Preparation of Tritium-Labeled Dibenzo-p-dioxin and 2,3,7,8

2

Preparation

of

Tritium-Labeled

Dibenzo-p-dioxin and

Downloaded by GEORGETOWN UNIV on August 29, 2015 | http://pubs.acs.org Publication Date: March 1, 1973 | doi: 10.1021/ba-1973-0120.ch002

2,3,7,8-Tetrachlorodibenzo-p-dioxin

J. H O W A R D

VINOPAL,

1

IZURU Y A M A M O T O ,

2

and J O H N E . CASIDA

Division of Entomology, University of California, Berkeley, Calif. 94720

1,6-Ditritiodibenzo-p-dioxin,

prepared by reductive dechlo-

rination of 1,6-dichlorodibenzo-p-dioxin chlorinated

in

chloroform

solution,

with tritium gas, is containing

catalytic

amounts of iodine and ferric chloride, to produce 1,6-ditritio2,3,7,8-tetrachlorodibenzo-p-dioxin.

'Tphe A

h i g h l y toxic

a n d teratogenic

2,3,7,8-tetrachlorodibenzo-p-dioxin

( 2 , 3 , 7 , 8 - C L r D B p D ) m a y o c c u r i n trace amounts i n the h e r b i c i d e , 2,4,5-

t r i c h l o r o p h e n o x y a c e t i c a c i d ( 2 , 4 , 5 - T ) (1, 2).

Radiolabeled preparations

of this d i o x i n are n e e d e d to f a c i l i t a t e studies of its d e g r a d a t i o n c h e m i s t r y , m e t a b o l i s m , a n d m o d e of a c t i o n . M u e l d e r a n d Shadoff ( 3 ) p r e p a r e d mmole)

b y c h l o r i n a t i o n of

potassium

14

14

1 4

C - 2 , 3 , 7 , 8 - C l - D B p D (0.9 m C i / 4

C-2,7-dichlorodibenzo-p-dioxin made from

C-2,4-dichlorophenate.

T h e p r e p a r a t i o n of t r i t i u m - l a b e l e d

2 , 3 , 7 , 8 - C l - D B p D is justified because the r a d i o l a b e l e d intermediates are 4

less expensive a n d m o r e accessible a n d because a h i g h e r specific a c t i v i t y is p o t e n t i a l l y attainable.

H e r e , w e consider t h e o p t i m a l c o n d i t i o n s f o r

the r e a c t i o n sequence d e s i g n e d to o b t a i n p r o d u c t s of h i g h c h e m i c a l a n d r a d i o c h e m i c a l p u r i t y s h o w n at t h e t o p of p . 8. Experimental Designation, Chromatographic Separation, and Analysis. T h e a b b r e v i a t i o n D B p D is u s e d f o r d i b e n z o - p - d i o x i n , a n d the positions of c h l o r i n e ' Present address: Entomological Sciences and Pesticide Division, U.S. Army Environmental Hygiene Agency, Edgewood Arsenal, Md. 21010. Present address: Department of Agricultural Chemistry, Tokyo University of Agriculture, Setagaya-ku, Tokyo, Japan. 2

7

In Chlorodioxins—Origin and Fate; Blair, E.; Advances in Chemistry; American Chemical Society: Washington, DC, 1973.

