Light-Activated Pesticides - American Chemical Society

Mechanisms of photooxidation of organic compounds are .... 1 ° 2. Substrate » Substrate • 0 2. Two major classes of singlet oxygen reactions are a...
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Chapter 2

Type I and Type II Mechanisms of Photodynamic Action

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Christopher S. Foote Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90024

Mechanisms of photooxidation of organic compounds are discussed, and methods of determining photooxidation mechanisms reviewed. Two cases that have been particularly well studied, cercosporin and a-terthienyl, are used to exemplify the techniques.

M a n y c h e m i c a l s , i n c l u d i n g natural c e l l c o n s t i t u e n t s , c a n a b s o r b light a n d p h o t o s e n s i t i z e d a m a g e to o r g a n i s m s . S o m e of t h e s e c o m p o u n d s a r e u s e d by o r g a n i s m s (including man) to attack o r d e f e n d a g a i n s t other o r g a n i s m s . T h i s p r o c e s s , c a l l e d " p h o t o d y n a m i c a c t i o n " , requires o x y g e n a n d d a m a g e s biological target m o l e c u l e s by photosensitized oxidation. B i o c h e m i c a l effects i n c l u d e e n z y m e d e a c t i v a t i o n (through d e s t r u c t i o n of s p e c i f i c a m i n o a c i d s , particularly methionine, histidine, a n d tryptophan), nucleic acid o x i d a t i o n (primarily of g u a n i n e ) , a n d m e m b r a n e d a m a g e (by o x i d a t i o n of unsaturated fatty a c i d s a n d cholesterol) (1. 2). M e c h a n i s m s of P h o t o o x y g e n a t i o n P h o t o s e n s i t i z e d oxidations are initiated by absorption of light by a sensitizer, w h i c h c a n be a d y e o r pigment, a ketone o r q u i n o n e , a n a r o m a t i c m o l e c u l e , or m a n y other t y p e s of c o m p o u n d . T h e s e n s i t i z e r ( S e n s ) is c o n v e r t e d to a n electronically e x c i t e d state by absorption of a photon. T h e initial product is a s h o r t - l i v e d s i n g l e t ( S e n s ) ; in m a n y c a s e s , this u n d e r g o e s i n t e r s y s t e m c r o s s i n g to t h e longer-lived triplet ( S e n s ) . B e c a u s e t h e singlet g e n e r a l l y h a s a very short lifetime, only reactants at relatively high c o n c e n t r a t i o n c a n interact with it before it d e c a y s ; h o w e v e r , m u c h l o w e r c o n c e n t r a t i o n s a r e sufficient to react with the longer-lived triplet state. 1

3

Sens

>

1

Sens

>

3

Sens

hv 0097-6156/87/0339-0022S06.00/0 © 1987 American Chemical Society

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

2.

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Mechanisms of Photodynamic Action

23

T h e r e are two m e c h a n i s m s of p h o t o s e n s i t i z e d oxidation, n a m e d " T y p e I" a n d " T y p e II" by G o l l n i c k (3) ( s e e F i g . 1). In the T y p e I p r o c e s s , substrate or s o l v e n t r e a c t s with the s e n s i t i z e r e x c i t e d s t a t e (either singlet or triplet, S e n s * ) to g i v e r a d i c a l s or radical i o n s , r e s p e c t i v e l y , by h y d r o g e n a t o m or electron transfer. R e a c t i o n of t h e s e r a d i c a l s with o x y g e n g i v e s o x y g e n a t e d products. In the T y p e II p r o c e s s , the e x c i t e d s e n s i t i z e r reacts with o x y g e n to form singlet m o l e c u l a r o x y g e n ( 0 2 ) , w h i c h t h e n r e a c t s with s u b s t r a t e to

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1

form t h e p r o d u c t s . T h e T y p e I a n d T y p e II m e c h a n i s m s a r e a l w a y s in competition; factors which govern the competition include o x y g e n c o n c e n t r a t i o n , the reactivities of the substrate a n d of the s e n s i t i z e r e x c i t e d state, the substrate concentration, a n d the singlet o x y g e n lifetime (4). T h e s e factors will be d i s c u s s e d in more detail in a s u b s e q u e n t s e c t i o n . Sens Type I Radicals •




2

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+

D0

2

DCA

T h e T y p e I reaction c a n a l s o result in h y d r o g e n abstraction, giving radical p r o d u c t s . T h e s e r a d i c a l s c a n react directly with o x y g e n to g i v e p e r o x i d e s or initiate r a d i c a l c h a i n a u t o x i d a t i o n . H y d r o g e n a b s t r a c t i o n is particularly c o m m o n with k e t o n e a n d q u i n o n e s e n s i t i z e r s , but a l s o o c c u r s with m a n y d y e s , a l t h o u g h u s u a l l y l e s s efficiently. S u b s t r a t e s that a r e g o o d h y d r o g e n d o n o r s promote this reaction (4). R C=0 2

