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Chemiluminescence of acridinium salts Richardson, Donald George 1969

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THE  CHEMILUMINESCENCE OF ACRIDINIUM SALTS by DONALD GEORGE RICHARDSON  B.Sc.  (Honours), U n i v e r s i t y  o f B r i t i s h Columbia, 1964  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE  REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  I n t h e Department of Chemis t r y  We a c c e p t t h i s  t h e s i s as conforming  to the required  THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1969  standard  In  presenting  an  advanced  the  Library  I further for  this degree shall  agree  scholarly  by  his  of  this  written  thesis  in partial  fulfilment  of  at  University  of  Columbia,  the  make that  i t freely  permission  purposes  may  representatives. thes.is  for  be It  financial  available for  of  The U n i v e r s i t y o f B r i t i s h V a n c o u v e r 8, Canada  by  the  is understood gain  Columbia  for  extensive  granted  permission.  Department  British  shall  Head  be  requirements  reference copying  that  not  the  of  and  of my  I agree  for that  Study.  this  thesis  Department  copying  or  allowed  without  or  publication my  TO ANNA  Without whose h e l p t h i s  thes  would not have been a r e a l i t y  iii  ABSTRACT  A s e r i e s o f 9 - s u b s t i t u t e d m e t h y l a c r i d i n i u m s a l t s have been d e v i s e d , chemiluminescent  i n a l k a l i n e hydrogen p e r o x i d e  T h e i r r e l a t i v e l i g h t i n t e n s i t i e s correspond  to t h e expected  of . . r e a c t i v i t y w i t h the n u c l e o p h i l i c h y d r o p e r o x i d e m e c h a n i s t i c evidence  solutions. order  a n i o n , and the  i s c o n s i s t e n t w i t h the requirement  f o r a four-  membered 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 as the d i r e c t p r e c u r s o r to chemiluminescence.  I n e v e r y case, the a c t u a l e m i t t e r i s N-methyl-  acridone, h 442 mu. max r  The b r i g h t e s t compounds a r e a s e r i e s o f s u b s t i t u t e d esters.  phenyl  They have l a r g e r a t e d i f f e r e n c e s ( w i t h a Hammett depend-  ence) b u t have equal quantum y i e l d s , e q u i v a l e n t to t h a t o f the closely-related  " c l a s s i c a l " l i g h t producer,  lucigenin.  iv  TABLE OF CONTENTS  page INTRODUCTION  1  EXPERIMENTAL  19  PART A:  SYNTHESIS  20  PART B:  PRODUCT STUDIES  34  PART C:  CHEMILUMINESCENCE AND FLUORESCENCE SPECTRA  35  CHEMILUMINESCENCE  37  PART D:  DISCUSSION  46  CONCLUSION  64  BIBLIOGRAPHY  65  LIST OF TABLES  page  I  II  III  CHEMILUMINESCENCE OF COMPOUNDS AT pH 12  40  KINETICS OF THE NITROPHENYL ESTERS AT pH 8  42  pH-DEPENDENT KINETICS OF 4-BROMO PHENYL ESTER 34d  IV  V  EFFECTS OF H O ESTER 34c  43 CONCENTRATION ON KINETICS OF  EFFECT OF SUBSTRATE CONCENTRATION ON THE KINETICS OF 34c ;  44  45  vi  LIST OF FIGURES page 1.  PRODUCT FORMATION IN CHEMILUMINESCENCE  2.  SYNTHETIC ROUTES TO THE CHEMILUMINESCENT COMPOUNDS ...  3.  CHEMILUMINESCENCE AND FLUORESCENCE SPECTRA FOR  7  19  SELECTED ACRIDINIUM SALTS  36  4.  LIGHT-MEASURING APPARATUS  37  5.  A TYPICAL CHEMILUMINESCENCE DECAY CURVE  39  6.  HAMMETT PLOT FOR THE SUBSTITUTED PHENYL ESTERS  41  7.  LOG k VS. pH, 4-BROMO PHENYL ESTER  43  vii  ACKNOWLEDGEMENT  I would  l i k e t o express my s i n c e r e g r a t i t u d e t o  Dr. Frank McCapra, whose a b i l i t i e s s e r v e as an i n s p i r a t i o n direction.  a s a chemist s h o u l d  t o anyone working under h i s  INTRODUCTION  i  INTRODUCTION Chemiluminescence i s t h e p r o c e s s formed i n an e x c i t e d s t a t e by chemical  of l i g h t reaction.  i n t e r e s t among chemists i s now becoming m a n i f e s t  emission  from m o l e c u l e s  Increasing  awareness and  by the i n c r e a s i n g number  of p u b l i c a t i o n s on the s u b j e c t which have appeared i n r e c e n t y e a r s .  Several  1-8 recent  reviews  c o n t a i n many o f the c u r r e n t views on the s u b j e c t , and the  9-13 o l d e r reviews f u r n i s h m a t e r i a l of h i s t o r i c a l i n t e r e s t . Many k i n d s o f chemical r e a c t i o n s emit l i g h t . Examples a r e gas reactions  14  and hydrocarbon o x i d a t i o n s -8  are  so s m a l l , i e 10  3 7 ' but o f t e n the quantum y i e l d s }  -15 t o 10  , that emission  cannot be d e t e c t e d  by the  naked eye. The  organic  r e a c t i o n s i n s o l u t i o n which emit v i s i b l e  l e s s common, and have aroused our i n t e r e s t . low,  10 ^ to 10  provide  , but nevertheless  a dramatic, v i s i b l e  l i g h t a r e much  The quantum y i e l d s a r e s t i l l  these r e a c t i o n s a r e e f f i c i e n t  enough to  d e m o n s t r a t i o n o f the energy r e l e a s e d .  perhaps b i o luminescence (about which v e r y  little  Excepting  i s known''") , i t i s p o s s i b l e  to c a t e g o r i s e most o f these r e a c t i o n s i n t o one of t h r e e g e n e r a l e l e c t r o n t r a n s f e r r e a c t i o n s , e x c i t e d oxygen f o r m a t i o n ,  types:  or peroxide  decom-  positions'. Electron-Transfer  Chemiluminescence  Chemiluminescence has been observed i n the e l e c t r o l y s i s o f p o l y c y c l i c aromatic hydrocarbons l i k e anthracene and rubrene i n the s o l v e n t s DMF o r 15 16 a c e t o n i t r i l e when a s u p p o r t i n g occurs  i n the v i c i n i t y  e l e c t r o l y t e i s present.  '  The e m i s s i o n  o f the cathode, and i t s wavelength corresponds to t h a t  of the hydrocarbon f l u o r e s c e n c e ,  i n d i c a t i n g t h a t the e m i t t e r i s probably  e x c i t e d s i n g l e t s t a t e o f t h e hydrocarbon.  an  + The  proposed mechanism i n v o l v e s the f o r m a t i o n o f r a d i c a l i o n s Ar*  and A r * a t the anode and cathode to the v i c i n i t y  o f the cathode,  an e l e c t r o n t r a n s f e r : + Ar* + A r *  *-  r e s p e c t i v e l y , the m i g r a t i o n o f the c a t i o n then a r a d i c a l a n n h i l a t i o n process  Ar + Ar*  I The  ^  A r + hv  c a t i o n c o u l d a c c e p t the e l e c t r o n i n t o a h i g h energy  r e s u l t i n g i n (most probably)  antibonding  t h e f o r m a t i o n o f an e x c i t e d  A more g e n e r a l type o f chemiluminescent among a r o m a t i c hydrocarbons  involving  singlet.  electron-transfer  has been d e s c r i b e d . ^  orbital,  reaction  T h i s i n v o l v e s the ad-  d i t i o n o f an e l e c t r o n t o a r a d i c a l c a t i o n o r the removal of an e l e c t r o n from a r a d i c a l a n i o n y i e l d i n g , excited  i n b o t h c a s e s , the parent hydrocarbon  state. I n a t y p i c a l example, the r a d i c a l a n i o n o f  (DPA)  i n an  i s generated  by treatment w i t h sodium.  9,10-diphenylanthracene  When mixed w i t h  9,10-dichloroI j  9,10-dihydro-9,10-diphenylanthracene  (DPACl,^) , probably r e a c t i n g as DPA  a b r i g h t chemiluminescence i s observed, The  and DPA i s produced  ,  i n very high y i e l d .  l i g h t e m i t t e d i s i d e n t i c a l t o the f l u o r e s c e n c e o f DPA. A r e a s o n a b l e e x p l a n a t i o n f o r the f o r m a t i o n o f e x c i t e d DPA (and hence  the e m i s s i o n o f l i g h t ) i s t h a t DPAC1* r e c e i v e s an e l e c t r o n from DPA lowest a n t i b o n d i n g o r b i t a l ,  r e s u l t i n g i n e x c i t e d DPAC1 .  into i t s  This species could  l o s e c h l o r i d e i o n to y i e l d , d i r e c t l y , e x c i t e d DPA w i t h an a n t i b o n d i n g which, when f a l l i n g  to a h a l f - f i l l e d bonding  . DPA" + DPAC1 2  DPAC1* + DPA" DPA*  orbital,  *  DPA + C l " + DPAC1'  > >  DPA + DPA* + C l " DPA + hV  e j e c t s a photon.  electron  3  Another chemiluminescent r e a c t i o n , t h a t o f DPA agent b e n z o y l p e r o x i d e , e l e c t r o n i n DPA  and the o x i d i s i n g  i s thought t o i n v o l v e the t r a n s f e r o f a bonding  t o a benzoate  radical: DPA* + PhCO  E x c i t e d Oxygen Formation Because most o f the o r g a n i c  chemiluminescent r e a c t i o n s  e i t h e r oxygen o r hydrogen p e r o x i d e , might be due t o some s p e c i e s  involve  s p e c u l a t i o n a r o s e that the e m i s s i o n  o f e x c i t e d oxygen.  Recent work by O g r y z l o jet al e m i s s i o n bands o f e x c i t e d oxygen.  18  identified  has  the p r i n c i p a l  The r e a c t i o n C l ^ + 0^2® 2  a r e d glow i n the r e g i o n 550 to 1300 mji.  The s t r o n g e s t  703 mp) a r e due t o a complex o f two e x c i t e d 0  *-  +  EM:  TS  bands (634 and  m o l e c u l e s undergoing the  These r e s u l t s appear t o e x c l u d e the p a r t i c i p a t i o n o f e x c i t e d oxygen i n the b r i g h t e r o r g a n i c more e n e r g e t i c , e m i t t i n g  chemiluminescent r e a c t i o n s , whose emissions a r e much l i g h t - i n the r e g i o n 400 t o 500 mu.  A  recent  19 attempt by two  t o i m p l i c a t e e x c i t e d oxygen i n these more e n e r g e t i c  emissions,  s t a t e , has been proven i n c o r r e c t f o r a t l e a s t  invoking a  c l a s s e s of compounds by work i n our l a b o r a t o r i e s . Organic P e r o x i d e Decompositions F o r a l l o f the b r i g h t e s t o r g a n i c  the e v i d e n c e so f a r o b t a i n e d  has e i t h e r proven o r , i n some cases,  i n f e r r e d that the light-producing organic  step  requires  strongly  t h e decomposition of an  p e r o x i d e to y i e l d a m o l e c u l e w i t h an e l e c t r o n i c a l l y e x c i t e d  containing escent  chemiluminescent r e a c t i o n s i n s o l u t i o n ,  chromophore.  B e f o r e examining some o f the best-known chemilumin-  r e a c t i o n s , some mention should be made o f the more g e n e r a l  common to a l l .  carbonyl-  aspects  4  That an e x c i t e d c a r b o n y l convincingly  i s responsible  f o r l i g h t e m i s s i o n has  been  demonstrated i n systems where the e m i s s i o n spectrum matches  e x a c t l y w i t h the f l u o r e s c e n c e  spectrum of a c a r b o n y l  under which these l i g h t - p r o d u c i n g  product.  r e a c t i o n s occur are  The  conditions  such t h a t the s i n g l e t  4  e x c i t e d s t a t e S a r e more l i k e l y than t r i p l e t s : (1)  The  s o l u t i o n s are n o r m a l l y at room temperature, where  v i b r a t i o n a l and i s very (2)  As  c o l l i s i o n a l d e a c t i v a t i o n of  triplets  pronounced.  Oxygen, a proven t r i p l e t quencher, i s o f t e n 4  fluorescence  i s known  present.  to i n v o l v e r a d i a t i v e t r a n s i t i o n s from an  s i n g l e t to a ground s t a t e s i n g l e t , i t i s r e a s o n a b l e to say chemiluminescent e m i t t e r fluorescence  Colored  t h a t the  actual  i s the e x c i t e d s t a t e of whichever p r o d u c t has  spectrum i d e n t i c a l to the  l i g h t emission  carbonyl  product  do not match, i d e n t i f i c a t i o n of the e m i t t e r i s more d i f f i c u l t .  byproducts can absorb some of the emitted  the e m i s s i o n ^  m a x  5  l i g h t and  hence  shift  a l t e r n a t i v e l y , energy t r a n s f e r from the e x c i t e d product  to a more h i g h l y f l u o r e s c e n t byproduct or i m p u r i t y e m i s s i o n spectrum.  In these more d i f f i c u l t  less self-absorption)  is helpful.  more s e n s i t i v e , spectrometers now  T h i s has  can  result in a  examples, h i g h d i l u t i o n  spurious (with  been made f e a s i b l e by the newer,  available.  The  problem of  interfering  f l u o r e s c e n t compounds r e q u i r e s  t h a t a r i g o r o u s product study be made i n  to i d e n t i f y the a c t u a l primary  emitter.  The  order  exact mechanism by which c h e m i c a l energy r e s u l t s i n an e l e c t r o n i c -  a l l y excited species, 20 by  a  spectrum.  In cases where the r e a c t i o n chemiluminescence and fluorescence  excited  our group  21 '  capable of e m i t t i n g  22 '  23 and  others  light,  i s not known.  However, work  24 '  has  y i e l d e d a r e a l i s t i c working hypo-  t h e s i s which accounts f o r much of the b e h a v i o u r observed f o r the b r i g h t e s t chemiluminescent r e a c t i o n s .  T h i s h y p o t h e s i s has  a l l o w e d us  to p r e d i c t  5  s u c c e s s f u l l y the r e l a t i v e b r i g h t n e s s of the new this investigation.  The mechanism e n v i s i o n s the c o n c e r t e d m u l t i p l e bond r e -  arrangement o r decomposition  o r an o r g a n i c p e r o x i d e t o y i e l d a c a r b o n y l  chromophore i n an e l e c t r o n i c a l l y e x c i t e d There a r e some important (1)  As  compounds s y n t h e s i z e d d u r i n g  state.  requirements  i n h e r e n t i n the proposed  the wavelength of the e m i t t e d l i g h t  mechanism:  i s u s u a l l y i n the  r e g i o n 400  to 500 mu,  c o r r e s p o n d i n g to c a . 70 k c a l mole ^  of energy,  the r e a c t i o n must be exothermic  by a t  least  25 t h i s amount.  P e r o x i d e s , w i t h t h e i r weak 0-0  ( r e q u i r i n g o n l y 33 k c a l  f o r cleavage) a r e  bond  especially  s u i t a b l e compounds. (2)  The bond rearrangement p r o c e s s must be a v e r y r a p i d  single  s t e p , p r e f e r a b l y c o n c e r t e d , because t h e r e i s no mechanism a v a i l a b l e f o r energy a c c u m u l a t i o n (3)  The  and  storage.  geometry of the bond rearrangement must be f a v o r a b l e  for e f f i c i e n t  f o r m a t i o n o f the e x c i t e d s t a t e .  r e s p e c t , the involvement  In  this  o f a t r a n s i e n t four-membered  cyclic  peroxide t r a n s i t i o n state or intermediate i s p a r t i c u l a r l y a t t r a c t i v e f o r s e v e r a l reasons: (a)  I t i s r i g i d and  compact, l e s s prone to v i b r a t i o n a l  deactivations; (b)  By 0-0 two  and C-C  bond cleavage i t y i e l d s  c a r b o n y l groups,  directly  o f t e n w i t h i n the same m o l e c u l e ;  one of them c o u l d w e l l have a c q u i r e d the n e c e s s a r y 70 k c a l of energy which i s r e q u i r e d ( t o t a l r e l e a s e d i n the c l e a v a g e : c a . 