8

CHLORODIOXINS

ci

isopropanol

l,6-Cl -DBpD

FATE

l,6-T -2,3J,8-Cl -DBpD

2

2

4

( C I ) or t r i t i u m ( T ) i n its d e r i v a t i v e s are i n d i c a t e d b y a p p r o p r i a t e designations—e.g., l , 6 - C l - D B p D , l , 6 - T - D B p D , a n d l,6-T -2,3,7,8-Cl DBpD. T h e c o m p o s i t i o n a n d p u r i t y of the u n l a b e l e d r e a c t i o n p r o d u c t s w e r e d e t e r m i n e d b y g a s - l i q u i d c h r o m a t o g r a p h y ( G L C ), u s i n g a flame i o n i z a t i o n detector u n d e r the c o n d i t i o n s g i v e n i n T a b l e I. R a d i o — G L C analysis of the l a b e l e d p r o d u c t s w a s m a d e b y the Z o e c o n C o r p . ( P a l o A l t o , C a l i f . ), u s i n g a B a r b e r - C o l e m a n i n s t r u m e n t ( m o d e l Selecta S y s t e m , series 5000; R o c k f o r d , 111.) a n d a glass c o l u m n (1 meter X 3 m m ) c o n t a i n i n g S E - 3 0 ( 3 . 8 % , w / w ) o n C h r o m o s o r b W (80/100 m e s h ) . A set of c o n d i t i o n s ( i n j e c t i o n a n d c o l u m n t e m p e r a t u r e , 1 1 0 ° C ; p r o g r a m m e d for 3 ° C increase i n t e m p e r a t u r e / m i n u t e ; H e , 60 m l / m i n ) , w h i c h separate D B p D , 1-C1D B p D , a n d l , 6 - C l - D B p D a n d another set of c o n d i t i o n s ( i n j e c t i o n a n d c o l u m n temperature, 2 2 0 ° C ; H e * , 60 m l / m i n ) , w h i c h separate C l - D B p D , 2 , 3 , 7 , 8 - C l - D B p D , a n d C l - D B p D , were used for r a d i o - G L C . T h i n layer c h r o m a t o g r a p h y ( T L C ) o n E a s t m a n 6061 s i l i c a g e l c h r o m a g r a m sheets (0.1 m m g e l t h i c k n e s s ) is u s e f u l i n s e p a r a t i n g the D B p D d e r i v a t i v e s ( h i g h R ) f r o m the c o r r e s p o n d i n g p h e n o l s ( l o w R ) b u t not f r o m each other be cause the R values are the same for the D B p D d e r i v a t i v e s ( D B p D , 1 - C l - D B p D , l , 6 - C l - D B p D , 2 , 7 - C l - D B p D , 2 , 3 , 7 , 8 - C l - D B p D ). T h e R values f o u n d w i t h e a c h of these d e r i v a t i v e s are 0.54-0.55 o n d e v e l o p m e n t w i t h c a r b o n t e t r a c h l o r i d e , 0.69-0.71 w i t h hexane, 0.81-0.82 w i t h acetone, 0.95 w i t h b e n z e n e , a n d 1.0 w i t h ether. U n l a b e l e d c o m p o u n d s w e r e de tected w i t h a silver n i t r a t e - h y d r o g e n p e r o x i d e reagent (4) (sensitive to 0.5 jug D B p D a n d 0.1 μg c h l o r o - D B p D d e r i v a t i v e ) , a n d l a b e l e d c o m pounds were traced b y radioautography. 2


AND

I

l,6-T -DBpD

2

ORIGIN

2

2

4

2

2

3

4

5

f

f

f

2

2

4

f

M a s s spectra ( M S ) w e r e d e t e r m i n e d w i t h d i r e c t inlet i n s e r t i o n of the samples i n a T y p e 2 1 - 1 0 3 C mass spectrometer ( C o n s o l i d a t e d E l e c t r o d y n a m i c s C o r p . , P a s a d e n a , C a l i f . ) . I n the analysis of samples c o n t a i n i n g a m i x t u r e of C l - D B p D , 2 , 3 , 7 , 8 - C l - D B p D , a n d C l - D B p D , the ratios of mass peak 285 ( o w i n g to C l - D B p D b u t not 2 , 3 , 7 , 8 - C l - D B p D ) a n d mass p e a k 358 ( o w i n g to C l > - D B p D ) to mass peak 322 ( f r o m 2 , 3 , 7 , 8 - C l D B p D ) w e r e d e t e r m i n e d a n d c o r r e c t e d for the sensitivity factors of the respective p a r e n t ions of these c o m p o u n d s . T h e a p p r o p r i a t e sensitivity factors w e r e p r o v i d e d b y t h e D o w C h e m i c a l C o . ( M i d l a n d , M i c h . ) ( 5 ) . T h e c r y s t a l structure studies, i n v o l v i n g x-ray d i f f r a c t i o n ( 6 ) , w e r e per f o r m e d b y the D o w C h e m i c a l C o . T h e c h e m i c a l p u r i t y a n d i d e n t i t y of the u n l a b e l e d a n d l a b e l e d c h e m i c a l s w e r e d e t e r m i n e d b y i n f r a r e d spectroscopy, mass spectrometry, G L C , and T L C . Unlabeled Chemicals. Samples of u n l a b e l e d D B p D d e r i v a t i v e s w e r e o b t a i n e d f r o m the f o l l o w i n g sources: 2 , 3 , 7 , 8 - C l - D B p D ( a n a l y t i c a l stand8

4

5

8

4

r

4

4


2.

viNOPAL

E T

Τritium-Labeled Dioxins

AL.

