65 to 75 percent of the t o t a l r e a c t i o n . Beyond t h i s p o i n t , the r e a c t i o n r a t e tended to slow down but i n a few cases, e s p e c i a l l y i n the more bas i c b u f f e r s , i t began to a c c e l e r a t e . The " i n f i n i t y " value o f a given s o l u t i o n measured at 420 mu u s u a l l y l a y somewhat above (•~10$) the value c a l c u l a t e d f o r remaining 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 organic species at t h i s wave-l e n g t h . In the case where the i n i t i a l r eactant c o n c e n t r a t i o n r a t i o i s 0.25, the r e a c t i o n followed second-order k i n e t i c s f o r approximately 40 percent o f the 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 . In t h i s p a r t i c u l a r case only-one h a l f the t o t a l amount o f potassium f e r r i c y a n i d e i n i t i a l l y present had been consumed. A t y p i c a l r a t e p l o t f o r the 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 the i n i t i a l r e a ctant concentrations are 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 Figure 20. S i m i l a r r a t e p l o t s were obtained f o r those r e a c t i o n s i n which n o n - s t o i c h i o m e t r i c amounts o f reac t a n t s were i n i t i a l l y present using the appropriate FIGURE 20 OXIDATION KINETICS TYPICAL RATE PLOT, SPECTROPHOTOMETRY METHOD 0 50 100 150 2 0 0 250 T l M E ^ S E C O N D S ) 1 5 7 k i n e t i c treatment o f the data. Concern that p o s s i b l e s i d e r e a c t i o n s of the organic s u b s t r a t e w i t h the 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 that 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 Table IX are the observed second-order r a t e constants, k^, p e r t a i n i n g to the disappearance 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 concentrations at various pH values. The data 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 concentrations of reactants are c o l l e c t e d i n Table 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 the degree of l i n e a r i t y of the p l o t t e d data. In ge n e r a l , the e r r o r s i n the reported r a t e constants are of the order o f 0 . 2 to 1 . 5 percent as estimated by comparison o f i d e n t i c a l runs. A p l o t o f the p e r t i n e n t data contained i n Tables IX and X versus 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 apparent 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 organic 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 of the oxidant i n the more basic pH regions i s i n d i c a t e d i n t h i s F i g u r e . When the observed r a t e constants are p l o t t e d a g a i n s t hydroxide i o n c o n c e n t r a t i o n , l i n e a r r e l a t i o n s h i p s are obtained (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 the hydroxide i o n concent r a t i o n i n 1 5 8 Table IX The e f f e c t of pH on the o x i d a t i o n r e a c t i o n of DHTML and potassium f e r r i c y a n i d e 0.050M K2HPC>4, u = 0 . 2 5 , T = 2 5 . 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 pH1" —A ( i • m o l e ~ l s e c • _ 1 ) ] 1 2 . 5 2 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 s o l u t i o n 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 A ( l , m o l e ~ 1 s e c . _ 1 ) r 1 2 . 5 2 4 1 . 8 6 0 . 9 9 8 3 1 2 . 0 1 1 7 . 8 7 O . 9 9 9 6 1 1 . 4 9 8 . 4 5 O . 9 9 8 1 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 c o n c e n t r a t i o n r a t i o DHTML/ferricyanide 2 J2S k (l.mole" sec. ) r 1 2 . 0 1 9.16 0 .9981 1 1 . 4 7 5 .29 0 .9994 1 1 . 0 2 3.46 0 .9994 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 . 2 5 the pH range 10 to 1 2 . 5 . The extreme r a p i d i t y o f the o x i d a t i o n r e a c t i o n i n more bas i c s o l u t i o n s prevented 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 process i n strong aqueous a l k a l i . E x t r a p o l a t i o n to zero hydroxide i o n co n c e n t r a t i o n In Figure 22 y i e l d s a r e l a t i v e l y s m a ll r a t e constant c o n s i s t e n t v/ith the trend toward slower rates f o r the 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 out i n more n e u t r a l b u f f e r s o l u t i o n s . order r a t e constants versus the negative logarithm o f the hydroxide i o n c o n c e n t r a t i o n (Figure 23) a f f o r d s a s t r a i g h t l i n e o f very n e a r l y u n i t slope In the case o f those.reactions 1 2 . 0 1 64 . 65 A p l o t o f the logarithms of the observed second-160 FIGURE 81 OXIDATION KINETICS 16.1 FIGURE 22 OXIDATION KINETICS RELATIONSHIP BETWEEN OXIDATION RATE AND HYDROXIDE ION CONCENTRATION 162 FIGURE 23 1.00 - L 0 G 1 0 [ 0 H " ] 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 ctant concentrations were employed. For the other 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 slopes 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 organic substrate increases 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 experiments which 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 . 0 5 0 M phosphate b u f f e r at pH 1 2 . 0 using s t o i c h i o m e t r i c i n i t i a l r e a ctant concentrations unless otherwise s p e c i f i e d . c_. A c t i v a t i o n Parameters The energy requirements of a given 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 or more d i f f e r e n t temperatures. In the usual manner, use i s made of the equation l o g k/T = 10.319 - 1 / T ( AH*A . 5 7 4J + As*/2*-574 (13 ) where k = measured r a t e constant, A H = enthalpy o f a c t i v a t i o n , As* = entropy o f 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 slope can be used to c a l c u l a t e AH*. A more accurate value o f A S i s u s u a l l y obtained by s u b s t i t u t i o n o f A H back i n t o equation (.13) r a t h e r than using the i n t e r c e p t of 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 , Ea, i s given by A H + RT, or over the range of temperatures near room temperature, 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 studied using the spectrophotometric technique. The temperature of the c e l l compartment o f the spectrophotometer was determined a c c u r a t e l y w i t h a Chromel-Alumel thermocouple (reference j u n c t i o n at 0°) and a Honeywell Model 2732 potentiometer. The r e s u l t s o f the a c t i v a t i o n parameter study are presented i n Table XI. Table XI A c t i v a t i o n parameters; o x i d a t i o n of DHTML w i t h potassium f e r r i c y a n i d e pH = 12.0, 0.050M K2HP04, u = 0 .25, DHTML/ferricyanide =0 . 5 Temperature T k„ l ° C . r ^ (°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.) versus - l o g k A/T 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 i l l u s t r a t e d i n Figure 24. From the slope 165 FIGURE 24 OXIDATION KINETICS 1 6 6 o f t h i s l i n e and equation (13) the c a l c u l a t e d magnitudes of the a c t i v a t i o n parameters are as f o l l o w s : A H * = 8 . 