The chemiluminescence of a 6,7-dihydroflavin and some related pteridines

vji t— t~> O IM O O 00 00 O

THE CHEMILUMINESCENCE OF A 6,7-DIHYDROFLAVIN

AND SOME RELATED PTERIDINES

Proefschrift

ter verkrijging van de graad van doctor

in de technische wetenschappen

aan de Technische Hogeschool Delft,

op gezag van de Rector Magnificus,

prof. ir. B.P.Th. Veltman,

in het openbaar te verdedigen ten overstaan van

het College van Dekanen o

op 24 mei 1984 te 16.00 uur

door

RUDOLF ADDINK

scheikundig ingenieur,

geboren te Vught

Dr. ir. H.I.X. Mager heeft in hoge mate bijgedragen

aan de begeleiding bij het totstandkomen van het

proefschrift en is als zodanig aangewezen door

het College van Dekanen

Cover photograph, from l e f t t o r i g h t : chemiluminescent r e a c t i o n s o l u t i o n s , photographed i n t h e i r own l i g h t ; 6 , 7 , 8 t r i m e t h y l -lumazine/DMF/t-BuOK; 7-hydroxy-6,7-dihydrolumiflavin/MeOH/NaOMe; 3,6,7,8-tetramethyllumazine/DMF/t-BuOK.

CONTENTS

L i s t o f a b b r e v i a t i o n s and formulae v i

CHAPTER I GENERAL INTRODUCTION 1

Mechanisms 4 U n i m o l e c u l a r d e c o m p o s i t i o n o f 1,2-dioxetanes 5 C h e m i c a l l y I n i t i a t e d E l e c t r o n Exchange Luminescence 8 B a c t e r i a l b i o l u m i n e s c e n c e 11 A p p l i c a t i o n s 17 Model systems 19 References 23

CHAPTER I I SYNTHESIS OF 7-HYDROXY-6,7-DIHYDROLUMIFLAVIN 27 R e p r i n t e d w i t h p e r m i s s i o n from

Tetrahedron 29, 879-881 (1973)

CHAPTER I I I THE AUTOXIDATION PRODUCT OF

7-HYDROXY-6,7-DIHYDROLUMIFLAVIN 31 R e p r i n t e d w i t h p e r m i s s i o n from

Tetrahedron 30, 75-78 (1974).

CHAPTER IV THE MECHANISM OF THE CHEMILUMINESCENT AUTOXIDATION OF 7-HYDROXY-6,7-DIHYDROLUMIFLAVIN

AND SOME RELATED PTERIDINES 43 R e p r i n t e d w i t h p e r m i s s i o n from

Tetrahedron 37, 833-841 (1981).

CHAPTER V THE AUTOXIDATION OF LUMIFLAVIN 60

CHAPTER VI CHEMILUMINESCENCE AND FLUORESCENCE MEASUREMENTS 69

SUMMARY 7 7

LIST OF ABBREVIATIONS AND FORMULAE AMP ATP BL t-BuOK CIEEL CL DMF DMSO ECL FMN NADH adenosine monophosphate adenosine t r i p h o s p h a t e bioluminescence p o t a s s i u m - t - b u t o x i d e c h e m i c a l l y i n i t i a t e d e l e c t r o n exchange luminescence chemiluminescence d i m e t h y l f ormamide d i m e t h y l s u l f o x i d e e l e c t r o g e n e r a t e d chemiluminescence f l a v i n e mononucleotide

n i c o t i n a m i d adenine d i n u c l e o t i d e , reduced form

f l a v i n F l a v i n •• 7 , 8 - d i m e t h y l b e n z o [ g ] p t e r i d i n e - 2 , 4 ( 3 H , 10H)-dione I s o a l l o x a z i n e benzo[g]pteridine-2,4(3H,1OH)-dione Lumazine p t e r i d i n e - 2 , 4 ( 1 H , 3 H ) - d i o n e or p t e r l d i n e - 2 , 4 ( 3 H , 8 H )-dione P t e r i n 2-amino-pteridine-4(3H)-one o r 2-amino-pteridine-4(8H)-one

CHAPTER I

GENERAL INTRODUCTION

The e m i s s i o n o f l i g h t i n a c h e m i c a l r e a c t i o n i s d e s i g n a t e d as chemiluminescence. The p r o d u c t i o n o f l i g h t by l i v i n g organisms i s c a l l e d b i o -luminescence. I t has f a s c i n a t e d men from t h e b e g i n n i n g o f time and f i l l e d him both w i t h wonder and d e l i g h t1. I t has always i n s p i r e d him t o i n v e s -t i g a -t e -t h i s phenomenon.

A l t h o u g h t h e e a r l i e s t d e s c r i p t i o n s a r e concerned w i t h luminous f l e s h , f i s h , wood, t h e f i r e f l y and t h e glowworm, most o f t h e l u m i n e s c i n g a n i m a l s can be found i n t h e s e a . At a depth o f over 7 0 0 m i t i s estima-t e d estima-t h a estima-t up estima-t o 90% o f a l l organisms a r e b i o l u m i n e s c e n estima-t . Ranging from u n i c e l l u l a r organisms t o s e v e r a l c l a s s e s o f f i s h e s . T h i s d i v e r s i t y has been e x t e n s i v e l y r e v i e w e d1 .

I t i s more than 3 0 0 years ago that Robert Boyle i n 1 6 6 7 concluded t h a t a i r , oxygen was not known a t t h a t t i m e , was a p r e r e q u i s i t e f o r t h e b i o l u m i n e s c e n c e e x h i b i t e d by r o t t e n f l e s h and wood; phenomena now known t o be caused by b a c t e r i a and f u n g i r e s p e c t i v e l y . The f i r s t h i n t t h a t t h i s luminescence might be due t o l i v i n g t h i n g s was g i v e n i n t h e m i d s t 18th

3

c e n t u r y , when i t was suggested t h a t the l i g h t might come from " a n i m a l -c u l e s " . However, i t l a s t e d u n t i l the end o f t h e 19th -c e n t u r y b e f o r e f u r t h e r b i o c h e m i c a l r e s e a r c h was d i r e c t e d a t these phenomena.

In 18861 Raphael Dubois ushered i n a new e r a w i t h t h e i s o l a t i o n o f two s u b s t a n c e s from t h e l i g h t o r g a n o f the luminous b e e t l e Pyrophorus. When t h e l i g h t o r g a n s were submerged i n hot water the luminescence was e x t i n g u i s h e d i m m e d i a t e l y . However, a c o l d water e x t r a c t , which had g r a d -u a l l y ceased t o emit l i g h t , c o -u l d be s t i m -u l a t e d t o l-uminesce a g a i n by a d d i n g t h e h o t water e x t r a c t .

T h i s l e d Dubois t o t h e c o n c l u s i o n t h a t i n t h e hot water e x t r a c t a r e l a t i v e l y h e a t - s t a b l e compound was o x i d i z e d under t h e i n f l u e n c e o f a c a t a l y s t p r e s e n t i n the c o l d water e x t r a c t . He named the h e a t - s t a b l e s u b s t r a t e " l u c i f e r i n " and the enzyme which c a t a l y z e d i t s o x i d a t i o n " l u c i f e r a s e " .

With the i s o l a t i o n o f t h e s e two components he n o t o n l y gave a c h e m i c a l b a s i s t o b i o l u m i n e s c e n c e , but he a l s o e s t a b l i s h e d i t s g e n e r a l p r i n c i p l e . Dubois concluded t h a t these two substances were s u f f i c i e n t f o r l i g h t p r o d u c t i o n . This appeared t o be an o v e r s i m p l i f i c a t i o n ; i n s e v e r a l b i o l u m i n e s c e n t systems a d d i t i o n a l c o f a c t o r s a r e r e q u i r e d . Never-t h e l e s s Never-t h e Never-terms l u c i f e r i n and l u c i f e r a s e a r e s Never-t i l l i n use Never-today. The p r i n c i p l e became the b a s i s f o r the c l a s s i c a l l u c i f e r i n - l u c i f e r a s e t e s t , which was r o u t i n e l y done on a l l new luminous organisms d i s c o v e r e d .

As t h e amount o f l u c i f e r i n s produced by t h e organisms i s so very s m a l l , i t l a s t e d u n t i l the development o f more advanced a n a l y t i c a l t e c h n i q u e s before s t r u c t u r a l e l u c i d a t i o n s were a c c o m p l i s h e d .

