Photochemical Genetics. II. The Kinetic Role of Water in the

30 Photochemical Genetics. II. The Kinetic Role of Water in the Photohydration of

Downloaded by UNIV LAVAL on July 14, 2016 | http://pubs.acs.org Publication Date: January 1, 1968 | doi: 10.1021/ba-1968-0081.ch030

Uracil and 1,3-Dimethyluracil J. G. BURR and E. H. PARK Science Center/Aerospace and Systems Group, North American Rockwell Corporation, Thousand Oaks, Calif. 91360

The rate of photohydration of uracil with light of 265 n.m. in mixtures of acetonitrile and water is a linear function of the water content of the mixture; the deuterium isotope effect, R /R , is 1.18. These observations are shown to be consistent with the mechanism for uracil photohydrate formation proposed to explain the p H dependence of the rates. The rate of photohydrate formation for 1,3-dimethyuracil is approximately proportional to the square of the water concentration in acetonitrile-H O mixtures, acetonitrile-D O, and in dioxane-water mixtures, R /R = 2.84. These data are also interpretable in terms of a common photohydration mechanism. H2O

D2O

2

2

H2o

D2o

he effect of p H changes a n d n e u t r a l salt c o n c e n t r a t i o n u p o n t h e rate A

of u r a c i l p h o t o h y d r a t i o n has b e e n r e p o r t e d (together w i t h s i m i l a r

d a t a f o r some 1 - a l k y l u r a c i l s a n d u r i d i n e ) i n t h e first p a p e r of the series (3).

It w a s c o n c l u d e d f r o m those observations that t h e p r o d u c t - f o r m i n g

reaction was one between a n excited ( U H ) * , formed b y e q u i l i b r i u m +

p r o t o n a t i o n of singlet e x c i t e d u r a c i l , a n d a n e u t r a l w a t e r species. T h e m o l e c u l a r i t y of this n e u t r a l w a t e r species has n o w b e e n e x a m i n e d b y m e a s u r i n g the rate of u r a c i l p h o t o h y d r a t i o n i n mixtures of acetonitrile a n d w a t e r , a n d acetonitrile a n d D 0 . P h o t o h y d r a t e f o r m a t i o n i n 1,32

d i m e t h y l u r a c i l ( D M U ) i n s i m i l a r mixtures, as w e l l as i n mixtures of d i o x a n e a n d w a t e r has also b e e n s t u d i e d . T h e results of these studies are r e p o r t e d i n this p a p e r . 435 Hart; Radiation Chemistry Advances in Chemistry; American Chemical Society: Washington, DC, 1968.