0 -± 0.4 k c a l . m o l e " 1 A s * = -24.6 ± 1.3 e.u. Ea = 8.6 k c a l . m o l e - 1 In any mechanistic study i t i s d e s i r a b l e to compare ex p e r i m e n t a l l y determined a c t i v a t i o n parameters w i t h values 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, there 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 data a v a i l a b l e on systems s i m i l a r to the present one and t h i s i s e s p e c i a l l y true 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 b u f f e r s o l u t i o n s (0.050M K^HPO^) at pH 1 2 . 0 were prepared c o n t a i n i n g amounts of added potassium f e r r o c y a n i d e ranging i n c o n c e n t r a t i o n from 0 to 1.0 x 10""%. The 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-fold excess over 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 maintained at 0 . 3 0 by the a d d i t i o n o f potassium c h l o r i d e . In 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 continued to e x h i b i t second-order k i n e t i c s although there was 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 percent completion o f the r e a c t i o n . A c o n t r o l experiment showed that potassium f e r r o c y a n i d e and 7,8,-dihydro-6 , 7,8-trimethyllumazine do not 167 r e a c t under the c o n d i t i o n s of t h i s experiment. S i m i l a r l y , 6 , 7 , 8 - t r i m e t h y l l u m a z i n e and potassium ferrocyanide do not appear to r e a c t . The r e s u l t s of the experiments c a r r i e d out i n the "doped" b u f f e r systems are c o l l e c t e d i n Table X I I . The reported second-order r a t e constants r e f e r to the f i r s t 50 percent o f the r e a c t i o n . Table X I I E f f e c t o f added potassium ferrocyanide on the o x i d a t i o n r e a c t i o n o f DHTML wi 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 , T = 2 5 . 0 ° I n i t i a l added K^Fe(CN)g n k A (moles. 1 ( l .mole - 1sec . - 1 ) 0 . 0 4 2 . 0 6 9 . 7 x 1 0 " 5 . 3 7 . 7 8 - 4 9 . 7 x 10 21 . 2 3 9 . 7 x 1 0 " 3 . 3 . 3 1 n - co r r e c t e d f o r d i l u t i o n by added reactant s o l u t i o n s . I t i s evident from the r e s u l t s i n Table X I I that f e r r o c y a n i d e Ion has an i n h i b i t o r y e f f e c t on the rat e o f the o x i d a t i o n r e a c t i o n . Therefore, the r a t e a c c e l e r a t i o n observed when nearing the completion of the r e a c t i o n s does not appear to be r e l a t e d d i r e c t l y to the presence of the hexacyano-f e r r a t e ( l l ' ) s p e c i e s . 168 A p l o t o f the r e c i p r o c a l s of the second-order r a t e constants 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 Figure 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 inverse p r o p o r t i o n a l i t y that e x i s t s between the o x i d a t i o n r a t e and the co n c e n t r a t i o n o f the hexa c y a n o f e r r a t e ( I I ) s p e c i e s . £• 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 at pH 12.0 were prepared i n which the i o n i c s t r e n g t h was adjusted to 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 . In t h i s manner, the anion o f the s a l t i s maintained at a constant value o f 0.050M while the t o t a l potassium i o n c o n c e n t r a t i o n i s O.167M unless otherwise noted. The r e s u l t s o f these experiments are presented i n the f o l l o w i n g Table. Table X I I I E f f e c t of anions on the DHTML-ferricyanide o x i d a t i o n r e a c t i o n pH = 12.0, 0.050M KgHPOip u = 0 . 2 5 , T = 25.0° Concentration'of anion = 0.050M, t o t a l potassium i o n c o n c e n t r a t i o n = O.167M unless otherwise noted. Potassium s a l t k^ ( l .mole _ 1sec . ""*") KC1 3 5 . 5 6 KN0 3 3 4 . 7 2 K B r 0 3 2 8 . 8 1 K 2 S 0 4 ° 1 9 . 6 6 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 that the l a r g e r and more h i g h l y charged anions have an i n h i b i t o r y e f f e c t on the o x i d a t i o n r e a c t i o n r a t e . The decrease observed i n the case of potassium s u l f a t e may be due, i n p a r t , to the t o t a l decreased amount o f potassium i o n of the b u f f e r s o l u t i o n r e l a t i v e to those b u f f e r s c o n t a i n i n g the other s a l t s . f. E f f e c t o f potassium i o n Many p o l y v a l e n t c a t i o n s a s s o c i a t e w i t h f e r r i c y a n i d e i n aqueous s o l u t i o n but of the u n i v a l e n t c a t i o n s only 166 l 6 ? potassium i o n and tetraalkyammonium ions are known to do so. This i s r a t h e r s u r p r i s i n g i n view o f the larg 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 anion and a c a t i o n . In c o n t r a s t , there i s evidence f o r a s s o c i a t i o n o f a larg e number of ca t i o n s w i t h . . 159,168 f e r r o c y a n i d e . -169 In a study of the k i n e t i c s of the e l e c t r o n exchange between hex 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 to depend markedly upon the nature and co n c e n t r a t i o n o f the c a t i o n s present. In the r e a c t i o n between potassium ferro c y a n i d e and potassium 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 rat e constants w i t h potassium 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 Io n i c s t r e n g t h at constant c a t i o n c o n c e n t r a t i o n . In p a r t i c u l a r , the r a t e increase w i t h i n c r e a s i n g potassium i o n co n c e n t r a t i o n 170 was concluded to be a r e s u l t of 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~-5 and K S 2 O 3 " , r a t h e r than between the corresponding unassociated s p e c i e s . At the r i s k of the r e s u l t s being l e s s than q u a n t i t a t i v e because of the apparent rate-decreasing e f f e c t of the s u l f a t e anion, the dependence of the o x i d a t i o n r e a c t i o n on i n c r e a s i n g potassium i o n was studied at pH 12.0. A s e r i e s of 0.050M b u f f e r s were prepared i n which varying amounts of KC1 and KgSO^ were added to increase the t o t a l potassium i o n c o n c e n t r a t i o n while maintaining the i o n i c s t r e n g t h constant. The r e s u l t s of 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 are presented In Table XIV. Table XIV E f f e c t o f potassium i o n on the DHTML-ferricyanide o x i d a t i o n r e a c t i o n pH 12.0, 0.050M KgHPO^, T = 25.0°, u = 0.40 T o t a l potassium i o n c o n c e n t r a t i o n k ^ (moles.I"-1-) ( 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 of the data i n t h i s Table 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 Figure 26. E x t r a p o l a t i o n o f the l i n e 172 to a concentration of potassium ion equal to 0.151M yield s a rate approximately of the order found for the experiment i n which potassium sulfate alone was used to adjust the ionic strength (see Table XIII). This indicates that the lower rate observed i n this l a t t e r experiment was probably a r e s u l t of the lower potassium ion concentration. A p l o t of the logarithms of the rate constants and potassium ion concentration (Figure27) yie l d s a f a i r l y s t r a i g h t l i n e of slope I . 