108  kcal);  energy  6  (c)  I t s conformation  resembles  t h a t of the  resulting  26  e x c i t e d c a r b o n y l group, t e t r a h e d r a l it  can be argued  .  Moreover,  on o r b i t a l symmetry grounds t h a t  a c o n c e r t e d decomposition  would y i e l d one  carbonyl  27  i n an a n t i b o n d i n g e x c i t e d (d)  state.  A 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 has been i n the n o n - c h e m i l u m i n e s c e n t i o x i d a t i o n s  • 28  (e)  ...  proposed of  <*-  29  j . 3 0 diketones , n i t r i l . e s . , and enammes ; Very r e c e n t l y , c y c l i c p e r o x i d e s have been i s o l a t e d /  and  c h a r a c t e r i s e d ; moreover, chemiluminescent  composition  i s observed when heated 31  i n the  de-  presence  of a f l u o r e s c e n t a c c e p t o r . (4)  F o r b r i g h t e m i s s i o n , the e x c i t e d c a r b o n y l chromophore should be i n d i r e c t c o n j u g a t i o n w i t h a f l u o r e s c e n t 7T-system.  Other  t h i n g s b e i n g e q u a l , the b r i g h t n e s s w i l l be d i r e c t l y p r o p o r t i o n a l to the f l u o r e s c e n c e e f f i c i e n c y 5, i e the f r a c t i o n of which the e x c i t e d ground s t a t e .  singlet  energy  r e l e a s e s i n a r a d i a t i v e decay to the  I n those cases where the c a r b o n y l product i s  not f l u o r e s c e n t , the c l o s e p r o x i m i t y of a f l u o r e s c e n t molecule can r e s u l t i n energy latter Although  t r a n s f e r and  subsequent e m i s s i o n by  molecule.  the exact mechanism of l i g h t p r o d u c t i o n i s s t i l l  unknown,  the f o l l o w i n g i s a reasonable p i c t u r e o f the p r i n c i p l e s i n v o l v e d . y i e l d Q i s governed by a t l e a s t t h r e e f a c t o r s : (1)  The  the  f l u o r e s c e n c e e f f i c i e n c y 3> f o r the e m i t t e r m o l e c u l e ,  The  quantum  7  (2)  The f r a c t i o n o f these m o l e c u l e s excited  (3)  f i n d i n g themselves  i n an  state  The e x t e n t o f "dark" s i d e r e a c t i o n s g i v i n g n o n - f l u o r e s c e n t by-products.  Some o f t h e " c l a s s i c a l " chemiluminescent y i e l d s o f a s i n g l e product,  the proven  reactions give q u a n t i t a t i v e  e m i t t e r , y e t have a Q of 17o o r l e s s . 32  U n l e s s the f l u o r e s c e n c e has been quenched by s o l v e n t o r some o t h e r then (2) must be o f c r u c i a l importance  i n obtaining a bright  solute,  emission.  P r o d u c t f o r m a t i o n can be e n v i s i o n e d as p r o c e e d i n g by e i t h e r o f the two r o u t e s : (1) A s o - c a l l e d " l i g h t " path l e a d i n g d i r e c t l y to t h e e x c i t e d p r o d u c t , and (2) a "dark" path r e s u l t i n g i n g r o u n d - s t a t e See F i g u r e I .  REOCTANT  P RODUCT  REACT\OM  Figure I:  Product F o r m a t i o n  COORD •  i n Chemiluminescence  product.  8  The unknown.  d i f f e r e n c e s , i f any,  The  i n the e a r l y p a r t of the two paths a r e  o v e r a l l r e a c t i o n mechanisms may  d i f f e r , o r , i f the same  mechanism i s shared by both paths, then unknown geometric play a part.  We  have o b t a i n e d evidence t h a t , i n our s e r i e s o f compounds,  both p o s s i b i l i t i e s Let  us now  chemiluminescent  f a c t o r s must  are c o n t r i b u t i n g f a c t o r s  (see d i s c u s s i o n ) .  a p p l y these c r i t e r i a to the b r i g h t e r o f the o r g a n i c  reactions.  Luminol Luminol  (5-amino-2,3-dihydro-l,4-phthalazinedione)  (1) i s chemi-  luminescent when an a l k a l i n e s o l u t i o n c o n t a i n i n g oxygen o r hydrogen p e r o x i d e is  t r e a t e d w i t h an o x i d i z i n g agent The  chemical r e a c t i o n and  such as potassium light  5 13 ferricyanide. '  e m i s s i o n step have been w e l l  char-  33 a c t e r i z e d by White and co-workers,  who  e s t a b l i s h e d the s t o i c h i o m e t r y but  not the mechanism f o r the r e a c t i o n i n d i m e t h y l s u l f o x i d e , where an agent  oxidizing  ( o t h e r than oxygen) i s not r e q u i r e d .  NH  2 NaOH DMSO  L  The aminophthalate  NH  t  O  a n i o n 2 was  NH,  unambiguously i d e n t i f i e d  as  the  e m i t t e r by the congruence of i t s f l u o r e s c e n c e spectrum w i t h the e m i s s i o n spectrum  (\  max  485mu). r  D e t a i l s o f the mechanism a r e not known, but the o x i -  d a t i v e a d d i t i o n o f 02 to the l u m i n o l d i a n i o n i s thought an adduct which, upon c l e a v a g e , c o u l d y i e l d  to proceed  through  t r i p l e t ground-state n i t r o g e n  9  and  a vibrationally  crossing  t r i p l :t aminophthalate m o l e c u l e .  excited  to an i s o e n e r g e t i c  l e v e l of the e x c i t e d s i n g l e t  sequent e m i s s i o n o f a photon comp etes  adduct has been s p e c u l a t e d  s t a t e and sub-  the p r o c e s s .  i n t e r m e d i a t e s have been i s o l a t e d ,  A l t h o u g h no  Intersystem  the nature o f the  3 or 4 are p o s s i b i l i t i e s .  upon"*;  o e  O  NH  3  O©  2  4-  Note t h a t b o t h a r e p e r o x i d e s , and t h a t 4 resembles the f o u r membered c y c l i c p e r o x i d e s d e s c r i b e d  earlier.  33 The  quantum y i e l d Q f o r l u m i n o l  i s about 57o i n DMSO  and about 17°  34 i n aqueous systems.  Electron-releasing  the a m i n o p h t h a l a t e a n i o n (107o the quantum y i e l d ,  ring substituents  35 36  w i t h no s u b s t i t u e n t s ) .  '  enhance $ f o r  This  increases  so d e r i v a t i v e s such as 5 a r e b r i g h t e r than the p a r e n t  luminol.  S  10  Lophine Lophine  ( 2 , 4 , 5 - t r i p h e n y l i m i d a z o l e ) ( 6 ) i s chemiluminescent  on r e a c t i o n  w i t h oxygen i n a l k a l i n e s o l u t i o n s , and the l i g h t i s enhanced by o x i d i z i n g agents.  37-  The s u g g e s t i o n  substantiated A-  ponding  39  38  t h a t a f r e e r a d i c a l may be i n v o l v e d has been  ; r e a c t i o n of t h i s r a d i c a l w i t h oxygen r e s u l t s i n the c o r r e s -  i j .. hydroperoxide  , 40, 41 7.  ph OOH  P^  - < 1  Pk  PK 7  Lophine  itself  i s weakly chemiluminescent  i n strongly alkaline  s o l u t i o n s c o n t a i n i n g oxygen and, p r e f e r a b l y , an o x i d i z i n g agent. d e g r a d a t i o n o c c u r s , making i d e n t i f i c a t i o n o f the e m i t t e r The  difficult.  c o r r e s p o n d i n g p e r o x i d e , however, i s much more e f f i c i e n t , r e -  q u i r i n g o n l y a m i l d base f o r b r i g h t e m i s s i o n . the p e r o x i d e decomposition itself  Considerable  The p r o d u c t s i s o l a t e d  a r e the d i b e n z o y l b e n z a m i d i n e  (20%).  O  O 1  9  from  8 (70%) and l o p h i n e  A series  24  o f r i n g - s u b s t i t u t e d l o p h i n e s , and t h e i r c o r r e s p o n d i n g  h y d r o p e r o x i d e s , a r e a l s o chemiluminescent. d e r i v a t i v e s exact matching spectrum emitter.  o f the amidine  With a t l e a s t  two of these  of the e m i s s i o n spectrum w i t h t h e f l u o r e s c e n c e  a n i o n product has proven t h a t the a n i o n i s the  Moreover, the l i g h t enhancement by e l e c t r o n - r e l e a s i n g  phenyl  r i n g s u b s t i t u e n t s a g a i n i n d i c a t e s that t h e f l u o r e s c e n c e e f f i c i e n c y o f the 42 p r o d u c t i s an important f a c t o r .  A s i m i l a r study  l o p h i n e s has e s t a b l i s h e d a Hammett r e l a t i o n s h i p but the r e s u l t s a r e almost  of ring-substituted f o r the l i g h t  intensities,  c e r t a i n l y due t o d i f f e r e n c e s i n f l u o r e s c e n t  e f f i c i e n c i e s and n o t t h e o x i d a t i o n r a t e s . The mechanism o f l o p h i n e p e r o x i d e decompositions d e p i c t e d as an i n t e r n a l rearrangement yielding directly  the amidine  can be c o n v e n i e n t l y  v i a the c y c l i c 4-membered p e r o x i d e ,  anion i n an e x c i t e d s i n g l e t  state,  resulting  i n emission of l i g h t . PH  N — C —  PK  M— C  PK  8 The  observation  24  that chemiluminescence  occurs i n anhydrous s o l v e n t s 23  i s e v i d e n c e t h a t an a l t e r n a t i v e mechanism by Rauhut e t al breakup does n o t operate h e r e .  f o r the p e r o x i d e  T h i s i s an important  1 *~\  8  PK  c o n c l u s i o n , and has d i r e c t b e a r i n g on our work.  (see d i s c u s s i o n . )  12  Indoles I n t e r e s t i n the chemiluminescence o f s u b s t i t u t e d i n d o l e s i s j u s t i f i e d by the r e c e n t s t r u c t u r a l e l u c i d a t i o n o f the b i o l u m i n e s c e n t l u c i f e r i n o f the c r u s t a c e a n C y p r i d i n a h i l g e n d o r f i i , •  ,  •  •  1  A  4  i n which an i n d o l e  3  moiety i s i n v o l v e d . 22 Work i n these  laboratories  has r e s u l t e d i n the s y n t h e s i s o f a  s e r i e s of i n d o l e n y l 3-hydroperoxides 9, chemilumiraescent sulfoxide/potassium  i n dimethyl  t-butoxide.  PRODS •  9a,R = b. R» v  The  io  cHj - 0 " *  *  -  N(cn ) 3  x  ,  OCH  3  ,  OH,  H,  Halogen  amides 10 have been i s o l a t e d i n h i g h y i e l d , and a r e the proven  e m i t t e r s by 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 i r a n i o n s , whose f l u o r e s c e n c e e f f i c i e n c i e s a r e d i r e c t l y p r o p o r t i o n a l to the chemiluminescence of the p a r e n t  intensities  peroxides. 18  The  p r e p a r a t i o n o f an  rearrangement, w i t h o u t  O-enriched  d i l u t i o n of l a b e l ,  p e r o x i d e and i t s subsequent to the c o r r e s p o n d i n g  o f f e r e d as d i r e c t p r o o f t h a t t h e four-membered c y c l i c  amide i s  t r a n s i t i o n state i s  i n v o l v e d , analogous to t h a t f o r l o p h i n e :  •*»..  10  13  In the p e r o x i d e s 9b, a Hammett r e l a t i o n s h i p has been e s t a b l i s h e d l i g h t i n t e n s i t i e s , most p r o b a b l y due t o the d i f f e r i n g  f o r the  fluorescence ef-  f i c i e n c i e s o f the amides. Secondary E m i s s i o n s As  previously  discussed,  newly formed e x c i t e d c a r b o n y l system.  bright  chemiluminescence may o c c u r i f the  group i s conjugated w i t h a f l u o r e s c e n t "iT-  I n t h e cases where t h i s product i s n o t f l u o r e s c e n t ,  t r a n s f e r to a f l u o r e s c e n t m o l e c u l e i n s i t u can y i e l d  then energy  light.  44 Thus, the remarkable r e s u l t s of Rauhut and co-workers  have e s t a b -  l i s h e d a whole new f a m i l y o f chemiluminescent r e a c t i o n s w i t h Q v a l u e s as h i g h as 23%. oxalate  One c l a s s o f r e a c t a n t s  esters  i s of s p e c i a l i n t e r e s t .  ( e g , 11) a r e v e r y e f f i c i e n t  Selected  aryl  l i g h t producers when t r e a t e d  w i t h hydrogen p e r o x i d e i n the p r e s e n c e o f a f l u o r e s c e n t a c c e p t o r l i k e DPA or  rubrene.  0 0 c y s i — °  ~^~  C-  0  ~ ~ ^ - 3 —  I! The  reaction stoichiometry  and e m i s s i o n s p e c t r a a r e c o n s i s t e n t w i t h the  f o l l o w i n g proposed mechanism:  00 " II R0-C-C-0R  u  n  2°2 — f »• g.lyme H  00 II Ii R0-C-C-00H  +  R0H  »  00 II |l C-C jj 0-0 12  12  fluorescor  ^  fluorescor*  I h?  +  2 CO^  + R0H  14  Moreover, t h e r e i s evidence t h a t c y c l i c p e r o x i d e 12, w h i l e not i s o l a b l e , is a discrete  intermediate.  Lucigenin The  i n v e s t i g a t i o n o f the l i g h t  e m i s s i o n by l u c i g e n i n  (10, 10'-dimethyl  9 , 0 - b i a c r i d i n i u m d i n i t r a t e ) ( 1 3 ) , have been, on the whole, r a t h e r factory.  The compound i s chemiluminescent i n aqueous a l k a l i n e  containing  oxygen o r , b e t t e r y e t , hydrogen p e r o x i d e .  unsatis-  solutions  The b l u e - g r e e n e m i s s i o n  i s enhanced by t h e a d d i t i o n o f a l c o h o l , p y r i d i n e , ammonia, and ( e s p e c i a l l y ) osmium t e t r o x i d e ; ^ ^ ^ adequately explained  but the f u n c t i o n o f these " c a t a l y s t s " has not been to date.  Most i n v e s t i g a t o r s have sought to i m p l i c a t e  various  b i a c r i d i n y l m o l e c u l e as the e m i t t e r ,  on the grounds that  reduced forms a r e g r e e n - f l u o r e s c e n t ,  comparable to ..the u s u a l  k i n d s of e x c i t e d  l u c i g e n i n and i t s emission  spectrum o f l u c i g e n i n (480-525 mp.) . 47 Gleu and P e t s c h ,  the d i s c o v e r e r s  of t h i s phenomenon, had  postulated  a mechanism i n v o l v i n g the f o r m a t i o n of a l u c i g e n i n d i c a r b i n o l pseudobase (14) which underwent o x i d a t i o n reduction  by H^O^  t o the c y c l i c p e r o x i d e ( 1 5 ) f o l l o w e d  from a second H 0 „ m o l e c u l e , the a c t u a l l i g h t e m i s s i o n o  O 16  step:  by  15  Decomposition was thought  of 15 to N-methylacridone  16, the o n l y i d e n t i f i a b l e p r o d u c t ,  to be a s i d e r e a c t i o n which t e r m i n a t e d the e m i s s i o n by des-  t r o y i n g the r e a c t i v e p e r o x i d e s u b s t r a t e . 48 Tamamushi  e n v i s i o n e d the o x i d a t i o n o f l u c i g e n i n by m o l e c u l a r  oxygen, r e s u l t i n g i n a 10,10'-dimethyl-9,9 then formed  the p e r o x i d e 15.  1  b i a c r i d i n y l d i r a d i c a l which  Subsequent r e d u c t i o n by  s u  PP  o s e a  ly  r e  ~  s u i t e d i n e x c i t e d pseudobase, the c l a i m e d e m i t t e r . 38 Kautsky and K a i s e r  then re-examined  t h e chemiluminescence  of l u c i g e n i n and found t h a t a t lower c o n c e n t r a t i o n s and h i g h e r the e m i s s i o n was b l u e , not green. ponded w i t h t h e N-methylacridone  temperatures  Moreover, the e m i s s i o n spectrum  corres-  f l u o r e s c e n c e , thus i n d i c a t i n g N-methyl-  a c r i d o n e was the primary e m i t t e r and t h a t the lower-energy escence a r o s e by energy  spectrum  green  chemilumin-  t r a n s f e r to a h i g h l y f l u o r e s c e n t s p e c i e s c o - o c c u r i n g .  T h i s s p e c i e s was assumed to be l u c i g e n i n i t s e l f .  