+

R'-H

R C-0H o

+

R'«

Radical Chain Reactions T h e T y p e II

Process

T h e T y p e II r e a c t i o n p r o d u c e s s i n g l e t m o l e c u l a r o x y g e n , w h i c h r e a c t s directly with s u b s t r a t e s to give o x y g e n a t e d products or d e c a y s to the g r o u n d state if it fails to react. T h e rate of d e c a y is strongly d e p e n d e n t on solvent: in w a t e r , the lifetime of singlet o x y g e n is a b o u t four m i c r o s e c o n d s , while in o r g a n i c s o l v e n t s (and p r e s u m a b l y a l s o in the lipid r e g i o n s of m e m b r a n e s ) , the lifetime is o n the order of ten to twenty t i m e s longer (8.9).

3

°2

*

1

°2

Substrate »

Substrate •

0

2

T w o major c l a s s e s of singlet o x y g e n r e a c t i o n s are additions to olefins with allylic h y d r o g e n s , giving allylic h y d r o p e r o x i d e s , with a shift in position of the d o u b l e b o n d (the " e n e " reaction, 1Q), a n d addition to d i e n e s , a r o m a t i c s , a n d h e t e r o c y c l e s , giving e n d o p e r o x i d e s (the D i e l s - A l d e r reaction, H ) .

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

LIGHT-ACTIVATED PESTICIDES

26

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O t h e r p r o c e s s e s include reaction of electron-rich olefins to give unstable f o u r - m e m b e r e d ring p e r o x i d e s c a l l e d d i o x e t a n e s ( 1 £ ) , oxidation of s u l f i d e s to s u l f o x i d e s ( v i a a n i n t e r m e d i a t e " p e r s u l f o x i d e " , 12), a n d r e a c t i o n of electron-rich p h e n o l s , including tocopherol, to unstable h y d r o p e r o x y d i e n o n e s (14).

1

0

2

+ R S

> R S+00-

2

2

>

2R S+0 2

M a n y c o m p o u n d s d e a c t i v a t e (i. e . q u e n c h ) singlet o x y g e n efficiently without reacting (15.). F o r e x a m p l e , p - c a r o t e n e inhibits p h o t o o x i d a t i o n of m a n y c o m p o u n d s efficiently at e v e n very l o w c o n c e n t r a t i o n s b y a n e n e r g y t r a n s f e r m e c h a n i s m , w i t h o u t b e i n g a p p r e c i a b l y o x i d i z e d itself. O t h e r c o m p o u n d s s u c h a s D A B C O (1,4-diazabicyclooctane) a n d a z i d e i o n q u e n c h singlet o x y g e n b y a c h a r g e - t r a n s f e r p r o c e s s . P h e n o l s a n d s u l f i d e s a l s o q u e n c h singlet o x y g e n , in competition with their oxidation. F o r e x a m p l e , cct o c o p h e r o l q u e n c h e s singlet o x y g e n at a high rate in all s o l v e n t s , but reacts rapidly only in protic s o l v e n t s (16-18). f

1

1

0

2

+ DABCO

0

2

+ Car

>

3Car

>0 -— D A B C O + 2

+

3

0

>

2

DABCO

+ 3(>

2

S i n g l e t o x y g e n i s a n electronically e x c i t e d m o l e c u l e , a n d c a n return to the g r o u n d state with e m i s s i o n of light ( I S ) . T h e r e a r e t w o t y p e s of singlet o x y g e n l u m i n e s c e n c e , from a single m o l e c u l e at 1.27 p.m, a n d d i m o l " lumin e s c e n c e at 6 3 4 a n d 7 0 4 n m . Both t y p e s of l u m i n e s c e n c e a r e very inefficient b e c a u s e t h e lifetime of singlet o x y g e n in solution is short c o m p a r e d to t h e radiative lifetime. B e c a u s e t h e d i m o l e m i s s i o n d e p e n d s o n a b i m o l e c u l a r collision b e t w e e n t w o short-lived s p e c i e s , its efficiency a l s o d e p e n d s o n the concentration of singlet o x y g e n . H

1

0

2

> hv (1.27n)

2(1Q ) 2

> hv

(634,704nm)

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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Determination of

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Mechanisms of Photodynamic Action Mechanism