9

a r d , > 9 9 % p u r e ) f r o m t h e D o w C h e m i c a l C o . ; 1 - C l - D B p D a n d 2,3,7,8C l - D B p D f r o m A . E . P o h l a n d ( D i v i s i o n o f C h e m i s t r y a n d P h y s i c s , U . S. F o o d a n d D r u g A d m i n i s t r a t i o n , W a s h i n g t o n , D . C . ). D B p D was p r e p a r e d b y s l o w l y h e a t i n g a m i x t u r e o f o - c h l o r o p h e n o l (480 m m o l e s ) , p o t a s s i u m c a r b o n a t e ( 2 4 0 m m o l e s ) , a n d p u r i f i e d c o p p e r p o w d e r ( 5 0 m m o l e s ) i n a 5 0 0 - m l E r l e n m e y e r flask to 1 6 0 ° - 1 8 0 ° C a n d m a i n t a i n i n g this t e m p e r a t u r e f o r 6 hours. D B p D , w h i c h s u b l i m e d t o t h e w a l l s o f t h e flask as i t was f o r m e d , was r e c o v e r e d b y s c r a p i n g a n d w a s r e c r y s t a l l i z e d f r o m absolute e t h a n o l ( 1 4 % y i e l d ; w h i t e needles, m p 1 1 9 ° - 1 2 0 ° C ; reported 1 2 0 ° - 1 2 2 ° C ( 7 ) ; elemental analyses—calcd. C = 78.26, H = 4.34, f o u n d C = 77.98, H = 4 . 4 8 ) . D B p D w a s also p r e p a r e d o n a s u b m i l l i m o l e - s c a l e b y r e d u c t i v e d e c h l o r i n a t i o n of l , 6 - C l - D B p D . I n this m e t h o d l , 6 - C l - D B p D (0.1 m m o l e ) in 2-propanol (5 m l ) containing 1 0 % palladium on powdered charcoal (25 m g ) w a s h y d r o g e n a t e d at a pressure o f 10 lbs/square i n c h w i t h s h a k i n g for 1 h o u r at 25 ° C i n the 2 0 0 - m l pressure bottle o f a c o n v e n t i o n a l hydrogénation a p p a r a t u s ( P a r r I n s t r u m e n t C o . , M o l i n e , 111.). T h e cata lyst was r e m o v e d b y f i l t r a t i o n a n d was w a s h e d w i t h 25 m l o f 2 - p r o p a n o l . T h e c o m b i n e d 2 - p r o p a n o l fractions w e r e e v a p o r a t e d onto F l o r i s i l ( 1 0 g r a m , 60/100 m e s h , F l o r i d i n C o . , B e r k e l e y S p r i n g s , W . V a . ) , a n d t h e m i x t u r e w a s a d d e d to a glass c o l u m n ( 4 . 5 c m d i a m e t e r ) c o n t a i n i n g 30 grams F l o r i s i l . T h e n 25 grams F l o r i s i l w e r e a d d e d to t h e t o p o f t h e c o l u m n , a n d D B p D was e l u t e d w i t h hexane w h i c h left the v e r y p o l a r ( p r o b ably phenolic) breakdown products o n the column. T h e appropriate fractions w e r e c o m b i n e d ( b a s e d o n T L C - a n d G L C - m o n i t o r i n g ) a n d e v a p o r a t e d u n d e r n i t r o g e n to g i v e p u r e D B p D ( 5 5 - 6 5 % y i e l d ; single compound b y G L C and T L C ; m p 119°C, without recrystallization). l , 6 - C l - D B p D w a s p r e p a r e d either b y h e a t i n g 2 , 6 - d i c h l o r o p h e n o l ( 150 m m o l e s ), p o t a s s i u m carbonate, a n d c o p p e r p o w d e r u n d e r t h e c o n d i t i o n s above for D B p D or b y h e a t i n g the p o t a s s i u m phenate ( 35 m m o l e s )


4

2

2

2

Table I.

G L C Characteristics of Dibenzo-/>-dioxin and its Chlorinated Derivatives Retention Time, min

Compound

1%

DBpD 1-Cl-DBpD 1.6- C 1 - D B D 2.7- C l o - D B p D 2,3,7-Cl -DBpD* 2,3,7,8-CL-DBpD 2

