29 Photochemical Genetics. I. The Ionic Nature of Uracil Photohydration
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J. G. BURR, B. R. GORDON, and E. H. PARK Science Center/Aerospace and Systems Group, North American Rockwell Corporation, Thousand Oaks, Calif. 91360
The rates of photohydration at 265 n.m. of uracil, 1-ethyluracil (EU), 1-cyclohexyluracil (CU), and 1-ribosyluracil (uridine) in oxygen-saturated water are found to be sigmoid functions of the p H over a p H range of 1.5 to 10. The rates of uracil photohydration (10 M solutions) are independent of the concentration of UH and are unaffected by added NaCl. A mechanism is postulated which includes an equi librium between singlet excited uracil molecules and hydro gen ions. The main product forming reaction in the p H range 1.5 to 7 is that between an excited (UH )* and a water molecule. At higher p H values, the reactive species seem to be excited uracil and a water molecule. It is inferred from these data that the pK* of the four excited (UH )-type species are approximately 3.5 for (UH+)*, 6.5 for (EUH )*, and 7.5 for (CUH )*. -4
+
+
+
+
+
T j l t r a v i o l e t i r r a d i a t i o n of aqueous ^
solutions of u r a c i l , cytosine, a n d
t h y m i n e d e r i v a t i v e s has b e e n s h o w n to l e a d to t h e f o r m a t i o n of
t w o k i n d s o f p h o t o p r o d u c t s (4, 13).
O n e t y p e is a d i m e r of the p y r i m i
d i n e ; several s u c h p r o d u c t s seem to b e f o r m e d b u t t h e best k n o w n are those c o n t a i n i n g a c y c l o b u t a n e r i n g system ( s h o w n i n I f o r u r a c i l ) .
Hart; Radiation Chemistry Advances in Chemistry; American Chemical Society: Washington, DC, 1968.
29.
BURR E T A L .
Uracil
419
Photohydration
F o r m a t i o n of s u c h d i m e r s i n t h e n u c l e i c acids of a n o r g a n i s m or v i r u s has b e e n s h o w n to b e a p r o b a b l e cause f o r the i n a c t i v a t i o n of t h e o r g a n i s m (17)
or v i r u s .
T h e i n t e r m e d i a t e responsible f o r f o r m a t i o n of s u c h
d i m e r s , at least i n s i m p l e p y r i m i d i n e s , is p r o b a b l y t h e t r i p l e t excited state of t h e p y r i m i d i n e , o n the basis of the effects of k n o w n t r i p l e t sensitizers a n d q u e n c h e r s (8, 10, 18). T h e other p h o t o p r o d u c t w h i c h is f o r m e d is one r e s u l t i n g f r o m the l i g h t - i n d u c e d a d d i t i o n of w a t e r across the 5,6-double b o n d of t h e p y r i m i d i n e , s h o w n i n I I f o r u r a c i l . T h i s p r o d u c t w i l l b e d e n o t e d i n this p a p e r b y t h e t e r m " p h o t o h y d r a t e . " L i t t l e has b e e n r e p o r t e d a b o u t the m e c h a
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n i s m of this r e a c t i o n , or a b o u t t h e n a t u r e of t h e responsible i n t e r m e d i a t e (4).
I t is the p u r p o s e of this p a p e r to r e p o r t some observations
about
the v a r i a t i o n w i t h p H a n d salt c o n c e n t r a t i o n of t h e rate of t h e p h o t o hydration
process
i n four uracil derivatives—(1)
(2) 1-ethyluracil ( E U , R =
uracil ( R =
H),
ethyl), (3) 1-cyclohexyluracil ( C U , R
=
cyclohexyl), and (4) uridine ( R = ribosyl). Experimental Materials. U r a c i l ( U ) , 1-ethyluracil ( E U ) , 1-cyclohexyluracil ( C U ) , and 1,3-dimethyluracil ( D M U ) were obtained from C y c l o C h e m i c a l C o r p o r a t i o n ; u r i d i n e w a s f r o m C a l b i o c h e m . T h e y w e r e u s e d as o b t a i n e d ; i n w o r k i n g w i t h t h e m , n o i m p u r i t i e s w e r e apparent, s p e c t r o s c o p i c a l l y or c h r o m a t o g r a p h i c a l l y . Solutions f o r p h o t o l y s i s at a p a r t i c u l a r p H w e r e p r e p a r e d b y d i l u t i n g a 1 0 ~ M t r i p l e - d i s t i l l e d w a t e r stock s o l u t i o n o f t h e u r a c i l d e r i v a t i v e t e n f o l d w i t h t h e a p p r o p r i a t e p h o s p h a t e buffer. C o n centrations w e r e d e t e r m i n e d b y a b s o r b a n c e measurement, u s i n g a P e r k i n E l m e r M o d e l 202 S p e c t r o p h o t o m e t e r . T h e values of A , i n n . m . , a n d e m e a s u r e d f o r t h e f o u r substances w e r e : u r a c i l : 259, 8.6 X 1 0 ; 1-ethyl u r a c i l : 267, 9 X 1 0 ; 1 - c y c l o h e x y l u r a c i l : 270, 9 X 1 0 ; u r i d i n e : 265, 10 X 10 . O p t i c s . T h e l i g h t source u s e d w a s a 1000 w a t t G E B H 6 or F H 6 , aircooled mercury arc supported vertically i n a modified O r i o n Optics l a m p h o l d e r . T h e l i g h t w a s passed t h r o u g h a B & L H i g h Intensity m o n o c h r o m a t o r . E x a m i n a t i o n w i t h a 500 m m . B & L m o n o c h r o m a t o r a n d p h o t o c e l l of t h e l i g h t e m i t t e d f r o m the H i g h Intensity m o n o c h r o m a t o r s h o w e d that scattered l i g h t w a s n e g l i g i b l e a n d that at a m o n o c h r o m a t o r setting of 270 n . m . t h e t r a n s m i t t e d l i g h t a c t u a l l y p e a k e d at 265 n . m . , w i t h a w i d t h at h a l f - h e i g h t of 9 n . m . T h e h a l f - w i d t h s of the a b s o r p t i o n peaks w e r e w i d e r — e . g . , 1 0 ~ M u r a c i l ( A = 1) h a d a p e a k centered at 259 n . m . w i t h a h a l f - w i d t h of 33 n . m . T w o different l a m p - m o n o c h r o m a t o r assem blies w e r e u s e d ; i t w a s f o u n d , b y m e t h o d s d i s c u s s e d b e l o w , that t h e f r a c t i o n of l i g h t a b s o r b e d b y t h e same d i m e t h y l u r a c i l s o l u t i o n w a s d i f ferent f o r e a c h assembly. 3
m a x
3
3
3
3
4
A c t i n o m e t r y . T h e l i g h t o u t p u t ( Z ) of e a c h l a m p - m o n o c h r o m a t o r assembly w a s m o n i t o r e d ( a t least d a i l y ) b y m e a s u r i n g t h e A A ( A A == a b s o r b a n c e c h a n g e f o r 15 m i n u t e s i r r a d i a t i o n ) of a s t a n d a r d d i m e t h y l u r a c i l s o l u t i o n ( A = 0.925, C = 1.075 X 1 0 " M ) . T h e s e A A i values 0
i 5
0
4
Hart; Radiation Chemistry Advances in Chemistry; American Chemical Society: Washington, DC, 1968.