A s m e n t i o n e d in the introduction, high s e n s i t i z e r reactivity, high s u b s t r a t e reactivity a n d c o n c e n t r a t i o n , low o x y g e n c o n c e n t r a t i o n , a n d short singlet o x y g e n lifetimes favor the T y p e I m e c h a n i s m , while the opposite factors favor the T y p e II. O n e of the m o s t direct m e t h o d s of d e t e r m i n i n g w h e t h e r a r e a c t i o n is p r o c e e d i n g v i a a T y p e I or a T y p e II m e c h a n i s m is to vary s u b s t r a t e a n d o x y g e n c o n c e n t r a t i o n a n d determine the a m o u n t of products f o r m e d u n d e r v a r i o u s c o n d i t i o n s . T h i s t e c h n i q u e is particularly u s e f u l in h o m o g e n e o u s s o l u t i o n , e s p e c i a l l y w h e r e there are distinct s e t s of p r o d u c t s from the two m e c h a n i s m s . At sufficiently high o x y g e n a n d / o r low s u b s t r a t e c o n c e n t r a t i o n , a r e a c t i o n c a n b e f o r c e d into a c l e a n T y p e II p a t h w a y , w h e r e a s the T y p e I p a t h w a y c a n b e f o r c e d u n d e r the o p p o s i t e c o n d i t i o n s . C h a n g i n g solvent to o n e in w h i c h the singlet o x y g e n lifetime is longer helps to f a v o r the T y p e II m e c h a n i s m . B i n d i n g of s e n s i t i z e r to s u b s t r a t e (e.g., m e m b r a n e , p r o t e i n , or n u c l e i c a c i d ) is p a r t i c u l a r l y c o m m o n in living o r g a n i s m s , a n d t e n d s to favor T y p e I m e c h a n i s m s b e c a u s e of the effective i n c r e a s e in substrate concentration (20.21). T h e r e are two e x a m p l e s w h e r e the competition b e t w e e n T y p e I a n d T y p e II m e c h a n i s m s has been particularly well d o c u m e n t e d , 1,1d i p h e n y l m e t h o x y e t h y l e n e ( D P M E , 22) a n d d i m e t h y l s t i l b e n e (£3). In both c a s e s , the reaction c a n be m a n i p u l a t e d by m e a n s of the factors d e s c r i b e d a b o v e to give d i o x e t a n e products v i a the electron-transfer p a t h w a y or DielsA l d e r or e n e products, respectively, v i a the T y p e II route.

O

Rearrangement Products

Oxygen DPME

OCH

3

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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E s t a b l i s h i n g the m e c h a n i s m of p h o t o s e n s i t i z e d o x i d a t i o n s in c o m p l e x s y s t e m s is a difficult t a s k ( 2 4 - 2 6 ) . Kinetic tests a n d the u s e of inhibitors for v a r i o u s r e a c t i v e s p e c i e s a r e m o r e a m b i g u o u s t h a n in h o m o g e n e o u s s o l u t i o n , b e c a u s e r e a g e n t s a r e often c o m p a r t m e n t a l i z e d , b o u n d , or l o c a l i z e d , a n d it is rarely p o s s i b l e to k n o w the local c o n c e n t r a t i o n s of v a r i o u s reacting s p e c i e s , s e n s i t i z e r s , q u e n c h e r s , a n d traps. M a n y w o r k e r s h a v e u s e d a l l e g e d l y s p e c i f i c t r a p s or q u e n c h e r s for v a r i o u s reactive s p e c i e s , including singlet o x y g e n , s u p e r o x i d e ion ( 0 - ) , 2

hydroxyl radical (OH-), peroxy radicals ( R O O - ) , a n d other oxidants. H o w e v e r , the specificity of traps a n d inhibitors for oxidants requires far more s t u d y t h a n it h a s r e c e i v e d . F o r i n s t a n c e , all r e a g e n t s a n d q u e n c h e r s for singlet o x y g e n h a v e low oxidation potentials a n d will a l s o interact with other oxidants. A l s o , almost all q u e n c h e r s of singlet o x y g e n c a n q u e n c h s e n s i t i z e r e x c i t e d s t a t e s a s w e l l . Q u e n c h i n g of s e n s i t i z e r e x c i t e d s t a t e s c a n be d i s t i n g u i s h e d from singlet o x y g e n q u e n c h i n g by d e t e r m i n i n g the d e g r e e of inhibition at s e v e r a l o x y g e n c o n c e n t r a t i o n s , s i n c e if singlet o x y g e n is being q u e n c h e d , t h e d e g r e e of i n h i b i t i o n will not d e p e n d o n t h e o x y g e n concentration. Interconversions a n d interactions a m o n g reactive s p e c i e s c o m p l i c a t e the p r o c e s s further. In both T y p e I a n d T y p e II reactions, the initial products are often p e r o x i d e s , w h i c h c a n b r e a k d o w n to i n d u c e free r a d i c a l r e a c t i o n s . S u c h s e c o n d a r y thermal reactions h a v e b e e n s h o w n to c a u s e m u c h of the p h o t o d y n a m i c d a m a g e o b s e r v e d in m e m b r a n e s u n d e r s o m e c o n d i t i o n s ( 2 7 . 2 8 ) . R a d i c a l c h a i n s c a n c a u s e the o x i d a t i o n of m a n y m o l e c u l e s of