3 6 . g_. Kinetic isotope e f f e c t The presence of 6 , 7 , 8-trimethyllumazine as an intermediate i n the oxidation reaction of DHTML i n basic media requires the rupture of the C-7 carbon-hydrogen bond i n the l a t t e r compound at some stage In the reaction. The oxidation of 7,8-dihydro-6,7>8-trlmethyllumazine-7-d was examined i n order to est 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 step. Measurements were made under conditions s i m i l a r to those previously employed i n the case of the protlo compound. The results of three runs at. pH 12.0 using the deuterated dihydrolumazine are presented i n Table XV. For comparison, the rate constants for the oxidation reaction of DHTML under i d e n t i c a l conditions are Included. 1 7 3 FIGURE 2 6 GO-OXIDATION KINETICS EFFECT OF POTASSIUM ION ON THE OXIDATION RATE DHTML/FERRICYANIDE = 0 . 5 u = 0 . 4 0 , T = 2 5 . 0 ° , pH = 1 2 . 0 0 . 0 5 0 M K2HPO4 5 5 -K A (l.mole~1sec. 1 ) 5 0 -4 5 -4 0 -3 5 -0.24 2 - 2 -1 SLOPE = 2 1 6 l.mole sec. 0 . 2 6 0 . 2 8 K "^ (mole.l" 1) 0 . 3 0 0 . 3 2 174 FIGURE 27 OXIDATION KINETICS RELATIONSHIP BETWEEN POTASSIUM ION CONCENTRATION AND OXIDATION RATE (same experimental c o n d i t i o n s as i n FIGURE 26) .72-168 LOG, 0K AJ SLOPE = 1 . 3 6 1.64-.60-060 0.56 - L0G 1 0 Q52 0.48 175 Table XV K i n e t i c isotope e f f e c t ; o x i d a t i o n of 7 , 8 - d l h y d r o - 6 , 7 , 8 -trimethyllumazine w i t h potassium f e r r i c y a n i d e pH = 12.0, 0.050M K 2HP04, u = 0 . 2 5 , T = 25.0° DHTML/ferricyanide = 0 . 5 k A (H) k A (D) k H / k D (l.mole" sec." ) (l.mole" sec. ) 35.82 35.26 36.13 30.61 28.94 30.14 1.17 1.22 1.20 Mean k H / k D = 1.2 The kjj/kj-j value i s much sm a l l e r than those that are o r d i n a r i l y considered primary isotope e f f e c t s . In the case of carbon-bound hydrogen, a k^/k^ value of more than 2.0 1^ 2 (at room temperature) suggests that the hydrogen (or deuterium) atom i s being 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 step of the r e a c t i o n . In the present case, the k^/k^. value l i e s o u tside the range o f experimental e r r o r normally encountered w i t h the spectrophotometric a n a l y s i s technique. This i s so even when allowances are made f o r the f a c t that a 4 percent i m p u r i t y of the p r o t i o compound i s present i n the samples of the deuterated dlhydrolumazine. 2. Potentiometry The o x i d a t i o n r e a c t i o n of DHTML w i t h f e r r i c y a n i d e was stud i e d i n the pH range 5 to 12 using the p r e v i o u s l y described 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 intended 176 purpose o f t h i s method v/as to o b t a i n r a t e data i n the pH range where the spectroscopic technique was not a p p l i c a b l e . ' However, the 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 . This i s c l e a r l y i l l u s t r a t e d i n the rat e p l o t s shown i n Figure 28. F o l l o w i n g 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 extent o f which depends upon the pH, a slower n e a r l y second-order disappearance 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 the rat e was.noted near the end o f the r e a c t i o n s . I t i s immediately evident that the expected d i r e c t comparison o f the data obtained by spectroscopic and po t e n t i o m e t r i c techniques i s not r e a l i z e d . I t i s noteworthy that at pH 5, where the organic substrate i s n e u t r a l r a t h e r than the mono-anion as i s the case i n more basic r e g i o n s , a marked increase i n the extent of the i n i t i a l r a t e i s observed. The second-order r a t e constants determined by the po t e n t i o m e t r i c technique, k^,, are presented i n Table XVI at r e p r e s e n t a t i v e pH. These constants have been c a l c u l a t e d from the slopes o f the s t r a i g h t l i n e p o r t i o n of the r a t e p l o t s . Table XVI Oxidati o n r e a c t i o n ; r a t e constants determined by potentiometry 0.050M K2BTO4, u =0 . 2 5 , T = 25.0°, DHTML/ferricyanide = 0 .5 pH k Ap ( l . m o l e - 1 s e c . - 1 ) 12.0 21.55 11.0 2.82 9.0 2.20 FIGURE 28 T IME ( S E C O N D S ) 1 7 8 Table XVI (continued) pH k Ap (l.mole sec." ) 7 . 0 1 .20 5 . 0 0 . 1 5 By comparison o f the above data w i t h data obtained by the spec t r o s c o p i c technique i t i s evident that k A - ~ 2 k A p . 3 . Reaction o f 6 , 7 , 8 - t r i m e t h y l l u m a z i n e w i t h f e r r i c y a n i d e -a. Spectrophotometry r e s u l t s The r e a c t i o n between TML and potassium f e r r i c y a n i d e was monitored at 420 mu under s i m i l a r c o n d i t i o n s to those used i n the o x i d a t i o n o f DHTML. At pH 7 . 0 , very slow changes i n the absorbance at 420 mu (and 404 mu) i n d i c a t e d that 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 reactants i n n e u t r a l s o l u t i o n . In s t r o n g l y basic media, however, TML. and f e r r i c y a n i d e reacted r a p i d l y to y i e l d u l t i m a t e l y 7-oxo-6 , 8-dimethyllumazine and ferrocy a n i d e i o n . By spec t r o s c o p i c a n a l y s i s i t was as c e r t a i n e d that two moles o f the hexacyano-f e r r a t e ( l l l ) i o n r e a c t s w i t h one mole of TML to produce one mole of the 7-oxo compound and two moles o f f e r r o c y a n i d e . This i s somewhat s u r p r i s i n g i n view o f the f a c t that formation o f the 7-oxolumazine by o x i d a t i v e demethylation o f the C-7 methyl group o f TML would be expected to r e q u i r e more than two equ i v a l e n t s o f f e r r i c y a n i d e . When equimolar amounts of f e r r i c y a n i d e and TML are allowed to re a c t at pH 1 2 . 