436


Materials

RADIATION CHEMISTRY

and

Sample

1

Preparation

D i o x a n e f r o m a v a r i e t y of m a n u f a c t u r e r s a n d of v a r i o u s degrees of p u r i t y w e r e a l l unsatisfactory w i t h o u t extensive p u r i f i c a t i o n ; the measure of q u a l i t y was a sharp u l t r a v i o l e t cutoff at 203 n . m . a n d a l o w a n d r e p r o d u c i b l e rate of D M U p h o t o l y s i s i n the neat solvent. T h i s c o u l d o n l y be a c h i e v e d i n d i o x a n e f r a c t i o n a t e d i n a n i t r o g e n atmosphere five times f r o m sodium through a ten plate packed c o l u m n a n d distilled immediately i n t o flask c o n t a i n i n g the p y r i m i d i n e . D i o x a n e w a s not c o n s i d e r e d a satisfactory solvent. Acetonitrile ( E a s t m a n Spectrograde) was used without further p u r i f i c a t i o n , since f r a c t i o n a l d i s t i l l a t i o n h a d little effect o n the s p e c t r u m of the solvent or u p o n t h e rate of p y r i m i d i n e p h o t o l y s i s i n the neat solvent. Some photolyses w e r e a t t e m p t e d w i t h bis ( d i m e t h o x y e t h y l ) ether a n d w i t h t e t r a h y d r o f u r a n , b u t these w e r e even m o r e unsatisfactory t h a n d i o x a n e a n d no r e l i a b l e q u a n t i t a t i v e measurements of p y r i m i d i n e p h o tolysis rates w e r e o b t a i n e d . U r a c i l a n d 1 , 3 - d i m e t h y l u r a c i l w e r e C y c l o C h e m i c a l C o m p a n y stock. Solutions w e r e p r e p a r e d i n the neat solvents at 1 0 ~ M , a n d d i l u t e d w i t h a d d i t i o n a l solvent to m a k e 1 0 ~ M solutions; these w e r e t h e n d i l u t e d w i t h the a p p r o p r i a t e a m o u n t of w a t e r . T h e densities of b o t h d i o x a n e - w a t e r a n d a c e t o n i t r i l e - w a t e r s h o w slight ( 4 % ) d e v i a t i o n s f r o m i d e a l i t y (12, 13), so that m i n o r corrections w e r e necessary i n c a l c u l a t i n g p y r i m i d i n e concentrations. B o t h d i o x a n e a n d acetonitrile are w e a k e r bases t h a n w a t e r ( a c e t o n i t r i l e is m u c h w e a k e r ) (8); acetonitrile has a h i g h d i e l e c t r i c constant (10) of 37.5 a n d d i o x a n e a l o w one of 2.209. If d i l u t i o n w e r e s i m p l y the o n l y f a c t o r the p H of 5 . 6 M w a t e r i n acetonitrile s h o u l d b e 8; a c t u a l l y the p H of u n b u f f e r e d o x y g e n - b u b b l e d w a t e r was 6.3 a n d that of o x y g e n - b u b b l e d 5 . 6 M (10 V o l . % ) w a t e r i n acetonitrile w a s 5 ( B e c k m a n p H M e t e r ) . T h i s a d m i t t e d l y c r u d e measurement is t a k e n o n l y to i n d i c a t e that large changes i n h y d r o g e n i o n a v a i l a b i l i t y d o not o c c u r w h e n w a t e r is d i l u t e d to this extent b y acetonitrile. T h e p r o p e r v a l u e w o u l d b e p a , as d e f i n e d b y R . G . Bates (2), b u t the necessary d a t a are not a v a i l a b l e f o r m i x t u r e s of acetonitrile a n d w a t e r . W e s h a l l assume that the p H changes i n the a c e t o n i t r i l e - w a t e r a n d i n the d i o x a n e - w a t e r system are insufficient to affect p h o t o h y d r a t i o n rates ( 3 ) . 3

4

H

Irradiations. T h e source of l i g h t w a s a 1000 w a t t G E B H 6 h i g h pressure m e r c u r y arc, filtered t h r o u g h a B a u s c h a n d L o m b H i g h Intensity M o n o c h r o m a t o r . D e t a i l s of this a p p a r a t u s a n d i r r a d i a t i o n p r o c e d u r e s are d e s c r i b e d i n the p r e v i o u s p a p e r (3). Product Analysis. A n a l y s i s of the p h o t o l y z e d solutions for p h o t o h y d r a t e content w a s c a r r i e d out b y a c i d i f i c a t i o n a n d h e a t i n g , as d e s c r i b e d p r e v i o u s l y (3). T h e f r a c t i o n of p h o t o h y d r a t e f o r m e d i n p h o t o l y s i s of D M U a n d u r a c i l i n air-saturated a c e t o n i t r i l e - w a t e r a n d a c e t o n i t r i l e - D 0 solutions thus d e t e r m i n e d is s h o w n i n F i g u r e 1. S u b s t a n t i a l a m o u n t s of p r o d u c t s , r e p o r t e d as d i m e r s ( 9 ) , are f o r m e d f r o m u r a c i l a n d t h y m i n e i n a c e t o n i t r i l e — t h a t f r o m t h y m i n e d i f f e r i n g f r o m the t h y m i n e d i m e r p r o d u c e d i n D N A or f r o z e n t h y m i n e solutions ( 9 ) . H o w e v e r , the rates of p h o t o l y s i s of u r a c i l , t h y m i n e a n d D M U i n neat acetonitrile f o l l o w first 2

Hart; Radiation Chemistry Advances in Chemistry; American Chemical Society: Washington, DC, 1968.


30.

BURR A N D PARK

Photochemical

437

Genetics

Figure 1. The fraction of photohydrate formed in mixtures of acetonitrile and H 0 or D 0; initial pyrimidine concentrations 10~ M 2