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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Mechanisms of Photodynamic Action

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starting material for e a c h primary product. F o r this r e a s o n , product a n a l y s i s m a y reflect m a i n l y s e c o n d a r y c h a i n p r o c e s s e s rather t h a n the primary reaction m e c h a n i s m . S i m i l a r c o m m e n t s apply to inhibition s t u d i e s . M e t h o d s of a s s e s s i n g the relative i m p o r t a n c e of v a r i o u s p r o c e s s e s are n e e d e d . D e t e c t i o n of a n i n t e r m e d i a t e is a n e c e s s a r y but not sufficient c o n d i t i o n for its h a v i n g a c a u s a t i v e role in a p r o c e s s . It d o e s little g o o d to s h o w that a reactive intermediate is present without b e i n g a b l e to estimate what fraction of the overall oxidation it c a u s e s . S u c h quantitation h a s rarely b e e n a c c o m p l i s h e d in h e t e r o g e n e o u s s y s t e m s . T e c h n i q u e s for

Characterizing Singlet

Oxygen

A large n u m b e r of t e c h n i q u e s h a v e b e e n d e v e l o p e d for detection of p o s s i b l e reactive intermediates in biological o x y g e n d a m a g e ( 2 4 . 29). F o r r e a s o n s of s p a c e , this report will c o n c e n t r a t e o n t e c h n i q u e s that a r e u s e f u l for the detection a n d characterization of singlet o x y g e n . C h e m i c a l T r a p s . A large n u m b e r of c o m p o u n d s h a v e b e e n a d d e d to r e a c t i n g s y s t e m s a s t r a p s for singlet o x y g e n , a n d t h e f o r m a t i o n of the s u p p o s e d l y characteristic products u s e d a s a n indication of the intermediacy of 0 2 - F o r e x a m p l e , dimethylfuran reacts with singlet o x y g e n to give the 1

d i k e t o n e s h o w n b e l o w a s the ultimate product. Unfortunately, s o d o a very large n u m b e r of other oxidants. In fact, furans are a prime e x a m p l e of very n o n s p e c i f i c singlet o x y g e n traps (24)-

A d i a g n o s t i c t r a p for s i n g l e t o x y g e n is c h o l e s t e r o l , w h i c h r e a c t s with singlet o x y g e n to g i v e the 5-cc h y d r o p e r o x i d e ; r e a c t i o n s with r a d i c a l a n d other o x i d a n t s g i v e c o m p l e x mixtures, but the 5-cc product is not a m o n g t h e m (20). T h i s s y s t e m is s o m e w h a t limited b e c a u s e of the low reactivity of c h o l e s t e r o l with singlet o x y g e n . A l t h o u g h c h o l e s t e r o l is not s o l u b l e in water, it c a n be b o u n d to m i c r o s p h e r e s , allowing its u s e in a q u e o u s s y s t e m s (31). R

HO

HO OOH

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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A s e c o n d t r a p p i n g s y s t e m w h i c h a l s o a p p e a r s to b e s p e c i f i c u s e s s u i t a b l y s u b s t i t u t e d a n t h r a c e n e s (32. 3 3 ) . A n t h r a c e n e d e r i v a t i v e s a r e c o n s i d e r a b l y m o r e reactive t h a n c h o l e s t e r o l . T h e s e c o m p o u n d s c a n b e m a d e s o l u b l e in a n y m e d i u m by s u i t a b l e c h o i c e of s u b s t i t u e n t s . O n e d r a w b a c k to this s y s t e m is that a n t h r a c e n e s are a l s o p h o t o s e n s i t i z e r s , s o that w h e n s m a l l a m o u n t s of product are f o r m e d , adventitious photooxidation must be carefully ruled out.

A third trapping s y s t e m m a k e s u s e of the fact that p o l y u n s a t u r a t e d fatty a c i d s r e a c t with s i n g l e t o x y g e n to g i v e a mixture of c o n j u g a t e d a n d u n c o n j u g a t e d i s o m e r s of the product h y d r o p e r o x i d e s , w h e r e a s only the c o n j u g a t e d i s o m e r s are f o r m e d o n radical attack (34). T h e u n c o n j u g a t e d p r o d u c t s t h u s s e r v e a s c h a r a c t e r i s t i c s i n g l e t o x y g e n fingerprints. This s y s t e m , like the c h o l e s t e r o l trap, is s o m e w h a t difficult to u s e , s i n c e the i s o m e r s must be s e p a r a t e d by H P L C .

A f u r t h e r s y s t e m is s u g g e s t e d b y C a d e t , w h o h a s i s o l a t e d t h e h y d r o x y l a c t a m s h o w n b e l o w f r o m p h o t o o x i d a t i o n of g u a n o s i n e , a n d h a s s h o w n that this c o m p o u n d c a n be u s e d a s a fingerprint for the p r e s e n c e of s i n g l e t o x y g e n (35.). T h i s c o m p o u n d i s p r o b a b l y t h e p r o d u c t of r e a r r a n g e m e n t of t h e initial p e r o x i d e , w h i c h is not s t a b l e at r o o m temperature.