In 1957 B i t l e r and McElroy were t h e f i r s t t o i s o l a t e a pure l u c i f e r i n . From about 15,000 f i r e f l y t a i l s they o b t a i n e d 9 mg f i r e f l y l u c i -f e r i n ^ . I n 1961 i t s s t r u c t u r e was e l u c i d a t e d and i t s s y n t h e s i s accom-p l i s h e d . I n t h e l a s t s t e accom-p o f t h e s y n t h e s i s c y s t e i n e i s used. With D - ( - ) - c y s t e i n e a l u c i f e r i n i s o b t a i n e d which has a l l t h e p r o p e r t i e s o f

7

n a t u r a l l u c i f e r i n . When L ( + ) c y s t e i n e i s used , t h e r e s u l t i n g l u c i -f e r i n i s i n a c t i v e i n t h e enzymatic l i g h t r e a c t i o n . A p p a r e n t l y , i n n a t u r a l l u c i f e r i n t h e a s y m e t r i c carbon atom has t h e D - c o n f i g u r a t i o n .

S i n c e then t h e s t r u c t u r e s o f o n l y a r e l a t i v e l y s m a l l number o f l u c i f e r i n s have been e l u c i d a t e d ( t a b l e I ) . The most r e c e n t one was t h a t o f Diplocaraia longa i n 1976. A g r e a t number o f b i o l u m i n e s c e n t systems a r e s t i l l under i n v e s t i g a t i o n .

With the e l u c i d a t i o n o f t h e s t r u c t u r e s o f t h e s e l u c i f e r i n s and t h e i r o x i d a t i o n p r o d u c t s , c o n s i d e r a b l e i n s i g h t was gained on t h e mech-anism by which t h e l i g h t p r o d u c i n g r e a c t i o n t a k e s p l a c e .

Next t o these b i o l u m i n e s c e n t systems a g r e a t number o f chemilumi-nescent systems a r e known. One o f t h e f i r s t compounds s t u d i e d was l o p h i n e ( 2 , * l , 5 - t r i p h e n y l i m i d a z o l e ) , i t s chemiluminescent a u t o x i d a t l o n 0 + h v 0 l o p h i n e Scheme 1

T A B L E 1 H i 9 6 r i C O O H D - ( - ) - f i r e f l y l u c i f e r i n C H , £ - c2h5 1966' N ^ ( C H2): H H J*" N H2 C H3 O ^ C - C2H5 N ^ N H

jl

T

16

was d i s c o v e r e d i n 1877 ; t h e n a t u r e o f t h e e m i t t e r i s s t i l l unknown. 1 7

In 1928 A l b r e c h t p u b l i s h e d t h e f i r s t paper on one o f t h e most i n t e n -s i v e l y i n v e -s t i g a t e d chemilumine-scent -sub-stance-s known, l u m i n o l

( 5 - a m i n o - 2 , 3 - d i h y d r o - 1 , 4 - p h t a l a z i n e d i o n e ) : Q H-n: huru/Hcr/cnt Q H20 : H202/ H 0 7 c a t NH or f ^ V ^ O " lu D M S O : 02/ H O - U L r + N> + h v

PC

These and many o t h e r systems have been d e s c r i b e d e x t e n s i v e l y i n a

18-20

number o f r e v i e w s

MECHANISMS

As mentioned above oxygen o r hydrogenperoxlde p l a y a major r o l e i n chemi- and b i o l u m i n e s c e n c e . I t i s now g e n e r a l l y accepted t h a t p e r o x i d e i n t e r m e d i a t e s a r e formed, and t h a t t h e i r d e c o m p o s i t i o n i s t h e p r i n c i p a l pathway t o e x c i t e d s t a t e s . In t h e i n v e s t i g a t i o n o f a chemiluminescent r e a c t i o n i n s o l u t i o n t h e r e a r e t h r e e main c o n c e r n s : 1. Energy An obvious necessary c o n d i t i o n f o r t h e f o r m a t i o n o f e x c i t e d s t a t e p r o d u c t s i n a c h e m i c a l r e a c t i o n i s t h a t t h e r e must be s u f f i c i e n t energy The f r e e energy o f t h e r e a c t i o n p l u s perhaps some f r a c t i o n o f t h e a c t i -v a t i o n energy has t o be equal o r g r e a t e r than t h e d i f f e r e n c e i n energy between t h e ground s t a t e and t h e e x c i t e d s t a t e o f t h e p r o d u c t .

The energy n e c e s s a r y f o r t h e p r o d u c t i o n o f l i g h t o f a g i v e n wave-l e n g t h i s g i v e n by: N = Avogadro's number h = P l a n c k ' s c o n s t a n t c = v e l o c i t y o f l i g h t X = wavelength N.h.v = N.h.c

In t h e v i s i b l e spectrum, from 400800 nm, t h e r e f o r e the c h e m i l u n i -nescent r e a c t i o n must r e l e a s e between 36 and 71 k c a l / m o l e .

2. E m i t t e r

The i d e n t i f i c a t i o n o f t h e e m i t t i n g s p e c i e s l e a d s t o t h e under-s t a n d i n g o f t h e c h e m i c a l mechaniunder-sm o f t h e l i g h t p r o d u c i n g path of t h e r e a c t i o n . The e m i t t e r may be formed e i t h e r d i r e c t l y as a product o f t h e

21

r e a c t i o n or i n d i r e c t l y v i a energy t r a n s f e r from t h e f i r s t formed e x c i -ted s t a t e .

3. The c h e m i e x c i t a t i o n s t e p

The mechanism which e x p l a i n s how t h e energy r e l e a s e d i n a r e a c t i o n l e a d s t o t h e f o r m a t i o n o f e x c i t e d s t a t e p r o d u c t s i s f o r many complex chemiluminescent systems s t i l l unknown. The a v a i l a b i l i t y o f s u f f i c i e n t energy does n o t n e c e s s a r i l y l e a d t o e f f i c i e n t e x c i t e d s t a t e

produc-22 23

t i o n ' . Only f o r two types o f r e a c t i o n t h i s q u e s t i o n seems t o be s o l v e d :

I . The u n i m o l e c u l a r d e c o m p o s i t i o n o f 1,2-dioxetanes.

I I . The C h e m i c a l l y I n i t i a t e d E l e c t r o n Exchange Luminescence (CIEEL). I . The u n i m o l e c u l a r d e c o m p o s i t i o n o f 1,2-dioxetanes.

In 1 9 6 4 four-membered c y c l i c peroxides (dioxetanes I and 1,2-d i o x e t a n o n e s I I ) were propose1,2-d as h i g h energy i n t e r m e 1,2-d i a t e s i n a number

0 — 0 0 — 0

- H - - K o

i n

S c h e m e 3

o f chemiluminescent r e a c t i o n s . In analogy a dioxetanone i n t e r m e d i a t e was 24 25

proposed a l s o i n t h e r e a c t i o n o f f i r e f l y b i o l u m i n e s c e n c e ' (scheme 4 ) . 2 6

In 1968 McCapra f i r s t suggested t h a t m o l e c u l a r o r b i t a l symmetry c o n s i d e r a t i o n s , a p p l i e d t o the d e c o m p o s i t i o n o f these d i o x e t a n e i n t e r -mediates, might g i v e t h e answer t o the q u e s t i o n why e l e c t r o n i c a l l y e x c i t e d s t a t e s a r e formed.

H „, H - O A M P

f^ifVW

s r V rRr°

28

a 1,2-dioxetanone was r e p o r t e d . The chemilumlnescence accompanying t h e t h e r m a l decomposition o f t h e s e compounds s t r e n g t h e n e d t h e proposed mecha-nism. I t soon appeared, t h a t t h e u n i m o l e c u l a r decomposition o f these mo-d e l compounmo-ds generatemo-d h i g h y i e l mo-d s o f t r i p l e t e x c i t e mo-d c a r b o n y l p r o mo-d u c t s

29-31

and o n l y s m a l l amounts o f s i n g l e t e x c i t e d p r o d u c t s . The r a t i o o f

0 — c - c - x / II H20 0 - H - c \ c . A oVo* I V I + x-—c—c ^ C = 0 * + X " + H20 * + C 02 /C=0* + C02 0 ' = 1 80 S c h e m e 5

t r i p l e t : s i n g l e t v a r i e s from one d i o x e t a n e t o another i n t h e range 10-32

1000 . T h i s i s c l e a r l y n o t i n agreement w i t h t h e y i e l d o f e x c i t e d s i n g -l e t s t a t e s i n f i r e f -l y b i o -l u m i n e s c e n c e , which i s known t o approach u n i t y .