0 , an orange-coloured intermediate ( ^  max ^ 2 m x > £ ~ 1 0 ^ ) appears during the course of the r e a c t i o n . This m a t e r i a l was unstable as i n d i c a t e d by a slow 179 change o f the spectrum to that c h a r a c t e r i s t i c of 7-oxo-6,8-dimethyllumazine, even a f t e r a l l o f the f e r r i c y a n i d e was presumably consumed. This intermediate was not detected when an i n i t i a l excess of the oxidant was used. The disappearance of the intermediate i s a c c e l e r a t e d i n the presence o f oxygen. When the r e a c t i o n was c a r r i e d out i n an e.s.r. tube under the same c o n d i t i o n s as described 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 could be detected. b_. Potentiometry The r e a c t i o n of TML and f e r r i c y a n i d e was studied under the same c o n d i t i o n s employed In the o x i d a t i o n of DHTML. The r a t e p l o t s of the TML-ferricyanide 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 curvature c h a r a c t e r i s t i c o f the o x i d a t i o n r e a c t i o n s of DHTML monitored by potentiometry. The r e s u l t s o f t h i s experiment are c o l l e c t e d i n Table XVII. The reported r a t e constants r e f e r to the 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 data when p l o t t e d i n a second-order manner. Table XVII TML-ferricyanide 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 the r e a c t i o n r a t e , TML/ferricyanide = 0.5 0.050M KpHPOip u - 0.25, T = 25.0° pH k Ap ( l . m o l e _ 1 s e c . ~ 1 ) 12.04 16.76 9.02 1.82 7.00 o . n q q - i n i t i a l r a t e ; remainder of r e a c t i o n extremely slow a f t e r one e q u i v a l e n t of f e r r i c y a n i d e consumed. 180 When equimolar amounts of f e r r i c y a n i d e and TML were allowed to r e a c t at pH 1 2 . 0 , the p o t e n t i a l of the 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 the orange-coloured intermediate was noted. 4. Miscellaneous r e a c t i o n s a. D i s p r o p o r t i o n a t i o n experiment Equlmolar mixtures 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 were examined i n phosphate b u f f e r systems under n i t r o g e n atmosphere over the pH range 1 to 1 2 . No apparent r e a c t i o n between the above lumazines was observed as shown by s p e c t r a l scans o f the s o l u t i o n s . In base, small changes i n the composite spectrum of the mixture could be a t t r i b u t e d to the slow decomposition o f the dihydrolumazine. Thus, 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 TML + DHTML 2HTML* analogous 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 leuco-117 f l a v i n - f l a v i n couple e v i d e n t l y does not occur here. b. 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 potassium permanganate produced 7~oxo - 6 , 8-dimethyllumazine • but the r e a c t i o n proved to be too ra p i d to measure w i t h the a v a i l a b l e techniques. 6 ,7,8-Trimethyllumazine was found to be an intermediate i n t h i s r e a c t i o n . 181 D. D i s c u s s i o n The o x i d a t i o n - r e d u c t i o n chemistry of 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 has r e c e n t l y been reviewed . 1 5 9 The r e a c t i o n s of 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 one or the other o f two c l a s s e s . The f i r s t comprises the c l a s s i c r e a c t i o n s which i n v o l v e no more than a simple 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 very r a p i d . The second group in c l u d e s those r e a c t i o n s which are r e l a t i v e l y slow and are c h a r a c t e r i z e d by complicated k i n e t i c s . An e x a m p l e 1 ^ of the l a t t e r group of r e a c t i o n s i s the o x i d a t i o n o f aldehydes, ketones and 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. These compounds appear to be o x i d i z e d through the anion of the e n o l -or a c i - f o r m and the r e a c t i o n proceeds i n s e v e r a l consecutive stages. Free organic r a d i c a l s could not be detected by the 160 v i n y l p o l y m e r i z a t i o n technique 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 anion of the s u b s t r a t e , which i s then attacked by a second f e r r i c y a n i d e i o n or another complex. I t i s evident from the r e s u l t s of the present study that the o x i d a t i o n of 1,8~dihydro-6,"J,8-trimethyllumazine w i t h f e r r i c y a n i d e belongs to the second group of r e a c t i o n s of t h i s o x i d a n t . In the pH range examined (with the exception of the experiment c a r r i e d out at pH 5) the dihydrotrimethyllumazine e x i s t s predominantly as the mono-anion. However, the product 182 a n a l y s i s shows that the p a r t i c u l a r product(s) formed i n the o x i d a t i o n r e a c t i o n depends on the i o n i z a t i o n o f the t r i m e t h y l -lumazine intermediate r a t h e r than on the i o n i z a t i o n of the dihydrolumazine p r e c u r s o r . That i s , above pH 10 where the trimet h y l l u m a z i n e e x i s t s as the hydroxylated anion (see s t r u c t u r e L I I I ; R = CH3), the r e a c t i o n proceeds to give u l t i m a t e l y 7-oxo - 6 , 8-dimethyllumazine. In the pH regi o n where the former compound i s n e u t r a l , the o x i d a t i o n of DHTML y i e l d s o n l y the trimethyllumazine i t s e l f . The s mall enthalpy and energy of 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 possessing r e l a t i v e l y low energy reguirements. The la r g e and negative entropy term i s c o n s i s t e n t w i t h the coulombic i n t e r a c t i o n s expected of two (or p o s s i b l y more) s i m i l a r l y charged species corning together 173 In the t r a n s i t i o n s t a t e of the r e a c t i o n . L a i d l e r deduces that f o r i o n i c r e a c t i o n s such as the present one the 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 cr u d e l y estimated from the f o l l o w i n g equation ( l 4 ) : A S L . - - 1 0 Z A Z B ^ where z^, z^ = e l e c t r o n i c charges present on the r e a c t i n g species A and B r e s p e c t i v e l y . This gives a value o f A S~j* = -30 e.u. f o r the case where the r e a c t i n g species are the 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 of DHTML However, the dependence o f the rat e on potassium i o n -2 c o n c e n t r a t i o n suggests that the asso c i a t e d s p e c i e s , KFe(CN)g ~ may be the r e a c t i v e oxidant under the c o n d i t i o n s employed. 183 This would lead to a value o f A s „ „ of -20 e.u., not f a r e. s . ± from the exp e r i m e n t a l l y determined value of A S . The small 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 suggests that the C-7 carbon-hydrogen bond i s not being broken i n the r a t e - c o n t r o l l i n g step of the r e a c t i o n i n ba 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 7,8-dihydro-6,7,8-t r i m e t h y l l u m a z i n e by f e r r i c y a n i d e are second-order, f i r s t -o rder i n oxidant and f i r s t - o r d e r In organic s u b s t r a t e . 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 at present. 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 can be i n f e r r e d from the r e s u l t s : v — J (1) DHTML + OH" 7. DHTML" + HpO very r a p i d •\ -4 (2) F e ( C N ) 6 " J + DHTML X + Fe(CN)g r e v e r s i b l e -3 -4 (3) Fe(CN)g ° + X . * TML + Fe(CN)g (4| OH" +; X —> X' Although not i n d i c a t e d i n the above sequence o f r e a c t i o n s , i t i s expected that some o f the f e r r i c y a n i d e w i l l r e a ct as the -2 a s s o c i a t e d species KFe(CN)g The nature o f X and X' have not been e s t a b l i s h e d w i t h c e r t a i n t y . I t i s noted that e l e c t r o n a b s t r a c t i o n from the anion o f DHTML w i l l generate the mesomeric r a d i c a l (LXXVII). 