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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2. FOOTE

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Two other trapping systems are used primarily for kinetic characterization of singlet oxygen; neither is likely to be useful in systems where there is more than one strong oxidant. One is a sensitive system using the production and ESR detection of the nitroxide radical from a tertiary amine (a process whose mechanism and stoichiometry are poorly understood) ( 3 6 ) . The second uses the bleaching of a p-nitrosodimethylaniline on reaction with the peroxide produced by singlet oxygen and histidine as a measure of singlet oxygen production (37). Inhibitors. As mentioned above, many compounds such as carotene, DABCO, and azide, are effective quenchers for singlet oxygen. These compounds, and others which react with singlet oxygen, are frequently used to inhibit reactions in which singlet oxygen is thought to be a reactive intermediate. Care must be taken in interpretation of the results, however, because of their lack of specificity, as discussed above. One way of using inhibitors that partly avoids this problem is to use a quantitative treatment, calculating the amount of singlet oxygen expected to be inhibited from known rate constants and comparing it with that observed ( 2 4 ) . The quantitative kinetic technique cannot be used in inhomogeneous solutions, where the local concentration of the inhibitor cannot be calculated. Dj>0 Effect- Singlet oxygen has a longer lifetime in D2O than in H2O (iL. 22). Thus many reactions of singlet oxygen proceed more efficiently in D2O than in H 0 . However, there are two important limitations to this technique. First, singlet oxygen reactions in the two solvents will differ in efficiency only if solvent quenching of singlet oxygen limits its lifetime; if substrate or quencher is already removing all the 0 2 , there will be no effect of deuteration on the lifetime. Secondly, it has been shown that 0 " also has a longer lifetime in D 0 than in H 0 (2£L), and reactions of superoxide ion would therefore also be expected to be more efficient in the deuterated solvent. The effect of solvent deuteration on other possible reactive species has not been shown. Thus, this effect cannot be used to distinguish between reactions of 0 a n d 0 " . 2

1

2

2

1

2

2

2

" C l e a n " S o u r c e s of Singlet O x y g e n , One useful technique for studying

suspected singlet oxygen reactions is to generate singlet oxygen under carefully defined conditions free of any other reactive species, and compare its effects with those of the susjpect system. Photochemical systems (using unreactive sensitizers, at high 6 pressure, and with low concentrations of substrates that are unreactive in the Type I reaction) can often be used. Another technique is to use a reverse Diels-Alder reaction, using a naphthalene endoperoxide (40); this technique can be used under very mild conditions (37 °C, neutral), and no side reactions have yet been reported. Most other known chemical sources of singlet oxygen {e. g., hypochlorite/H 02, phosphite ozonides (4JJ) involve very strong oxidants which can react with singlet oxygen substrates. 2

2

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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L u m i n e s c e n c e . D i m o l (visible) l u m i n e s c e n c e m a y b e s p e c i f i c for singlet o x y g e n , if the w a v e l e n g t h is carefully m e a s u r e d (42), but c a n not b e easily u s e d to determine the a m o u n t of singlet o x y g e n present, s i n c e it d e p e n d s on a s e c o n d - o r d e r r e a c t i o n b e t w e e n two s i n g l e t o x y g e n m o l e c u l e s . It is e s s e n t i a l that the w a v e l e n g t h of the e m i s s i o n b e carefully d e t e r m i n e d ; in m a n y c a s e s , the s o u r c e of light e m i s s i o n w a s s u b s e q u e n t l y f o u n d to be s o m e t h i n g other than singlet o x y g e n w h e n the w a v e l e n g t h w a s d e t e r m i n e d . D i m o l e m i s s i o n is a l s o difficult to interpret b e c a u s e the extreme sensitivity of photomultipliers a l l o w s the m e a s u r e m e n t of tiny a m o u n t s of light that m a y h a v e little r e l a t i o n s h i p to the m a j o r c h e m i c a l p r o c e s s e s g o i n g o n . T h e infrared l u m i n e s c e n c e of singlet o x y g e n c a n be quantitatively related to the a m o u n t of s i n g l e t o x y g e n p r o d u c e d , a n d c a n l e n d c o n f i d e n c e to its identification if the w a v e l e n g t h is carefully e s t a b l i s h e d (43. 44). A short p u l s e of l a s e r light c a n be u s e d to excite singlet o x y g e n s e n s i t i z e r s , a n d the resulting intensity a n d d e c a y rate of the 1.27^im l u m i n e s c e n c e of singlet o x y g e n c a n b e d e t e c t e d by a g e r m a n i u m p h o t o d i o d e with a lown o i s e a m p l i f i e r a n d a digitizer with s i g n a l a v e r a g i n g ; a s c h e m a t i c of the a p p a r a t u s is s h o w n in F i g . 3 (9. 4 5 . 4 6 ) . T h e a m o u n t of singlet o x y g e n p r o d u c e d a n d its lifetime c a n b e m e a s u r e d very e a s i l y this w a y . T h i s t e c h n i q u e p r o v i d e s a definitive a n d quantitative m e t h o d of c h a r a c t e r i z i n g s i n g l e t o x y g e n p r o d u c e d in p h o t o c h e m i c a l s y s t e m s . F u r t h e r m o r e , by m e a s u r i n g the c h a n g e of lifetime of 0 w h e n a reagent is a d d e d , the rate of its reaction with 0 2 c a n be s i m p l y a n d rapidly d e t e r m i n e d . 1