In o r d e r t o c l a r i f y the s i t u a t i o n a number o f i n v e s t i g a t o r s t r i e d to g i v e evidence f o r o r a g a i n s t these c y c l i c p e r o x i d e i n t e r m e d i a t e s i n the b i o l u m i n e s c e n c e systems o f t h e f i r e f l y , Renilla and Cyprictina. I n

18 18

l a b e l i n g experiments w i t h 0^ o r 0 i t was t r i e d t o d i s t i n g u i s h be-tween a l i n e a r and a c y c l i c mechanism (scheme 5 ) .

I n i t i a l l y the outcome of t h e experiments was c o n t r a d i c t o r y , but i n 1978 t h e q u e s t i o n was f i n a l l y s e t t l e d i n f a v o r o f a dioxetanone

Interme-33 34 35 d i a t e . This was confirmed by Shimomura and Johnson i n 1979 , who

concluded t h a t t h e dioxetanone mechanism i s t h e o n l y pathway i n t h e b i o l u m i n e s c e n t systems o f both f i r e f l y and Cypriaina.

I t has alway been the c h e m i s t ' s aim t o immitate n a t u r e i n t h e l a b -o r a t -o r y , and p r e f e r r a b l y i n a manner a t l e a s t e q u a l t -o n a t u r e . T h i s i s i l l u s t r a t e d by a "chemist's p r a y e r " : Oh L o r d , I f a l l upon my knees And pray t h a t a l l my s y n t h e s e s , May no l o n g e r be i n f e r i o r To those conducted by b a c t e r i a .

In 1967 Rauhut e t a l . succeeded t o r e a c h t h i s aim w i t h a very e f -f i c i e n t chemiluminescent system c o n s i s t i n g o -f an o x a l i c e s t e r , hydrogen p e r o x i d e and a f l u o r e s c i n g dye: M M b a s e C _ c + H202 *• 2 R O " + C — C R 0/ \ > R 0 0 — 0 d y e h v « d y e * + 2 C 02 S c h e m e 6

With the 2 , 4 - d i n i t r o p h e n y l e s t e r and rubrene as the f l u o r e s c e n t dye a 37

quantum y i e l d of 0. 2 3 was reached , thus surpassing the * k i = 0.1 of b a c t e r i a l b i o l u m i n e s c e n c e i n v i t r o . By changing the n a t u r e o f the e s t e r , r e c e n t l y even a * .= 0.34 was o b t a i n e d

37

A 1,2-dioxetanedione was proposed as the c r u c i a l intermediate i n the r e a c t i o n . Although mass s p e c t r a l evidence was p r e s e n t e d f o r t h i s

39 32 i n t e r m e d i a t e , H a s t i n g s and W i l s o n concluded i n a r e c e n t r e v i e w t h a t :

" i n the r e a c t i o n o f o x a l i c e s t e r s , the m y t h i c a l i n t e r m e d i a t e d i o x e t a n e -dione remains as s e d u c t i v e and e l u s i v e as ever. The chemiluminescence community s h o u l d o f f e r a reward f o r i t s c a p t u r e " .

Although i n many d i o x e t a n e model systems m a i n l y t r i p l e t e x c i t e d c a r b o n y l compounds are formed, d e c o m p o s i t i o n of a dioxetanone i n t e r m e d i -a t e -as i n f i r e f l y b i o l u m i n e s c e n c e l e -a d s t o -a s i n g l e t e x c i t e d p r o d u c t . In

40

a d d i t i o n , i n 1974 i t was found t h a t a t l e a s t dioxetanone models c o u l d produce s i n g l e t e x c i t e d s t a t e s when c e r t a i n f l u o r e s c e n t compounds l i k e rubrene were p r e s e n t i n the r e a c t i o n m i x t u r e . As i n the case o f o x a l i c e s t e r chemiluminescence the amount o f l i g h t was dependent on the n a t u r e o f the added f l u o r e s c e n t compound.

A s a t i s f a c t o r y e x p l a n a t i o n f o r these phenomena i s g i v e n by:

I I . The C h e m i c a l l y I n i t i a t e d E l e c t r o n Exchange Luminescence mechanism. In 1977 S c h u s t e r and coworkers f o r m u l a t e d t h i s mechanism f o r the f o r m a t i o n o f s i n g l e t e x c i t e d s t a t e s , which may be the e x c i t a t i o n

mecha-22 nism u n d e r l y i n g the chemi- en b i o l u m i n e s c e n c e o f many systems

I t was f o r m u l a t e d f o r the decomposition of d i p h e n o y l p e r o x i d e i n the presence of c e r t a i n a r o m a t i c hydrocarbons (scheme 7) y i e l d i n g chemi-luminescence which was i d e n t i c a l to the f l u o r e s c e n c e o f the added

hydro-22 23

carbon ' . (Although s u f f i c i e n t energy was a v a i l a b l e , no e x c i t e d s t a t e s were d e t e c t e d i n the u n i m o l e c u l a r d e c o m p o s i t i o n o f the p e r o x i d e ) .

The f i r s t and r a t e d e t e r m i n i n g s t e p i n the CIEEL p r o c e s s i s an e l e c t r o n t r a n s f e r from the hydrocarbon c a t a l y s t t o the p e r o x i d e . The d i p h e n o y l p e r o x i d e r a d i c a l a n i o n formed i s u n s t a b l e and d e c a r b o x y l a t e s . Ring c l o s u r e of the r e m a i n i n g fragment g i v e s the r a d i c a l a n i o n of benzocoumarin. Back e l e c t r o n t r a n s f e r from t h i s r a d i c a l a n i o n t o the

S c h e m e 7

r a d i c a l c a t i o n o f the a r o m a t i c hydrocarbon r e s u l t s , when s u f f i c i e n t energy i s a v a i l a b l e , i n the g e n e r a t i o n of the e l e c t r o n i c a l l y e x c i t e d s i n g l e t s t a t e o f the hydrocarbon. The r a t e o f t h e p e r o x i d e d e c o m p o s i t i o n r e a c t i o n and the amount o f chemiluminescence produced a r e r e l a t e d t o the redox p o t e n t i a l of the hydrocarbon c a t a l y s t . The lower i t s redox

p o t e n t i a l the f a s t e r the r e a c t i o n proceeds and the more chemilumines-cence i s produced. The e x c i t e d s t a t e f o r m a t i o n i n e l e c t r o n t r a n s f e r p r o c e s s e s was f i r s t d i s c o v e r e d i n 1964 i n r a d i c a l c a t i o n a n i o n a n n i h i l a t i o n r e a c -41, 42 t i o n s R* + R~ *~ 1R* + R >• 3R* + R

These r a d i c a l s were generated by e l e c t r o l y s i s and the r e s u l t i n g l i g h t e m i s s i o n i s r e f e r r e d t o as e l e c t r o g e n e r a t e d chemiluminescence (ECL).

43-46 E x t e n s i v e r e v i e w s on t h i s s u b j e c t have been p u b l i s h e d

The energy r e l e a s e d i n t h i s redox p r o c e s s i s g i v e n by the d i f f e r -ence of the s t a n d a r d o x i d a t i o n - r e d u c t i o n p o t e n t i a l s o f the donor and

the a c c e p t o r ,43, 47

A G° = E° (H/BÏ) - E° (H/Rt) " C ( l n eV)

Where t h e l a s t term r e p r e s e n t s a c o r r e c t i o n f o r t h e coulombic a t t r a c t i o n i n t h e r a d i c a l i o n p a i r , which amounts t o about 0.06 eV.

In a system t h a t produces s u f f i c i e n t energy t h e r e a c t i o n may g i v e s i n g l e t and/or t r i p l e t e x c i t e d s t a t e s . In an energy d e f i c i e n t system, when o n l y t r i p l e t s can be formed s t i l l s i n g l e t e m i s s i o n i s o b s e r v e d . T h i s i s e x p l a i n e d by t r i p l e t - t r i p l e t a n n i h i l a t i o n . Formation o f t h e e x c i t e d s t a t e by e l e c t r o n t r a n s f e r can be r e p r e -sented s c h e m a t i c a l l y by t h e f o l l o w i n g s i m p l i f i e d m o l e c u l a r o r b i t a l diagram. R ' R- R 'R S c h e m e 8 The c a t a l y t i c a c t i o n o f f l u o r e s c e n t hydrocarbons on t h e c h e m i l u -minescent d e c o m p o s i t i o n o f a c t i v a t e d o x a l a t e e s t e r s and dioxetanones was e x p l a i n e d i n t h e same way. Not o n l y c y c l i c p e r o x i d e s a r e s u b j e c t t o the CIEEL mechanism, but a l s o l i n e a r p e r o x i d e s l i k e secondary

peroxy-48

e s t e r s appear t o r e a c t by t h i s mechanism (scheme 9 ) .

hSC H 0XO_O> - C H 3 + ArH hv A r H * + A 0 C H3 \ \ - o ^ " 3 + A r H' • C H 3 C O O H 0 7 +

JL

This e l e c t r o n t r a n s f e r t h e o r y has been a p p l i e d a l s o t o s e v e r a l b i o l u m i n e s c e n t systems. For t h e f i r e f l y and Cypriaina b i o l u m i n e s c e n c e an

48-50

i n t r a m o l e c u l a r CIEEL mechanism has been proposed , thus s o l v i n g t h e i n c o n g r u i t y mentioned above.

