Luminescence Applications - American Chemical Society

Chapter 12

Use of Fluorescent 1,3-Disubstituted 2(1H)Pyridones for Environmental Analysis

Downloaded by UNIV OF SYDNEY on May 3, 2015 | http://pubs.acs.org Publication Date: December 30, 1989 | doi: 10.1021/bk-1989-0383.ch012

David A. Nelson, James H. Bush, James R. Beckett, Douglas M. Lenz, and David W. Rowe Department of Chemistry, University of Wyoming, Laramie, WY 82071 Fluorescent derivatives were prepared from a series of alkyl halides and alcohols using derivatizing reagents prepared from 3-carbamoyl-2(1H)pyridone and 3-phenyl-2(1H)pyridone. These derivatives were designed to be analyzed by high-performance liquid chromatography (HPLC) using fluorescence detection, and they could be measured at the picomole level. The derivatization chemistry is applicable to a wide variety of organic compounds. Derivatives were also prepared from amines, phenols, acrylonitrile, and heptachlor. A series of six pyridone sulfonates was synthesized for use as fluorescent, site-specific, water-tracing compounds. The development o f f l u o r e s c e n t d e r i v a t i z i n g reagents f o r h i g h performance l i q u i d chromatography (HPLC) i s an area o f c o n s i d e r a b l e i n t e r e s t , m a i n l y because o f the i n h e r e n t s e n s i t i v i t y g a i n o f f l u o rescence d e t e c t i o n o v e r u l t r a v i o l e t d e t e c t i o n and because o f the a v a i l a b i l i t y of reasonably p r i c e d , s e n s i t i v e fluorescence detectors. The use o f l a s e r - i n d u c e d f l u o r e s c e n c e d e t e c t o r s f o r HPLC has a l r e a d y been r e p o r t e d by s e v e r a l r e s e a r c h groups ( 1 - 3 ) , and no doubt such d e t e c t o r s w i l l soon be c o m m e r c i a l l y a v a i l a b l e . When used w i t h m i c r o bore HPLC t e c h n o l o g y , such d e t e c t o r s can a c h i e v e e x t r e m e l y s e n s i t i v e l e v e l s o f d e t e c t i o n (4). New f l u o r e s c e n t r e a g e n t s t a i l o r e d f o r s p e c i f i c purposes are l i k e l y t o become i n c r e a s i n g l y i m p o r t a n t f o r environmental analyses. R e p o r t s on the use o f f l u o r e s c e n t d e r i v a t i v e s abound ( 5 ) . Some reagents have become w i d e l y u s e d . The d a n s y l group i s p r o b a b l y the most t h o r o u g h l y s t u d i e d . D a n s y l c h l o r i d e has been w i d e l y used as a f l u o r e s c e n t d e r i v a t i z i n g reagent f o r HPLC ( 6 , 7 ) . I t reacts r e a d i l y w i t h p r i m a r y and secondary amino groups (7) and w i t h phenols ( 8 ) , but forms d e r i v a t i v e s o f a l c o h o l s v e r y s l o w l y ( 9 ) . The lower d e t e c t i o n l i m i t f o r d a n s y l d e r i v a t i v e s o f a l i p h a t i c amines i s i n the range o f 300 femtomoles p e r i n j e c t i o n . D a n s y l c h l o r i d e does have some drawbacks when used w i t h r e v e r s e phase HPLC. One o f t h e s e i s t h a t the quantum y i e l d o f f l u o r e s c e n c e v a r i e s g r e a t l y w i t h the p o l a r i t y o f the s o l v e n t f o r d a n s y l d e r i v a t i v e s ( 1 0 , 2 1 ) . As the p o l a r i t y o f the s o l v e n t i n c r e a s e s , the 0097-6156/89/0383-O206$06.50A) ° 1989 American Chemical Society

In Luminescence Applications; Goldberg, M.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.


12.

NELSON ET AL.