184 <—> etc (LXXVII) In s o l u t i o n s where the dihydrolumazine i s n e u t r a l (e.g. pH 5) 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 which, upon l o s s o f a proton, gives the same r a d i c a l (LXXVII). Further e l e c t r o n a b s t r a c t i o n from (LXXVII) can lead to the production o f 6 , 7 , 8-trircethyIlumazine, as In d i c a t e d below. This corresponds to step (3) of the r e a c t i o n sequence and i s l i k e l y the r a t e - c o n t r o l l i n g step f o r the o x i d a t i o n r e a c t i o n i n the n e u t r a l pH re g i o n s . This i m p l i e s that the e q u i l i b r i u m (2) i s s h i f t e d to the right-hand side i n order that the r e a c t i o n be f i r s t - o r d e r w i t h respect to f e r r i c y a n i d e i o n . CH-H 3 C CH 8 8 iH k CH-z I ° H ^ ( ^ K > k / Q o 0 185 A concurrent r e a c t i o n o f X wit h hydroxide i o n (step 4) may be o c c u r r i n g which a f f e c t s the rate of f e r r i -cyanide consumption i n the more bas 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) to the r i g h t . This does not deny the p o s s i b i l i t y of 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 i t s e l f . The p o s s i b i l i t y of d i m e r i z a t i o n of (LXXVIIJ to produce (LXXVTII) e x i s t s . Proton removal from the 7- or 7 ' - p o s i t i o n of t h i s species by base can lead to the formation of DHTML anion and tr i m e t h y l l u m a z i n e . C H 3 H 3 C (LXXVIIIj D i m e r i z a t i o n o f r a d i c a l intermediates i n the re a c t i o n s o f c e r t a i n organic compounds v/ith 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 formulated. P o t e n t i o m e t r i c a n a l y s i s of the o x i d a t i o n r e a c t i o n I n d i c a t e s that f e r r i c y a n i d e i s consumed r a p i d l y In the i n i t i a l stages o f the r e a c t i o n over the range of 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 at pH 5, where the 186 dihydrotrirnethyllumazine e x i s t s predominantly as the n e u t r a l s p e c i e s , i s l i k e l y due 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 of oxidant i s not i n d i c a t e d by spectrophotometry a n a l y s i s , however. I t i s i n f e r r e d that the production of X causes a c o n t r i b u t i o n to the absorbance at 420 mu i n a manner which masks the I n i t i a l uptake of f e r r i c y a n i d e . I f X possesses the r a d i c a l s t r u c t u r e (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 an excess of the organic s u b s t r a t e w i l l cause the observed r e t a r d i n g e f f e c t on the r e a c t i o n r a t e . A s i m i l a r e f f e c t , a l s o unexplained, was encountered b y ' S t e i n and Tendeloo 1^^ i n a study of the r e a c t i o n between p h l o r o g l u c i n o l and f e r r i c y a n i d e . The o x i d a t i o n of 6 , 7 , 8-trimethyllumazine v/ith f e r r i c y a n i d e produces 7-oxo - 6 , 8-dimethyllumazine In basic media v i a a h i g h l y - c o l o u r e d , unstable, int e r m e d i a t e . This l a t t e r species i s l i k e l y a r a d i c a l but t h i s could not be confirmed. Apparently, on l y two e q u i v a l e n t s of the o x i d i z i n g agent are required to complete the r e a c t i o n . This i s l e s s than what would be expected f o r the removal of the C-7 methyl group of TML by o x i d a t i o n to the corresponding c a r b o x y l i c a c i d followed by d e c a r b o x y l a t i o n . Although s u f f i c i e n t i n f o r m a t i o n to e l u c i d a t e the r e a c t i o n mechanism f u l l y i s s t i l l 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 : 187 (5) TML + OH" C H 3 (6) H 3 C q H f < k > K C H 3 j^ TMLOH" H H 3e P H + Pe(CN) 6 -3 f a s t 11 O X" + Fe(CN) -4 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 the hydroxylated anion of t r i m e t h y l -lumazine. This would account f o r the orange c o l o u r of the intermediate observed i n the r e a c t i o n . a H , N k / ^ 0 • O ^ H N x ^ ^ o 1 > o . (XXXIX) etc Further r e a c t i o n s o f (LXXIX) are p o s s i b l e . For example; I . One-electron a b s t r a c t i o n from (LXXIX) by a second mole of f e r r i c y a n i d e can lead to the c a t i o n (LXXX). R i n g - c l o s u r e , rearrangement of the C-7 methyl 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 of methanol. A s i m i l a r rearrangement of a c a t i o n occurs i n the chromic a c i d o x i d a t i o n of c e r t a i n t e r t i a r y 179 a l c o h o l s . The c a t i o n (LXXX) i s probably never f u l l y formed, the methyl 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 l e a d i n g to t h i s i o n . i i . 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 (LXXIX) leads to (LXXXI). Reaction of t h i s l a t t e r r a d i c a l w i t h any oxygen present i n s o l u t i o n gives the corresponding peroxide r a d i c a l which, upon r e a c t i o n w i t h water, y i e l d s the peroxide i t s e l f and hydrogen peroxide. E l i m i n a t i o n of formaldehyde and water from the organic peroxide produces 7-oxo - 6 , 8-dimethyl-lumazine. (LXXX) CH O (LXXXI) 189 The f a s t e r r a t e of disappearance of the coloured r e a c t i o n intermediate i n the presence of oxygen Is c o n s i s t e n t w i t h the preceding r e a c t i o n sequence. Attempts to detect formaldehyde and methanol i n the f i n a l s o l u t i o n s of the TML-ferricyanide r e a c t i o n by gas chromatography, as w e l l as attempts to detect C0 2 by ad s o r p t i o n onto A s c a r i t e , have not been s u c c e s s f u l . This 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 of the small q u a n t i t i e s of reactants used. That TML and f e r r i c y a n i d e r e a c t only s l o w l y under c o n d i t i o n s where the organic compound i s n e u t r a l i n d i c a t e s that increased, a c t i v a t i o n of the C-7 methyl group as w e l l as formation o f the hydroxylated anion are necessary requirements 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 s i m i l a r i t y between the k i n e t i c s of the o x i d a t i o n of the present model 177 system and those of 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 proceed 1 0 5 , 1 0 9 through a r e l a t i v e l y s t a b l e semiquinone int e r m e d i a t e . E x t r a s t a b i l i z a t i o n of the r a d i c a l intermediate i s provided 104 by the benzene r i n g i n the f l a v i n system. The l a c k of 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 regard to the i n s t a b i l i t y of r a d i c a l intermediates i n the dihydro-lumazine-lumazine system. 