No e v i d e n c e as yet has been found t h a t s i m p l e t e t r a a l k y l s u b s t i t u t e d 51

1,2-dioxetanes e x h i b i t e l e c t r o n exchange chemiluminescence . A p p a r e n t l y t h e i r redox p o t e n t i a l s a r e t o o low f o r t h e t r a n s f e r o f an e l e c t r o n , from t h e hydrocarbon t o compete w i t h normal t h e r m a l d e c o m p o s i t i o n .

Only s p e c i a l s u b s t i t u t i o n o f the 1,2dioxetane r i n g system w i t h a r o -m a t i c r e s i d u e s see-ms t o -make p o s s i b l e t h e e f f i c i e n t f o r -m a t i o n o f e x c i t e d

50 52 53 s i n g l e t s t a t e s v i a an i n t r a m o l e c u l a r e l e c t r o n exchange pathway ' ' BACTERIAL BIOLUMINESCENCE

As mentioned b e f o r e the n e c e s s i t y f o r oxygen i s a l r e a d y known f o r more than 300 y e a r s . The requirement f o r FMN i n c o m b i n a t i o n w i t h NADH

54 55

however, was o n l y r e c o g n i z e d i n 1953 ' , NADH a c t i n g as an e l e c t r o n donor f o r FMN r e d u c t a s e t o g i v e FMNH^. I n t h e same year i t was found t h a t l o n g c h a i n f a t t y aldehydes enormously s t i m u l a t e d t h e b a c t e r i a l b i o l u m i n e s c e n c e ^ . Many i n v e s t i g a t i o n s were c a r r i e d o u t t o f i n d even more e s s e n

-57

t i a l f a c t o r s , but i n 1955 McElroy and Green f o r m u l a t e d t h a t the absol u t e r e q u i r e m e n t s f o r b a c t e r i a absol b i o absol u m i n e s c e n c e a r e : t h e enzyme absol u c i f e r -ase, reduced FMN, oxygen and a l o n g c h a i n a l d e h y d e . They suggested t h a t the aldehyde i s c o n v e r t e d i n t o t h e c o r r e s p o n d i n g a c i d , t h e f l a v i n p l a y i n g the r o l e o f o x i d a n t coupled t o oxygen, but no r e a c t i o n mechanism was f o r m u l a t e d .

U n t i l t h a t t i m e , i n b i o l o g i c a l o x i d a t i o n s , oxygen was o n l y c o n s i d -ered t o a c t as a t e r m i n a l e l e c t r o n a c c e p t o r . This concept was r e j e c t e d when i n 1955 the a c t u a l i n c o r p o r a t i o n o f m o l e c u l a r oxygen i n t o a

sub-58 59 s t r a t e was e s t a b l i s h e d by Mason e t a l and H a y a i s h i e t a l .

Thermodynamically oxygen i s a very r e a c t i v e element; most r e a c t i o n s w i t h o r g a n i c s u b s t r a t e s are h i g h l y e x e r g o n i c . Under normal c o n d i t i o n s , t h a t i s room temperature and atmospheric p r e s s u r e , no v i o l e n t r e a c t i o n s occur due t o t h e h i g h a c t i v a t i o n energy (which i s very f o r t u n a t e f o r man-k i n d ) . This i s r a t i o n a l i z e d by t h e f a c t t h a t oxygen i n the ground s t a t e

i s a t r i p l e t , and hence a r e a c t i o n w i t h a s i n g l e t s u b s t r a t e i s a " f o r -b i d d e n " o n e ^ . N e v e r t h e l e s s i n many -b i o c h e m i c a l systems enzymes, c a l l e d monooxygenases, mediate the i n c o r p o r a t i o n o f one atom o f t h e oxygen m o l e c u l e i n t o the s u b s t r a t e . This c a l l s f o r a mechanism t o e x p l a i n t h e l o w e r i n g o f t h e a c t i v a t i o n energy; i n other words a mechanism f o r oxygen a c t i v a t i o n .

An i m p o r t a n t c l a s s o f monooxygenases have d i h y d r o f l a v i n s o r t e t r a -h y d r o p t e r i d i n e s as c o f a c t o r s . I n our l a b o r a t o r y i n v e s t i g a t i o n s on t -h e a u t o x i d a t i v e behaviour o f t h e i s o l a t e d c o f a c t o r s l e d t o the development o f a theory on t h e c h e m i c a l a s p e c t s o f the mechanism o f a c t i v a t i o n and t r a n s f e r o f oxygen, which was f i r s t p u b l i s h e d by Mager^1 i n 1965. I t was p o s t u l a t e d t h a t m o l e c u l a r oxygen i s a c t i v a t e d by c o v a l e n t l i n k a g e a t t h e 4a, o r 1 0 a ( 8 a ) b r i d g e p o s i t i o n o f t h e c o f a c t o r s t o g i v e d i h y d r o f l a v i n -and t e t r a h y d r o p t e r i d i n e p e r o x i d e s a c t i n g as key i n t e r m e d i a t e s i n t h e oxy-gen t r a n s f e r . T h e i r o c c u r r e n c e i s now g e n e r a l l y accepted i n t h e c h e m i s t r y o f monooxygenation. * o d i h y d r o f l a v i n or t e t r a h y d r o p t e n d i n e 0 H S c h e m e 10 * B i o c h e m i c a l and B i o p h y s i c a l L a b o r a t o r y , D e l f t U n i v e r s i t y o f Technology.

Based on t h i s and t h e a b s o l u t e requirements d e s c r i b e d above, i n 62

1972 Eberhard and H a s t i n g p o s t u l a t e d a r e a c t i o n scheme f o r the bac-t e r i a l b i o l u m i n e s c e n c e :

The r e a c t i o n s are a l l t a k i n g p l a c e on the enzyme l u c i f e r a s e . The sequence i s q u i t e d i f f e r e n t from other b i o l u m i n e s c e n c e r e a c t i o n s , s i n c e no c y c l i c p e r o x i d e s are i n v o l v e d and no c a r b o n d i o x i d e i s formed.

I t took many y e a r s t o f i l l i n most gaps i n the evidence f o r t h i s scheme. The q u e s t i o n , which product i s formed i n the e x c i t e d s t a t e has s t i l l not been s o l v e d .

6 3

In 1973 McCapra and Hysert proved that the aldehyde was converted i n t o the c a r b o x y l i c a c i d .

At about the same time a l u c i f e r a s e - F M N H -0 complex ( i n t e r m e d i a t e 64 I I , scheme 11) was i s o l a t e d and proven t o be an important intermediate

65 as r e a c t i o n w i t h aldehyde o n l y produced l i g h t . In 1978 G h i s l a et a l

1 3

c o n c l u d e d from C-NMR s p e c t r a o f t h i s complex, t h a t i t c o n s i s t e d o f a l u c i f e r a s e bound 4a-hydroperoxydihydro-FMN. In 1979 i t was found t h a t

15

t e t r a d e c a n a l was the n a t u r a l l o n g c h a i n aldehyde

In 1983 i t was shown that molecular oxygen i s incorporated i n t o the c a r b o x y l i c a c i d , thus p r o v i n g the monooxygenase a c t i o n o f b a c t e r i a l

, 66 l u c i f e r a s e

With the a i d o f s t r u c t u r a l analogs o f FMN, H a s t i n g s e t a l 67

H

3

Y

V

Y

Y°

i n d i c a t e d t h a t the e m i t t e r i n t h e b a c t e r i a l b i o l u m i n e s c e n c e very w e l l c o u l d be a 4 a - s u b s t i t u t e d FMN d e r i v a t i v e . He p r e f e r s t h e f o r m a t i o n o f a 4a-hydroxy-FMN i n t h e e x c i t e d s t a t e .