207

Fluorescent 1,3-Disubstituted 2(lU)Pyridones

f l u o r e s c e n c e quantum y i e l d d e c r e a s e s , and i t i s s m a l l e s t i n w a t e r , a common s o l v e n t f o r r e v e r s e - p h a s e l i q u i d chromatography. F o r example, the v a l u e o f the quantum y i e l d o f f l u o r e s c e n c e f o r d a n s y l - d l - t r y p t o phan i n water i s 0 . 0 6 8 , compared t o 0.70 i n d i o x a n e , and the wave l e n g t h o f the e m i s s i o n maximum f o r the t r y p t o p h a n d e r i v a t i v e can v a r y as much as 78 nm w i t h v a r y i n g s o l v e n t s ( I I ) . I f solvent pro gramming i s b e i n g used t o develop the HPLC chromatogram, t h i s d i f f e r e n c e i n e m i s s i o n maximum c o u l d r e s u l t i n a w i d e l y v a r y i n g s i g n a l . The f l u o r e s c e n t s i g n a l w i l l change w i t h v a r i a t i o n i n quantum y i e l d o f f l u o r e s c e n c e and w i t h molar a b s o r p t i v i t y . Not o n l y do f l u o r e s c e n c e quantum y i e l d s v a r y w i t h the d i f f e r e n t d a n s y l d e r i v a t i v e s formed, b u t so do the molar a b s o r p t i v i t i e s ( 1 2 ) . A n o t h e r problem i s e x e m p l i f i e d by the 30-nm d i f f e r e n c e i n the e m i s s i o n maxima o f the d a n s y l d e r i v a t i v e s o f p h e n o l and 2 , 4 , 5 - t r i c h l o r o p h e n o l ( 1 3 ) . I t would be c o n v e n i e n t i f the quantum y i e l d s and molar a b s o r p t i v i t i e s were c o n s t a n t w i t h i n a s e t o f d e r i v a t i v e s . F i n a l l y , the f l u o r e s c e n t s i g n a l from d a n s y l d e r i v a t i v e s decreases i n a c i d i c s o l u t i o n (13) due t o p r o t o n a t i o n o f the dimethylamino g r o u p . Other l i m i t a t i o n s found i n f l u o r e s c e n t d e r i v a t i v e s i n c l u d e quenching by oxygen, m e t a l s , o r o t h e r s p e c i e s , c h e m i c a l o r p h o t o c h e m i c a l i n s t a b i l i t y , and b i o d e g r a d a b i l i t y . The f l u o r e s c e n c e o f c e r t a i n 1 , 3 - d i s u b s t i t u t e d 2 ( l H ) p y r i d o n e s has been noted i n the l i t e r a t u r e ( 1 4 - 1 6 ) , but l i t t l e s p e c t r o s c o p i c i n f o r m a t i o n has been r e p o r t e d . We have found t h a t p y r i d o n e compounds h a v i n g a 1 - a l k y l group and an e l e c t r o n - w i t h d r a w i n g 3 - g r o u p , such as - C 0 N H , -C00R, - C N , o r - C H , absorb a t about 320 nm and emit a t about 380 nm w i t h a h i g h quantum e f f i c i e n c y . S t u d i e s made w i t h l - m e t h y l - 3 - c a r b a m o y l - 2 ( l H ) p y r i d o n e show t h a t i t s f l u o r e s c e n c e e f f i c i e n c y i s e s s e n t i a l l y c o n s t a n t over the range o f pH 1-12, and c o n s t a n t i n aqueous methanol o r aqueous a c e t o n i t r i l e m i x t u r e s i n w h i c h the p e r c e n t a g e o f o r g a n i c s o l v e n t v a r i e s from 0 t o 75% ( T a b l e I ) . The p y r i d o n e r i n g system i s q u i t e s t a b l e c h e m i c a l l y . We i n f e r r e d t h a t t h e s e p r o p e r t i e s might be e x p l o i t e d i n a s e r i e s o f unique d e r i v a t i z i n g r e a g e n t s d e s i g n e d s p e c i f i c a l l y f o r t r a c e a n a l y s i s o f o r g a n i c compounds u s i n g HPLC s e p a r a t i o n and f l u o r e s c e n c e detection. The use o f these p y r i d o n e s f o r the a n a l y t i c a l purposes r e p o r t e d here i s based on t h e i r a c i d i c p r o p e r t i e s . Treatment o f a Ι Η - 2 - p y r i d o n e w i t h a base c o n v e r t s the p y r i d o n e t o i t s s a l t . 2