199 • SUGGESTIONS FOR FURTHER WORK The o x i d a t i o n of 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 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 areas i s required to e l u c i d a t e the r e a c t i o n mechanism f u l l y . For i n s t a n c e , a study of the e f f e c t s o f added fe r r o c y a n i d e i o n on the r e a c t i o n r a t e and measurement of the isotope e f f e c t i n the n e u t r a l pH regions i s necessary. I t would be j u d i c i o u s to study the o x i d a t i o n r e a c t i o n w i t h another one-electron reagent i n order that a comparison of r e s u l t s using the two oxidants can be made. Manganic a c e t a t e , which i s known to re a c t by one-electron t r a n s f e r , seems a s u i t a b l e reagent f o r t h i s purpose. In the case of the 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 basic media, a study of the e f f e c t of added fer r o c y a n i d e on the r e a c t i o n r a t e i s required i n order to e s t a b l i s h whether the i n i t i a l step (6) of the r e a c t i o n i s r e v e r s i b l e . Confirmation o f the r a d i c a l nature of the orange-coloured intermediate might be achieved by attempting to polymerize a c r y l o n i t r i l e 1 ^ i n i t s presence. In conjunction with the exchange study o f DHTML, a d d i t i o n of hydrogen peroxide to a n e u t r a l s o l u t i o n of the compound i n D 2 0 was employed to produce 6 , 7 , 8 ~ t r i m e t h y l l u m a z i n e . A r e l a t i v e l y s t a b l e intermediate of t h i s r e a c t i o n was detected and 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 that i t may be the hydro-peroxide (see s t r u c t u r e LXI) or the corresponding hydroxylated 191 compound of DHTML. An i n v e s t i g a t i o n of the o x i d a t i o n of 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 hydrogen peroxide under a wide v a r i e t y o f c o n d i t i o n s seems p o s s i b l e using both p.m.r. techniques and spectrophotometry a n a l y s i s . I t would be of i n t e r e s t to determine the e f f e c t i v e n e s s of a c i d s and bases on the exchange r e a c t i o n s of both TML and DHTML i n more q u a n t i t a t i v e terms. For example, use of a s e r i e s of mono-protic a c i d s , such as the c h l o r o a c e t i c a c i d s , r a t h e r than the phosphate species might allow more accurate c a l c u l a t i o n o f the cC parameter i n terms of the Br/nsted 142 r e l a t i o n s . An I n t e r e s t i n g companion study to the t r l 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 would be the corresponding r e a c t i o n between 6 ,7-diphenyl~8-methyllumazine and f e r r i c y a n i d e . 34 I t has been shown J that o x i d a t i v e removal of the 7-phenyl group does not occur when 6 , 7-diphenyl - 8-methyl-2-oxopteridine and potassium permanganate are present together i n ac i d and a l k a l i n e s o l u t i o n . 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 occur between the 6 , 7-diphenyl-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 occurs w i t h the anion o f t h i s p a r t i c u l a r lumazine d e r i v a t i v e . Observations could be made using both p o t e n t i o m e t r i c and spectroscopic techniques. An ambitious program to synthesize 7 , 8-dihydro - 6 , 7 -diphenyl - 8-methyllumazine d e r i v a t i v e s w i t h various s u b s t i t u e n t s i n the phenyl r i n g s would a l l o w a study of the o x i d a t i o n 192 r e a c t i o n i n terms of s u b s t i t u e n t e f f e c t s . I t i s expected that 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 have a c o n s i d e r a b l e e f f e c t on the energy of the anion of the lumazine. This e f f e c t would be r e f l e c t e d i n the energy required to a b s t r a c t an e l e c t r o n from t h i s anion and a l s o i n the energy requirements of a r a d i c a l intermediate analogous to (LXXVTI). The r e l a t i v e s t a b i l i t y o f diphenylmethyllumazine i n aqueous s o l u t i o n commends i t s use i n such a study. I t would be i n t e r e s t i n g to study the r e a c t i o n o f DHTML w i t h oxygen, and f o r that matter other o x i d a n t s , under 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 of the dihydrolumazine does not i n t e r f e r w i t h the r e s u l t s . The l a t t e r problem may be circumvented by studying the o x i d a t i o n r e a c t i o n s i n anhydrous medium. P r e l i m i n a r y t e s t s i n d i c a t e that d i m e t h y l s u l f o x i d e provides a s u i t a b l e solvent system f o r such a study. 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 Ring Index", Second e d i t i o n , American Chemical 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 (1952) . 3 . M. Gates, Chem. Revs. 4 l , 63 (1947) . 4 . "The Chemistry and Biology of P t e r i d i n e s " , Ciba Foundation Symposium, J.A. C h u r c h i l l L t d . , London, 1954. 5. A. A l b e r t , F o r t s c h r . Chem. org. N a t u r s t o f f e , 11 , 350 (1954) . 6. W. P f l e i d e r e r , Angew. Chem. 75 , 933 (1963); I n t e r n . Ed. 3, 114 (1964) . 7- " P t e r i d i n e Chemistry", Third I n t e r n a t i o n a l P t e r i d i n e Symposium, S t u t t g a r t , 1962. Permagon Press, Oxford, 1964 - The Macmillan Company, New York, 1964. 8. " H e t e r o c y c l i c Compounds", R.C. E l d e r f i e l d ed., J . Wiley and Sons Inc., New York, 1967. Volume 9 . 9 . A. V e i l l a r d and B. Pullman, Compte. rend. 353, 24 l8 (1961) . 10. B. Pullman and A. Pullman, Proc. N a t l . Acad. S c i . U.S. 44, 1197 (1958) . 11 . T.H. Goodwin and A.L. P o r t e , J . Chem. Soc. 3595 (1956) . 12. 0 . Chalvet and C. Sandorfy, Compt. rend. 228, 566 (1949) . 13. S.F. Mason, i n reference 4 , page 74 . 14. D.J. Brown and S.F. Mason, J . Chem. Soc. 3443 (1956) . 15. V/. P f l e i d e r e r , J . Bunting, D.D. P e r r i n and G. Nubel, Chem. Ber. 99 , 3503 (1966) . 1.6. C.H. Winestock and G.W.E. P l a u t , J . Org. Chem. 26 , 4456 (1961) . 17. E.C. Tay l o r J r . , i n reference 4 , page 2 . -l 8 a . A. A l b e r t , D.J. Brown and G. Cheeseman, J . Chem. Soc. 474 (1951) . b. A. A l b e r t , D.J. Brown and G. Cheeseman, J . Chem. Soc. 1620 (1952) . 194 19 . J . W e i j l a r d , M. T i s h l e r and A.E. E r i c k s o n , J . Am. Chem. Soc. 67 , 802 (1945) . 20 . A. A l b e r t , J . Chem. Soc. 2690 (1955) . 2 1 . E.C. Tay l o r J r . , J . Am. Chem. Soc. 74 , 1651 (1952) . 22 . T.A. Hamor and J.M. Robertson, J . Chem. S o c , 3586 (1956) . 23 . A. A l b e r t , Angew. Chem. I n t . Ed. 6, 919 (1967) . 24. A. A l b e r t , i n reference 7, page 111. 25 . D.D. P e r r i n , i n "Advances i n H e t e r o c y c l i c Chemistry", A.R. K a t r i t z k y ed., Academic Press, New York., 1965. Volume 4 , pp. 4 3 - 7 3 . 26 . A. A l b e r t and G. B a r l i n , J . Chem. Soc. 5156 (1963) . 