Although t h e f i n a l f o r m a t i o n o f a 4a-hydroxy FMN i s v e r y w e l l pos-s i b l e , t h e f o r m a t i o n o f a c a r b o x y l i c a c i d apos-s one o f t h e p r o d u c t pos-s pos-suggepos-st a B a y e r V i l l i g e r l i k e rearrangement o f t h e i n t e r m e d i a t e h y d r o x y a l k y l p e r

-68

o x y f l a v i n t o a 4a-FMN-ester as t h e primary p r o d u c t . We wondered

whether t h i s compound might be formed i n t h e e x c i t e d s t a t e . I n a subsequent s t e p t h e c a r b o x y l i c a c i d and FMN can be formed d i r e c t l y o r i n d i -r e c t l y v i a t h e 4a-pseudobase (scheme 12).

A l t e r n a t i v e l y a 4a-hydroxy-FMN e x c i t e d s t a t e c o u l d be formed by an 48

i n t r a m o l e c u l a r CIEEL pathway as suggested by S c h u s t e r (scheme 13).

R ' C O O H + ( R ' C O O H )1 R = - C H2- ( C H O H )3- C H2O P C > 3 H2 R'= - C i3H2 7 S c h e m e 13 1 The e a s i l y o x i d i z a b l e f l a v i n r e s i d u e donates an e l e c t r o n t o t h e p e r o x i d e bond. T h i s r e s u l t s i n t h e f o r m a t i o n o f a r a d i c a l p a i r . Loss o f a p r o t o n f o l l o w e d by back e l e c t r o n t r a n s f e r t o t h e f l a v i n r a d i c a l c a t i o n might then r e s u l t i n t h e f o r m a t i o n o f t h e e x c i t e d s t a t e o f 4a-hydroxy-FMN.

R e c e n t l y 69a, b the o c c u r r e n c e o f 4 a - s u b s t i t u t e d - d i h y d r o f l a v i n r a d i c a l c a t i o n s was i n d i c a t e d i n a study on t h e f o r m a t i o n o f f l a v i n r a d i c a l s . T h i s might open new e x p e r i m e n t a l pathways t o the development o f chemiluminescent model systems.

The b i o l u m i n e s c e n c e e m i s s i o n maximum i n the b a c t e r i u m Vibrio harveyi s t u d i e d by H a s t i n g s and coworkers, i s c e n t e r e d around 490 nm. Depending on t h e type of b a c t e r i u m however, t h e b i o l u m i n e s c e n c e can have maxima

70

r a n g i n g from 472 545 nm . At one end o f the s c a l e t h e i n v i v o b i o l u -minescence o f Photobacterium phosphoreum i s found around 476 nm and a t

the o t h e r end t h e i n v i v o b i o l u m i n e s c e n c e o f a c e r t a i n s t r a i n o f Photo-71

bacterium fischeri i s found around 545 nm

The i n v i t r o b i o l u m i n e s c e n c e i n both cases however, i s maximal around 495 nm, very c l o s e t o t h e b i o l u m i n e s c e n c e e m i s s i o n o f Vibrio har-veyi. The i n v i t r o b i o l u m i n e s c e n c e from a l u c i f e r a s e p r e p a r a t i o n o f V.harveyi i s i d e n t i c a l w i t h t h e i n v i v o e m i s s i o n o f t h i s b a c t e r i u m .

72

R e c e n t l y i t has been shown t h a t t h e i n v i v o b i o l u m i n e s c e n c e emiss i o n i n P.phoemissphoreum can be a t t r i b u t e d t o t h e h i g h l y f l u o r e emiss c e n t p r o emiss -t h e -t i c group o f an enzyme c l o s e l y a s s o c i a -t e d w i -t h -t h e l u c i f e r a s e . The p r o s t h e t i c group appeared t o be 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 (scheme

In t h e P.fischeri s t r a i n a s i m i l a r enzyme i s found w i t h a f l a v i n as a 6 7

p r o s t h e t i c group . These f l u o r e s c e n t p r o t e i n s a r e s e p a r a t e d from the l u c i f e r a s e s i n t h e i n v i t r o p r e p a r a t i o n s , and t h i s r e s u l t s i n a d i f f e r e n t i n v i t r o e m i s s i o n . A d d i t i o n o f the f l u o r e s c e n t p r o t e i n s t o t h e i n v i t r o system s h i f t s t h e e m i s s i o n s p e c t r a back t o the f l u o r e s c e n c e o f these p r o t e i n s .

In t h e case of P.phosporeum t h i s s h i f t i s t o s h o r t e r wavelength and one might w e l l ask whether t h e i n v i t r o e m i s s i o n can be a t t r i b u t e d t o a p r i m a r y e m i t t e r . That i s , an e m i t t e r formed d i r e c t l y i n t h e e x c i t e d

14).

H2C - ( C H 0 H )3- C H2O H

0 S c h e m e 14

s t a t e i n the c h e m i c a l r e a c t i o n . I t i s argued by Lee and coworkers ' t h a t t h i s s h i f t t o h i g h e r energy can o n l y be e x p l a i n e d by assuming the f o r m a t i o n of a h i g h energy e x c i t e d s t a t e , which can t r a n s f e r i t s energy e i t h e r t o the lumazine p r o t e i n or t o the i n v i t r o e m i t t e r . The l a t t e r

67

might be the 4a-FMN d e r i v a t i v e d e s c r i b e d by H a s t i n g s et a l but t h i s compound does not n e c e s s a r i l y have to be the primary e x c i t e d s t a t e .

Only very r e c e n t l y a new a d d i t i o n a l t r a n s i e n t has been d e s c r i b e d , i n the i n v i t r o b i o l u m i n e s c e n c e of V.harveyi, whose f l u o r e s c e n c e spectrum

74

i s i d e n t i c a l w i t h the b i o l u m i n e s c e n c e spectrum . No i d e a t o i t s chem-i c a l s t r u c t u r e was g chem-i v e n however.

S t u d i e s on model 4 a - ( a l k y l p e r o x y ) f l a v i n d e r i v a t i v e s by Shepherd and 75

B r u i c e a l s o show the p o s s i b i l i t y of energy t r a n s f e r . Chemiluminescence

o f t h i s model i s due t o the f o r m a t i o n of the e x c i t e d s t a t e of the parent f l a v i n ( F l ). A d d i t i o n of a v a r i e t y of f l u o r e s c e r s , i n c l u d i n g F l ,

ox ox l e a d s not o n l y t o the f o r m a t i o n of e x c i t e d f l u o r e s c e r , but a l s o to a

h i g h e r y i e l d of l i g h t . T h i s proves among o t h e r s , t h a t e x c i t e d F 1Q X i s not produced d i r e c t l y , but t h a t i n t h i s case a l s o the f o r m a t i o n of an as y e t unknown e x c i t e d i n t e r m e d i a t e must be assumed.

APPLICATIONS

Apart from the s c i e n t i f i c i n t e r e s t i n the phenomena of chemi- and b i o l u m i n e s c e n c e , many a p p l i c a t i o n s have been found f o r these l i g h t g i v i n g systems.

E d u c a t i o n a l purposes

Many l e c t u r e r s t r y t o c a t c h the a t t e n t i o n o f t h e i r s t u d e n t s by t h e f a s c i n a t i n g l i g h t g i v i n g r e a c t i o n s . Most o f t h e a r t i c l e s , w r i t t e n w i t h t h i s purpose i n mind have c o n s e q u e n t l y appeared i n t h e J o u r n a l o f

Chem-. Chem-. _Chem-. Chem-.Chem-. 76-82 i c a l E d u c a t i o n

L i g h t g i v i n g purposes

The most e f f i c i e n t chemiluminescent r e a c t i o n o f hydrogen p e r o x i d e w i t h o x a l i c e s t e r s ' , as d e s c r i b e d on p. 7, has been developed f o r the p r o d u c t i o n o f an emergency s a f e t y l i g h t . The American Cyanamid Co produces t h e C y a l u m e - l i g h t s t i c k , a p o l y e t h y l e n e tube c o n t a i n i n g a s o l u t i o n o f an o x a l i c e s t e r and a f l u o r e s c e n t dye and a g l a s s ampoule w i t h a hydrogenperoxide s o l u t i o n

When t h e r e a g e n t s a r e mixed by b r e a k i n g t h e g l a s s ampoule an e f f i -c i e n t l i g h t sour-ce i s o b t a i n e d l a s t i n g f o r about 4-8 h.

A n a l y t i c a l purposes

The m a j o r i t y o f a p p l i c a t i o n s a r e found i n t h e a n a l y t i c a l f i e l d . As t h e r e have been developed very s e n s i t i v e i n s t r u m e n t s t o d e t e c t l i g h t , i t i s c l e a r t h a t t h e c o n s t i t u e n t s o f any chemi o r b i o l u m i n e s c e n t r e a c -t i o n can be de-termined very s e n s i -t i v e l y .