2

4

order kinetics, F i g u r e 2, a g r e e i n g w i t h the observations of L a m o l a a n d M i t t a l ( 9 ) . F i r s t o r d e r k i n e t i c s f o r d i m e r f o r m a t i o n i n acetonitrile are difficult to u n d e r s t a n d since i n o u r photolysis system p h o t o h y d r a t e f o r m a t i o n i n u r a c i l f o l l o w e d s i m i l i a r first o r d e r k i n e t i c s ; d i m e r f o r m a t i o n thus s h o u l d b e i n i t i a l l y at least second order i n p y r i m i d i n e c o n c e n t r a t i o n as o b s e r v e d b y S z t u m p f - K u l i k o w s k a et al, (11) f o r orotic a c i d d i m e r i z a t i o n , a n d b y us f o r u r a c i l d i m e r i z a t i o n i n w e l l degassed aqueous s o l u t i o n ( 3 ) . G r e e n s t o c k et al, i n d i c a t e that p r o d u c t s other t h a n d i m e r s a r e f o r m e d i n acetonitrile (7); w e h a v e some i n d i c a t i o n that acetonitrile m a y be r e a c t i n g w i t h the excited p y r i m i d i n e m o l e c u l e s u n d e r c e r t a i n c o n d i tions. W e are c o n t i n u i n g o u r i n v e s t i g a t i o n of the n a t u r e of these p r o d u c t s , w i t h the t h o u g h t that t h e y m a y not b e the p h o t o d i m e r . T h e nature of the p r o d u c t s f o r m e d f r o m D M U i n d i o x a n e w i t h less than 4 0 % water were complicated. Products were formed i n pure d i oxane w h i c h o n a c i d i f i c a t i o n a n d h e a t i n g r e v e r t e d to D M U b u t w h i c h d i d not have the same R as D M U o n p a p e r c h r o m a t o g r a m s . T h e s e p r o d u c t s persisted u p to about 30 to 4 0 % w a t e r ; above this w a t e r c o n centration, the p h o t o p r o d u c t w a s e n t i r e l y the k n o w n p h o t o h y d r a t e . It is p r o b a b l e that c a r b i n o l s a n d peroxides present i n the d i o x a n e w e r e react i n g w i t h the excited D M U molecules, since c a r b i n o l s a n d other n e u t r a l n u c l e o p h i l e s are k n o w n to b e c a p a b l e of s u c h reactions (4). f

Calculation of Rates.

T h e relative rates of p y r i m i d i n e p h o t o l y s i s

i n these solvent m i x t u r e s g i v e n i n F i g u r e s 3 a n d 4 w e r e o b t a i n e d b y


438

RADIATION

CHEMISTRY

1

o b s e r v i n g t h e a b s o r b a n c e changes f o r a s t a n d a r d photolysis t i m e ( u s u a l l y 20 m i n u t e s ) , after i t w a s d e t e r m i n e d that t h e reactions w e r e first o r d e r i n p y r i m i d i n e — i . e . , plots of l o g A /A Q

values of A A coefficient. X

10

2 0

2 0

were linear w i t h time.

These

w e r e c o n v e r t e d to rates, u s i n g k n o w n values of the e x t i n c t i o n

T h e rates w e r e n o r m a l i z e d t o a s t a n d a r d v a l u e of I

quanta liter

- 1

G

== 5.36

m i n . " , as i n t h e p r e v i o u s s t u d y ( 3 ) . N o r m a l i z e d 1

s t a n d a r d rates of p h o t o h y d r a t e f o r m a t i o n w e r e t h e n o b t a i n e d b y m u l t i p l y i n g t h e photolysis rate at e a c h solvent c o m p o s i t i o n b y t h e f r a c t i o n of p h o t o h y d r a t e f o r m e d i n a m i x t u r e of that c o m p o s i t i o n , t a k e n f r o m F i g u r e 1. Rates of D M U p h o t o h y d r a t e f o r m a t i o n i n t h e r e g i o n 0 to 4 0 % w a t e r


c o u l d o n l y b e estimated f r o m t h e p a p e r c h r o m a t o g r a m s ; t h e v a r i a t i o n of p h o t o h y d r a t e f o r m a t i o n rate w a s a p p r o x i m a t e l y l i n e a r w i t h w a t e r c o n c e n t r a t i o n i n this range.

A s c a n b e seen f r o m F i g u r e 4, some p r o d u c t

y i e l d u n c e r t a i n t y i n this r e g i o n o f w a t e r c o n c e n t r a t i o n d i d n o t h a v e m u c h effect o n t h e final result.