The o v e r a l l r e a c t i o n was  proposed a s :  <r3  CH,  H  H,0 2^1  Z OH  14,  In s p i t e o f these f i n d i n g s , mechanisms i n v o l v i n g  l a t e r workers c o n t i n u e d to d e v i s e  excited b i a c r i d i n y l  s p e c i e s i n o r d e r to account f o r  49 the green e m i s s i o n .  Kurtz  proposed  l o s s o f 0^ by the c y c l i c p e r o x i d e 15  would y i e l d an e x c i t e d t r i p l e t of N , N - d i m e t h y l b i a c r i d e n e  17.  16  HO,  13  IS  4- Km  CH  The  same p r o c e s s was  phorescence  of 17 was  suggested  5  by K a r y a k i n , ^ ^ who  o c c u r r i n g ( a t room  c l a i m e d t h a t phos-  temperatures).  Kroh e n v i s i o n e d f o r m a t i o n of the pseudobase 14 from the b i a c r i d e n e 17 o r i t s oxide t o be the l i g h t e m i s s i o n  step.  52a Erdey  had o r i g i n a l l y  supported  the mechanism o f G l e u , but more  5 2b recently r e i t e r a t e d the K u r t z p r o p o s a l , i n which the e x c i t e d b i a c r i d e n e t r a n s f e r r e d energy to an added f l u o r e s c e n t a c c e p t o r m o l e c u l e (eg, f l u o r e s c i n ) 53 G r i g o r o v s k y and Simenov o x i d e , and  found  s y n t h e s i z e d the b i a c r i d e n e 17 and i t s  them to be spontaneously  chemiluminescent  s o l v e n t s w i t h o u t b e n e f i t of base o r o x i d i z i n g agent. was  o b t a i n e d w i t h added B.^)^, and  f o r m a t i o n of N-methylacridone.  i n organic  B r i g h t e r emission  the compounds remained unchanged without  E v i d e n c e was  p r e s e n t e d to i n d i c a t e t h a t  these compounds were merely c a t a l y s t s f o r the e x e r g o n i c breakdown of hydrogen peroxide. The  c l a i m e d spontaneous chemiluminescence of 17 was  also reported  54 by T o t t e r  .  performing  the r e a c t i o n i n mixed s o l v e n t s (aqueous p y r i d i n e or a l c o h o l )  at  He  identified  the primary e m i t t e r as N-methylacridone  low l u c i g e n i n c o n c e n t r a t i o n s .  when r e a c t i n g w i t h oxygen l e d him  The n e c e s s i t y f o r r e d u c t i o n o f to propose an e l e v e n step  by  lucigenin  enzymatic  17  reduction  p r o c e s s w i t h complex r a t e e q u a t i o n s , t o t a l l y w i t h o u t  verification.  I n a subsequent c o n s i d e r a t i o n 55  enzymatic r e d u c t i o n , T o t t e r  and  L** + 2 NH^OH + OH"  ^  L" " + H0 "  LH  1-1  N  2  With the e x c e p t i o n  emitter,  LH  N  +  +  0  of l u c i g e n i n (L  | | )  t o the r e a c t i o n s +  2  + N H  4  +  3  H^  .  o f Kautsky and K a i s e r ,  i g a t i o n s of l u c i g e n i n reported The  of t h e r e a c t i o n without  proposed t h e r e d u c t i o n  by ammonia o r hydrogen p e r o x i d e a c c o r d i n g  experimental  we c o n s i d e r  here as too c o n t r a d i c t o r y  l u c i g e n i n r e a c t i o n i s a complex one.  N-methylacridone, a r e o n l y 50% a t b e s t .  a l l the i n v e s t -  t o be r e l i a b l e .  The y i e l d s o f the proven The a l k a l i n e p e r o x i d e  s o l u t i o n i s g e n e r a l l y y e l l o w and has a green f l u o r e s c e n c e ,  i n d i c a t i n g the  p o s s i b i l i t y o f a reduced b i a c r i d a n s p e c i e s , a r i s i n g perhaps by an unknown disproportionation to be i n v o l v e d ,  process.  C e r t a i n l y we might expect the|pseudobase 14  since acridinium  s a l t s a r e known t o r e a c t r e a d i l y i n t h i s  56 way.  T r a n s f o r m a t i o n s t h e r e a f t e r have n o t been p r o p e r l y  investigated to  date. We i n t e r p r e t t h e chemiluminescent p o r t i o n of the l u c i g e n i n  reaction  as b e i n g a n u c l e o p h i l i c a t t a c k by hydrogen p e r o x i d e y i e l d i n g , u l t i m a t e l y , the c y c l i c p e r o x i d e 15 which undergoes c l e a v a g e t o g i v e N-methylacridone 16 and  light.  The exact d e t a i l s o f t h i s r e a c t i o n a r e not y e t c l e a r , but a  p a r t i a l p i c t u r e now e x i s t s and w i l l be d i s c u s s e d i n s i g h t was gained by analogy w i t h t h e a c r i d i n i u m  later.  C e r t a i n l y , much  s a l t s devised  and s t u d i e d  in this investigation. Our the  i n i t i a l approach t o t h e l u c i g e n i n problem was a s f o l l o w s :  chemiluminescence o f b i a c r i d i n i u m  i f  s a l t s i s accompanied by c o m p l i c a t e d  s i d e r e a c t i o n s t h a t obscure t h e s a l i e n t f e a t u r e s  o f the l i g h t - p r o d u c i n g  steps,  18  then the s y n t h e s i s of a s u i t a b l e model compound, a 9 - s u b s t i t u t e d N-methylacridinium salt hydroperoxide  (whose 9 - s u b s t i t u e n t  i s prone to n u c l e o p h i l i c a t t a c k by  a n i o n ) , might r e s u l t i n a chemiluminescent compound whose  b e h a v i o r under the same r e a c t i o n c o n d i t i o n s would be  less  complicated.  S y n t h e s i s of 9-cyano-10-methylacridinium n i t r a t e 23 confirmed p r e d i c t i o n s i n every  respect.  The  compound y i e l d e d l i g h t and,  our  in virtually  q u a n t i t a t i v e y i e l d N-methylacridone, i d e n t i f i e d unambiguously as the  emitter.  20 Encouraged by t h i s e a r l y s u c c e s s ,  we  have now  of t h i r t e e n a d d i t i o n a l r e l a t e d compounds whose r e l a t i v e  prepared light  a  intensities  obey p e r f e c t l y t h e i r p r e d i c t e d r e a c t i v i t i e s w i t h hydrogen p e r o x i d e , v i d i n g s t r o n g evidence  series  pro-  f o r the g e n e r a l r e a c t i o n mechanism e n v i s i o n e d f o r  them.  9  EXPERIMENTAL  19  FIGURE 2:  S y n t h e t i c r o u t e s t o the chemiluminescent  compounds.  20  PART A:  Figure  SYNTHESIS The  synthetic routes  to t h e chemiluminescent compounds a r e shown i n  2.  F o r the most p a r t , standard  p e c t e d r e s u l t s were u s u a l l y o b t a i n e d . showed u n u s u a l f e a t u r e s  that m e r i t  methods were employed, and the exO c c a s i o n a l l y , however, the r e a c t i o n s  s p e c i a l comment.  I n the N - m e t h y l a t i o n o f the a c r i d i n e phenyl e s t e r s 33, the r e a c t i v i t y towards d i m e t h y l s u l f a t e decreased markedly when there was an e l e c t r o n withdrawing r i n g s u b s t i t u e n t . d i r e c t conjugation therefore,  T h i s e f f e c t was n o t expected, as t h e r e  between the f u n c t i o n a l groups i n v o l v e d .  t h a t a l a r g e degree of i n d u c t i v e t r a n s m i s s i o n  i s no  I t would appear,  through the e s t e r  oxygen e x i s t s . Similarly, sulfate. group  the a c r i d i n e a c i d 27 was u n r e a c t i v e  towards h o t neat d i m e t h y l  The reasons f o r t h i s a r e even l e s s c l e a r , because t h e  i s e s s e n t i a l l y un-ionized  i n the a c i d i c medium  carboxylate  of d i m e t h y l s u l f a t e .  Consequently, the d e s i r e d a c r i d i n i u m a c i d 31 was o b t a i n e d  by a l k a l i n e h y d r o l -  y s i s o f the c o r r e s p o n d i n g methyl e s t e r 30. For  some o f the a c r i d i n i u m  determined. melting  p o i n t s havenot been p r e c i s e l y  There i s e v i d e n c e of slow decomposition upon h e a t i n g ,  points v a r i e d according  satisfactory  s a l t s , melting  determination  to how r a p i d l y they  and the  were approached.  c o u l d p r o b a b l y be made u s i n g d i f f e r e n t i a l  A more thermal  analysis. U n l e s s o t h e r w i s e noted, a l l i n f r a r e d s p e c t r a were measured on a P e r k i n Elmer 137 I n f r a c o r d Beckman DK-2  ( n u j o l m u l l s ) , and u l t r a v i o l e t  spectrometer ( 9 5 % e t h a n o l ,  measured on a K o f l e r h o t stage,  and a r e  s p e c t r a were recorded  distilled). uncorrected.  The m e l t i n g  on a  p o i n t s were  21  9-Benzylacridine  18  The B e r n t h s e n yield, acid,  reaction  of 9 - b e n z y l a c r i d i n e . 24 gm  58  was  utilized  A mixture  f o r the f o r m a t i o n , i n poor  of 30 gm  (0.14 moles) diphenylamine,  (0.22 moles) p h e n y l a c e t i c  and 40 gm  (0.30 moles) f r e s h l y  f u s e d z i n c c h l o r i d e were heated a t 200° f o r 15 hours w i t h o c c a s i o n a l s t i r ring.  , The  r e s u l t i n g b l a c k t a r was  poured  i n t o 300 ml  of c o n c e n t r a t e d  ammonia; a b r i g h t y e l l o w s o l i d and a b l a c k t a r p r e c i p i t a t e d out. d e c a n t a t i o n of the y e l l o w compound, f o l l o w e d by washing w i t h water, gave ( a f t e r d r y i n g ) 9.5  gm  Careful  suction f i l t r a t i o n (25%)  and  of crude p r o d u c t which, o  a f t e r r e p e a t e d r e c r y s t a l l i z a t i o n from benzene, had mp mp  A mixture  19 of 5 gm  sodium dichromate  (0.185 moles) 9 - b e n z y l a c r i d i n e and 4 gm  i n 100 ml  g l a c i a l a c e t i c a c i d was  the r e s u l t i n g orange s o l u t i o n was The  y e l l o w s o l i d was c h l o r o f o r m and form and  then adding e t h a n o l .  6 0  mp  A f t e r b o i l i n g o f f most of the c h l o r o -  of 2 gm  s e m i s o l i d was  c o l l e c t e d ; 3.7  gm  (70%),  214-216°).  20  (0.007 moles) 9 - b e n z o y l a c r i d i n e and  0.0215 moles) methyl s u l f a t e was resulting  torr) a bright  then r e c r y s t a l l i z e d by d i s s o l v i n g up i n  10- M e t h y l - 9 - b e n z o y l a c r i d i n i u m m e t h o s u l f a t e A mixture  hours,  s u c t i o n f i l t e r e d and washed w e l l  c o o l i n g , a b r i g h t y e l l o w mass o f prisms was  214-216° ( l i t .  r e f l u x e d f o r 2.5  Upon s u b l i m a t i o n (220°, 0.2  c o l l e c t e d which was  (0.135 moles)  d i l u t e d f i v e - f o l d w i t h water.  i n s o l u b l e r e d d i s h p r o d u c t was  w i t h water, then a i r - d r i e d .  mp  170-173 ( l i t .  173°).  9- B e n z o y l a c r i d i n e  and  59  heated on  triturated  2 ml  (2.7  the steambath f o r 2 hours.  s e v e r a l times w i t h e t h e r , then  i n methanol and p r e c i p i t a t e d out w i t h e t h e r .  gm, The  dissolved  22  Only a p o r t i o n was  w a t e r - s o l u b l e , so the p r o d u c t was  water and the aqueous washings were evaporated c r y s t a l l i z e d from methanol-ether, mp  293-295° was  to d r y n e s s .  a bright yellow s o l i d ,  Again, re-  1.2  gm  (40%),  obtained.  anal.Calcd f o r C  H N0 S: i g  5  found  l>  1665  X  242 mp  max  washed w i t h  cm'  1  (sh)  :  C, 64.54;  H, 4.68;  N,  3.42%  C, 64.32;  H,  N,  3.04%  4.27;  (CO)  (e 43,000),  N-methylacridinium methosulfate  248  (sh) (64,000),  350  (10,800),  365  253  (97,000),  (13,000)  21  A m i x t u r e of 25 gm a c r i d i n e (K & K C h e m i c a l s ) , 5 ml of methyl and  15 ml o f benzene were warmed i n a f l a s k equipped w i t h a r e f l u x  The exothermic  r e a c t i o n was  sulfate,  condenser.  o c c a s i o n a l l y moderated by b r i e f c o o l i n g w i t h an  ice bath. A f t e r 30 minutes, and  filtered.  the b r i g h t y e l l o w s o l i d was  Washed w e l l w i t h e t h e r and then w i t h acetone,  p r o d u c t , a s t r o n g l a c h r y m a t o r , was The  t r i t u r a t e d with ether  p r o d u c t , 14 gm  (33%) was  the b r i g h t y e l l o w  d r i e d i n vacuo to a v o i d a i r o x i d a t i o n .  used without  further purification.  I t blackens  w i t h o u t m e l t i n g above 300°.  9-cyano-10-methylacridan  22  To an aqueous s o l u t i o n o f N - m e t h y l a c r i d i n i u m m e t h o s u l f a t e was dropwise,  a s a t u r a t e d s o l u t i o n of potassium  the i n s o l u b l e product was  complete.  added,  c y a n i d e u n t i l p r e c i p i t a t i o n of  23  I t was  f i l t e r e d by  then r e c r y s t a l l i z e d a t 160°, was  s u c t i o n and washed w e l l w i t h water,  from acetone; c o l o r l e s s p r i s m s , mp  obtained  ( l i t . ' ' " mp, N  2  found  V A  max max  2280 cm" 278 mu  :  C, 81.79;  H, 5.49;  N,  12.72%  :  C, 81.83;  H,  N,  12.67%  8,000)  9-cyano-10-methylacridinium The a c r i d a n (10 gm)  nitrate was  which was  combined w i t h the f i r s t gm  (75%,) of orange  crop and  found  264 mu  9-cyanoacridine To 45 gm  (26,900),  crystals  368  :  from e t h a n o l .  160-162° (dec.) was  isolated.  C, 64.04;  H,  3.94;  N,  14.94%  C, 64.27;  H, 4.15;  N,  14.81%  (2,820),  386  A  (6,000)  24 (0.25 moles) a c r i d i n e and  200 ml of e t h a n o l was The  recrystallized  n e e d l e s , mp  a n a l . C a l c d f o r C-.H-N.O. : 14 8 3 3  of water.  Upon c o o l i n g , l a r g e orange  and  C o n c e n t r a t i n g the mother l i q u o r s a f f o r d e d a l i t t l e more p r o d u c t ,  t o t a l o f 9.5  '\  23  d i s s o l v e d i n 50 ml of d i l u t e n i t r i c a c i d  warmed on the steambath f o r 1 hour. deposited.  5.29;  (CN)  1  ( e  r  110-112°, r e m e l t i n g  143°).  6  anal. Calcd f o r C^H  d r i e d i n vacuo,  added 25 gm  s o l u t i o n was  16 ml o f g l a c i a l a c e t i c a c i d i n  (0.40 moles) potassium c y a n i d e i n 35 ml  heated to r e f l u x w i t h m e c h a n i c a l s t i r r i n g ;  w i t h i n a s h o r t time, the product began t o c r y s t a l l i z e  out.  and,  24  After  1 hour o f r e f l u x i n g , the r e a c t i o n m i x t u r e was  suction f i l t e r e d .  The y e l l o w product was  c o o l e d to 0 and  washed w i t h a l i t t l e ethanol,  w i t h l a r g e amounts of water t o remove t r a c e s of c y a n i d e i o n . y e l l o w s o l i d was  a l l o w e d t o a i r - d r y f o r s e v e r a l days u n t i l  crystallized  resulting  t h e r e was no  t r e a t e d w i t h cone. HC1.  l o n g e r a green c o l o r v i s i b l e when a sample was Taken up i n c h l o r o f o r m and  The  t r e a t e d s e v e r a l times w i t h N o r i t , then r e -  from a c h l o r o f o r m - e t h a n o l s o l v e n t p a i r , 28 gm (52%) was i s o l a t e d  as f i n e , b r i g h t y e l l o w n e e d l e s , mp 1 8 2 ° ( l i t .  acridine-9-carboxyllic acid T h i s compound"* was  mp 1 8 1 ° ) .  6 1  27 12 gm (0.056 moles) 9-cyano  prepared by d i s s o l v i n g  a c r i d i n e i n 100 ml o f cone, s u l f u r i c a c i d on the steambath f o r 2 hours, c o o l i n g to 0 ° .  