i 5
5
420
RADIATION CHEMISTRY
1
w e r e c o n v e r t e d to n u m e r i c a l estimates of I i n q u a n t a l i t e r m i n . , b y use of a r e m e a s u r e d v a l u e f o r t h e q u a n t u m y i e l d f o r p h o t o h y d r a t i o n of d i m e t h y l u r a c i l . R e m e a s u r e m e n t of this v a l u e r e s o l v e d a n existing a n o m a l y i n t h e literature of p y r i m i d i n e p h o t o c h e m i s t r y . It h a s - b e e n r e p o r t e d (4, 13) that t h e q u a n t u m y i e l d s f o r p h o t o l y s i s of several u r a c i l d e r i v a t i v e s , i n c l u d i n g that of d i m e t h y l u r a c i l , w h i c h is k n o w n at these values of t o t a l dose to f o r m o n l y the p h o t o h y d r a t e , v a r i e d w i t h c o n c e n t r a t i o n t o such a n extent that t h e a p p a r e n t q u a n t u m y i e l d f o r d i m e t h y l u r a c i l v a r i e d d u r i n g t h e course o f a p h o t o l y s i s . T h e s e a p p a r e n t variations i n t h e o r d e r of a s u p p o s e d l y first order process h a v e e l i c i t e d attempts at m e c h a n i s t i c i n t e r p r e t a t i o n (13). - 1
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0
- 1
A c c o r d i n g l y , w e d e t e r m i n e d the q u a n t u m y i e l d of t h e p h o t o h y d r a t e of 1 , 3 - d i m e t h y l u r a c i l ( D M U ) f o r i n i t i a l D M U concentrations of 1 X 1 0 M , a n d 1 X 1 0 " M i n u n b u f f e r e d t r i p l e - d i s t i l l e d w a t e r . T h e measure ments w e r e m a d e a c c o r d i n g to the c o n v e n t i o n a l d o u b l e c e l l t e c h n i q u e ( 1 2 ) , u s i n g u r a n y l oxalate as t h e actinometer. I n this m e t h o d , t h e t o t a l n u m b e r of i n c i d e n t a n d t r a n s m i t t e d q u a n t a are m e a s u r e d b y c h e m i c a l actinometry. T h e quantum yield for D M U disappearance was f o u n d to be 3.93 X M T at 1 X 1 0 " M D M U a n d 3.79 X 10" at 1 X 1 0 " M D M U ( s i n g l e d e t e r m i n a t i o n s ) , a n d is thus i n d e p e n d e n t of c o n c e n t r a t i o n i n this range. _ 3
4
3
4
3
3
T h e a b s o r b a n c e change d u r i n g p h o t o l y s i s of t h e 1 0 " M s o l u t i o n w a s f r o m 1.0 to 0.5. T h e q u a n t u m y i e l d c a l c u l a t e d f r o m this c o n c e n t r a t i o n change, u s i n g t h e c o n v e n t i o n a l expression, I = J ( 1 — 10" ), to esti m a t e the n u m b e r of q u a n t a a b s o r b e d w a s i n d e e d f o u n d to v a r y d u r i n g the course of a r u n , a n d thus to v a r y w i t h c o n c e n t r a t i o n . H o w e v e r , the a c t u a l f r a c t i o n of J a b s o r b e d i n the s o l u t i o n d u r i n g t h e r u n w a s m e a s u r e d b y c h e m i c a l a c t i n o m e t r y to b e 0.31; that estimated b y the e x p o n e n t i a l expression w a s a b o u t 0.8. T h i s d i s c r e p a n c y is caused, o f course, b y t h e f a c t that t h e s i m p l e e x p o n e n t i a l expression is v a l i d o n l y i f I is m o n o c h r o m a t i c ; t h e a c t u a l h a l f - h e i g h t b e a m w i d t h w a s about 9 n . m . T h e a m o u n t of l i g h t a b s o r b e d is thus expressed b y t h e i n t e g r a l e q u a t i o n 4
a
A
0
0
Q
I t= a
J
P
2
Ai J
C
h
h
I (A)[l-10-^ v
p
a
-dC/dt=