0

10

20

30

40

50

60

70

Minutes

Figure 2. Rates of photolysis of several pyrimidines in neat acetonitrile; initial pyrimidine concentrations about 10 *M Results and Discussion T h e rate of u r a c i l p h o t o h y d r a t e f o r m a t i o n is a l i n e a r f u n c t i o n of t h e H 0 o r t h e D 0 c o n c e n t r a t i o n i n acetonitrile, F i g u r e 3. T h i s v a r i a t i o n i n rate is n o t c a u s e d b y a v a r i a t i o n i n h y d r o g e n ion—i.e., s o l v a t e d h y d r o g e n i o n — b e c a u s e t h e estimated v a r i a t i o n i n p H over this solvent range ( 1 2

2


30.

BURR AND PARK

Fhotochemicol

—I

I —

1

— I

50 Volume

— I — 100

f—

60 HgO

70

R~

1

40

m

1

40 50 Volume %

20

439

Genetics

60 %

70

80

D 0 2

O

Uracil in Acetonitrile - H 0

•

Uracil in Acetonitrile - D 0

2

2

2


o

20

30

60

40

W , Moles per Liter

Figure 3. Rates of photohydration of uracil (10~*M) in mixtures of acetonitrile and H 0 or D 0. The rates are all normalized to l = 5.36 X 10 quanta liter' min.' 2

0

2

20

1

1

u n i t ) is insufficient to cause s u c h a rate v a r i a t i o n ( 3 ) ; f u r t h e r m o r e , the t r e n d is opposite to that w h i c h w o u l d be p r e d i c t e d b y the p H v a r i a t i o n . T h e l i n e a r i t y a n d d i r e c t i o n of the t r e n d i n rate is not i n c o n f o r m i t y w i t h trends p r e d i c t e d for variations of c a t i o n - d i p o l e r e a c t i o n rates or d i p o l e d i p o l e r e a c t i o n rates w i t h v a r i a t i o n of d i e l e c t r i c strength.

T h e fastest

p r o d u c t - f o r m i n g step i n u r a c i l p h o t o h y d r a t i o n was s h o w n i n the

first

p a p e r of this series ( 3 ) to b e p r o p o r t i o n a l to the c o n c e n t r a t i o n of e x c i t e d p r o t o n a t e d u r a c i l ( U H ) * a n d a n e u t r a l w a t e r species. +

T h e rate c o n

stants of reactions b e t w e e n cations a n d n e u t r a l d i p o l a r molecules s h o u l d increase

as the d i e l e c t r i c constant of the m e d i u m decreases ( I ) .

The

effects of d i e l e c t r i c constant o n reactions b e t w e e n t w o n e u t r a l d i p o l a r m o l e c u l e s — i . e . , b e t w e e n excited u r a c i l , U * , a n d a n e u t r a l w a t e r s p e c i e s — are d i f f i c u l t to d i s t i n g u i s h f r o m other solvent a n d s t r u c t u r a l effects, b u t i f this c a n be done, t h e n the l o g of the rate constant s h o u l d b e i n v e r s e l y p r o p o r t i o n a l to the d i e l e c t r i c constant

of the m e d i u m ( I ) .

the v a r i a t i o n of the p h o t o h y d r a t i o n rate of D M U w i t h w a t e r

However, concentra-


440

RADIATION CHEMISTRY

1

t i o n ( F i g u r e 4 ) is the same i n mixtures of d i o x a n e - w a t e r , D == 2.21-78.54, as i t is i n m i x t u r e s of acetonitrile a n d w a t e r , D = 37.5-78.54. T h i s c o i n c i dence

also excludes m a n y other properties of the m i x e d solvents as

sources of the rate v a r i a t i o n s . T h e rate trends i n d i c a t e d i n F i g u r e s 3 a n d 4 m a y b e u s e d w i t h g o o d assurance, therefore, to d e t e r m i n e the m o l e c u l a r i t y of the n e u t r a l w a t e r species c o n c e r n e d i n t h e p h o t o h y d r a t i o n reactions.

T h e linear variation

of u r a c i l p h o t o h y d r a t i o n rate w i t h w a t e r c o n c e n t r a t i o n indicates that this r e a c t i o n is first o r d e r i n w a t e r c o n c e n t r a t i o n .

T h e p r o d u c t f o r m i n g step

i n this r e a c t i o n c a n n o w b e w r i t t e n w i t h assurance, e m p l o y i n g the nota


t i o n f r o m o u r first p a p e r ( 3 ) , as E q u a t i o n 1. (dU )/dt

fc,(UH )*(H 0)

=

mo

+

(1)

2

Figure 4. Rates of photohydration of DMU (10~ M) in mixtures of water with dioxane or acetonitrile and in mixtures of acetonitrile and D 0; I = 5.36 X 10 quanta liter' min' 4

2

0

20

1

1


30.