To the b r i g h t r e d s o l u t i o n was  s u b s i d e d , the r e a c t i o n m i x t u r e was  then  c a u t i o u s l y added, i n s m a l l  p o r t i o n s , 40 gm (0.50 moles) o f s o l i d sodium n i t r i t e . r e a c t i o n had  then  . A f t e r the v i g o r o u s  heated  f o r another  2 hours,  t h e n d i l u t e d w i t h water to p r e c i p i t a t e out the p r o d u c t . The  finely  d i v i d e d , b r i g h t y e l l o w s o l i d was  w e l l w i t h water, then was suction f i l t e r i n g  p u r i f i e d by r e p e a t e d l y d i s s o l v i n g up i n 107o a l k a l i ,  t o remove any b a s e - i n s o l u b l e m a t e r i a l s , and  s t r o n g l y a c i d i c w i t h cone. HC1 to p r e c i p i t a t e the The  s u c t i o n f i l t e r e d and washed  crude y i e l d of p r o d u c t was  product.  10.6 gm (75%), not r e c r y s t a l l i z e d  to i t s i n s o l u b i l i t y i n a l l t h e common o r g a n i c s o l v e n t s . (lit.  6 2  The mp was  due  above 3 0 0 °  mp, 3 0 0 ° ) .  Acridine~9-carbonamide  25  A s m a l l p o r t i o n of the h o t s u l f u r i c a c i d was  f i n a l l y making  s o l u t i o n of  9-cyanoacridine  d i l u t e d w i t h water, p r e c i p i t a t i n g out the i n t e r m e d i a t e amide.  yellow f i n e l y  d i v i d e d s o l i d was  recrystallized  The  pale  from a c e t i c a c i d - w a t e r , w i t h  25  mp 263-265° ( l i t .  1?  mp 263-264°).  3400 cm" (NH); 1  max  1700 and 1660 cm" (CO). 1  lO-methylacridinium-9-carbonamide  methosulfate  26  0.5 gm (2.2 m moles) 25 was m a g n e t i c a l l y s t i r r e d w i t h 1 m l (1.35 gm, .01 moles) d i m e t h y l s u l f a t e .  A f t e r two hours o n l y a s m a l l amount had gone  i n t o s o l u t i o n , so 5 ml d r y methanol was added; w i t h i n a few minutes t h e s t a r t i n g m a t e r i a l was a l l i n s o l u t i o n .  S t i r r i n g was c o n t i n u e d o v e r n i g h t , r e -  s u l t i n g i n a b r i g h t y e l l o w p r e c i p i t a t e and a y e l l o w s o l u t i o n .  Adding  ether  threw out a l i t t l e more s o l i d , then the e n t i r e amonant was c o l l e c t e d by s u c t i o n and washed w e l l w i t h e t h e r . The  product  (0.55 gm) was r e c r y s t a l l i z e d from methanol:  p o s i t i n g golden y e l l o w p r i s m s , mp 2 4 9 - 2 5 0 ° , 507o e t h y l a c e t a t e i n c h l o r o f o r m ) . anal. Calcd f o r C , H , N 0 S  16 16 2 5 1  1  o  c  found  1?  max  Phenyl  3400 c m ( N - H ) , _1  e t h e r , de-  homogenous on t h i n l a y e r  R e c r y s t a l l i z e d y i e l d , 0.5 gm  (70%).  : C, 55.17 ;  H, 4.63; N, 8.04%  C, 55.35 ;  H, 4.80; N, 7.85%  :  (silica:  1690 (C=0).  e s t e r syntheses. The a c r i d i n i u m phenyl e s t e r s 34a to 34e were prepared by the f o l -  lowing g e n e r a l  route:  a c r i d i n e - 9 - c a r b o x y l l i c a c i d 27 was c o n v e r t e d t o i t s  a c i d c h l o r i d e 28 by s t i r r i n g a suspension o f 27 i n neat, b o i l i n g , c h l o r i d e (10-fold e x c e s s ) .  A f t e r a l l the a c i d had gone i n t o  thionyl  solution  ( g e n e r a l l y w i t h i n one h o u r ) , t h e excess SOCl^ was c a r e f u l l y removed by vacuum d i s t i l l a t i o n  ( 8 0 ° , 100 mm).  The s o l i d r e s i d u e was crushed and t r i -  t u r a t e d s e v e r a l times w i t h dry^benzene t o remove r e s i d u a l S0C1 , and t h e  26  r e s u l t i n g b r i g h t y e l l o w s o l i d was  the a c i d c h l o r i d e 28,  sufficiently  pure  f o r the next s t e p . E s t e r i f i c a t i o n was anhydrous  accomplished by a d d i n g , t o the a c i d c h l o r i d e i n  p y r i d i n e , an e q u i v a l e n t amount o f the a p p r o p r i a t e phenol  s o l v e d i n 'dry p y r i d i n e .  A f t e r b r i e f h e a t i n g the r e a c t i o n m i x t u r e became  a homogenous s o l u t i o n which was sodium h y d r o x i d e . was  c o o l e d and then s l o w l y d i l u t e d w i t h  washed w e l l w i t h water, and a i r d r i e d .  After recrystallization,  the e s t e r 33 was  N-methylated  by h e a t i n g  w i t h an excess of neat d i m e t h y l s u l f a t e i n the u s u a l manner. s u i t a b l e time the a c r i d i n i u m e s t e r 34 was  9-carbo  o f f , and  10%  The d e s i r e d a c r i d i n e phenyl e s t e r 33 p r e c i p i t a t e d out,  c o l l e c t e d on the Buchner,  filtered  dis-  After a  p r e c i p i t a t e d out w i t h e t h e r ,  purified.  (4-methyl) p h e n o x y a c r i d i n e  33a -2  The  r e a c t i o n between 2.5  gm  (10  moles) of a c i d c h l o r i d e 28 and  -2 1.08  gm  (10  phenyl e s t e r .  moles) p - c r e s o l y i e l d e d I t was  chloroform-methanol anal.  ~y>  max  9-carbo  sublimed (.200°, 0.1  t o g i v e 1.5  Calcd for C H 2]|  1750  cm"  1  gm  gm  2  (53%) of the crude 4-methyl  t o r r ) and c r y s t a l l i z e d  gm of f l u f f y ,  N 0  found (C=0)  gm  :  C, 80.49;  H, 4.83;  N,  4.47%  :  C, 80.71;  H, 5.09;  N,  4.36%  170-171°.  34a  (4.65 m moles) 33a a t 100° f o r 2 hours gave  (71%.) of a b r i g h t y e l l o w s o l i d .  e t h e r to g i v e y e l l o w n e e d l e s , mp i n s t a n t l y i f dropped  from  o f f - w h i t e n e e d l e s , mp  (4-methyl) phenoxy-10-methylacridinium m e t h o s u l f a t e M e t h y l a t i o n of 1.45  1.55  1.65  I t was  r e c r y s t a l l i z e d from  methanol-  237-242° w i t h p r i o r s i n t e r i n g , m e l t i n g  on the hot s t a g e a t 200°.  27  anal.  Calcd f o r C  H N 23 21 0 0  0,S : 6  0 1  found V  max  ^max  9-carbo  1750 cm" 2  6  0  m  P  :  H, 4.82;  N, 3.197.  C, 63.02 ; H, 5.G2; N,  e  91,500),  350  (4-methyl) p h e n o x y a c r i d i n e  (11,300)  365  (12,700)  33b  -2 The  3.297.  (C=0)  1  <  C, 62.86;  r e a c t i o n o f 2.5 gm  -2  ( 1 0 - moles)  28 and 1.24 gm (10  moles)  hydroquinone monomethyl e t h e r gave 1.85 gm (567.) o f crude e s t e r as a l i g h t brown s o l i d . was mp  A f t e r s u b l i m a t i o n (200°, 0.1 t o r r ) ,  recrystallized  from c h l o r o f o r m - m e t h a n o l , g i v i n g c o l o r l e s s f l u f f y n e e d l e s ,  200-202°. anal.  V  9-carbo  max  Calcd f o r C  1755 cm"  2 ]  H  1 5  N0  3  found (C=0)  1  : C, 76.58;  H, 4.59;  N, 4.25%  : C, 76.85;  H, 4.88;  N, 4.16%  (4-methoxy) phenoxy-10-methylacridinium m e t h o s u l f a t e M e t h y l a t i o n o f 1.4 gm  at  t h e pure m a t e r i a l (1.45 gm)  (4.25 m moles) 33b w i t h 5 ml d i m e t h y l s u l f a t e  100° f o r 2 hours gave 1.9 gm  crystallized anal.  (987.) o f a b r i g h t orange s o l i d .  from methanol:ether to g i v e b r i g h t orange Calcd f o r C H 2 3  2 ] |  N0 S 7  found  •0  'max  A  m9.x  1750 cm' 242 mu »  1  34b  I t was r e -  t a b l e t s , mp  :  C, 60.66;  H, 4.65;  N, 3.08%  :  C, 59.85;  H, 5.05;  N, 3.29%  167-172°.  (C=0) (e 260  26,000),  248 (sh) (50,000),  (105,000),  350 (9,100),  253 (sh) (82,000),  365  (11,400)  28  9-carboxyphenoxyacridine 5.0  33c  gm (.0225 moles o f a c i d 27 was  c o n v e r t e d t o the a c i d c h l o r i d e 28,  and to t h i s was added 2.1 gm (.0225 moles) phenol i n the u s u a l manner. Routine workup gave 4.5 gm (677o) o f f l u f f y , o f f - w h i t e needles from c h l o r o form: methanol, anal.  mp 189-190°. c  Calcd for  o i3 °2 H  2  N  :  found  V  max  1750  cm"  C  »  8 0  *  2 5  »  H  > 4  : C, 80.43;  3 8  ;  N  »  4.68% N, 4.507  H, 4.48;  o  (C=0)  1  9-carbophenoxy-10-methylacridinium H e a t i n g 4.5 gm (.015  m e t h o s u l f a t e 34c  moles) 33c i n 5 ml d i m e t h y l s u l f a t e a t 100° f o r  2 hours r e s u l t e d i n 5.9 gm (927o) of a b r i g h t y e l l o w s o l i d .  Crystallization  from e t h a n o l a f f o r d e d b r i g h t y e l l o w p r i s m s , mp 228-230° a f t e r d r y i n g i n vacuo anal.  C a l c d f o r C H N 0 , S : C, 62.12; o o  1 o  found -|? max A  max  1752  cm"  N, 3.29%  H, 4.76;  N, 3.21%  (C=0)  1  260 mu '  : C, 62.00;  H, 4.50;  ( e 106,000),  350 (11,300),  365  (14,000).  9-carbo (4-bromo) p h e n o x y a c r i d i n e 33d -2 The  r e a c t i o n between 2.5 gm  (10  ~2 moles) o f 28 and 1.73  gm  (10 mol  p-bromophenol y i e l d e d 2.2 gm (58%,) o f crude p r o d u c t which, a f t e r s u b l i m a t i o n 200°, 0.1 t o r r ) and r e c r y s t a l l i z a t i o n from chloroform-methanol, a f f o r d e d 2.0 gm o f c o l o r l e s s n e e d l e s , mp 1 9 0 - 1 9 1 ° . anal.  Calcd for  c  o i2 H  2  N 0  2  found  B  r  : C  '  6 3  '  5 3 ;  H  > '  : C,63.25;  3  4 7  '  N  '  3  H, 3.48;  -  7  1  %  N, 3.44%  29  9-carbo  (4-bromo) phenoxy-10-methylacridinium The m e t h y l a t i o n of 1.95  gm  methosulfate  (5.15 m moles) of 33d i n 5 ml neat  d i m e t h y l s u l f a t e r e q u i r e d 5 hours on the steambath s o l v e d up.  34d  b e f o r e a l l had  A f t e r p r e c i p i t a t i o n w i t h e t h e r and r e c r y s t a l l i z a t i o n  methanol:ether,  2.0  gm  (777») of b r i g h t y e l l o w n e e d l e s , mp  disfrom  261-263°, were  obtained. anal.  Calcd f o r C  o o  H  1 o  N 0 , Br S : found  V max  1755  A  9-carbo  cm"  :  C, 52.39;  H, 3.60;  N, 2.787»  C, 52.89;  H, 3.88;  N,  2.85%  (C=0)  1  262 mu  (£  93,000),  (3-nitro) phenoxyacridine  350  (10,000),  365  (12,500)  33e  -4 The  r e a c t i o n of 0.6  gm  (2.4 x 10  moles) a c i d c h l o r i d e 28 and  0.27  -4 gm  (1.95 x 10  I t was  moles) 3 - n i t r o p h e n o l r e a d i l y a f f o r d e d the d e s i r e d  recrystallized  pale yellow f l u f f y The  from chloroform:methanol  f i b r e s , mp  t o g i v e 0.43  gm  compound.  (647 ) o f 0  170-172°.  sample p r e p a r e d f o r a n a l y s i s was,  u n f o r t u n a t e l y , used up i n  a n a l y s e s where the equipment was m a l f u n c t i o n i n g . But a m o l e c u l a r weight d e t e r m i n a t i o n u s i n g the h i g h - r e s o l u t i o n MS-9 a p a r e n t peak o f m/e 344.076658. 0 12 2°4 Vmax 1750 (C=0) C  H  N  2  9-carbo  r  mass spectrometer i n d i c a t e d l / 344.079700. e  c  u  i  r  e  s  m  e  ( 3 - n i t r o ) phenoxy-10-methylacridinium m e t h o s u l f a t e 34e A m i x t u r e of 0.4  s u l f a t e was  gm  (1.16 m moles) 33e and 1.0  heated a t 150° f o r 15 h o u r s .  w i t h a m e l t i n g p o i n t range of 170-230°,  gm  (180 m moles) d i m e t h y l  T r i t u r a t i o n with ether l e f t a evidence o f a m i x t u r e .  f r a c t i o n a l c r y s t a l l i z a t i o n from e t h a n o l c e t h e r a f f o r d e d 50 mg  solid  Repeated  of a yellow s o l i d  30  which was then r e c r y s t a l l i z e d from minimum e t h a n o l t o g i v e dark y e l l o w p r i s m s , mp 245-247° w i t h some p r i o r s i n t e r i n g . hot stage a t 210° m e l t s The  The compound dropped  on a  instantly.  remaining m a t e r i a l , a g e l a t i n o u s mass, c o n t a i n e d l a r g e amounts  o f the d e s i r e d p r o d u c t , a c c o r d i n g t o IR; b u t i t was n o t p r a c t i c a l t o i s o late . anal.  C a l c d f o r C__H.. N. 0 Q  S  o  found V  K  max  ^max  1760 cm" 2  6  0  m  C, 56.17;  H, 3.86; N, 5.96%  :  C, 56.35;  H, 4.17;  N, 5.85%  (C=0)  1  F 365  :  108,000),  280 (sh) (27,500),  350 (11,000)  (11,000)  9-carbo ( 4 - n i t r o ) p h e n o x y a c r i d i n e  33f  From 10 gm (.045 moles) of a c i d 27, the e s t e r i f i c a t i o n w i t h 6.2 gm (.045  moles) p - n i t r o p h e n o l f o l l o w e d by r e c r y s t a l l i z a t i o n from c h l o r o f o r m :  methanol y i e l d e d 8.9 gm (58%,) o f p a l e y e l l o w , f i b r o u s p r o d u c t , mp a n a l . C a l c d f o r C 2 1 2 2 0^ : H  N  2  found  V  max  1750 cm"  1  :  C, 69.76;  H, 3.51; N, 8.14%  C, 69.84;  H, 3.87; N, 8.25%  (C=0)  9-carbo ( 4 - n i t r o ) phenoxy-10-methylacridinium A mixture  188-190°.  methosulfate  34f  of 8.0 gm (.0233 moles) 33f and 5.0' gm (.040 moles) d i m e t h y l !  s u l f a t e i n 150 ml d r y benzene was m e c h a n i c a l l y s t i r r e d and r e f l u x e d f o r 20 hours.  The r e s u l t i n g m i x t u r e was h o t f i l t e r e d ; upon c o o l i n g , the f i l t r a t e  d e p o s i t e d a l a r g e amount of s t a r t i n g m a t e r i a l a c c o r d i n g to t h e i n f r a r e d .  31  The b r i g h t y e l l o w b e n z e n e - i n s o l u b l e s o l i d f i l t r a t i o n was taken up i n 200 ml methanol,  c o l l e c t e d during the hot  and upon adding 50 ml water a  l i t t l e more s t a r t i n g m a t e r i a l was thrown down and removed by f i l t r a t i o n . The  f i l t r a t e was evaporated t o d r y n e s s , and t h e b r i g h t y e l l o w r e s i d u e was  r e c r y s t a l l i z e d from methanol t o g i v e 1.9 gm (17%) of p r i s m s , mp anal. Calcd for C  2 2  H  l g  N  2  0  g  S  :  found: T> X  243-248°.  C, 56.17;  H, 3.86;  N, 5.96%  C, 56.49;  H, 4.28;  N, 5.64%  1750 cm" (C=0) 1  max  248 mu  (6  r  352  58,500),  253  (12,000),  (97,000),  260(126,000),  365 (13,500).  9-carbo ( 2 , 4 r d i n i t r o ) p h e n o x y a c r i d i n e 33g -2 The  r e a c t i o n o f 3.2 gm (1.28 x 10  moles) a c i d  c h l o r i d e 28 and 3.0 gm  _2 (1.63 x 10  moles) 2 , 4 - d i n i t r o p h e n o l gave 3.0 gm o f a dark brown amorphous  powder i n s o l u b l e i n a l l t h e common o r g a n i c s o l v e n t s . by s u b l i m a t i o n (250°, 0.05 t o r r , mp  263-269°, anal.  ~i)  max  20 h o u r s ) , and t h e p a l e y e l l o w s u b l i m a t e had  C a l c d f o r C-.H-.N. 0, : 20 11 3 6 found :  C, 61.70; C,  at  H, 2.85; N, 10.79%  61.85; H, 3.22;  N, 10.45%  1765 cm" (C=0) 1  9-carbo ( 2 , 4 - d i n i t r o ) phenoxy-10-methylacridinium 0.35  A sample was p u r i f i e d  methosulfate  34g  gm o f the sublimed 33g and 2 ml neat d i m e t h y l s u l f a t e were heated  150° f o r 5 h o u r s .  The r e s u l t i n g m i x t u r e was t r i t u r a t e d  e t h e r t o remove excess d i m e t h y l s u l f a t e .  s e v e r a l times w i t h  The e t h e r - i n s o l u b l e r e s i d u e was  leached w i t h b o i l i n g methanol t h r e e times, and t h e combined methanol washings ( b r i g h t y e l l o w ) were taken t o d r y n e s s .  The r e s i d u e (0.25 gm) was r e c r y s t a l -  l i z e d t w i c e from e t h a n o l t o g i v e golden y e l l o w p r i s m s , mp  174-176°.  32  anal.  