The numerous systems t h a t have been developed cannot be t r e a t e d here p r o f u s e l y . Only a few examples w i l l be g i v e n . For f u r t h e r i n f o r -mation some r e c e n t r e c i e w s should be c o n s u l t e d8* * '

In 1902 B e y e r i n c k was t h e f i r s t t o use b i o l u m i n e s c e n t b a c t e r i a t o

3

t e s t f o r oxygen e v o l u t i o n i n p h o t o s y n t h e s i s . T h i s method has been work-ed o u t r e c e n t l y f o r t h e d e t e r m i n a t i o n o f oxygen c o n c e n t r a t i o n s i n t h e range o f 5 . 1 0- 9 - 10 "6 M8 6.

With the a i d o f t h e i s o l a t e d l u c i f e r a s e , compounds as NADH, FMNH,,, aldehydes and a l l o t h e r r e a c t i o n s p r o d u c i n g o r consuming t h e s e components can be determined.

The f i r e f l y l u m i n e s c e n t system (scheme 4) i s s p e c i f i c f o r t h e de--13

t e r m i n a t i o n o f ATP. The d e t e c t i o n l i m i t i s i n t h e o r d e r o f 10 g. A l l l i v i n g c e l l s c o n t a i n ATP. By t h i s method t h e r e f o r e l i v i n g c e l l s

can be counted. T h i s can be used f o r example f o r the d e t e c t i o n of bac-t e r i a l c o n bac-t a m i n a bac-t i o n of s u r f a c e wabac-ters or of f o o d , or o f i n f e c bac-t i o n s l i k e u r e t e r i n f l a m m a t i o n . The advantage of t h i s method i s t h a t the r e s u l t s are a v a i l a b l e much f a s t e r then by c o n v e n t i o n a l methods.

Luminol ( 5 - a m i n o - 2 , 3 - d i h y d r o - 1 , 4 - p h t a l a z i n e d i o n e ) i s one of the b e s t s t u d i e d chemiluminescent compounds (scheme 2 ) . With t h i s compound f o r example hydrogenperoxide and numerous m e t a l i o n s , which s p e c i f i c a l l y enhance the chemiluminescence of the r e a c t i o n , can be d e t e c t e d .

In c l i n i c a l c h e m i s t r y chemiluminescent l a b e l s a r e used i n immuno-a s s immuno-a y . One of t h e most f r e q u e n t l y used l immuno-a b e l s i s i s o l u m i n o l (6-immuno-amino- (6-amino-2 , 3 - d i h y d r o - 1 , 4 - p h t a l a z i n e d i o n e ) . These l a b e l s r e p l a c e the r a d i o a c t i v e ones used up t o now. The chemiluminescence i s a t l e a s t as s e n s i t i v e , much e a s i e r t o d e t e c t and l e s s h a r m f u l t o the environment.,

A d e t a i l e d survey of the a n a l y t i c a l a p p l i c a t i o n s o f chemi en b i o -luminescence can be found i n the p r o c e e d i n g s o f two r e c e n t l y h e l d

sym-. sym-. sym-.87, 88 p o s i a on t h i s s u b j e c t

The b a c t e r i a l and f i r e f l y l u c i f e r i n - l u c i f e r a s e systems are commer-c i a l l y a v a i l a b l e . S e v e r a l commer-companies have developed s p e commer-c i a l luminometers to measure the l i g h t produced i n these r e a c t i o n s .

These v e r y s e n s i t i v e methods w i l l g r a d u a l l y t a k e a p l a c e i n a l l b i o c h e m i c a l and c l i n i c a l l a b o r a t o r i e s .

MODEL SYSTEMS

In t h e c o u r s e of t h e i r work on b a c t e r i a l b i o l u m i n e s c e n c e S t r e h l e r 89

and Shoup i n 1953 d e s c r i b e d the chemiluminescence e n t i c e d from the non-enzymic system r i b o f l a v i n - h y d r o g e n p e r o x i d e . T h i s was the f i r s t time t h a t f l a v i n was brought t o chemiluminescence. The weak chemiluminescence c o u l d be enhanced by t h e a d d i t i o n o f m e t a l i o n s l i k e copper ( I I ) or i r o n ( I I ) . (A FMN-hydrogenperoxide system has been d e s c r i b e d f o r the s e l e c t i v e chemiluminescent d e t e r m i n a t i o n o f copper ( I I ) w i t h a d e t e c t i o n l i m i t o f 30 pg ).

In the n i n e t e e n s i x t i e s a s e r i e s of papers on t h e chemiluminescence 90-97

S t r e h l e r and Shoup o n l y suggested t h a t the chemiluminescence e m i s s i o n (X , ca 550 nm) i s s i m i l a r i f not i d e n t i c a l w i t h t h e f l u o r e s c e n c e

emis-max

s i o n o f r i b o f l a v i n . However, the chemiluminescence e m i s s i o n o f a r i b o -97

f l a v i n - h y d r o g e n p e r o x i d e - O s C l ^ system , was c e n t e r e d around 495 nm

s i m i l a r t o t h e maximum observed f o r b a c t e r i a l b i o l u m i n e s c e n c e , i n d i c a t i n g t h a t r i b o f l a v i n i s n o t t h e e m i t t e r .

The chemiluminescence o f t h e r i b o f l a v i n - h y d r o g e n p e r o x i d e system was 98

brought i n c o n n e c t i o n w i t h a r e p o r t o f Mager on a t e t r a h y d r o p y r a z i n e -p e r o x i d e . T h i s com-pound was formed through t h e a d d i t i o n o f hydrogen-p e r o x i d e t o t h e C=N double bond o f a d i h y d r o hydrogen-p y r a z i n e .

In analogy, we proposed t h e f o r m a t i o n o f f l a v i n h y d r o p e r o x i d e s by the a d d i t i o n o f hydrogenperoxide, l e a d i n g t o t h e f o r m a t i o n o f a 4a- o r

10a-hydroperoxide, as a c r u c i a l s t e p i n t h e p r o d u c t i o n o f e x c i t e d s t a t e s (scheme 16).

S c h e m e 16

That f o r m a t i o n o f p e r o x i d e s i n t h i s manner i s p o s s i b l e was proven 61 99

from t h e work o f Mager ' w i t h model compounds and s e v e r a l y e a r s l a t e r by Kemal and B r u i c e1 1^ . H a s t i n g s e t a l1<^ o b t a i n e d b i o l u m i n e s c e n c e i n a system c o n s i s t i n g o f l u c i f e r a s e - F M N - h y d r o g e n p e r o x i d e - a l d e h y d e , s u g g e s t i n g the f o r m a t i o n o f a f l a v i n p e r o x i d e by d i r e c t a t t a c k o f hydrogenperoxide on FMN.

The aim o f t h i s t h e s i s i s t o c o n t r i b u t e t o a b e t t e r u n d e r s t a n d i n g o f r.he p o s s i b l e mechanisms o f some chemiluminescent r e a c t i o n s r e l a t e d t o t h e s e f l a v i n - p e r o x i d e systems.

In r e p e a t i n g t h e experiments o f S t r e h l e r and Shoup, indeed a s m a l l f l a s h o f l i g h t was observed when a s o l u t i o n o f r i b o f l a v i n was t r e a t e d w i t h hydrogen p e r o x i d e . The a d d i t i o n o f aldehyde d i d not seem t o i n f l u -ence t h i s weak chemiluminesc-ence.

As t h e key i n t e r m e d i a t e was thought t o be a p e r o x i d e formed a t t h e C4 - or C10 - b r i d g e carbon atoms o f the f l a v i n m o l e c u l e , the benzene

a a °

n u c l e u s d i d n o t seem c r u c i a l i n t h i s model i n v e s t i g a t i o n .

T h e r e f o r e we t r i e d t o o b t a i n chemiluminescence a l s o by t r e a t i n g some 8-substituted-lumazine d e r i v a t i v e s w i t h hydrogen peroxide. In a c i d i c s o l u t i o n t h i s r e s u l t e d indeed i n chemiluminescence.