2

1

T h e y i e l d of singlet o x y g e n p h o t o s e n s i t i z e d by p h o t o d y n a m i c s e n s i t i z e r s c a n b e m e a s u r e d u s i n g this a p p a r a t u s . T h e intensity of the 0 2 l u m i n e s c e n c e is c o m p a r e d with that of a s e n s i t i z e r of k n o w n singlet o x y g e n yield u n d e r c o n d i t i o n s w h e r e the two s e n s i t i z e r s h a v e e q u a l o p t i c a l d e n s i t y . T h e s e v a l u e s are c h e c k e d by m e a s u r i n g the amount of a w e l l - c h a r a c t e r i z e d singlet o x y g e n s u b s t r a t e p h o t o l y z e d in a g i v e n time. W i t h correction for the inefficiency of singlet o x y g e n t r a p p i n g (which c a n be c a l c u l a t e d f r o m the k n o w n rate of reaction of the substrate a n d the d e c a y rate of singlet o x y g e n in the s o l v e n t ) , the a m o u n t of singlet o x y g e n p r o d u c e d in a g i v e n time c a n be c a l c u l a t e d . T h i s v a l u e c a n c o n v e r t e d to a q u a n t u m y i e l d by m e a s u r i n g t h e n u m b e r of q u a n t a a b s o r b e d f r o m t h e l a m p in a g i v e n t i m e by c o n v e n t i o n a l actinometry. T h e infrared l u m i n e s c e n c e determination m e a s u r e s the l o s s of singlet o x y g e n a n d nothing e l s e , s o that it is p o s s i b l e to m e a s u r e a b s o l u t e rates of s i n g l e t o x y g e n r e a c t i o n s with b i o l o g i c a l a c c e p t o r s with c o n f i d e n c e a n d 1

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Mechanisms of Photodynamic Action

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simplicity. T h e sensitivity a n d time r e s p o n s e must be o p t i m i z e d for the timer e s o l v e d s y s t e m to be u s a b l e in a q u e o u s m e d i a , w h e r e the lifetime of singlet o x y g e n is m u c h shorter than in o r g a n i c s o l v e n t s . Transient Absorption Spectroscopy. Transient absorption s p e c t r o s c o p y is useful for m e a s u r i n g m e a s u r i n g the absorption of both radical ions a n d triplet m o l e c u l e s o n a n a n o s e c o n d time s c a l e (47. 48). T h e s e n s i t i z e r is e x c i t e d by a short p u l s e of light, usually from a laser, a n d the a b s o r b a n c e of the transient s p e c i e s m e a s u r e d by a l a m p / p h o t o d e t e c t o r s y s t e m , s h o w n s c h e m a t i c a l l y in F i g . 4. T h i s a p p a r a t u s is u s e f u l for o b s e r v i n g transient intermediates from p h o t o d y n a m i c s e n s i t i z e r s or a c c e p t o r s u n d e r g o i n g T y p e I r e a c t i o n if either the r e d u c e d s e n s i t i z e r o r the o x i d i z e d a c c e p t o r h a s a m e a s u r a b l e a b s o r b a n c e , a s most do. C o n d u c t i v i t y . T i m e - r e s o l v e d c o n d u c t i v i t y m e a s u r e m e n t s h a v e not p r e v i o u s l y b e e n u s e d m u c h in this field, but s h o u l d b e v e r y u s e f u l for the study of electron-transfer T y p e I m e c h a n i s m s . T h e a p p a r a t u s s h o w n in F i g . 5 is w i d e l y u s e d in p u l s e r a d i o l y s i s ( 4 9 ) . F o r p h o t o c h e m i c a l w o r k , t h e s e n s i t i z e r is e x c i t e d by a p u l s e d light s o u r c e , a n d the c h a n g e in conductivity m e a s u r e d a s a f u n c t i o n of t i m e . T h i s a p p a r a t u s c a n b e u s e d o n a m i c r o s e c o n d o r n a n o s e c o n d t i m e s c a l e by s l i g h t m o d i f i c a t i o n s . T h e sensitivity for detection of i o n s is excellent, in fact better than that of optical techniques. Examples T w o e x a m p l e s of m e c h a n i s t i c s t u d i e s o n p h o t o d y n a m i c p e s t i c i d e s that h a v e b e e n s t u d i e d in u n u s u a l detail will be p r e s e n t e d to illustrate the u s e s of s o m e of the t e c h n i q u e s d e s c r i b e d in this article. C e r c o s p o r i n . T h e f u n g a l p i g m e n t c e r c o s p o r i n , the structure of w h i c h is s h o w n b e l o w , a c t s p h o t o d y n a m i c a l l y o n plant t i s s u e s , c a u s i n g electrolyte l e a k a g e a n d o t h e r d a m a g e ; t h e s e effects p r o b a b l y a i d t h e attack of the f u n g u s o n the plant ( 5 0 . 5 1 ) . T h i s pigment c a u s e s lipid p e r o x i d a t i o n in the p r e s e n c e of light a n d o x y g e n , a n d the a c t i o n s p e c t r u m for the d a m a g i n g effects is the s a m e a s the absorption s p e c t r u m of c e r c o s p o r i n (52). OH