P

3

SE-30

a

1.5 3.0 5.9 5.9

C

C C c

0.7 0.7 1.3 2.5

d d

10%

OV-17

b

3.0 6.0 11.0 10.0

e e e e

0.8' 1.4' 2.4' 2.2'

d d

33.5 , 6 . 8 ' e

On Chromosorb Ρ (100/120 mesh), 1.5 m Χ 3 mm stainless steel column On Chromosorb W (80/100 mesh), 1.5 m X 3 mm stainless steel column Injection temperature = 245°C, column temperature = 160°C, N = 25 ml/min Injection temperature = 280°C, column temperature = 195°C, N = 66 ml/min Injection temperature = 280°C, column temperature = 250°C. N = 25 ml/min ' Injection temperature = 280°C, column temperature = 270°C, N = 66 ml/min Obtained by irradiating an ethanol solution of 2,3,7,8-Cl4-DBpD with short-wave length ultraviolet light. a

b c

d

e

2

2

2

2

0


10

CHLORODIOXINS

ORIGIN

AND

FATE

d i r e c t l y w i t h c o p p e r p o w d e r . T h e d e s i r e d p r o d u c t s u b l i m e d i n the f o r m e r m e t h o d at 2 4 0 ° - 2 6 0 ° C a n d i n the latter one at 3 0 0 ° - 3 2 0 ° C . A f t e r r e c r y s t a l l i z a t i o n f r o m m e t h a n o l , the p r o d u c t s o b t a i n e d h a d the f o l l o w i n g characteristics: b y the p h e n o l m e t h o d — 4 . 1 % y i e l d ; w h i t e plates, m p 1 9 7 ° - 1 9 9 ° C ; e l e m e n t a l a n a l y s e s — c a l c d . C — 56.91, H = 2.37, C l = 28.06, f o u n d C = 57.04, H = 2.52, C l = 28.18, a n d b y the p o t a s s i u m phenate p r o c e d u r e — 7 . 8 % y i e l d ; w h i t e needles, m p 1 9 8 ° - 2 0 0 ° C ; e l e m e n t a l a n a l y s e s — f o u n d C = 56.92, H — 2.30, C l = 27.88. 2 , 7 - C l - D B p D was p r e p a r e d b y the same t w o p r o c e d u r e s u s e d for 1 , 6 - C L - D B p D i n v o l v i n g 2 , 4 - d i c h l o r o p h e n o l (75 m m o l e s ) a n d s u b l i m a t i o n at 2 0 0 ° - 2 2 0 ° C or p o t a s s i u m 2 , 4 - d i c h l o r o p h e n a t e (63 m m o l e s ) a n d sub l i m a t i o n at 2 2 0 ° - 2 4 0 ° C . A f t e r r e c r y s t a l l i z a t i o n f r o m p e t r o l e u m ether, the p r o d u c t s o b t a i n e d h a d the f o l l o w i n g characteristics: b y the p h e n o l p r o c e d u r e — 4 . 5 % y i e l d ; w h i t e needles, m p 2 0 6 ° - 2 0 8 ° C ; e l e m e n t a l a n a l y s e s — c a l c d . C = 56.91, H = 2.37, C l = 28.06, f o u n d C = 56.64, H = 2.40, C l = 28.10, a n d b y the p o t a s s i u m p h e n a t e p r o c e d u r e — 5 . 7 % y i e l d ; w h i t e needles, m p 2 0 7 ° - 2 0 8 ° C ; e l e m e n t a l a n a l y s e s — f o u n d C = 56.92, H = 2.52, C l = 28.12 ( 2 , 7 - C L - D B p D p r e p a r e d p r e v i o u s l y f r o m s o d i u m 2 , 4 - d i c h l o r o p h e n a t e gave m p 2 0 1 ° - 2 0 2 ° C ( 8 ) a n d b y c h l o r i n a t i o n of D B p D gave m p 2 0 3 ° C (7) and m p 1 9 5 ° - 1 9 7 ° C (9)). 