BURR A N D PARK

Photochemical

441

Genetics

T h i s c o n c l u s i o n is r e i n f o r c e d b y the d i r e c t i o n of t h e d e u t e r i u m iso tope effects f o r p h o t o h y d r a t e f o r m a t i o n i n b o t h u r a c i l a n d D M U :

fc An H

is 1.18 f o r u r a c i l p h o t o h y d r a t i o n a n d 2.8 f o r D M U p h o t o h y d r a t i o n ( F i g ures 3 a n d 4 ) . T h e f a c t that

fc An

is greater t h a n 1.0 f o r u r a c i l p h o t o

H

h y d r a t i o n is significant because i n that photolysis t h e p r i n c i p a l p r o d u c t forming

reaction

rium between

mechanism

was shown

(2)

to i n c l u d e

excited uracil a n d hydrogen ion.

a equilib

T h i s is n o w s h o w n

U * + H ^± ( U H ) * +

(UH )* + H +

2

+

O ^ U

H

2

+ H

0

+


to b e f o l l o w e d b y r e a c t i o n b e t w e e n the e x c i t e d p r o t o n a t e d u r a c i l a n d H 0.

Ionic

2

reactions

such

as this

i n w h i c h the water

is i n v o l v e d

o n l y i n e q u i l i b r i u m p r o t o n a t i o n of a base, a n d i n w h i c h t h e p r o d u c t for m a t i o n f r o m the p r o t o n a t e d base is r e l a t i v e l y s l o w e r t h a n t h e p r o t o n a t i o n —i.e., a c i d - c a t a l y z e d reactions w i t h a " p r e - e q u i l i b r i u m , " g e n e r a l l y a r e faster i n D 0 t h a n i n H 0 because D 0 is a better p r o t o n d o n o r t h a n H 0 2

(pK

I ) 2 0

=

2

14.955, p K

H 2

2

o =

p r o t o n a t e d base is thus h i g h e r i n D (5)—i.e., k /k H

B

2

13.997 at 2 5 ° C . ) 2

( 6 ) ; the concentration of

0 t h a n i n H 0 , a n d t h e rate is faster 2

is less t h a n 1.0. T h e m a g n i t u d e of t h e isotope effects i n

these photolyses is, h o w e v e r , characteristic of a k i n e t i c isotope effect i n w h i c h m o l e c u l a r w a t e r w o u l d b e i n v o l v e d i n t h e p r o d u c t - f o r m i n g step. T h e m a g n i t u d e of the v a l u e f o r u r a c i l is that of a secondary isotope effect, a n d m a g n i t u d e f o r D M U is that n o r m a l l y f o u n d i n a p r i m a r y isotope effect. T h e rate of p h o t o h y d r a t i o n of D M U i n either

dioxane-water or

acetonitrile-water is, h o w e v e r , n o t l i n e a r i n t h e w a t e r c o n c e n t r a t i o n b u t m o r e n e a r l y p r o p o r t i o n a l to t h e square of t h e w a t e r c o n c e n t r a t i o n .

It

s h o u l d b e s a i d at once that t h e reasons f o r t h e differences b e t w e e n t h e k i n e t i c b e h a v i o r of u r a c i l a n d D M U i n these solvent mixtures is n o t c o m p l e t e l y u n d e r s t o o d , a n d m a y e v e n t u a l l y b e f o u n d to reflect s i m p l y u n d i s c o v e r e d features of photolyses i n s u c h m i x e d solvent systems. nevertheless

I t is

i n s t r u c t i v e to examine these differences i n t h e l i g h t o f t h e a U

l

U *

b 0 !U* -» U(+ U* -» U) 2

3

c *U* + H ^ d +

(UH )* +

e ( U H ) * -> U + H +

f ( U H ) * + H 0 -> U +

2

H 2 G

+

+H

+


442

RADIATION CHEMISTRY

1

r e a c t i o n m e c h a n i s m w h i c h has b e e n p r o p o s e d f o r u r a c i l p h o t o h y d r a t i o n (3),

c o n s i s t i n g o f r e a c t i o n steps ( a ) - ( f ) .

T h e o v e r a l l rate of p h o t o

h y d r a t i o n , w r i t t e n as E q u a t i o n 2 i n t h e earlier p a p e r , c a n b e r e w r i t t e n as E q u a t i o n 3. (H )

k W

Photochemical Genetics. II. The Kinetic Role of Water in the

Recommend Documents