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In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

LIGHT-ACTIVATED PESTICIDES

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Digitizer Signal Avg.

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Filters

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Fig. 3. Singlet Oxygen Detection

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In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

2.

FOOTE

35

Mechanisms of Photodynamic Action

T h e p r o d u c t s of p h o t o o x i d a t i o n of o l e i c a n d l i n o l e b a c i d s a n d of c h o l e s t e r o l s e n s i t i z e d by this pigment w e r e identical to t t o s e with singlet o x i d a t i o n (53. 54). T h e oxidation is inhibited by carotenaids a n d D A B C O (52). D a m a g e is a l s o inhibited by v a r i o u s p h e n o l i c antioxidants (53), but this m a y b e c a u s e d by inhibition of r a d i c a l c h a i n a u t o x i d a t b n of the lipids by b r e a k d o w n of the initial p e r o x i d e s . T h e c h a r a c t e r i s t i c 1.27 n.m singlet o x y g e n e m i s s i o r is readily o b s e r v e d w h e n c e r c o s p o r i n s o l u t i o n s in C D are irradiated (59© q u a n t u m yield of singlet o x y g e n is 0 . 8 1 , a s d e t e r m i n e d by comparison with m e s o - p o r p h y r i n IX d i m e t h y l ester. T h i s v a l u e w a s c o n f i r m e d by 2-rrethyl-2-pentene photo oxidation. T h

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6

6

T e r t h i e n y L a - T e r t h i e n y l ( a - T ) is a m e m b e r of a c l a s s of p h o t o d y n a m i c s e n s i t i z e r s , t h e p o l y a c e t y l e n e s , w h i c h a r e present in a n u m b e r of plant s p e c i e s (5£). T h e plants a p p a r e n t l y u s e t h e s * c o m p o u n d s a s to protect a g a i n s t insect attack; after ingesting the m a t e i a l s , i n s e c t s or their o v a are killed p h o t o d y n a m i c a l l y . ( H o w e v e r , the nemacocidal activity o b s e r v e d with this c o m p o u n d is difficult to e x p l a i n o n n e b a s i s of a p h o t o d y n a m i c m e c h a n i s m , b e c a u s e no a p p r e c i a b l e light would be e x p e c t e d to penetrate the soil to the depth of the n e m a t o d e s . ) a - T h a s b e e n s h o w n to kill a wide variety of c e l l s , a n d , a l t h o u g h there h a s b e e n s o m e d i s a g r e e m e n t o n this score, t h e r e is a r e q u i r e m e n t for o x y g e n (57. 58). T h e action s p e c t r u m fcr the d a m a g i n g effects is the s a m e a s the absorption s p e c t r u m of a - T .

T h e m e c h a n i s m of a c t i o n of ;his c o m p o u n d h a s b e e n r e v i e w e d (57). T h e r e is c o n s i d e r a b l e c h e m i c a l evidence that singlet o x y g e n is p r o d u c e d by a - T o n irradiation with n e a r - U V Ight. Inhibition of the effects by inhibitors of o t h e r r e a c t i v e o x y g e n s p e c i e s is not o b s e r v e d , but a v a r i e t y of s i n g l e t o x y g e n q u e n c h e r s p r o t e c t a g a i n s t d e a c t i v a t i o n of e n z y m e s by t h i s c o m p o u n d , a n d t h e r e is a p o s i t i v e D 0 effect o n t h e d e a c t i v a t i o n of 2