2 , 3 , 7 , 8 - C l - D B p D was p r e p a r e d o n a s u b m i l l i m o l e - s c a l e b y c h l o r i n a t i o n of D B p D u n d e r c a r e f u l l y s t a n d a r d i z e d c o n d i t i o n s . T r a c e amounts of i o d i n e a n d f e r r i c c h l o r i d e w e r e a d d e d to a s o l u t i o n of p u r e D B p D (0.22-0.24 m m o l e ) i n c h l o r o f o r m (0.5 m l ) i n a 2 5 - m l c o n i c a l test t u b e . A f t e r c o o l i n g to 0 ° - 5 ° C , c h l o r i n e gas w a s b u b b l e d s l o w l y t h r o u g h the c h l o r o f o r m m i x t u r e for 5 - 6 m i n , f o r m i n g a copious p r e c i p i t a t e . T h e p r e c i p i t a t e i n the r e a c t i o n m i x t u r e was w a s h e d t w i c e w i t h 1.5-ml portions of c h l o r o f o r m at 2 5 ° C , u s i n g c e n t r i f u g a t i o n for m a x i m u m separation, to r e m o v e the u n w a n t e d c h l o r o f o r m - s o l u b l e materials. T h e p r e c i p i t a t e w a s t h e n treated b y s h a k i n g it w i t h c h l o r o f o r m ( 5 m l ) a n d w a t e r (1 m l ) , a n d the w a t e r - s o l u b l e p r o d u c t s ( s u c h as f e r r i c c h l o r i d e ) w e r e r e m o v e d , f o l l o w i n g c e n t r i f u g a t i o n . T h e c h l o r o f o r m phase, c o n t a i n i n g some u n d i s s o l v e d m a t e r i a l , w a s e v a p o r a t e d to dryness u n d e r a n i t r o g e n stream, a n d the r e s i d u e was transferred to a 2 5 - m l E r l e n m e y e r flask to w h i c h w a s a d d e d anisole ( 1 0 m l ) . T h e anisole i n the flask w a s h e a t e d to reflux to dissolve most of the r e s i d u e , a n d the u n d i s s o l v e d p o r t i o n was r e m o v e d b y filtering the hot anisole s o l u t i o n . C r y s t a l l i z a t i o n w a s a c h i e v e d b y a l l o w i n g the anisole s o l u t i o n to c o o l to 25 ° C , p u t t i n g i t i n the r e f r i g e r a t o r o v e r n i g h t , a n d filtering to recover 0.0093-0.0185 m m o l e of m a t e r i a l w h i c h c o n t a i n e d ( G L C a n d M S ) 9 0 - 9 4 % 2 , 3 , 7 , 8 - C l - D B p D ( r a n g e f r o m sev eral different p r e p a r a t i o n s ) , the r e m a i n d e r b e i n g m o s t l y C l - a n d C l D B p D . I n one s t u d y three batches of m a t e r i a l (0.050 m m o l e ) f r o m the anisole r e c r y s t a l l i z a t i o n w e r e c o m b i n e d a n d w a s h e d w i t h hot c h l o r o f o r m (8 m l ) . T h e c h l o r o f o r m - i n s o l u b l e p o r t i o n (0.022 m m o l e ) consisted of a single c r y s t a l l i n e m a t e r i a l w h i c h gave a x-ray d i f f r a c t i o n p a t t e r n i d e n t i c a l to that of k n o w n 2 , 3 , 7 , 8 - C l - D B p D , i n d i c a t i n g that the p r o d u c t consisted o n l y of 2 , 3 , 7 , 8 - C l - D B p D a n d d i d not c o n t a i n a n y other i s o m e r i c C l D B p D . ( T h e p r o d u c t also c o n t a i n e d some glass, p o s s i b l y f r o m the coarse f r i t t e d d i s k i n the B u c h n e r f u n n e l u s e d i n filtrations. ) I n another s t u d y the p r o d u c t f r o m the anisole r e c r y s t a l l i z a t i o n (0.013 m m o l e ) w a s c o m b i n e d w i t h a u t h e n t i c 2 , 3 , 7 , 8 - C l - D B p D (0.037 m m o l e , > 9 9 % p u r e ) to