C H3

0

S c h e m e 17

I n v e s t i g a t i o n o f t h i s phenomenon however, soon r e v e a l e d the presen-ce o f a c o n t a m i n a t i n g compound, which was r e s p o n s i b l e f o r the chemiluminescence o b s e r v e d . T h i s compound appeared t o be 7 h y d r o x y 6 , 7 d i h y d r o -l u m i f -l a v i n ( I , scheme 1 8 ) , which not on-ly gave chemi-luminescence with h y d r o g e n p e r o x i d e , but i n a l k a l i n e medium a l s o w i t h oxygen:

0 2 , p H > 7 . 0. H3C H ( 4 8 8 n m ) NH 0 H H S c h e m e 18

The f l u o r e s c e n c e spectrum o f t h e product I I proved t o be i d e n t i c a l w i t h t h e chemiluminescence spectrum o f t h e r e a c t i o n . I t i s s u r p r i s i n g , t h a t t h e chemiluminescence maximum a t 488 nm i s almost the same as the maximum observed i n t h e i n v i t r o b a c t e r i a l b i o l u m i n e s c e n c e .

N o t w i t h s t a n d i n g , t h e lumazines gave t r i b u t e t o t h e i r name y e t , by g i v i n g chemiluminescence when t r e a t e d w i t h a s t r o n g base i n a p o l a r w a t e r f r e e s o l v e n t . Ç H3 Ç H3 H3C v ^ N DMF|p2 ^ C y N N 0 H3C - ^ N ^ V -N H « -Bu0K

H

C ^ N -

V

T h i s appeared not t o be r e s t r i c t e d t o l u m a z i n e s . G e n e r a l l y 8-sub-s t i t u t e d p t e r i d i n e 8-sub-s b e a r i n g a 7-methyl 8-sub-s u b 8-sub-s t i t u e n t gave chemilumine8-sub-scence under these c o n d i t i o n s . Again the f l u o r e s c e n c e s p e c t r a o f t h e p r o d u c t s were i d e n t i c a l w i t h t h e chemiluminescence s p e c t r a o f t h e r e a c t i o n s .

F i n a l l y the i n v e s t i g a t i o n o f t h e a u t o x i d a t i o n o f a f u l l y o x i d i z e d f l a v i n , l i k e l u m i f l a v i n under these same c o n d i t i o n s , a g a i n r e v e a l e d the

t - B u O K

S c h e m e 20

o x i d a t i o n o f a methyl group, i n t h i s case t o a c a r b o x y l i c a c i d . T h i s r e a c t i o n however, was not accompanied by chemiluminescence. I n these cases t h e presumption t h a t t h e 4a and/or 1 O a - b r i d g e p o s i t i o n s would par-t i c i p a par-t e i n p e r o x i d e f o r m a par-t i o n i s a p p a r e n par-t l y nopar-t r i g h par-t . I n s par-t e a d par-the a t t a c k o f m o l e c u l a r oxygen r e s u l t s i n the f o r m a t i o n o f p e r o x i d e i n t e r -mediates a t the methyl s u b s t i t u e n t s .

The d e t a i l e d i n v e s t i g a t i o n s o f these r e a c t i o n s a r e d e s c r i b e d i n the next c h a p t e r s .

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84. W.R. S e i t z , C r i t . Rev. i n A n a l . Chem. 13, 1 (1981). 8 5 . E.L. Wehry, A n a l . Chem. ¿ 4 , 131R ( 1 9 8 2 ) .

8 6 . B. Chance, R. Oshino and N. Oshino, Methods Enzymol. ¿ 4 , 4 9 9 ( 1 9 7 8 ) .

8 7 . I n t . Symp. on A n a l . A p p l i c . o f B i o - and Chemiluminescence (E. Schräm and P. S t a n l e y , Eds.) S t a t e P r i n t i n g & P u b l . I n c . C a l i f o r n i a

1979-88. 2nd Symp. on B i o - and Chemiluminescence, L a J o l l a 1980 (M. DeLuca

and W.D. M c E l r o y , E d s . ) , Academic P r e s s 1 9 8 1 .

8 9 . B.L. S t r e h l e r and C S . Shoup, Arch. Biochem. Biophys. 4 7 , 8 (1953).

9 0 . R.H. S t e e l e , B i o c h e m i s t r y 2, 529 ( 1 9 6 3 ) .

9 1 . J.R. W i l l i a m s and R.H. S t e e l e , B i o c h e m i s t r y _4, 814 ( 1 9 6 5 ) .

9 2 . J.E. Vorhaben and R.H. S t e e l e , Biochem. Biophys. Res. Comm.

1 9 , 1 8 7 ( 1 9 6 5 ) .

93. J.E. Vorhaben and R.H. S t e e l e , B i o c h e m i s t r y 6 , 1404 ( 1 9 6 7 ) .

94. M.O. Stone, J.E. Vorhaben and R.H. S t e e l e , Biochem. B i o p h y s . Res. Comm. 3 6 , 502 ( 1 9 6 9 ) .

9 5 . M.O. Stone and R.H. S t e e l e , Fed. P r o c . 2 8 , 9 0 0 ( 1 9 6 9 ) . 9 6 . M.O. Stone and R.H. S t e e l e , B i o c h e m i s t r y £, 4 3 4 3 (1970).

9 7 . R.D. Towner, H.A. Neufeld and P.B. S h e v l i n , Arch. Biochem. Biophys. 137, 102 (1970).

9 8 . H.I.X. Mager ana W. Berends, Ree. Trav. Chim. 84, 314 ( 1 9 6 5 ) .

9 9 - H.I.X. Mager, R. Addink and W. Berends, Ree. Trav. Chim.

86> 8 3 3 ( 1 9 6 7 ) .

1 0 0 . C. Kemal and T.C. B r u i c e , P r o c . N a t l . Acad. S e i . USA. 7 3 , 9 9 5 ( 1 9 7 6 ) .

1 0 1 . J.W. H a s t i n g s , S.C. Tu, J.E. Becvar and R.P. Presswood, Photochem. P h o t o b i o l . 2 9 , 3 8 3 ( 1 9 7 9 ) .

CHAPTER I I

SYNTHESIS OF 7-HYDR0XY-6,7-DIHYDR0LUMIFLAVIN *

In t h e course of i n v e s t i g a t i o n s on t h e a u t o x i d a t i o n o f reduced p t e r -1

i d i n e s and f l a v i n s , we were c o n f r o n t e d w i t h a chemiluminescence phenom-enon. When an a c i d s o l u 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 (3_) was t r e a t e d w i t h hydrogen p e r o x i d e , a f l a s h o f l i g h t c o u l d be o b s e r v e d . E x t e n s i v e r e c r y s t a l l i z a t i o n o f t h e compound decreased t h e amount o f chemilumines-cence c o n s i d e r a b l y . A p p a r e n t l y a c o n t a m i n a t i n g compound formed d u r i n g the s y n t h e s i s was r e s p o n s i b l e f o r t h e chemiluminescence.

2 3

The s y n t h e s i s o f 3_ was c a r r i e d out by c o n d e n s a t i o n o f d i a c e t y l w i t h 5-amino-6-(methylamino)-2,4(1H,3H)-pyrimidinedione (J_). The d i a c e t y l appeared t o c o n t a i n a s m a l l amount of i t s dimer (2_). T h i s dimer c o u l d be o b t a i n e d on a p r e p a r a t i v e s c a l e by c a r r y i n g out an a l d o l condensation

4 5 w i t h d i a c e t y l ' .

2 6

The condensation o f 2_ w i t h J_ has been d e s c r i b e d , ' but no d e f i n i t e s t r u c t u r e s o f t h e products were g i v e n .

R e i n v e s t i g a t i o n o f t h i s r e a c t i o n , i n d i c a t e d a s t r o n g dependence on a c i d i t y . By c a r e f u l l y a d j u s t i n g t h e pH t o the v a l u e s i n d i c a t e d i n t h e Scheme, compounds 4_, 5_ and 6_ c o u l d be i s o l a t e d .

To t h e "pH 5" product we a s s i g n e d s t r u c t u r e 5_ on account o f : 1 . Comparison o f t h e NMR s p e c t r a of 5_ and 3_ showed that the 7-Me group

was s t i l l present i n 5.

2. UV a b s o r p t i o n s p e c t r a showed t h a t 5_ was c o n v e r t e d q u a n t i t a t i v e l y i n t o 4_ i n a l k a l i n e medium. T h i s f u r t h e r e s t a b l i s h e d t h e a c i d c h a r a c t e r o f the 7-Me group, which was a l r e a d y known from deuterium exchange ,

7 - 9 experiments To t h e "pH 6" product we a s s i g n e d s t r u c t u r e 4_ on account o f : 1 . The compound 4_ i s c o n v e r t e d q u a n t i t a t i v e l y i n t o l u m i f l a v i n (J_) by r e f l u x i n g i n aqueous a c i d s o l u t i o n . 2. Mass s p e c t r a o f 1_ and 4_ a r e i d e n t i c a l w i t h e x c e p t i o n o f t h e m o l e c u l a r i o n peak o f 4_ (J_ M+ = 256; 4_ M+ = 2 7 2 ) .