C a l c d f o r C^EL^  0 S  N  lQ  found  V  max  A.  1765  cm"  :  C, 51.26;  H, 3.32;  N,  8.15%  :  C, 51.39;  H,  N,  8.20%  3.55;  (C=0)  1  260 mu  (£  9-carbomethoxyacridine  29  T h i s compound was  90,000),  355,  (9,600)  made i n the same manner as the p h e n y l  esters,  t h i s time a d d i n g anhydrous methanol to the a c i d c h l o r i d e 28 and working i n the u s u a l manner. a c i d 27 was  mp  recrystallized  126.5-127.5 ) . 1) max  5.8  1725  cm"  The y i e l d o f pure m a t e r i a l was  gm  methosulfate  (.0245 moles) 29 was  gm  anal.  I t was  Calcd f o r C^H  A  1725  cm"  260 mu  7.5  gm  crude  126-128°  (50%).  N 0  2  30  c o o l e d and t r i t u r a t e d w i t h e t h e r r e recrystallized  (86%) o f b r i g h t y e l l o w n e e d l e s , mp S  found max  The  heated i n 25 ml neat d i m e t h y l s u l f a t e  i n a b r i g h t y e l l o w s o l i d , which was  V  moles) of  (C=0)  1  on the steambath f o r 3 h o u r s .  to g i v e 7.7  (.063  from methanol as p a l e y e l l o w n e e d l e s , mp  9-carbomethoxy-10-methylacridinium  sulting  gm  c o n v e r t e d to 28 then t r e a t e d w i t h 100 ml d r y methanol.  e s t e r 29 was (lit.  I n a t y p i c a l p r e p a r a t i o n , 14.0  up  from  methanol:ether  228-229°.  :  C, 56.18;  H, 4.77;  N,  3.85%  :  C, 56.40;  H, 4.65;  N,  3.70%  350  (10,800),  (C=0)  1  (e.  92,000),  365  (12,200)  33 9-carboxy-10-methylacridinium 6.7  gm  chloride  31  (.0185 moles) of 30 i n 70 ml water was  sodium h y d r o x i d e .  The p u r p l e s o l u t i o n was  3 hours, then f i l t e r e d  hot.  The  added to 70 ml  10%  heated on the steambath f o r  f i l t r a t e was  a c i d i f i e d w i t h cone.  HC1,  g i v i n g a green s o l u t i o n and a mass o f y e l l o w - g r e e n n e e d l e s , weighing 4.45  gm  (887c) a f t e r d r y i n g  i n a i r . The p r o d u c t was  methanol as y e l l o w p r i s m s , mp V 3400 cm" *max  242-244° ( l i t . " ^ mp  (br)(0-H),  1  1730  A sample of 31, d r i e d i n vacuo over KOH, thionyl chloride  time a l l the suspended  (freshly distilled)  material  above 205°  32 was  suspended  f o r 15 minutes.  a l k a l i n e peroxide. peak f o r the 0-H  The  chemiluminescent  i n f r a r e d (nujol)  1790  cm"  1  l u c i g e n i n bromide 8.0 filtered  gm hot.  i n water. eared.  of K&K  Two  By  this o f the  yellow  containing  showed a s m a l l but s i g n i f i c a n t  i n extensive darkening.  above 210° w i t h much decomposition 1> max  i n solutions  of the p a r e n t a c i d ; but a l l attempts  the compound r e s u l t e d  i n neat  had gone i n t o s o l u t i o n ; removal  excess t h i o n y l c h l o r i d e under reduced p r e s s u r e l e f t a b r i g h t amorphous s o l i d b r i l l i a n t l y  dec.)  (C=0)  9-chlorocarbonyl-10-methylacridinium chloride  boiling  r e c r y s t a l l i z e d from  ( l i t . " ^ mp  to r e c r y s t a l l i z e  The mp was  broad,  starting  175-178°)  (C=0) 13 l u c i g e n i n was  dissolved  To the hot f i l t r a t e was  Upon c o o l i n g  up i n 250 ml hot water and  added a hot s o l u t i o n o f 80 gm  to room temp a mass of golden y e l l o w p l a t e s  crops of p r o d u c t , 7.4  gm,  mp  above 300° were o b t a i n e d .  KBr app-  34  PART B: (a)  PRODUCT STUDIES  9-cyano-10-methylacridan  (22)  -3 0.60 gm (2.7 x 10 ethanol.  moles) o f 22 were d i s s o l v e d i n 75 ml 95%  To t h i s was added a s o l u t i o n o f KOH (0.3 gm) i n e t h a n o l (10 m l ) .  Upon b u b b l i n g oxygen through  the s o l u t i o n , a steady b l u e glow was observed.  A f t e r t h e l i g h t r e a c t i o n was over, some p a l e golden n e e d l e s had appeared; they were i s o l a t e d by s u c t i o n , f i l t r a t i o n , washed w e l l w i t h water, and d r i e d i n vacuo. under reduced  The f i l t r a t e was d i l u t e d w i t h 30 ml water, c o n c e n t r a t e d  p r e s s u r e , and e x t r a c t e d w i t h 4 x 10 ml c h l o r o f o r m .  The com-  b i n e d c h l o r o f o r m e x t r a c t s were d r i e d over sodium s u l f a t e and taken t o dryness on t h e r o t o v a p , g i v i n g a p a l e y e l l o w s o l i d .  T h i s , too, was d r i e d i n  vacuo. The ical  two crops o f s o l i d  infrared  (178 mg and 383 mg r e s p e c t i v e l y ) had i d e n t -  s p e c t r a , and m e l t i n g p o i n t s of 201-202° and 202-203°  mp o f N-methylacridone,  201-203°).  The y i e l d  of N-methylacridone  (lit ^ 6  was 561 mg  (95%). (b)  9-cyano-10-methylacridinium In  n i t r a t e (23)  a s i m i l a r manner, 0.5 gn of, 23 i n 50 ml 25%. aqueous e t h a n o l was t r e a t e d 3S3  dropwise w i t h a s o l u t i o n o f 0.5 ml 3% ^2^2 A f t e r the b r i g h t ,  m  l  s h o r t - l i v e d e m i s s i o n had ceased,  c o n c e n t r a t e d and worked up as b e f o r e .  1  ^^° ^ 'soc  :  uni  hydroxide.  the r e a c t i o n s o l u t i o n was  The i s o l a t e d N-methylacridone,  mp  200-202°, weighed 480 mg ( 9 6 % ) . (c)  9-carbo (4-methyl) phenoxy-10-methylacridinium  m e t h o s u l f a t e (34a)  A s o l u t i o n o f 50 mg 34a i n 10 ml 507 aqueous e t h a n o l was t r e a t e d o  w i t h a l k a l i n e ^2^2 ^> a f t e r chemiluminescence ceased, was c o n c e n t r a t e d an<  under reduced p r e s s u r e .  The r e s i d u e was leached w i t h c h l o r o f o r m s e v e r a l  times, then d i l u t e d w i t h 10%, HC1.  The r e s u l t i n g a c i d i c  tracted with several portions of chloroform.  s o l u t i o n was ex-  35  Both c h l o r o f o r m silica,  e x t r a c t s were examined by t h i n - l a y e r ( f l u o r e s c e n t  50% e t h y l a c e t a t e i n c h l o r o f o r m ) .  methylacridone;  the l a t t e r had mainly  The former c o n t a i n e d o n l y N-  para-cresol with  some  N-methylacri-  done and a l s o a s m a l l amount of y e l l o w m a t e r i a l a t t h e o r i g i n . r e l a t i v e amount o f t h i s y e l l o w substance  was i n c r e a s e d when the workup  i n v o l v e d c o n c e n t r a t i n g the r e a c t i o n s o l u t i o n a t h i g h e r In the p r e c e d i n g  The  temperature.  experiments, no attempts were made to i d e n t i f y  the o t h e r p o s t u l a t e d p r o d u c t s ,  cyanate i o n ( i n (a) and (b)) and CO^  ( i n (c) ) . PART C:  CHEMILUMINESCENCE AND FLUORESCENCE The  23,  chemiluminescent e m i s s i o n  SPECTRA  spectrum o f t h r e e b r i g h t compounds,  34c and 32, were measured and compared w i t h the f l u o r e s c e n c e of N-  methylacridone  16.  These measurements were made w i t h an Aminco-Bowman  s p e c t r o f l u o r i m e t e r and X-Y p l o t t e r . c h l o r i d e 32, i t was n e c e s s a r y  F o r the b r i g h t e s t compound,- the a c i d  t o use f a s t scan;  the o t h e r s , r e a c t i n g more  s l o w l y , a l l o w e d use o f the b e t t e r r e s o l u t i o n slow scan. congruence o f the e m i s s i o n  spectrum w i t h  I n each  case,  the f l u o r e s c e n c e o f 16 i d e n t i f i e d  i t as the e m i t t e r .  (See F i g u r e 3)  The  f l u o r e s c e n c e e f f i c i e n c y $ o f very pure N-methylacridone, 10  -4  -4 i n e t h a n o l , was found by c a l i b r a t i o n a g a i n s t a known standard quinine s u l f a t e i n 1 N^H^Sfj) The  06  (10  M  u s i n g the method i n C a l v e r t and P i t t s .  v a l u e o f 0.85 was u n a f f e c t e d by added water or a l k a l i .  6 7  M  36  (a) : E m i s s i o n  of 32 v s . f l u o r e s c e n c e spectrum of _3  N-methylacridone 16 The  (both  10  M) , f a s t  curves have been n o r m a l l i z e d by  scan.  adjusting  the a m p l i f i e r g a i n . _3  (b)  Emission  of 34c  escence of 16; F i g u r e 3:  or 23 slow  Chemiluminescence and  (10  M),  vs.  fluor-  scan. fluorescence  acridinium  spectra f o r selected  salts.  37  PART D:  CHEMILUMINESCENCE S o l u t i o n s of reagents  A 4.4 x 10 ^M  were ^prepared  s o l u t i o n of H 2 O 2 was  f o r the l i g h t e m i s s i o n  made up by d i l u t i n g o . l ml  to 25 ml w i t h 50%. aqueous e t h a n o l .  T h i s c o n c e n t r a t i o n was  iodometric  t i t r a t i o n w i t h 0.1  solutions,  i n u n i t pH v a l u e s from 7 to 12, were made up  u t i o n s of 0.1  M HC1,  tables.Their  ml  and  found  e t h a n o l , the pH v a l u e d i d not The  The  by  buffer  from s t o c k  sol-  "borax" a c c o r d i n g to p u b l i s h e d  to be a c c u r a t e  of any b u f f e r s o l u t i o n was  ^2^2  verified  solution.  checked a g a i n s t known standards  on a Beckman pH meter and 0.1  KR^PO^,  NaOH,  pH was  N sodium t h i o s u l f a t e  30%  studies.  (pH 7.0  and  10!o)  to w i t h i n + 0.05  pH.  When  added to a 1 ml  s o l u t i o n of 50% aqueous  change.  compounds were examined f o r l i g h t e m i s s i o n i n the f o l l o w i n g -4  manner. H 0 2  A 50%. aqueous ethanols§lution c o n t a i n i n g s u b s t r a t e s (10  ( 4 . 4 x 10 M) was _2  2  q u a r t z c u v e t t e was light.  The  prepared.  added 0.1  l i g h t output was  To  ml o f pH  1.0  ml of t h i s  M)  and  s o l u t i o n i n a 1 cm  12 b u f f e r , r e s u l t i n g i n a f l a s h of  monitored w i t h  the apparatus i n F i g u r e  4.  1P2&  O U  CUV  TEKTRONMV  5-64-  T o poweR •suPri-y  F i g u r e 4: The  Light-Measuring  Apparatus  c u v e t t e , i t s upper p o r t i o n masked to overcome s p l a s h e f f e c t s ,  was  mounted i n a clamp which c o u l d be moved a l o n g the o p t i c a l bench, maint a i n i n g a f i x e d geometric  r e l a t i o n s h i p to the phototube w h i l e a l l o w i n g  38  a v a r i a b l e path  length.  i n b l a c k c l o t h and  I t was  necessary  to p r o v i d e a low  to shroud the e n t i r e apparatus  l e v e l red i l l u m i n a t i o n f o r the  operator. The 60 cm  path  l e n g t h s chosen were 11 cm  f o r the s t r o n g e s t .  The  f o r the weaker compounds  i n t e n s i t i e s measured at the l o n g e r  and  path  were r e l a t e d to the o t h e r s by u s i n g a moderately s t r o n g compound,  the  4-methyl phenyl e s t e r 34a,  lengths;  the a t t e n u a t i o n was  as a c a l i b r a t i o n  found to be n e a r l y  standard  100.  The most b r i l l i a n t compounds o v e r l o a d e d l o n g e r path  l e n g t h , so t h e i r  light  output was  the l i g h t path, a n e u t r a l d e n s i t y f i l t e r a t t e n u a t i o n f a c t o r of 30,  a t both path  determined by  the phototube even a t attenuated  of O.D. the use  1.7  the  by p l a c i n g , i n  which a f f o r d e d  of a s m a l l  an  incandescent  68 bulb a t low v o l t a g e . the.entire v i s i b l e r e g i o n was The  still  This f i l t e r  was  spectrum, e n s u r i n g  known to t r a n s m i t  linearly  t h a t i t s a t t e n u a t i o n i n the  to the i n p u t o f a T e k t r o n i x 564  phototube, whose  was  fed d i r e c t l y  the  'scope i n p u t impedance (1 Megohm) a c t e d as a l o a d f o r the  and  i t s v o l t a g e decrease as a f u n c t i o n of time was Moreover-; . the instrument  was  The  scanning r e s u l t was  imprinted  s e t so t h a t the  phototube, on the  initial  a r e c o r d of the l i g h t seconds and  i n t e n s i t y decay curve,  light and  over a time range measured by  ( v o l t a g e ) d e f l e c t i o n was  stopwatch.  s u p p l i e d by John M c i n t o s h o f t h i s  ac-  2 minutes, over a wide range  F o r . slower r e a c t i o n s , beyond the c a p a b i l i t i e s o f  h o r i z o n t a l scan r a t e , the v e r t i c a l  * Kindly,  face  circuits.  c u r a t e f o r r e a c t i o n s between 0.1 of i n t e n s i t i e s .  output  storage o s c i l l o s c o p e ,  b u r s t , r e s u l t i n g from the a d d i t i o n o f base, t r i g g e r e d the v e r t i c a l horizontal  blue  30.  i n t e n s i t i e s were measured w i t h a 1P28  of the c r t .  over  department.  the  monitored  39  The most r a p i d r e a c t i o n s (0.1 seconds and l e s s ) posed a k i n d o f problem, because reaction lifetime.  the m i x i n g time became comparable  special  to the  T h i s caused, i n g e n e r a l , a b r o a d e n i n g o f the decay  curve and a much lower i n i t i a l  i n t e n s i t y than would be observed f o r  p e r f e c t , instantaneous mixing.  The m i x i n g method found to be most  f a c t o r y was  i n j e c t i o n o f 0.1 ml of b u f f e r from a 1 ml  the r a p i d , f o r c e f u l  s y r i n g e equipped w i t h a l o n g 20-gauge n e e d l e whose t i p was s u r f a c e o f the r e a c t i o n s o l u t i o n . bar was  below the  The a d d i t i o n of a magnetic  n o t h e l p f u l ; i n f a c t , i t o n l y lengthened the m i x i n g The pH o f the r e a c t i o n s o l u t i o n was  which had been added; and, because trations  (10 M 2  and 4.4  x 10  satis-  stirring  time.  the same as t h a t o f the b u f f e r  the f i n a l base and p e r o x i d e concen-  r e s p e c t i v e l y ) were i n l a r g e excess w i t h -4  r e s p e c t to the s u b s t r a t e (10 first  M),  expected to be psuedo  order i n substrate. T h i s proved to be c o r r e c t .  was  the r e a c t i o n was  found by p l o t t i n g I a g a i n s t  The  time-dependence o f l o g  time on semi-log paper.  most r a p i d r e a c t i o n s , a l i n e a r r e l a t i o n s h i p was  1  (intensity)  In a l l but the  obtained f o r at l e a s t three  69 half-lives,  i n d i c a t i n g a t r u e e x p o n e n t i a l decay and  I  first  order k i n e t i c s .  lo I 3  2 F i g u r e 5:  2.  3  A t y p i c a l chemiluminescence  decay  curve.  3  40  COMPOUND  No. t j X s e c )  Me e s t e r  30  0.038  Benzoyl  20  2.1  Nitrile  23  Lucigenin bromide  13  Phenyl esters  34  4-OCH„  b  37  4-CH„  a  k^sec  )  18.4  4,500  0.33  80  0.11  0.023  5.6  0.5  169  0.004  1  7.6  0.0187  4.6  35  35,  0.020  5.0  45  10.8  0.064  1.635  0.42  a  a  0.015 5 . 8 x l 0 "  6  10"  0.020 2 . 9 x l 0 "  4  10-'  2  10  4.6  0.98  5.9  1.15  154  20.3  1.20  4300  565  1.35  a ,b  3-NO  r  0.007  25,000  100  20,000°'° 2 6 5 0 ' C  d  4-NO„  (a) (b) (c) (d)  phototube supply v o l t a g e 900V, path l e n g t h 11cm phototube supply v o l t a g e 700V, p a t h l e n g t h 60cm :not a c c u r a t e because o f mixing problems c o n d i t i o n s as i n ( b ) , w i t h n e u t r a l d e n s i t y f i l t e r added  Table I: f_ s u b s t r a t e ^ [H 0 2  2  t_0H~]  ]  Chemiluminescence of compounds a t pH 12  10  -4  M  4.4 x 1 0 M _2  10 M _2  i n 50% aqueous e t h a n o l  8  6  0.066 1 . 