e n z y m e s . T h e d i o x e t a n e , a typical singlet o x y g e n product, c a n be f o r m e d by a - T - s e n s i t i z e d photooxidation of a d a m a n t y l i d e n e a d a m a n t a n e . D i f f e r e n c e s b e t w e e n the b i o l o g i c a l activities of a - T a n d the singlet o x y g e n s e n s i t i z e r m e t h y l e n e blue h a v e been o b s e r v e d , but they m a y be d u e to d i f f e r e n c e s in localization b e t w e e n the lipophilic a - T a n d the polar m e t h y l e n e blue. T h e f l u o r e s c e n c e yield of a - T in v a r i o u s s o l v e n t s is l e s s t h a n 0 . 1 , a n d the triplet yield is sub&'antial, o n the order of 0.2. T h e singlet o x y g e n yield in e t h a n o l w a s reported to be b e t w e e n 0.15 a n d 0.2 (58). Singlet o x y g e n production by a - T is o b s e r v e d by 1.27 [im e m i s s i o n (R. K a n n e r a n d C . S . F o o t e , in p r e p a r a t i o n ) . T h e q u a n t u m y i e l d of s i n g l e t o x y g e n production is high in b e n z e n e , a s e s t a b l i s h e d by c o m p a r i s o n of the l u m i n e s c e n c e yield with that of s e v e r a l s e n s i t i z e r s with k n o w n q u a n t u m

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

36

LIGHT-ACTIVATED PESTICIDES

y i e l d s of s i n g l e t o x y g e n production. W e a r e currently attempting to determine this q u a n t u m yi*ld m o r e p r e c i s e l y , but p r e s e n t results s u g g e s t t h e y i e l d is around 0.8. It s not certain w h y o u r results differ from t h o s e of K a g a n , S a n t u s et a l ; the s o l v e n t is different, a n d t h e s e a u t h o r s u s e d a s o m e w h a t i n d i r e c t m e t h o d of d e t e r m i n i n g t h e q u a n t u m y i e l d of s i n g l e t o x y g e n formation, the d i s a p p e a r a n c e of d i p h e n y l i s o b e n z o f u r a n . A very recent p a p e r h a s r e p o r t e d t h e kinglet lifetime of a - T t o b e v e r y s h o r t , a n d h a s c h a r a c t e r i z e d t h e p t o t o p h y s i c a l p r o p e r t i e s of both t h e singlet a n d triplet

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(52). Summary P r o d u c t i o n of singlet oxygen from t h e s e both c e r c o s p o r i n a n d a - T h a s b e e n u n e q u i v o c a l l y demonstrated. S i n c e in both c a s e s , t h e p h y s i o l o g i c a l effects of t h e p h o t o d y n a m i c actio* of both c o m p o u n d s h a v e b e e n s h o w n to b e i n h i b i t e d b y s i n g l e t o x y g e i q u e n c h e r s , b o t h n e c e s s a r y a n d sufficient c o n d i t i o n s for t h e i n t e r m e d k c y of s i n g l e t o x y g e n in t h e a c t i o n of t h e s e c o m p o u n d s a p p e a r to b e present. Acknowledgments T h e original work reported in this p a p e r w a s s u p p o r t e d by grants from t h e NIH a n d N S F .

Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

Straight, R. C.; Spikes, J. D. In Singlet O ; Frimer, A. A. Ed.; C R C : Boca Raton, Fla. 1985; Vol. IV, 85-144. Elstner, E. F. Ann. Rev. Plant Physiol. 1982, 33, 73-96. Gollnick, K. Advan. Photochem. 1968, 6, 1-122. Foote, C. S. Free Radicals in Biology 1976, 2, 85-133. Mattes, S. L . ; Farid, S. Science 1984, 226, 917-21. Mattes, S. L . ; Farid, S. In Organic Photochemistry: Padwa, A. Ed.; Marcel Dekker: New York, 1983; 233-326. Foote, C. S. Tetrahedron 1985. 41, 2221-7. Wilkinson, F . ; Brummer, J. G. J. Phys Chem. Ref. Dat. 1981, 10, 809-1000. Monroe, B. In Singlet O ; Frimer, A. A. E d . ; CRC: Boca Raton, Fla. 1985; Vol. I, pp 177-224. Gollnick, K . ; Kuhn, H. J. In Singlet Oxygen: Wasserman, H. H . ; Murray, R. W . , Eds.; Academic: New York, 1979; pp 287-429. Frimer, A. A. In The Chemistry of Peroxides. Patai. S., E d . ; J. Wiley and Sons:, New York, 1983; Chapter 7. Bartlett, P. D . ; Landis, M. In Singlet Oxygen: Wasserman, H. H . ; Murray, R. W . , Eds.; Academic: New York , 1979; pp 244-86. Ando, W . ; Takata, T. In Singlet O ; Frimer, A. A. E d . ; CRC: Boca Raton, Fla. 1985; Vol. III, pp 1-118. 2

2

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In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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