Me Me

3 6 8 SCHEME I

3. R e f l u x i n g 5_ i n d i l u t e d i d e u t e r o s u l f u r i c a c i d a f f o r d e d 7_ i n which the

a r o m a t i c p r o t o n on the 9 - p o s i t i o n , o r i g i n a t i n g from the 7-Me group o f

5_, was exchanged f o r deuterium.

4. R e f l u x i n g 4_ i n d i l u t e d i d e u t e r o s u l f u r i c a c i d a f f o r d e d 7 i n which the

a r o m a t i c protons were s t i l l p r e s e n t . I f 4_ bears a methylene moiety i n the 9 - p o s i t i o n one s h o u l d expect i n c o r p o r a t i o n o f deuterium. As t h i s was not observed the methylene moiety of _4 i s supposed t o be i n the

6 r - p o s i t i o n .

C a r r y i n g out the c o n d e n s a t i o n o f J_ w i t h 2_ a t pH < 3 gave a m i x t u r e of 5 and 6.

R e f l u x i n g an aqueous a c i d s o l u t i o n o f 5_ a f f o r d e d a m i x t u r e o f 6^ and 7- In a s i m i l a r experiment C r e s w e l l and Wood o b t a i n e d a compound t o which they t e n t a t i v e l y a s s i g n e d s t r u c t u r e 8_. Although 6 c o n t a i n e d one mole o f water o f c r y s t a l l i z a t i o n i n s t e a d of h a l f a mole, comparison of UV a b s o r p t i o n d a t a and chromatographic behaviour showed we had i s o l a t e d the same compound.

However we had t o r e j e c t s t r u c t u r e 8. Our f i n d i n g s mentioned below

11

a r e i n f a v o u r o f s t r u c t u r e 6_ f o r the "pH < 3 " product:

1. In an NMR spectrum of 6_ the f o l l o w i n g s i g n a l s are present: two 3 -p r o t o n s i n g l e t s (N-Me s h i f t e d t o h i g h e r f i e l d i n r e l a t i o n t o 4_ and 5_, i n d i c a t i n g a more s a t u r a t e d c h a r a c t e r , and a Me group r e s o n a t i n g i n the a l i p h a t i c r e g i o n ) , one 3-proton doublet coupled with a one-proton q u a r t e t ( a l l y l i c c o u p l i n g ) , an AB system ( J = 15 Hz, s u g g e s t i n g e i t h e r an open c h a i n o r a r i n g ) , and two exchangeable p r o t o n s (presumably p r e s e n t i n t h e p y r i m i d i n e p a r t o f the m o l e c u l e , which were c o n f i r m e d by m e t h y l a t i o n experiments i n which 6^ took up two Me groups g i v i n g two i s o m e r i c d i m e t h y l d e r i v a t i v e s ) .

2. Hydrogénation o f a d i m e t h y l d e r i v a t i v e o f 5_ r e s u l t e d i n t h e uptake o f t h r e e moles o f hydrogen per mole o f compound, r u l i n g out t h e p o s s i b i -l i t y o f t h e above mentioned open c h a i n .

3 . 2,4-Dinitrophenylhydrazones were obtained from 6_ as w e l l as from i t s two d i m e t h y l d e r i v a t i v e s . T h i s i n d i c a t e s t h e presence o f a CO group, which was s u b s t a n t i a t e d by IR d a t a .

4 . R e f l u x i n g 5_ i n d i l u t e dideutero s u l f u r i c a c i d a f f o r d e d 6_ i n which the Me group r e s o n a t i n g a t h i g h e s t f i e l d was d e u t e r a t e d . The protons o f the methylene group were a l s o exchanged p a r t i a l l y f o r deuterium. 5. When 6_ was d i s s o l v e d i n e i t h e r d i d e u t e r o s u l f u r i c a c i d o r sodium

d e u t e r o o x i d e o n l y exchange o f t h e protons o f t h e methylene group was observed.

From t h e s e d a t a we conclude t h a t t h e Me group r e s o n a t i n g i n t h e a l i p h a t i c r e g i o n ( CQ -Me) o r i g i n a t e d from t h e 7-Me group o f 5. The

ya

d e u t e r a t i o n o f the methylene group was caused by t h e a d j a c e n t CO group. In a c i d medium o n l y compounds 4^ and 5_ produced l i g h t when t r e a t e d w i t h hydrogen p e r o x i d e . In a l k a l i n e medium chemiluminescence o c c u r r e d w i t h oxygen as a r e a c t a n t . As compound 5_ i s c o n v e r t e d i n t o 4_ under these c o n d i t i o n s , compound 4_ i s chemiluminescing i n a l k a l i n e medium. D e t a i l s of the l a t t e r r e a c t i o n w i l l be d e s c r i b e d i n t h e next p a r t o f these s e r i e s .

EXPERIMENTAL

NMR s p e c t r a were recorded on a V a r i a n A-60, or where i n d i c a t e d on a V a r l a n XL-100 s p e c t r o m e t e r , w i t h TMS as an i n t e r n a l s t a n d a r d .

6,7,8-Trimethyllumazine (3_). This compound was prepared according t o a method d e s c r i b e d '3; NMR (dg-DMSO); 6 = 2.53 ( s , 3, C&-Me); 2.64 ( s , 3, C?-Me); 3-99 ( s , 3, Ng-Me).

5-Amino-6-(methyl ami no)-2, 4 (1H, 3H)-pyrimidinedione hydrochloride ( 1 ) . 6 - ( M e t h y l a m i n o ) - 5 - n i t r o - 2 , 4 ( 1 H , 3 H ) - p y r i m i d i n e d i o n e was s y n t h e s i z e d a c c o r d i n g t o C r e s s w e l l and Wood^. Reduction of t h e n i t r o compound t o the 5-formylamino d e r i v a t i v e , and subsequent d e f o r m y l a t i o n was c a r r i e d out a c c o r d i n g t o P f l e i d e r e r and N i i b e l1 0.

5-Acetyl-2-hydroxy-2,5-dimethyl-4, 5-dihydro-3(2H)-furanone ( 2 ) . T h i s dimer of d i a c e t y l was prepared a c c o r d i n g t o D i e l s et al. The p r o d -u c t was 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 C C l ^ . Two d i a s t e r e o - i s o m e r s c o u l d be i s o l a t e d : (a) m.p. 38.5-40.5°; NMR (CDC13): 6 = 1.52 ( s , 3 ) ; 1.65(s,3); 2.35(s,3); 2.69 and 3-02 (AB, J = 19.5 Hz,2); 3-90(s,1); (b) m.p. 48-50°; NMR ( C D C l ^ : 6 r 1.48 and 1.49 ( 2 s , 6 ) ; 2 . 3 3 ( 8 , 3 ) ; 2.22 and 3-35 (AB, J = 19 Hz,2); 3.91-4.16 (1H). Both isomers gave t h e same p r o d u c t s i n r e a c t i o n w i t h

7Hydroxy6,7dihydrolumiflavin ( 4 ) . Compound 1 (385 g) was s u s -pended i n water (100 m l ) . The pH o f t h e suspension was brought t o 6.0 w i t h 5 0 % NaOH, and then 2 (3.5 g) was added. A f t e r s t i r r i n g and r e a d j u s t i n g the pH t o 6.0 t h e deep orange s o l n was p l a c e d i n t h e r e f r i g e r -a t o r . The s o l n soon bec-ame d-ark green -and d-ark green c r y s t -a l s s e p -a r -a t e d . A f t e r s e v e r a l days the s o l i d was f i l t e r e d o f f and washed w i t h w a t e r , y i e l d 3-90 g. D r y i n g i n a vacuum d e s i c c a t o r over P.O.. r e s u l t e d i n the

2 5

l o s s o f one mole o f water o f c r y s t a l l i z a t i o n , which was taken up a g a i n upon exposure t o a i r . R e c r y s t a l l i z a t i o n from water (200 ml) y i e l d e d 3.06 g (52%) m.p.> 300°, ( C ^ H ^ N ^ . H ^ (292.30) C a l c d : C, 53-42; H, 5-52; N, 1 9. 1 7 ; Found: C, 53-3; H, 5-7; N, 19.0%); NMR, 100 MHz

(d6-DMS0): 6 = 1 . 2 4 ( s,3,C?- M e ) ; 2.19(d,J = 1.5Hz,3,Cg-Me); 3.00 and 3-15 (AB, J = 1 6 H Z , 2 , C6- H2) ; 3.90(s,3,N-Me); 5-37(s,1,OH); 6.79(q,J = 1.5Hz,