2

4

4

3

r >

4

4

4

4


2.

VINOPAL

E T

11

Tritium-Labeled Dioxins

AL.

o b t a i n sufficient m a t e r i a l to w a s h t w i c e w i t h hot c h l o r o f o r m (8 m l a n d 3 m l ) . T h e final p r o d u c t w a s > 9 9 % 2 , 3 , 7 , 8 - C l - D B p D , b a s e d o n M S analysis. Labeled Chemicals. l , 6 - T - D B p D was s y n t h e s i z e d essentially b y the same p r o c e d u r e u s e d to p r e p a r e u n l a b e l e d D B p D o n a s u b m i l l i m o l e scale b y r e d u c t i v e d e c h l o r i n a t i o n ( a b o v e ) w i t h the f o l l o w i n g exceptions. A f t e r r e d u c t i o n of 1 , 6 - C L - D B p D (0.1 m m o l e ) i n 2 - p r o p a n o l ( 5 m l ) for 1 h o u r b y s t i r r i n g w i t h t r i t i u m gas ( 5 C i ) a n d h y d r o g e n gas (0.5 m l ) at atmos p h e r i c pressure, the r e a c t i o n m i x t u r e was w a s h e d w i t h 2 - p r o p a n o l (2 X 10 m l ) to r e m o v e l a b i l e t r i t i u m ; the catalyst w a s r e m o v e d b y filtration; the solvent was r e m o v e d b y v a c u u m d i s t i l l a t i o n , a n d the p r o d u c t s w e r e r e d i s s o l v e d i n 2 - p r o p a n o l (10 m l ) . ( T h i s p o r t i o n of the p r o c e d u r e w a s c a r r i e d out b y N e w E n g l a n d N u c l e a r C o r p . ( B o s t o n , M a s s . ) . ) T h e t r i t i a t e d m a t e r i a l w a s d i l u t e d ( to o b t a i n an a p p r o p r i a t e scale of r e a c t i o n i n s u b s e q u e n t steps) b y a d d i n g p u r e u n l a b e l e d D B p D (0.082 m m o l e ) i n 2 - p r o p a n o l (25 m l ) , a n d the r e s u l t i n g m i x t u r e w a s c h r o m a t o g r a p h e d (as a b o v e ) to isolate c r u d e l , 6 - T - D B p D (0.17 m m o l e , 1.1 C i / m m o l e ) . A n a l yses b y M S a n d G L C i n d i c a t e d the presence of trace amounts of a c o n t a m i n a n t , l - C l - 6 - T - D B p D , i n the c r u d e l , 6 - T - D B p D . T h e presence of this k i n d of c o n t a m i n a n t i n t h e l a b e l e d p r e p a r a t i o n b u t not i n the u n l a b e l e d p r e p a r a t i o n r e s u l t e d p r o b a b l y f r o m the l o w e r pressure u s e d d u r i n g the r e d u c t i o n step—i.e. 10 lbs/square i n c h f o r the u n l a b e l e d p r e p a r a t i o n a n d a t m o s p h e r i c pressure for the t r i t i u m - p r e p a r a t i o n . A s m a l l p o r t i o n (0.015 m m o l e ) of the c r u d e l , 6 - T - D B p D was r e d u c e d to r e m o v e the trace l e v e l of m o n o c h l o r o i m p u r i t y b y d i s s o l v i n g the sample i n 2 - p r o p a n o l (3.5 m l ) , a d d i n g 1 0 % p a l l a d i u m o n p o w d e r e d c h a r c o a l (3.8 m g ) , a n d s h a k i n g w i t h h y d r o g e n gas at 8 lbs/square i n c h for 15 m i n at 2 5 ° C . T h e catalyst w a s r e m o v e d b y filtration, a n d an a d d i t i o n a l p o r t i o n of p u r e u n l a b e l e d D B p D (0.054 m m o l e ) i n 2 - p r o p a n o l (10 m l ) w a s a d d e d b e f o r e c h r o m a t o g r a p h i c isolation of l , 6 - T - D B p D (138 m C i / m m o l e ; single r a d i o a c t i v e c o m p o u n d b y T L C a n d r a d i o - G L C ; no 1-C1D B p D d e t e c t e d b y M S ) . T h e c a l c u l a t e d specific a c t i v i t y , a s s u m i n g that there is no loss of c o m p o u n d o n r e d u c t i o n , is 239 m C i / m m o l e , i n d i c a t i n g that a p o r t i o n of the l a b e l e d m a t e r i a l is lost d u r i n g the process of the second r e d u c t i o n . 4


2

2

2

2

2

l , 6 - T - 2 , 3 , 7 , 8 - C l - D B p D was p r e p a r e d b y c h l o r i n a t i o n of 1,6-T D B p D (0.23 m m o l e , a m i x t u r e of 0.15 m m o l e of the c r u d e l , 6 - T - D B p D d e s c r i b e d above a n d 0.08 m m o l e of u n l a b e l e d D B p D ) i n the same m a n n e r d e s c r i b e d p r e v i o u s l y for p r e p a r i n g u n l a b e l e d 2 , 3 , 7 , 8 - C l - D B p D . T h i s re s u l t e d i n the i s o l a t i o n of c r u d e l , 6 - T - 2 , 3 , 7 , 8 - C l - D B p D (0.0115 m m o l e , c a l c u l a t e d as the tetrachloro d e r i v a t i v e a l t h o u g h some C l D B p D a n d a smaller a m o u n t of C l - D B p D w e r e p r e s e n t ) f o l l o w i n g r e c r y s t a l l i z a t i o n f r o m anisole. U n l a b e l e d 2 , 3 , 7 , 8 - C l - D B p D (0.043 m m o l e ) was t h e n a d d e d to the c r u d e l a b e l e d p r o d u c t , a n d the m i x t u r e was w a s h e d w i t h hot c h l o r o f o r m (8 m l ) , as d e s c r i b e d a b o v e for the u n l a b e l e d 2 , 3 , 7 , 8 - C l - D B p D , filtered, a n d w a s h e d a g a i n w i t h hot c h l o r o f o r m ( 3 m l ) to o b t a i n the final l , 6 - T - 2 , 3 , 7 , 8 - C l - D B p D p r e p a r a t i o n (0.011 m m o l e , 107 m C i / m m o l e ) . T h e specific a c t i v i t y is b e l o w the t h e o r e t i c a l v a l u e of 152 m C i / m m o l e , c a l c u l a t e d f r o m the v a r i o u s d i l u t i o n factors, p r o b a b l y as a result of i n a c curacies i n w e i g h i n g s or of a s m a l l a m o u n t of glass i n the s a m p l e f r o m the f r i t t e d glass filter. T h e p r o d u c t c o n t a i n e d no r a d i o l a b e l e d i m p u r i t i e s 2