2 5 x l 0 " 1 0 ~  C  15.6  400  ( a )  0.15  30  4-Br  r e l . a b s . Q.Y Q.Y  r e l . a b s . r e l rate I (V) I max max  10  4  41  In  T a b l e I the v a r i o u s compounds a r e ranked i n o r d e r o f observed  quantum e f f i c i e n c y  r e l a t i v e to l u c i g e n i n bromide.  I n a l l c a s e s , the r a t e  c o n s t a n t s were o b t a i n e d from the s l o p e s o f the l o g I-time p l o t s , and the initial In  intensity J  (I ) v a l u e s were found by e x t r a p o l a t i o n to zero time. max J  a l l but two c a s e s , these corresponded ' r  The  initial  intensities  c l o s e l y t o the observed I max J  (measured i n output v o l t a g e o f the phototube)  were  then computed on an a b s o l u t e b a s i s , u s i n g the weakest compound as r e f e r e n c e p o i n t and a c c o u n t i n g f o r any subsequent  attenuations.  The r e l a t i v e quantum  y i e l d s were o b t a i n e d by graphing the a b s o l u t e i n t e n s i t y decay  curves and  comparing  the a r e a s under the curves w i t h t h a t of l u c i g e n i n bromide. 54 a b s o l u t e quantum y i e l d s a r e based on l u c i g e n i n . The to  r a t e c o n s t a n t s o f the s u b s t i t u t e d phenyl e s t e r s 34 were compared  see i f a Hammett r e l a t i o n s h i p e x i s t e d between them.  the s u b s t i t u e n t e f f e c t i s shown.  F i g u r e 6:  The  The cr-values  Thus, i n F i g u r e 6  a r e from J a f f e e ' s  Hammett p l o t f o r the s u b s t i t u t e d phenyl  esters.  review.^  42  The described  highly reactive nitrophenyl earlier;  i n f a c t , a l l three  r e a c t i o n r a t e s a t pH 12.  Re-testing  e s t e r s had the m i x i n g problems compounds had e s s e n t i a l l y i d e n t i c a l  of these e s t e r s a t pH 8 d i s c l o s e d  t h a t t h e i r r a t e s , now slower by a f a c t o r o f 20, d i d indeed show t h e d i f ference  predicted  f o r them.  Phenyl e s t e r  3-N0 4-N0  4.1  2  5.5 2  Table I I :  The  )  12.8  2  10 M; C ^ o j , _4  1  2  2,4-(N0 )  [substrate!,  k^(sec  4.4 x 1 0 M ; [0H~] , 1 0 M -2  _6  K i n e t i c s of the n i t r o p h e n y l  e s t e r s a t pH 8.  pH-dependent k i n e t i c s o f one p h e n y l e s t e r a r e shown i n T a b l e I I I  and  Figure  7.  The c o n c e n t r a t i o n s  o f s u b s t r a t e and p e r o x i d e were as b e f o r e ,  but  t h e b u f f e r s added were o f pH 7 t o 12 i n c l u s i v e , and double the q u a n t i t y  (0.2 ml) was added t o ensure t h a t a c c u r a t e Again, the rate constants and  pH c o n d i t i o n s were m a i n t a i n e d .  were determined from s l o p e s  of the l o g I p l o t s ,  r e l a t i v e quantum y i e l d s ( i n a r b i t r a r y u n i t s ) were found by measuring t h e  areas under the a b s o l u t e  i n t e n s i t y decay  curves.  43  k^(sec  pH  ^)  Quantum Y i e l d (arbitrary  .00168  8  .023  98  9  .154  -  10  .71  ,100  11  .92  -  _4  7  Table I I I :  -2  to 10 M _2  (0.2 ml)  pH-dependent k i n e t i c s o f 4-bromo p h e n y l e s t e r 34d  -I  1  1  V  7  8  <d  |0  Figure  93  5.8  1 0 M , [ H ^ ] , 4.4 x 1 0 M , (1 ml 50% a q . etOH)  [substrate], [0H"]= 1 0 "  -  7  12  units)  7:  :  »  [|  1—  12  Log k v s . pH, 4-bromo p h e n y l  ester  44  There i s . a s p e c i f i c requirement producing H^O^  f o r hydrogen p e r o x i d e  r e a c t i o n , so the k i n e t i c s and  c o n c e n t r a t i o n were of i n t e r e s t .  e s t e r 34c  (10 M)  w i t h v a r i o u s ^2^2  4  i n the  light-  quantum y i e l d as a f u n c t i o n of  I n T a b l e IV,  the u n s u b s t i t u t e d  c o n c e n t r a t i o n s was  the  phenyl  t r e a t e d w i t h pH  12  -4 buffer.  In the r e a c t i o n s , the phenyl  of ^ 2 ® 2  l  n  a  c  l  u  of b u f f e r was  e  o  u  s  e t  hanol  T a b l e IV:  an equal volume  e t c . ) were pre-mixed, then 0.1  10"  2  0.21  100  x  10"  3  0.49  105  x  10"  4  0.66  61  x  10"  5  0.62  13  x  10"  6  0.21  2  1 0 M ; [ h y D ^ , as above;[OH~3, _4  10 M -2  E f f e c t s of H„0„ c o n c e n t r a t i o n on k i n e t i c s o f e s t e r 34c  e f f e c t of s u b s t r a t e c o n c e n t r a t i o n on the k i n e t i c s and examined ( T a b l e V) by v a r y i n g the amount of phenyl  M HO  ml  Q Output (arbitrary units)  k ^ ( s e c •*")  -2 4 x 10  and  x  [substrate],  y i e l d was  (M)  2  2.2  The  x 10  M)  added.  H 0 conc. 2  (4.4  e s t e r (2 x 10  and  t r e a t i n g w i t h the pH  12 b u f f e r .  quantum  e s t e r 34c  in  45  substrate  IO"  k ^ ( s e c •*")  Q.Y.  4  0.22  100  5 x  IO  - 5  0.39  32  2 x  IO  - 5  0.56  14  0.66  5  IO"  5  [ e s t e r ] - as above; [ H ^ ]  T a b l e V:  Rel.  -  4 x 10 M _ 2  ; [oH J_  10 M _ 2  E f f e c t of s u b s t r a t e c o n c e n t r a t i o n on the k i n e t i c s  of  34c  46  I t i s now groups:  apparent  t h a t the compounds can be d i v i d e d i n t o t h r e e  those which emit no  light at a l l ,  the b r i g h t compounds whose quantum y i e l d The non-chemiluminescent and  the c o r r e s p o n d i n g amide 26.  r a p i d l y y i e l d s N-methylacridone  those which emit weakly, and i s about t h a t of  lucigenin.  compounds a r e the a c r i d i n i u m 9 - a c i d 31 The a c i d i n an a l k a l i n e p e r o x i d e without any  solution  l i g h t e m i s s i o n , p r o b a b l y by  the f o l l o w i n g mechanism:  31  16  A s o l u t i o n of the y e l l o w a c r i d i n i u m carbonamide 26 i s r a p i d l y  decolorized  by an a l k a l i n e p e r o x i d e s o l u t i o n , but t h e r e i s no N-methylacridone proven by the absence  of i t s c h a r a c t e r i s t i c b l u e f l u o r e s c e n c e .  formed,  It is  l i k e l y t h a t the r e a c t i v e 9 - p o s i t i o n has been a t t a c k e d by base o r (more l i k e l y ) h y d r o p e r o x i d e a n i o n , r e s u l t i n g i n a m e t a s t a b l e pseudobase which i s not  l i k e l y to react  The most s i g n i f i c a n t  further.  f a c t o r here i s t h a t n e i t h e r the a c i d nor the amide i s  l i k e l y to have i t s c a r b o n y l group a t t a c k e d by h y d r o p e r o x i d e a n i o n , and f o r t h i s r e a s o n we  d i d n o t expect l i g h t e m i s s i o n .  F o r t u n a t e l y , n o n e was  observed.  DISCUSSION  47  The weakest e m i t t e r s a r e t h e methyl e s t e r 30, t h e b e n z o y l compound 20, and the n i t r i l e 23, i n o r d e r o f i n c r e a s i n g b r i g h t n e s s . a r e extremely  —8 10  weak, w i t h quantum y i e l d s  The f i r s t two  ( u s i n g l u c i g e n i n as standard) o f  —6 and 10  r e s p e c t i v e l y , and c a n be seen o n l y i n . t o t a l darkness.  (See  Table I ) . The methyl e s t e r should be e s s e n t i a l l y u n r e a c t i v e t o h y d r o p e r o x i d e a n i o n , because the l a t t e r i s a weaker base than methoxide a n i o n . we c l a i m ) the h y d r o l y s i s by hydroperoxide  anion i s necessary  I f (as  for light  emission, the r e a c t i o n  -  V  R - n - O M e  +•  _  HOO  ^ :  I  R-C-OMe  \(  1  1 OOH  K_,  >  |l  K-C-OOH I  -v o^Ac  ligKt * reactio  would have k ^ » k^ f o r t h e methyl e s t e r . ? 1  1  Indeed, e t h y l a c e t a t e i s known  72 to be u n r e a c t i v e t o hydroperoxide  anion.  Moreover, the c h a r a c t e r i s t i c  y e l l o w c o l o r o f the a c r i d i n i u m methyl e s t e r i s r a p i d l y d i s c h a r g e d , but t h e r e i s no p e r c e p t i b l e N-methyl a c r i d o n e f l u o r e s c e n c e .  The b e h a v i o r  o f the methyl  e s t e r may, t h e r e f o r e , be s i m i l a r to t h a t o f t h e amide; and the very f e e b l e light  i s an i n d i c a t i o n o f t h e s m a l l degree t o which t h e hydrogen p e r o x i d e  h y d r o l y s i s occurs,  on t h e o t h e r hand, one might expect  slow b u t continuous  process, y i e l d i n g  l i g h t u n t i l the s u b s t r a t e had been com-  p l e t e l y d e s t r o y e d by t h i s o r dark processes v e r y b r i e f f l a s h observed  the h y d r o l y s i s to be  (eg, a l k a l i n e h y d r o l y s i s ) .  The  suggests a t r a c e i m p u r i t y may be r e s p o n s i b l e , or  t h a t perhaps the s u b s t r a t e i s transformed i n c a p a b l e o f chemiluminescence.  almost  instantly into a  substance  48  S i m i l a r b e h a v i o r i s shown by the b e n z o y l compound 20: is  100-times s t r o n g e r  A g a i n , there  i t s emission  than t h a t o f the methyl e s t e r , but i s s t i l l v e r y  i s no p e r c e p t i b l e N-methyl a c r i d o n e  d e c o l o r i z a t i o n of the a c r i d i n i u m  s a l t occurs.  base i s h i g h l y p r o b a b l e , as i n the p r e v i o u s  weak.  formed, a l t h o u g h r a p i d  Thus, a m e t a s t a b l e pseudo-  cases.  The l i g h t - p r o d u c i n g  r e a c t i o n almost s u r e l y i n v o l v e s a t t a c k by h y d r o p e r o x i d e on t h e b e n z o y l bonyl,  but the subsequent mechanism d i f f e r s  because there later. ring,  from that o f t h e methyl e s t e r More w i l l be s a i d about  this  The b e n z o y l compound, w i t h i t s e l e c t r o n - w i t h d r a w i n g aromatic i s expected t o be more r e a c t i v e t o n u c l e o p h i l e s  ester. still  i s no l e a v i n g group a v a i l a b l e .  car-  than i s t h e methyl  T h i s p r e d i c t i o n i s borne o u t , but 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 i s a very unfavorable The  process.  f e e b l e l i g h t output o f t h e methyl e s t e r and b e n z o y l compound  i n d i c a t e d t h a t a d e t a i l e d study o f t h e r e a c t i o n s i n v o l v e d would not y i e l d much u s e f u l i n f o r m a t i o n processes,  about the n a t u r e of e f f i c i e n t  chemiluminescent  so a t t h i s p o i n t t h e i n v e s t i g a t i o n o f these compounds was term-  inated. The  acridinium n i t r i l e  23 i s t h e most e f f i c i e n t o f t h r e e weak  emitters,  -4 w i t h a quantum y i e l d o f ca.  10  .  Its reactivity  toward h y d r o p e r o x i d e  a n i o n i s , t h e r e f o r e , a t l e a s t 10,000 times t h a t o f the methyl e s t e r , and 100 times t h a t o f the b e n z o y l compound. In c o n t r a s t w i t h the weaker compounds, the n i t r i l e r e a c t s w i t h a l k a l i n e 1^02 t o g i v e N-methylacridone i n y i e l d s o f >95%. cleaner  Thus, the r e a c t i o n i s much  than t h a t o f l u c i g e n i n ( v i d e i n f r a ) , but p a r a d o x i c a l l y has a much  s m a l l e r quantum y i e l d .  We i n t e r p r e t t h i s as an i n d i c a t i o n of s i d e  p r o d u c i n g N-methylacridone by dark pathways.  reactions  Two p o s s i b l e routes a r e :  49  E v i d e n c e of these dark r e a c t i o n s n i t r i l e and  H^C^  i n 50%  N-methylacridone ( b l u e evolved. duration  i s the o b s e r v a t i o n  aqueous e t h a n o l g i v e s fluorescence) within  quantities  l i g h t , but  the  no  Moreover, the k i n e t i c s of  cleanly f i r s t - o r d e r ; non-linear  pH v a l u e s .  This  more than one  i s t r e a t e d by  indicates  that  the  the  the of  light is  intensity  is;much reduced i n comparison to t h a t o f a f r e s h s o l u t i o n .  reverse order.  reagents i n  and Similar  the  the n i t r i l e chemiluminescence  are  p l o t s are o b t a i n e d , e s p e c i a l l y a t h i g h substrate  i s being p a r t i t i o n e d  into  r e a c t i o n pathway.  E v i d e n c e w i l l be  p r e s e n t e d l a t e r s u g g e s t i n g t h a t the  mechanism i s d i f f e r e n t from those d e p i c t e d catalyzed  considerable  15 minutes, but  Subsequent a d d i t i o n o f base g i v e s  b e h a v i o r i s observed i f the n i t r i l e  not  t h a t a s o l u t i o n of  above.  r e a c t i o n between hydrogen p e r o x i d e and  The  light-producing  k i n e t i c s of the  base-  a r o m a t i c n i t r i l e s were shown  73 by Wiberg  t o be  first  o r d e r i n each o f the  t h a t a d d i t i o n o f h y d r o p e r o x i d e i o n t o the  t h r e e reactanfcs  n i t r i l e was  the  step.  rate  F u r t h e r i n v e s t i g a t i o n of the r e a c t i o n d i s c l o s e d t h a t 4 i o n was 10 times more n u c l e o p h i l i c than h y d r o x i d e i o n , the These f i n d i n g s are  consistent  w i t h our  proposed mechanism.  involved,  and  determining  hydroperoxide s t r o n g e r base.  71  50  A s i m i l a r chemiluminescence  i s g i v e n by 9-cyano-10-methylacridan  22,  which i s o x i d i z e d t o N-methylacridone when an a l k a l i n e e t h a n o l s o l u t i o n i s t r e a t e d w i t h m o l e c u l a r oxygen. r a d i c a l s , because  The  r e a c t i o n does not appear t o i n v o l v e  t h e r e i s no enhancement w i t h p o t a s s i u m f e r r i c y a n i d e ,  s u l f a t e , o r c u p r i c bromide,  74  c y a n i d e i o n , or hydroquinone.  and no i n h i b i t i o n w i t h a r s e n i o u s o x i d e , The p r o c e s s c o u l d be e n v i s i o n e d as an  d a t i o n t o the a c r i d i n i u m n i t r i l e  free ferrous  75 oxi-  23 and h y d r o p e r o x i d e a n i o n , which can then  interact. The  s t a n d a r d of comparison  was  l u c i g e n i n bromide  a c r i d i n i u m dibromide, 13, so chosen because  (N,N -dimethyl1  i t c o u l d be c o n v e n i e n t l y r e -  c r y s t a l l i z e d and made much more pure than the commercially a v a i l a b l e genin (the d i n i t r a t e ) .  Besides t h i s ,  N,N--dimethylbiacridene,17,  nearly  t h r e e minutes  a b s o l u t e quantum y i e l d was  easily  reduced to  a compound of c o n s i d e r a b l e i n t e r e s t .  L u c i g e n i n bromide has a f a i r l y l i f e was  t h e bromide was  luci-  strong, durable emission.  Its half-  under the c o n d i t i o n s s e l e c t e d , and i t s  assumed to be t h a t o f l u c i g e n i n i t s e l f :  10  -2  ,  54 a c c o r d i n g to T o t t e r .  The  l i g h t was  g r e e n i s h i n s t e a d o f the b l u e c o l o r  shown by a l l the o t h e r compounds i n t h i s s e r i e s , and the r e s u l t i n g was  y e l l o w i n s t e a d o f water-white.  