2 7 . A. A l b e r t , W.L.P. Amarego and E Spinner, J . Chem. Soc. 2689, 5267 (1961) . 28 . A. A l b e r t , C.F. Howell and E. Spinner, J . Chem. Soc. 2595 (1962) . 29 . A. A l b e r t and P. Reich, J . Chem. Soc. 127 ( 1 9 6 l ) . 30. J . C l a r k and D.D. P e r r i n , Quart. Revs. Chem. Soc. 18, 295 (1964); G.B. B a r l i n and D.D. P e r r i n , I b i d . 20 , 75 (1966) . 31 . R.N. Jones, J . Am. Chem. Soc. 67 , 2127 (1945) . 32. Y. Inoue and D.D. P e r r i n , J . Phys. Chem. 66, 1689 (1962) . 33 . A. A l b e r t , Y. Inoue and- D.D. P e r r i n , J . Chem. Soc. 5151 (1963) . 34. N. Jacobsen, J . Chem. Soc. (c) IO65 (1966) . 35 . 0 . P h i l l l p o w , J . Pra k t . Chem. 93 , 162 (1916) . 36. T. Rowan, H.C.S. Wood and P. Hemmerich, Proc. Chem. Soc. 260 (1961) . 37 . R.M. Cr e s s w e l l and H.C.S. Wood, J . Chem. Soc. 4768 ( i 9 6 0 ) . 38 . E.C. Ta y l o r and W.R. Sherman, J . Am. Chem. Soc. 8 l , 2464 (1959) . 39. M. Polonovski and M. Pesson, F i r s t I n t e r n . Congr. Biochem. Cambridge, 1949. page 231 . 195 40. A. A l b e r t and S. Matsuura, J . Chem. Soc. 5131 (1961) . 4 1 . A. A l b e r t and S. Matsuura, J . Chem. Soc. 2162 (1962) . 42 . A. A l b e r t and D.J. Brown, J . Chem. Soc. 74 (1953) . 43 . M. V i s c o n t i n i and S. Huwyler, Helv.. Chim. Acta. 48, 764 (1965) . 44 . M. Pesson, B u l l . soc. chim. France, 963 (1948); I b i d . 423, 458 (1951); M. Po l o n o v s k i , M. Pesson and A. P u i s t e r , I b i d . 521 (1951) . 45. G.B. E l l o n , i n reference 4 , page 49 f f . 46. H. Wieland and R. Purrmann, Ann. 544, 146 (1944) . 4 7 . B.L. O'Dell, J.M. Vanderbelt, E.S. Bloom, and J . J . P f i f f n e r , J . Am. Chem. Soc. 69 , 250 (1947) . 48. G.B. E l i o n and G.B. H i t c h i n g s , J . Am. Chem. Soc. 74 , 3877 (1952) . 49 . T. Rowan and H.C.S. Wood, J . Chem. Soc. (C) 452 (1968) . 50. M. V i s c o n t i n i and H.R. Wellenmann, Helv. Chim. Acta. 42, 1854 (1959); I b i d . 4 1 / 2 1 7 0 (1958) . 51 . A. S t u a r t , H.C.S. Wood and D. Duncan, J . Chem. Soc. (C) 285 (1966) . 52. M. V i s c o n t i n i and A. Bobst, Helv. Chim. Acta. 48, 816 (1965) . 53. G.B. E l i o n , A.E. L i g h t and G.B. H i t c h i n g s , J . Am. Chem. Soc. 7 1 , 741 (1949) . 54 . J.H. Boothe et a l , J . Am. Chem. Soc. 70 , 27 (1948) . 55. J.R. T r o t t e r , J . B i o l . Chem. 154, 105 (1944) . 56. A. Ahrenberg, P. Hemmerich, F. M u l l e r , T. Okada and M. V i s c o n t i n i , Helv. Chim. Acta. 50, 4 l l (1967) . 57. M. V i s c o n t i n i and T. Okada, Helv. Chim. Acta. 50 , 1492 (1967) . 58 . A. Bobst, Helv. Chim. Acta. 50, 2222 (1967) . 59. H.I.X. Mager, R. Addink and W. Berends, Rec. Trav. Chim. Pays-Bas, 86, 833 (1967) . 196 60. D.J. Vonderschmitt and K.G. Scrlmgeour, Biochem. Biophys. Res. Comm. 28 , 302 (1967). 61. H. Isay, Chem. Ber. 3 9 / 250 (1906).• 62. G.M. Timmis, Nature (London) l 6 4 , 133 (1949); U.S. Patent 2 ,581 ,889 (1952) . 63. H.S. F o r r e s t and J . Walker, J . Chem. Soc. 2077 (1949). 6 4 . W.R. Boon and W.G.M. Jones, J . Chem. Soc. 591 (1951) . 65. V/.R. Boon, W.G.M. Jones and G.R. Ramage, J . Chem. Soc. 96, (1951) . 66. W.R. Boon and T. Leigh, J . Chem. Soc. 1497 (1951). 67. E.C. Tay l o r J r . , i n reference 4 , page 104. 68. E.C. T a y l o r , J.A. Carbon and D.R. Hoff, J . Am. Chem. Soc. 75 , 1904 (1953) . 69. E.C. Ta y l o r , R.B. Garland and C F . Howell, J . Am. Chem. Soc. 78 , 210 (1956); W.B. Wright and J.M.. Smith, I b i d . 77 , 3927 (I955)i G.P. Dick and H.C.S. Wood, J . Chem. Soc. 1379 (1955) . 70. T.S. Osdene and E.C. Taylor J r . , J. Am. Chem. Soc. 78 , 5451 (1956) . . •71. H.K. M i t c h e l l , E.E. S n e l l and R.J. W i l l i a m s , J . Am. Chem. Soc. 63, 2284 (1941); I b i d . 66, 267 (1944) . 72 . F.A. Robinson, "The Vitamin B Complex", John Wiley and Sons Inc., New York, 1951. page 457. 73 . A. Haddow, B r i t . Med. B u l l . 4 , 338 (1947) . 74. E.E. S n e l l and W.B. Wright, Ann. Rev. Biochem. 19, 277 (1950) . 75 . D.M. Greenberg, J . Pharmacology, 97 , 4 8 4 (1949) . 76 . T. Masuda, Pharm. B u l l . (Tokyo) 4 , 71 (1956); I b i d . 5 , 28 (1957) . 7 7 . T. Masuda, T. K i s h i and M. A s a i , Pharm. B u l l . (Tokyo) 5, 598 (1957) ; I b i d . 6, 291 (1958) . 197 7 8 . G.P. Maley and G.W.E. Plaut,. J . B i o l o g . Chem. 234, 64l (1959) . 7 9 . W. P f l e l d e r e r and G. Nubel, Chem. Ber. 93 , l 4o6 ( i 9 6 0 ) . 80. G.W.E. P l a u t and G.F. Maley, Arch. Biochem. Biophys. 80, 219 (1959) . 81 . T. Masuda, Pharm. B u l l . (Tokyo) 5, 136 (1957) . 82. J . D a v o l l and D.D. Evans, J . Chem. Soc. 504l ( i960) and references c i t e d t h e r e i n . 83 . G.W.E. P l a u t , i n reference 7 , page 443; J . B i o l o g . Chem. 235, PC4l ( i 9 6 0 ) . 84. T. Rowan and H.C.S. Wood, Proc. Chem. Soc. (London) 2 1 , (1963) . 85. R.M. C r e s s w e l l , T. Ne i l s o n and H.C.S. Wood, J . Chem. Soc. 4776 (I960). 86 . A.J. B i r c h , Proc. Chem. Soc. (London) 11 (1962) . 87. A. A l b e r t , Biochem. J . 65 , 124 (1957) . 88 . R.K. Robins, i n "H e t e r o c y c l i c Compounds", R.C. E l d e r f i e l d ed., John Wiley and Sons Inc., New York, 1967. Volume 8 , page 162 and c i t e d r e f e r e n c e s . 89. G.R. Penzer and G.K. Radda, Quart. Revs. Chem. Soc. 21 , 43 (1967) . 90. R.M. Acheson, "An I n t r o d u c t i o n to the Chemistry of Ple t e r o c y c l i c Compounds", I n t e r s c i e n c e Inc., New York, I960, pp 3 0 6 . 91 . H.S. F o r r e s t and S. Nawa, i n reference 7 , page 2 8 l . 92. A. S t u a r t and H.C.S. Wood, Proc. Chem. Soc. (London) 151 (1961) . 93. J.R. T r o t t e r , i n "Annual Reviews of Biochemistry", ' J.M. Luck, F.W. A l l e n and G. MacKinney eds., Annual Reviews Inc., Vol.. 26 .(1957), page 192; H.P. B r o q u i s t , I b i d . V o l . 27 (1958) , page 285 . 94. J.M. Peters and D.M. Greenberg, J . Am. Chem. Soc. 80 , 6679 (1958) . 198 95. R.L. B l a k l e y , i n "Current Trends In H e t e r o c y c l i c Chemistry", A. A l b e r t , G.M. Badger and C.W. Shopee eds., Butterworths, London, 1958 . page 140. 9 6 . A. A l b e r t , i n " P h y s i c a l Methods i n H e t e r o c y c l i c Chemistry", A.R. K a t r i t z k y ed., Academic Press, 1963 , Volume 1, page 1 2 . 97. R.J. W i l l i a m s and H.M. K i r b y , Science 107 , 48l ( 1 9 4 8 ) . 9 8 . E. King and T. King, J . Chem. Soc. 726 ( 1 9 4 7 ) . 9 9 . W. Winkelmann, J . Pr a k t . Chem. 115 , 292 ( 1 9 2 7 ) . 1 0 0 . A.J. B i r c h and C.J. Moye, J. Chem. Soc. 2623 ( 1 9 5 8 ) . 1 0 1 . D.J. Brown and N.W. Jacobsen, J . Chem. Soc. 4 4 l 6 ( 1 9 6 1 ) . 1 0 2 . A. A l b e r t , Biochem. J . 47, 531 ( 1 9 5 0 ) ; I b i d , 54, 646 ( 1 9 5 3 ) . 103. J.H. L i s t e r and G.R. Ramage, J . Chem. Soc. 2234 ( 1 9 5 3 ) . 104. R. Stewart, "Oxidation Mechanisms: A p p l i c a t i o n to Organic Chemistry", W.A. Benjamin Inc., 1964, Chapter 1 1 , pages 140 to 1 4 4 . 