4

2

2

4

2

4

; r

5

4

4

2

4


12

CHLORODIOXINS

ORIGIN

A N D

FATE

d e t e c t a b l e b y T L C a n d r a d i o a u t o g r a p h y or r a d i o - G L C ; there w e r e n o significant u n l a b e l e d c o n t a m i n a n t s e v i d e n t o n G L C , a n d M S i n d i c a t e d a c h e m i c a l p u r i t y of > 9 9 % . Discussion Three

routes

investigated.

for preparing tritium-labeled 2 , 3 , 7 , 8 - C l - D B p D 4

were

T h e first i n v o l v e d a n exchange r e a c t i o n b e t w e e n t r i t i u m

water a n d 2,4,5-trichlorophenol

to o b t a i n t r i t i u m - l a b e l e d

2,4,5-trichloro-

p h e n o l , u s e f u l i n s y n t h e s i z i n g 2 , 3 , 7 , 8 - C l - D B p D b y the salt f u s i o n reac 4


tion.

P r e l i m i n a r y studies w i t h d e u t e r i u m oxide, i n v o l v i n g p r o t o n m a g

netic resonance m o n i t o r i n g , i n d i c a t e d that extensive exchange of a r o m a t i c protons c a n be a c h i e v e d u n d e r basic b u t n o t a c i d i c c o n d i t i o n s .

However,

the degree of exchange does n o t seem to be sufficiently great to p r e p a r e h i g h specific a c t i v i t y m a t e r i a l u p o n r e p l a c i n g the d e u t e r i u m o x i d e w i t h t r i t i u m water.

T h e second route started w i t h l , 6 - C l - D B p D w h i c h w a s 2

c o n v e r t e d to the 1 , 6 - d i l i t h i u m d e r i v a t i v e , a n d this w a s h y d r o l y z e d w i t h t r i t i u m w a t e r to p r o d u c e l , 6 - T - D B p D .

T h e D B p D p r e p a r e d b y this

2

method was always contaminated w i t h about 3 0 % 1 - C l - D B p D , indicating that the o v e r a l l r e a c t i o n w a s n o t sufficiently c o m p l e t e .

A l s o , the separa

t i o n of D B p D a n d 1 - C l - D B p D w a s v e r y difficult. T h e route selected uses t r i t i u m gas instead of t r i t i u m w a t e r f o r l a b e l i n g , a n d this c h a n g e a l l o w s c o m p o u n d s of h i g h e r specific a c t i v i t y t o b e p r e p a r e d . Reductive

d e c h l o r i n a t i o n of l , 6 - C l - D B p D , u s i n g t r i t i u m gas a n d 2

s u b s e q u e n t c h l o r i n a t i o n of t h e l , 6 - T - D B p D , is a c o n v e n i e n t m e t h o d f o r 2

p r e p a r i n g l , 6 - T - 2 , 3 , 7 , 8 - C l - D B p D , b u t care must b e exercised i n several 2

4

steps i n v o l v e d i n this s u b m i l l i m o l e - s c a l e radiosynthesis.

Insufficient re

d u c t i o n of l , 6 - C l - D B p D results i n c o n t a m i n a t i o n b y 1 - C l - D B p D w h i l e 2

the a m o u n t of p o l a r p r o d u c t s increases i f the r e d u c t i o n is too drastic. U s i n g exactly 0.5 m l of p r e c o o l e d c h l o r o f o r m f o r the c h l o r i n a t i o n step results i n a m i x t u r e of t h e f o l l o w i n g components, as d e t e r m i n e d b y G L C chromatography 2-Cl-DBpD);

and M S : D B p D ;

a monochloro

2 , 7 - C l - D B p D ; another 2

isomeric

derivative

Cl -DBpD; 2

(probably a

trichloro

d e r i v a t i v e , a s s u m e d to b e 2 , 3 , 7 - C l D B p D ; 2 , 3 , 7 , 8 - C l - D B p D , a n d C l , ; r

DBpD.

4

r

N o h i g h e r c h l o r i n a t e d d e r i v a t i v e s of D B p D w e r e detected.