The wavelength  solution  shift i s a natural  sequence o f the s e l f - a b s o r p t i o n p r o p e r t i e s o f c o l o r e d  con-  solutions.  T h i n - l a y e r examination of the r e a c t i o n m i x t u r e i n d i c a t e d the presence of  s e v e r a l o t h e r compounds b e s i d e s N-'methylacridone;  o t h e r p r o d u c t s was  not  investigated.  Thus, l u c i g e n i n bromide gave 100 but the r e a c t i o n was the energy  but the n a t u r e o f these  not as c l e a n .  times more l i g h t  than t h e n i t r i l e  T h i s c o u l d be i n t e r p r e t e d to mean t h a t  t r a n s f e r p r o c e s s r e s u l t i n g i n e x c i t e d N-methylacridone  more e f f i c i e n t f o r l u c i g e n i n bromide than f o r the n i t r i l e .  i s much  Especially  23,  51  s i g n i f i c a n t i s the o b s e r v a t i o n t h a t , a c c o r d i n g to our mechanism, the r e a c t i o n of each m o l e c u l e of l u c i g e n i n bromide w i t h one of hydrogen y i e l d s two m o l e c u l e s y i e l d s o n l y one. i s greater with  of N-methylacridone, whereas the c o r r e s p o n d i n g  Thus, the p r o b a b i l i t y o f forming l u c i g e n i n bromide, a l t h o u g h  a c t i o n s i s unknown.  the extent o f "dark" s i d e r e -  do not know t h e i r  c o u l d be i n v o l v e d w i t h  phenyl  c o n d i t i o n s chosen.  e s t e r s 34 a r e b r i l l i a n t e m i t t e r s under  Their i n i t i a l  brightness r e l a t i v e  l u c i g e n i n bromide i s anywhere from f i v e to 4000 times  is,  as s t r o n g , but  a t the expense o f d u r a t i o n .  .We  have a l r e a d y  t o the a c r i d i n i u m phenyl  esters.  curve)  i n i t i a l b r i g h t n e s s i s gained  s a i d that l u c i g e n i n bromide i s  to have a h i g h e r p r o b a b i l i t y of e x c i t e d N-methylacridone the a c r i d i n i u m n i t r i l e  their  t h a t of l u c i g e n i n bromide.  Thus, o n l y a k i n e t i c f a c t o r i s i n v o l v e d h e r e , and  than w i l l  to  (as measured by the t o t a l a r e a under the e m i s s i o n  t o a l l i n t e n t s and purposes, the same as  expected  l u c i g e n i n bromide,  extent.  The N - m e t h y l a c r i d i n i u m  t o t a l quantum y i e l d  chemilumin-  i s the l a r g e amount of N-methylacridone p r o d u c t i o n  by dark r o u t e s ; s i m i l a r p r o c e s s e s  the standard  nitrile  e x c i t e d N-methylacridone  An a d d i t i o n a l f a c t o r p r e v e n t i n g e f f i c i e n t  escence of the n i t r i l e  but we  peroxide  formation  23. A s i m i l a r comparison should be a p p l i e d T h i s p o i n t w i l l be a m p l i f i e d a f t e r  the  proposed chemiluminescent mechanism has been d i s c u s s e d . The most s i g n i f i c a n t p r o p e r t y of the s u b s t i t u t e d phenyl that  t h e i r r e a c t i o n r a t e s obey a Hammett r e l a t i o n s h i p  T a b l e I ) ; and, moreover, t h i s i s the f i r s t t h a t i s dependent on r a t e s o n l y .  esters i s  (See F i g u r e  6,  such s e r i e s i n chemiluminescence  Throughout the  s e r i e s we  have the same  e m i t t e r , N-methylacridone, whose f l u o r e s c e n c e e f f i c i e n c y i s c o n s t a n t . we  have a v o i d e d  the problem of s u b s t i t u e n t e f f e c t s on the f l u o r e s c e n c e e f -  f i c i e n c y o f the e m i t t e r , a f a c t o r which f o r the l o p h i n e and was  not  Thus,  determined.  (See  Introduction.)  indole  peroxides  52  A t pH  12, the r e a c t i o n l i f e t i m e s ranged from 4 minutes  phenyl e s t e r ) down t o 0.05  seconds  (the various nitrophenyl e s t e r s ) .  the f a s t e s t r e a c t i o n s , the m i x i n g time was  comparable t o the t o t a l  time, and t h i s r e s u l t e d i n a g e n e r a l broadening i a t i o n s from t r u e f i r s t - o r d e r k i n e t i c s .  compounds, now  of the decay  Indeed,  had e s s e n t i a l l y i d e n t i c a l r e a c t i o n r a t e s a t pH predicted reactivity differences.  ( t h e 4-methoxyl For  reaction  curve and  dev-  the t h r e e n i t r o p h e n y l e s t e r s  12, i n c o n t r a d i c t i o n to the  Re-measurement a t pH 8 d i s c l o s e d t h a t  these  r e a c t i n g more s l o w l y , d i d indeed f o l l o w the expected o r d e r of  r e a c t i v i t y , with 3-nitro < 4-nitro <2,4-dinitro.  Thus, e x p e r i m e n t a l  dif-  f i c u l t i e s w i t h the f a s t e s t - r e a c t i n g phenyl e s t e r s has made the Hammett p l o t a t pH 12 somewhat l e s s p r e c i s e i n the r e g i o n of l a r g e  <r, but the t r e n d i s  clear. U n f o r t u n a t e l y , the r e a c t i o n c o n d i t i o n s chosen f o r t e s t i n g a l l the chemiluminescent  compounds were i d e a l  f o r the b r i g h t p h e n y l e s t e r s .  f o r the weaker ones but too v i g o r o u s  T h i s p r e v e n t s us from drawing more c o n c l u s i o n s  about the r e a c t i v i t y of those p h e n y l e s t e r s ; f o r example, the 3 - n i t r o compound,.might be expected to obey a l i n e a r r e l a t i o n s h i p more c l o s e l y than the 4n i t r o isomer.  We  were not a b l e t o c o n f i r m t h i s , however.  the 4-methoxy e s t e r 34b would expect.  i s p e r p l e x i n g ; i t r e a c t s more r a p i d l y than  There i s c l e a r l y no e x t r a resonance-donating  the nominal  deviation.  Perhaps  6 the  one  e f f e c t , however,  yg  _r_ because  The b e h a v i o r of  v a l u e of -0.78  (from Brown  value f o r this p a r t i c u l a r  ) g i v e s an even  larger  f u n c t i o n a l group  i s more  p r o p e r l y d e f i n e d by the s o - c a l l e d " c t v a l u e s " of van Bekkum e t a l . ^ n  attempted  to extend the Hammett p l o t by s y n t h e s i s o f a 4-N,N-dimethylamino  phenyl e s t e r , w i t h <T -0.83; but d i f f i c u l t i e s acridinium salt report.  We  encountered  i n the N-methyl-  f o r m a t i o n have p r e v e n t e d our i n c l u d i n g t h i s compound i n the  .53  The  R v a l u e was  found to be 4.4;  of p h e n o l s , whose p c o n s t a n t i s 2.1. ^ We  ionization  T h i s i l l u s t r a t e s the extreme  7  of the p e r o x i d e h y d r o l y s i s .  compare t h i s w i t h the  facility  should p o i n t out t h a t the phenyl e s t e r s were  expected t o r e a c t r e a d i l y w i t h h y d r o p e r o x i d e a n i o n , p r e c i s e l y because  their  b e h a v i o r w i t h r e s p e c t to n u c l e o p h i l i c a t t a c k i s d i a m e t r i c a l l y o p p o s i t e to t h a t o f the methyl l e a v i n g group  ester described previously.  (phenoxide  Here, the presence o f a good  i o n , a weaker base than h y d r o p e r o x i d e ion) a l l o w s  us to p r e d i c t t h a t e s t e r h y d r o l y s i s by h y d r o p e r o x i d e would be c o n s i d e r a b l y more f a c i l e .  Indeed,  the p r e d i c t i o n has been c o m p l e t e l y s u b s t a n t i a t e d .  C l e a r l y then, the chemiluminescence  of the a c r i d i n i u m phenyl  34 has, as i t s r a t e - d e t e r m i n i n g s t e p , the a t t a c k by hydrogen the e s t e r c a r b o n y l .  The  esters  p e r o x i d e upon  involvement of f r e e h y d r o p e r o x i d e a n i o n , however,  has been r u l e d out by the f o l l o w i n g experiment:  i f (say) the p h e n y l e s t e r  i n aqueous e t h a n o l i s t r e a t e d w i t h a weak base,  slow d e c o l o r i z a t i o n of the  yellow s o l u t i o n occurs, r e s u l t i n g ultraviolet  spectrum.^  i n the pseudobase 35 as proven by i t s  ( T h i s i s an e q u i l i b r i u m p r o c e s s ; the a c r i d i n i u m  form can be r e g e n e r a t e d by a d d i n g m i n e r a l a c i d . ) now  34c  added t o thespseudobase,  I f hydrogen  peroxide i s  t h e r e i s a f e e b l e but v e r y l o n g - l a s t i n g  light  emission. I n c o n t r a s t , when a s o l u t i o n of the a c r i d i n i u m phenyl e s t e r i s t r e a t e d i n the r e v e r s e o r d e r (^O^,  f o l l o w e d by b a s e ) , i n s t a n t d e c o l o r i z a t i o n and a  b r i g h t , s h o r t - l i v e d chemiluminescence The i n t e r p r e t a t i o n n i s c l e a r .  i s observed. In the former case, the  9-hydroxyl  pseudobase 35 i s c a p a b l e of r e a c t i n g w i t h f r e e h y d r o p e r o x i d e i o n a t the e s t e r c a r b o n y l , but t h i s process does not r e s u l t i n r a p i d , e f f i c i e n t production.  In the second experiment,  c o m p e t i t i o n between h y d r o x i d e i o n and  the much more n u c l e o p h i l i c h y d r o p e r o x i d e i o n i s expected t o produce p e r o x i d e 36 which can then y i e l d b r i g h t  light  light.  the a c r i d a n  54  The  '&2®2  f e e b l e l i g h t r e s u l t i n g from die-prior a d d i t i o n of base  cannot be  i n t e r p r e t e d i n terms of simple a l k a l i n e h y d r o l y s i s of  e s t e r , because a f t e r s t a n d i n g pseudobase 35  f o r 15 minutes i n a b u f f e r a t pH  can be a c i d i f i e d g e n e r a t i n g  upon a d d i n g a l k a l i n e ^ C ^ , ution.  Thus, we  gives  anion, very  an a c r i d i n i u m  The  light  conceivably  s p e c i e s which, sol-  a r i s e s only a f t e r  of the pseudobase has undergone exchange w i t h h y d r o p e r o x i d e  slow p r o c e s s due  t o the  p o s i t i o n of the  t h i s i s expected to be  a  equilibrium.  do not b e l i e v e t h a t a d e c o m p o s i t i o n of the p e r a c i d 37,  y i e l d i n g N-methylacridone, i s chemiluminescent.  the  the  ( v i s u a l l y ) as much l i g h t as a f r e s h  p r o b a b l y v i a the a c r i d i n i u m e s t e r 34c;  We  12  see t h a t the p h e n y l e s t e r s a r e q u i t e r e s i s t a n t to base  h y d r o l y s i s a t room temperature. the h y d r o x y l  before  Indeed, the  while  acridinium  55  a c i d 31 was shown t o g i v e N-methylacridone by a dark r o u t e :  2>\  o©  We admit t h e s t r u c t u r e s a r e n o t the same, b u t they do share one common feature:  they b o t h y i e l d N-methylacridone by an a c y c l i c p r o c e s s .  e v i d e n c e to d i s q u a l i f y  the p e r a c i d i s the analogous  of l o p h i n e , r u l e d out as a n e c e s s a r y i n t e r m e d i a t e . reason f o r the absence o f l i g h t  Further  «C-hydroxy1 h y d r o p e r o x i d e (See I n t r o d u c t i o n . ) The  i s not r e a d i l y apparent, b u t i t c o u l d w e l l  i n v o l v e an u n f a v o r a b l e t r a n s i t i o n s t a t e geometry p r e v e n t i n g energy t o the newly-forming N-methylacridone  transfer  carbonyl.  The o b s e r v a t i o n t h a t hydrogen p e r o x i d e i s i n v o l v e d both a t C-9 and a t the e s t e r c a r b o n y l leads i n e v i t a b l y to our proposed  chemiluminescence  mechanism f o r the a c r i d i n i u m s a l t s t h a t was suggested i n the I n t r o d u c t i o n ; the n e c e s s i t y f o r the d e c o m p o s i t i o n o f a c y c l i c p e r o x i d e t r a n s i t i o n or i n t e r m e d i a t e i n the f o r m a t i o n o f e x c i t e d N-methylacridone.  O  16  #  state  56  Moreover, t h i s p a r t i c u l a r mechanism i s the most r e a s o n a b l e one r e c o n c i l e s a l l the k i n e t i c data p r e s e n t l y The  l e s s e f f i c i e n t compounds a r e  luminescence mechanism. light-producing  in addition  step the  The  nitrile  sink! has  same type chemi-  f o r example, should have f o r i t s  reaction  to the dark r e a c t i o n s  of an e l e c t r o n  available.  expected to have the  23,  which  described  made p o s s i b l e  the  previously.  Here, the  utilization  f o r m a t i o n of a s u i t a b l e t r a n s i t i o n  state. Like manner as  the n i t r i l e ,  the b e n z o y l compound 20 cannot behave i n the same  the phenyl e s t e r s because there i s no  a t i v e route,using  an  electron  leaving  sink, i s again a v a i l a b l e .  group.  The  altern-  Subsequent breakup  o f the 4-membered t r a n s i t i o n s t a t e y i e l d s benzoate i o n .  An  attempt was  a c i d i n the  made u s i n g  reaction mixture.  We  t h i n l a y e r chromatography, to d e t e c t were unable to observe any,  but  -6 hardly  s u r p r i s i n g i n view of the observed quantum y i e l d  (10  ).  benzoic  this is  The methyl e s t e r has a l e a v i n g group, a l b e i t a v e r y poor one, on the c a r b o n y l carbon.  The f e e b l e l i g h t p r o d u c t i o n may t h e r e f o r e be an  i n d i c a t i o n o f the e x t e n t o f t h i s r e a c t i o n , a l t h o u g h f i c i e n t data a v a i l a b l e f o r d e f i n i t i v e The  comment.  r e l a t i v e l i g h t output o f the phenyl  bromide i s r o u g h l y  the same.  t h e r e i s not y e t s u f -  e s t e r s 34 and o f l u c i g e n i n  T h i s comparison can now be made i n the same  manner as f o r t h e n i t r i l e 23. As b e f o r e , the p r o b a b i l i t y o f forming  e x c i t e d N-methylacridone  (NMA)  s h o u l d be h i g h e s t f o r l u c i g e n i n bromide, because ( n e g l e c t i n g s i d e r e a c t i o n s ) i t y i e l d s two m o l e c u l e s  o f product  f o r every molecule  of reactant.  But,  whereas t h e n i t r i l e had o n l y l / 1 0 0 t h the Q of l u c i g e n i n bromide, the phenyl e s t e r s have an equal amount. the n i t r i l e ) g i v e NMA  A l s o , s i g n i f i c a n t l y , the phenyl  esters  (like  quantitatively.  The most r e a s o n a b l e e x p l a n a t i o n f o r the i n c r e a s e d e f f i c i e n c y t h e r e a r e fewer "dark" pathways f o r NMA r o u t e s would be through  production.  The two most  the p e r a c i d 37 and the h y d r o p e r o x i d e  37 38  i s that  obvious  c a r b o x y l a t e 38.  58  N e i t h e r path i s expected  to make a s i g n i f i c a n t c o n t r i b u t i o n .  f o r m a t i o n of 37 i s p r e d i c a t e d on h y d r o x y l i o n winning n u c l e o p h i l i c hydroperoxide  The  out over the much more  a n i o n i n c o m p e t i t i o n f o r the r e a c t i v e 9 - p o s i t i o n ,  f o l l o w e d by p e r o x i d e a t t a c k a t the e s t e r c a r b o n y l .  