1 0 5 . H. B e i n e r t , i n "The Enzymes", P.D. Boyer, H. Lardy and K. Myrback eds ., .... 1 0 6 . P. Hemmerich, C. Veeger and H.C.S. Wood, Angew. Chem. 7 7 , 1 ( 1 9 6 5 ) . 107. J . P. Lamboy, i n reference 8, page. .172. . . 1 0 8 . I.M. Gascoigne and G.K. Radda, Chem. Comm. 1 1 , 211 ( 1 9 6 5 ) . 1 0 9 . H. B e i n e r t , J . Am. Chem. Soc. 7 8 , 5323 (1956) and references c i t e d t h e r e i n . 1 1 0 . H. Gibson, V. Massey and N.M. Atherton, Biochem. J . 8 5 , 369 ( 1 9 6 2 ) . 1 1 1 . H. B e i n e r t and R.H. Sands, i n "Free R a d i c a l s i n B i o l o g i c a l Systems", M.S. B l o i s J r . , H.W. Brown, R.M. Lemmon, R.O Lindblom and M. Weisbluth eds.. Academic Pre s s , New York, 1961. Chapter 2 . 199 1 1 2 . G.P. Burn and J.R.P. O'Brien, Biochim. Biophys. Acta 3 1 , 328 ( 1 9 5 9 ) . 1 1 3 . C.H. S u e l t e r and D.E. M e t z l e r , Biochim. Biophys. Acta 4 4 , 23 ( I 9 6 0 ) . 1 1 4 . T.P. Singer and E.B. Kearney, J . B i o l . Chem. 1 8 3 , 409 ( 1 9 5 0 ) . 1 1 5 . G.K. Radda and M. C a l v i n , Biochemistry 3 / 3 8 4 ( 1 9 6 4 ) . 1 1 6 . J.L. Fo.x and G. T o l l i n , Biochemistry 5 , 3865 ( 1 9 6 6 ) . 117. Q.H. Gibson and J.W. Hastings, Biochem. J . 8 3 , 3 6 8 ( 1 9 6 2 ) . 1 1 8 . W. Berends, J . Posthuma, J.S. Sussenbach and H.I.X. Mager, i n " F l a v i n s and F l a v o p r o t e i n s " , B.B.A. L i b r a r y -Volume 8, E.C. S l a t e r ed., E l s e v i e r P u b l . Co. 1966 , pages 2 2 - 3 6 . 1 1 9 . G. Strauss and W.J. Nickerson, J . Am. Chem. Soc. 8 3 , 3187 ( 1 9 6 1 ) . . > 1 2 0 . L.P. Vernon, Biochim. Biophys. Acta 36 , 177 ( 1 9 5 9 ) . 1 2 1 . W.M. Moore, J.T. Spence, F.A. Raymond and D.S. Colson, J . Am. Chem. Soc. 8 5 , 3367 ( 1 9 6 7 ) . 1 2 2 . B. Holrnstom and G. Oster, J . Am. Chem. Soc. 8 3 , 1867 ( 1 9 6 1 ) . 1 2 3 . E.C. Smith and D.E. M e t z l e r , J . Am. Chem. Soc. 8 5 , 3285 ( 1 9 6 3 ) . 124. W.E. F i d l e r and H.C.S. Wood, J. Chem. Soc. 3980 ( 1 9 5 7 ) . 1 2 5 . W. P f l e i d e r e r , J . Bunting, D.D. P e r r i n and G. Nubel, Chem. Ber. 1 0 1 , 1072 ( 1 9 6 8 ) . 1 2 6 . A. A l b e r t and E.P. Serj e a n t , J . Chem. Soc. 3357 ( 1 9 6 4 ) . 1 2 7 . D.D. P e r r i n , i n " D i s s o c i a t i o n Constants of Organic Bases i n Aqueous S o l u t i o n ' 1 , Butterworths, London, 1965 . 1 2 8 . G. Machell, J . Chem. Soc. 683 ( i 9 6 0 ) . 1 2 9 . G.W.E. P l a u t , J . B i o l . Chem. 2 3 8 , 2225 ( 1 9 6 3 ) . 1 3 0 . M. V i s c o n t i n i and A, Bobst, Helv. Chim. Acta 4 9 , 1-815 ( 1 9 6 6 ) . •• _ 200 131. D.H. Wadke and D.E. Guttman, J. Pharm. S c i . 55, 1088 (1966) . 132. H.A. Flaschka, "E.D.T.A. T i t r a t i o n s " , Permagon Press Inc., 1959. pages 36,70. 133. J.E.B. Randies and J.M. Tedder, J. Chem. Soc. 1218 (1955). 134. "Handbook of Chemistry and Physics", The Chemical Rubber Co., Cleveland, Ohio. 45th e d i t i o n (1964) page E-48. 135. J . HIne, G. Hampton and B.C. Menon, J . Am. Chem. Soc. 89, 2664 (1967); S.W. Benson, "The Foundations of Chemical K i n e t i c s " , McGraw-Hill Inc., i960, page 16. 136. H.H. Jaf f e , Chem. Rev. 53, 191 (1953). 137. A. Dieffenbacher, R. Mondelli and W. von philipsborn , Helv. Chiro. Acta 49, 1355 (1966). " " """ 138. S. Matsuura and T. Goto, J. Chem. Soc. 1773 (1963); Ibid, 623 (1965). 139. K.J. La i d l e r , "Reaction K i n e t i c s " , Volume 2 - Reactions i n Solution, Permagon Press, 1963. page 54. 140. F.J. Bullock and 0. Jardetzky, J. Org. Chem. 30, 2056 (1965). 141. H. A. Laitinen, "Chemical Analysis", McGraw-Hill Inc., i960, pages 35-37. 142. J . N. Brpfasted and K.J. Pederson, Z. physik Chem. 108, 185, (1923). 143. P. Hemmerich, i n r e f e r e n c e d , page 143. 144. H.E.A. Kramer and R. Gompper, Tet. Let. 15, 969 (1963). 145. N.J. Leonard and J.A. Adamik, J. Am. Chem. Soc. 8 l , 595 (1959). 146. S. Kaufmann, J . Biolog. Chem. 236, 807 (1961). 147. J.M. Whiteley and F.M. Huennekens, Biochemistry 6, 2620 (1967) . 148. P. Hemmerich, In reference 7, page 162. 149. A. Bobst, Helv. Chim. Acta 50, 1480 (1967). 150. D.D. Per r i n , J . Chem. Soc. 645 (1962). 201 151. Y. Inoue and D.D. Perrin, J. Chem. Soc. 2648 (1963) . 152. D.D. Pe r r i n and Y. Inoue, Proc. Chem. Soc. (London) 342 ( i 9 6 0 ) ; Ibid, J. Chem. Soc. 2600 (1962) . 153. N.C. Deno, i n "Progress i n Physical Organic Chemistry", S.G. Cohen, A. Streitweiser J r . and R.W. Taft eds., Interscience Publishers 1964. Volume 2, page 163. 1 5 4 ^ W. von Philipsborn, H. S t i e r l i n , , and W. Traber, i n reference 7, page 173. i 155. W. A. Waters, "Mechanisms of Oxidation of Organic Compounds", John Wiley and Sons Inc., New York, N.Y. 1964. 156. T.A. Turney, "Oxidation Mechanisms", Butterworths Inc., Washington D.C. 1965. 157. B.S. Thyagarajan, Chem. Rev. 58, 439 (1958). 158. J . B. Conant and M.P. Pratt, J . Am. Chem. Soc. 48, 3178 (1926) . 159. I.R. Wilson, Rev. Pure and Appl. Chem. 16, 103 (1966) . 160. P.T. Speakman and W.A. Waters, J. Chem. Soc. 40 (1955) . 161. E.J. Meehan, I.M. Kolthoff and H. Kakiuchi, J. Phys. Chem. 66, 1238 (1962) . 162. A. Vogel, "A Text Book of Quantitative Inorganic Analysis", Longmans, Green and Company, 1939. page 436. 163. Radiometer B u l l e t i n No. ST40, "Redox Measurements,. Their Theory and Technique", Copenhagen, Denmark. 164. A. Albert, i n "Heterocyclic Chemistry", Athalone Press, London, 1959. page 350. 165. S.W. Benson, "The Foundations of Chemical K i n e t i c s " , McGraw-Hill Inc., New York, i 9 6 0 , pages 18-21 . 166. C.W. Davies, "Ion Association", Butterworths Inc., Washington D.C, 1962. pages 169 to 171. 167. D. Larsen and A.C. Wahl, Inorg. Chem. 4 , 1 2 8 l (1965) . o • • 168. S.R. Cohen and R.A. Plane, J. Phys. 'Chem. 6 l , 1096 (1957) . 2 0 2 l o 9 . R.J. Campion, C P . Deck, P. King J r . and A.C. Wahl, Inorg. Chem. 6 , 6 7 2 ( 1 9 6 7 ) . 1 7 0 . R.W. Chlebek and M.W. L i s t e r , Can. J . Chem. Soc. 4 4 , 4 3 7 ( 1 9 6 6 ) . 1 7 1 . J . H o l l u t a and W. Herman, Z. Physik Chem. L e i p z i g A 1 6 6 , 5 4 3 ( 1 9 3 3 ) . 1 7 2 . J . Hine, " P h y s i c a l 0rp:anic Chemistry", Second E d i t i o n , McGraw-Hill Inc ., ' 1 9 6 2 . . page 7 2 . 1 7 3 . K.J. L a i d l e r , i n reference 1 3 9 , page 1 2 . 1 7 4 . H.N. S t e i n and H.J.C. Tendeloo, Rec. t r a v . chim. 7 4 , 9 0 5 ( 1 9 5 5 ) . 1 7 5 . I.M. K o l t h o f f , E .J. Meehan, M.S. Tsao and Q.W. Choi, J . Phys. Chem. 6 6 , 1 2 3 3 ( 1 9 6 2 ) . 1 7 6 . P.J. A n d r u l l s J r . , M.J.S. Dewar, R. D i e t z and R.L. Hunt, J . Am. Chem. Soc. 8 8 , 5 4 7 3 ( 1 9 6 6 ) . 177. J.H. Swinehart, J . Am. Chem. Soc. 8 8 , 1 0 5 6 ( 1 9 6 6 ) ; I b i d . 8 7 , 9 0 4 ( 1 9 6 5 ) . 1 7 8 . R. Stewart, OxidationJ?yJ^erjTian_g;anate_, i n "Oxidation i n Organic ChemTstry"', K.BT Wiberg "e'dT'J Academic Pr e s s , Mew York, 1 9 6 5 . 1 7 9 . R. Stewart, i n reference 1 0 4 , pages 4 6 t o 4 8 . 

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