The

contaminants i n the m i x t u r e are r e m o v e d l a r g e l y i n various p u r i f i c a t i o n stages. W a s h i n g t h e c r u d e r e a c t i o n m i x t u r e w i t h c h l o r o f o r m removes a l l u n r e a c t e d D B p D , most of the 1 - C l - D B p D , a n d some of t h e C l - a n d CI32

D B p D d e r i v a t i v e s . T h e next step of w a s h i n g the c h l o r o f o r m w i t h w a t e r removes the f e r r i c c h l o r i d e catalyst. the

chloroform-soluble

products,

F o l l o w i n g anisole r e c r y s t a l l i z a t i o n of the material

2 , 3 , 7 , 8 - C L i - D B p D a n d 6 - 1 0 % of C l

: r

consists

and C l - D B p D . 5

of 9 0 - 9 4 %

of

T h e final c h l o r o

f o r m washes r e m o v e almost a l l r e s i d u a l C l - a n d C l - D B p D , r e s u l t i n g i n 8

5

very p u r e 2 , 3 , 7 , 8 - C l - D B p D . 4


2.

viNOPAL

E T

13

Τritium-Labeled Dioxins

AL.

T h e samples of l , 6 - T - D B p D a n d l , 6 - T - 2 , 3 , 7 , 8 - C l - D B p D are u s e f u l 2

2

4

i n m e t a b o l i s m a n d m o d e of a c t i o n studies. F o r e x a m p l e , w h e n i n c u b a t e d w i t h r a b b i t l i v e r microsomes, l , 6 - T - D B p D is extensively m e t a b o l i z e d to 2

polar product(s)

b u t o n l y w h e n these p r e p a r a t i o n s

reduced nicotinamide-adenine dinucleotide phosphate. conditions l , 6 - T - 2 , 3 , 7 , 8 - C l - D B p D 2

4

are f o r t i f i e d w i t h U n d e r the same

is c o m p l e t e l y resistant

to m e t a b o l i c

attack. I n some types of studies, a h i g h e r specific a c t i v i t y p o s s i b l y is d e sirable (i.e., > 1 C i / m m o l e ) , a n d this c a n b e a c h i e v e d , w i t h the m e t h o d ology a l r e a d y

d e v e l o p e d , b y u s i n g larger

amounts

of t r i t i u m gas or


w o r k i n g o n a larger synthetic scale so that it is not necessary to a d d u n l a b e l e d materials

to assist i n c r y s t a l l i z a t i o n steps w h e r e

a

certain

m i n i m u m a m o u n t of c o m p o u n d is necessary. Acknowledgment T h i s w o r k w a s a i d e d b y P H S grant E S 00049 a n d A E C C o n t r a c t N o . A T ( 0 4 - 3 ) - 3 4 , project agreement N o . 113. T h e authors are i n d e b t e d to J u n e T u r l e y a n d L e w i s Shadoff ( C h e m i c a l P h y s i c s R e s e a r c h L a b o r a t o r y ) and Warren Crummett (Analytical Laboratory, T h e D o w Chemical C o . , M i d l a n d , M i c h . ) a n d to L o r e n D u n h a m ( Z o e c o n C o r p . , P a l o A l t o , C a l i f . ) for a d v i c e a n d assistance i n p r o d u c t analysis. Literature

Cited

1. Wilson, J. G . , et al., "Report of the Advisory Committee on 2,4,5-T to the Administrator of the Environmental Protection Agency" (May 7, 1971). 2. Schwetz, Β. Α., Norris, J. M . , Sparschu, G. L., Rowe, V. K., Gehring, P. J., Emerson, J. L., Gerbig, C . G . , A D V A N . C H E M . SER. (1973) 120, 55. 3. Muelder, W. W., Shadoff, L . Α., A D V A N . C H E M . SER. (1973) 120, 1. 4. Kovacs, M . F . , Jr., J. Ass. Offic. Agr. Chem. (1963) 46, 884. 5. Dow Chemical Co., private communication. 6. Boer, F. P., Neuman, Μ. Α., van Remoortere, F. P., North, P. P., Rinn, H . W., ADVAN.

C H E M . SER. (1973) 120,

14.

7. Gilman, H . , Dietrich, J. J., J. Amer. Chem. Soc. (1957) 79, 1439. 8. Julia, M . , Baillarge, M., Bull. Soc. Chim. France (1953) 644. 9. Tomita, M . , Ueda, S., Narisada, M . , Yakugaku Zasshi (1959) 79, 186. R E C E I V E D February 18,

1972.


Preparation of Tritium-Labeled Dibenzo-p-dioxin and 2,3,7,8

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