The  second path would  i n v o l v e a l k a l i n e h y d r o l y s i s of 36 b e f o r e i n t e r n a l a t t a c k , a much more f a v o r able process, could occur.  In b o t h c a s e s , h y d r o x y l i o n would be f u n c t i o n i n g  as a r e a c t a n t i n s t e a d of a c a t a l y s t . pronounced a t pH v a l u e s  lower  While such an e f f e c t might be more  than the pK of hydrogen p e r o x i d e  (11.5), we  do  not b e l i e v e t h a t under the c o n d i t i o n s s e l e c t e d (pH 12, where the c o n c e n t r a t i o n of h y d r o p e r o x i d e  anion i s very high with  e f f e c t i v e c o m p e t i t i o n by h y d r o x y l i o n . action i s l i k e l y reactions.  Why  In s h o r t , the l i g h t - p r o d u c i n g r e -  to be much f a s t e r than e i t h e r of the c o n c e i v a b l e then i s the quantum y i e l d  There i s no c l e a r answer to t h i s . ( f o r the phenyl  r e s p e c t to s u b s t r a t e ) t h e r e i s an  o n l y 1% ? I f we  c y c l i c p e r o x i d e , then the low quantum y i e l d  state.  can c o r r e c t l y assume t h a t  e s t e r s a t l e a s t ) the m a j o r i t y of NMA  product m o l e c u l e s  "dark"  f o r m a t i o n goes v i a the  r e f l e c t s the p o p u l a t i o n of  f i n d i n g themselves i n an 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  Quenching e f f e c t s can be r u l e d out, because the f l u o r e s c e n c e e f -  f i c i e n c y 3> f o r NMA water and b a s e ) .  i s 857» i r r e s p e c t i v e of s o l v e n t ( e t h a n o l , w i t h or Moreover, the h i g h 9> v a l u e means t h a t we  cannot  without  significantly  enhance the quantum y i e l d by making a c r i d i n i u m s a l t s w i t h an e l e c t r o n releasing ring i s decreased process.  The  s u b s t i t u e n t , as w i t h l u m i n o l .  by h a v i n g o n l y one NMA o t h e r fragment, CO^,  molecule  C e r t a i n l y the quantum y i e l d formed i n the bond  c o u l d w e l l a c q u i r e the excess  l o s e i t (most probably) by c o l l i s i o n s w i t h s o l v e n t . v a r i a b l e s a r e accounted product  f o r , we  still  e x c i t a t i o n s t e p , and how  i s o b t a i n e d the q u e s t i o n s w i l l  cleavage energy  and  But a f t e r a l l the known  can o n l y s p e c u l a t e on the n a t u r e of  i t can be o p t i m i z e d .  remain unanswered.  the  U n t i l more i n f o r m a t i o n  59  The  b r i g h t e s t compound i n the whole s e r i e s i s the  a c i d c h l o r i d e 32.  Unfortunately,  to p u r i f y i t were o n l y p a r t i a l l y our  standard  hydrolyze  conditions  and  i t i s so h i g h l y r e a c t i v e t h a t attempts successful.  chemiluminesce ;too soon.  The  brightness  o f much s h o r t e r  duration.  studied i t s reactions.  r e a c t i o n , 32 had and  was  visibly  t h i s p o i n t , a paper appeared d e s c r i b i n g the independent  of t h i s same c o m p o u n d . R a u h u t and and  Moreover, when s u b j e c t e d  gave NMA  They found t h a t , i n a t y p i c a l  a l o n g w i t h other  our  the a c r i d i n i u m a c i d 31,  s e r i e s , the e m i t t e r was '  (comparable to our phenyl  products.  These other  carbon d i o x i d e , and  NMA N  (A 440 'max  A chemiluminescent mechanism was p e r a c i d 37a  as a key  intermediate.  discovery 32  chemiluminescent  thought to o c c u r v i a aqueous or a l k a l i n e h y d r o l y s i s of 32, t o be  greater  co-workers a t Cyanamid prepared  a quantum y i e l d of 1%  (52%)  to  f o r l i g h t measurement, the compound began to  than the phenyl e s t e r s , but At  N-methylacridinium  esters)  p r o d u c t s were and  turned  carbon monoxide.  As  out in  mu). ' proposed, and  E v i d e n c e f o r 37a  i n v o l v e d the hydroxywas  - (a) -R^U C=Q  $>  c  I  t-Bu  37 based on  several factors:  (1)  An  i n f r a r e d study of 32  t a i n i n g an e q u i v a l e n t a c i d c h l o r i d e was  i n anhydrous 1,2-dimethoxyethane con-  amount of 98% hydrogen p e r o x i d e showed the  f a i r l y r e s i s t a n t to a t t a c k  ( t , : 25 m i n u t e s ) ;  acridinium  60  (2)  light  e m i s s i o n was f a s t e r a t h i g h e r pH, o r w i t h added water;  (3)  weak chemiluminescence was observed when t - b u t y l  hydroperoxide  was s u b s t i t u t e d f o r hydrogen p e r o x i d e , s u g g e s t i n g 37b as the analogous intermediate. Our work on 32 has been l e s s e x t e n s i v e , but n e v e r t h e l e s s we the c o n c l u s i o n s drawn by the o t h e r group a r e v a l i d  only i n p a r t .  feel Their  mechanism i s p r e d i c a t e d on the a t t a c k by p e r o x i d e a n i o n on the c a r b o n y l , f o l l o w e d by hydroxy pseudobase f o r m a t i o n and a c y c l i c bond c l e a v a g e .  Sim-  23 i l a r p r o c e s s e s a r e p o s s i b l e i n a s e r i e s of a c y l c h l o r i d e s  ; and, w h i l e 32  may r e a c t i n a s i m i l a r f a s h i o n , the phenyl e s t e r s show p r o p e r t i e s incomp a t i b l e w i t h t h i s scheme. w i t h a l k a l i n e potassium  Most s i g n i f i c a n t l y , 32 i s weakly  chemiluminescent  t - b u t y l h y d r o p e r o x i d e , whereas the phenyl e s t e r s  a r e much l e s s s o . I t c o u l d be argued t - b u t y l hydroperoxide  That  t h a t the l a c k o f b r i g h t chemiluminescence u s i n g  does n o t r u l e out p e r a c i d 37a as the key i n t e r m e d i a t e . 78  i s , the low n u c l e o p h i l i c power  o f t - b u t y l hydroperoxide  might  prevent  its  s u c c e s s f u l a t t a c k of the e s t e r c a r b o n y l ; o r , i f the p e r a c i d d i d form,  its  f a c i l e decomposition  t-BuO  might be r e t a r d e d by the poor l e a v i n g a b i l i t y o f  a n i o n ( i n 37a) as compared to OH  i o n ( i n 37b).  These o b j e c t i o n s can now be c o u n t e r e d . t r e a t e d w i t h methyl h y d r o p e r o x i d e  n u c l e o p h i l i c as hydrogen p e r o x i d e ,  The phenyl e s t e r 34c  i n a l k a l i n e ethanol. 79  has been  T h i s reagent, as  y i e l d e d only a feeble l i g h t  (due per-  haps to t r a c e s o f H „ 0 ) ; and, moreover, NMA formed smoothly and r a p i d l y . * K i n d l y s u p p l i e d by P r o f . W. Jencks, B r a n d e i s U n i v e r s i t y . 9  The NMA  f o r m a t i o n can most r e a s o n a b l y be w r i t t e n as an a c y c l i c  i n v o l v i n g the e x p u l s i o n of CH^O pulsion) .  Most important,  a n i o n (even  l e s s f a v o r e d than t-BuO  the i m p o s s i b i l i t y of forming a c y c l i c  i n t e r m e d i a t e seems to have prevented b r i g h t  e s t e r 34d  i s shown.  ex-  peroxide  chemiluminescence.  In T a b l e I I I , the e f f e c t of pH on the r e a c t i o n r a t e and e m i s s i o n of phenyl  decomposition,  F o r three a r b i t r a r i l y  the  light  chosen pH  v a l u e s , the t o t a l amount of l i g h t i s ( w i t h i n e x p e r i m e n t a l e r r o r ) the same. Thus, i t would appear t h a t the r a t i o o f " l i g h t " i n f l u e n c e d by the h y d r o x y l i o n c o n c e n t r a t i o n . around 1%,  t h a t we  do not wish  to "dark" pathways i s not The  quantum y i e l d i s so  t o s p e c u l a t e on the exact n a t u r e of the  v a r i o u s competing "dark" r e a c t i o n s o c c u r r i n g a t d i f f e r e n t pH v a l u e s . the involvement  of h y d r o p e r o x i d e  a n i o n i n l i g h t p r o d u c t i o n was  the l o g a r i t h m i c p l o t of the r a t e c o n s t a n t s a g a i n s t the pH s l o p e 0 was  Br0nsted  10 t o 12, and (pKll.5)  1.0  But  shown i n  (Figure 7).  The  a t low pH v a l u e s , l e v e l l i n g o f f i n the pH r e g i o n  c o n s i s t e n t w i t h the requirement  i n chemiluminescence.  f o r i o n i z e d hydrogen p e r o x i d e  . W i t h i n t h i s simple framework, however,  t h e r e a r e more complex r e l a t i o n s h i p s . f o r the 9-hydroperoxide  low,  Evidence has a l r e a d y been p r e s e n t e d  36 as the chemiluminescence p r e c u r s o r .  Formation  of t h i s s p e c i e s would depend on the c o n c e n t r a t i o n of h y d r o p e r o x i d e  anion  and a l s o on the e q u i l i b r i u m c o n s t a n t i n v o l v e d .  the  High pH w i l l d r i v e  e q u i l i b r i u m towards the f i g h t , and w i l l a l s o c a t a l y z e the f u r t h e r of 36 because a l k y l . h y d r o p e r o x i d e s H 0 2  2  That  second  ionization  a r e known to have pK v a l u e s near  i o n i z a t i o n would be expected  t h a t of  to result i n internal attack  of the e s t e r group, g i v i n g the c y c l i c p e r o x i d e and chemiluminescence,  and i t  62  i s reasonable particular  to suppose t h a t the second step i s r a t e - d e t e r m i n i n g .  s e t o f runs,  I n our  t h e chemiluminescence l i f e t i m e s ranged from 20  minutes (pH 7) t o 1 second (pH 1 2 ) .  The  e f f e c t o f hydrogen p e r o x i d e  c o n c e n t r a t i o n was o f s p e c i a l  interest  i n l i g h t of the c l a i m s made f o r an e x c i t e d oxygen dimer as the energy  source  19 f o r chemiluminescence.  F o r t h i s t o be v a l i d ,  square dependence on t h e p e r o x i d e Our  one would expect t o f i n d a  concentration.  r e s u l t s ( T a b l e IV) do n o t s u b s t a n t i a t e these  range where hydrogen p e r o x i d e  claims.  I n the  i s i n l a r g e excess over s u b s t r a t e , the amount  of l i g h t i s unchanged upon a 1 0 - f o l d d i l u t i o n of p e r o x i d e . the square dependence so n e c e s s a r y  T h i s r u l e s out  f o r the e x c i t e d oxygen concept.  I n the runs where t h e i n i t i a l p e r o x i d e  c o n c e n t r a t i o n i s below t h a t o f  -4 the s u b s t r a t e  (10  M), we see a r e d u c t i o n i n l i g h t output,  i s o n l y about h a l f as g r e a t as e x p e c t e d of p e r o x i d e .  but the r e d u c t i o n  f o r each s u c c e s s i v e 1 0 - f o l d  We have no e x p l a n a t i o n a t the moment, although  chemiluminescence quantum y i e l d  dilution  p o s s i b l y the  ( i e , degree of e x c i t e d product  formation)  might be i n c r e a s i n g as t h e 1^02 c o n c e n t r a t i o n becomes low. The  v a r i a t i o n i n r a t e constants  i s also interesting.  At high  peroxide  c o n c e n t r a t i o n the r e a c t i o n has slowed down, and t h i s c o u l d be caused by t h e a t t a c k on s u b s t r a t e by two m o l e c u l e s o f p e r o x i d e , reacting  intermediate:  I  giving a relatively  slow-  63  With lower p e r o x i d e  concentration the rate i s progressively faster,  r e a c h i n g a maximum where the two r e a c t a n t s a r e approximately Below t h i s p o i n t , the r a t e s a g a i n decrease; due  equimolar.  t h i s phenomenon i s probably  t o the low c o n c e n t r a t i o n of b o t h r e a c t a n t s , a t which p o i n t the mag-  n i t u d e o f the "adduct" e q u i l i b r i u m c o n s t a n t becomes m e a n i n g f u l . evidence  h e r e i s t h a t the two weakest p e r o x i d e  luminescence curves  The  having  concentrations  g i v e chemi-  an i n d u c t i o n p e r i o d .  e f f e c t o f s u b s t r a t e c o n c e n t r a t i o n on the k i n e t i c s and l i g h t  of t h e phenyl e s t e r 34c has been s t u d i e d ( T a b l e V ) . t r a t i o n s used, w i t h p e r o x i d e pected  Additional  emission  I n t h e range o f concen-  and base i n l a r g e excess,  the r e a c t i o n was ex-  to be pseudo f i r s t - o r d e r i n s u b s t r a t e ; the l i n e a r r e l a t i o n s h i p o f  l o g I v s . time c o n f i r m s  t h i s f o r a l l the runs i n t h a t  We n o t i c e a moderate r a t e i n c r e a s e w i t h tration.  series.  decreasing  substrate  concen-  T h i s c o u l d be i n t e r p r e t e d as an e f f e c t of s i d e r e a c t i o n s becoming  more prominent as the s u b s t r a t e i s p r o g r e s s i v e l y d i l u t e d ; b u t , w i t h i n each run,  the s i d e r e a c t i o n s a r e n o t c a u s i n g d e v i a t i o n s from l i n e a r  kinetics.  There i s a p a r a l l e l e f f e c t on the t o t a l  larger loss i n l i g h t emission of d i l u t i o n .  we see a  simply as the e f f e c t  i n l i g h t output  ( i e , e f f i c i e n c y o f e x c i t e d product  substrate concentrations, process  than c o u l d be e x p l a i n e d  A l t e r n a t i v e l y , the discrepancy  enhanced quantum y i e l d  l i g h t output;  first-order  c o u l d be due t o  formation)  a t higher  due e i t h e r t o a more e f f i c i e n t energy t r a n s f e r  o r an absence o f quenching e f f e c t s .  a v a i l a b l e t o r e s o l v e these q u e s t i o n s ,  There i s n o t s u f f i c i e n t  however.  data  CONCLUSION  64  CONCLUSION  T h i s i n v e s t i g a t i o n has y i e l d e d c o n s i d e r a b l e i n f o r m a t i o n about the g e n e r a l mechanism proposed cesses.  That  f o r e f f i c i e n t chemiluminescent  i s , the involvement  of a 4-membered c y c l i c  pro-  peroxide  t r a n s i t i o n s t a t e or d i s c r e t e i n t e r m e d i a t e seems n e c e s s a r y , and f o r b r i g h t l i g h t production, a high r e a c t i v i t y with hydroperoxide appears which we  t o be n e c e s s a r y .  anion  But t h e r e a r e many a s p e c t s o f the r e a c t i o n s  do n o t y e t f u l l y u n d e r s t a n d .  F o r example, i t was  found  water, i n amounts f a r g r e a t e r than k i n e t i c a l l y s i g n i f i c a n t , was sary f o r b r i g h t  rapid  l i g h t emission.  but f u r t h e r evidence i s l a c k i n g .  T h i s may  The b r i g h t e s t compounds, the a c r i -  hydrogen p e r o x i d e ; t r a c e amounts o f ^2^2  m a  ^  but a g a i n , f u r t h e r work i s needed t o c l a r i f y  i n t h e absence o f  have been r e s p o n s i b l e , t h i s matter.  compounds i n the s e r i e s show anomalous p r o p e r t i e s and  In t h i s  neces-  be a s o l v a t i o n phenomena,  dinium phenyl e s t e r s , were weakly chemiluminescent  they emit may  that  The weakest  the l i g h t  that  not indeed be a r e s u l t o f h y d r o l y s i s by hydrogen p e r o x i d e .  r e s p e c t , a d e t a i l e d p r o d u c t study appears  i n order.  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