6

Η

5

Να*

I R

Scheme I

The s a l t i s an ambient n u c l e o p h i l e and can undergo e i t h e r Ν o r 0 alkylation. Chung and T i e c k e l m a n n (17) have shown t h a t u n s u b s t i t u t e d p y r i d o n e sodium s a l t s i n a c e t o n i t r i l e o r dimethylformamide s o l v e n t form p r i m a r i l y the tf-alkylated product. Using 3-substituted


208

LUMINESCENCE APPLICATIONS

I H II


I

(CH ) S0 Na a n =2 VI b n=3 c n=4 l

n

3

e

e

ce

(CHOnSofNa® a n=2 VII b n=3 c n=4


12.

NELSON ET AL.

Table I for

209

Fluorescent 1,3-Disubstituted 2(lR)Pyridones

F l u o r e s c e n c e as a F u n c t i o n o f S o l v e n t C o m p o s i t i o n Two 3 - S u b s t i t u t e d 2 ( 1 H ) - P y r i d o n e Compounds

Compound I :

R=CH , R'=C0NH 3

2

Compound V a : R=CH C H C00H, R ' = C H


2

Solvent 100% w a t e r - Methanol: 25% 50% 75% 100% Acetonitrile: 25% 50% 75% 100%

6

4

6

5

R e l a t i v e Fluorescence Va I 0.40 1.00

—

-

0.82 0.76 0.72 0.56

0.42 0.43 0.46 0.38

0.81 0.77 0.68 0.49

0.43 0.42 0.40 0.24

p y r i d o n e s under the c o n d i t i o n s r e p o r t e d i n t h i s s t u d y , tf-alkylated p r o d u c t s were formed e x c l u s i v e l y . The c h e m i s t r y d e s c r i b e d above has been e x p l o i t e d i n t h r e e ways: 1. D i r e c t use o f p y r i d o n e sodium s a l t s as f l u o r e s c e n t d e r i v a t i z i n g reagents. 2. S y n t h e s i s o f a more s p e c i f i c f l u o r e s c e n t a c i d c h l o r i d e d e r i v a t i z i n g reagent. 3. S y n t h e s i s o f p y r i d o n e s u l f o n a t e s as w a t e r - t r a c i n g compounds. Results

and

Discussion

1. Pyridone Sodium Salts as Nucleophilic Fluorescent Derivatizing Reagents. A d e r i v a t i z i n g reagent was p r e p a r e d by c o n v e r t i n g 3 - c a r b a m o y l - 2 ( l H ) p y r i d o n e t o i t s sodium s a l t ( I I I ) w i t h sodium h y d r o x i d e . Reagent I I I i s r e a s o n a b l y s t a b l e but must be used under anhydrous c o n d i t i o n s . The r e a g e n t can be used t o d e r i v a t i z e a wide v a r i e t y o f o r g a n i c compounds, based on the g e n e r a l p r i n c i p l e s o f n u c l e o p h i l i c s u b s t i t u t i o n and a d d i t i o n r e a c t i o n s . F i g u r e 1 shows the s e p a r a t i o n o f a s e r i e s o f d e r i v a t i v e s formed from a l k y l bromides w i t h Reagent I I I . The r e a c t i o n g i v e n i n F i g u r e 2 shows the s e l e c t i v e r e a c t i o n o f I I I w i t h the i n s e c t i c i d e h e p t a c h l o r . Only one o f the seven c h l o r i n e atoms i s r e a c t i v e . M i c h a e l a d d i t i o n s o f I I I t o r e a c t i v e a c c e p t o r s appear f e a s i b l e . D e r i v a t i v e s were formed from a c r y l o n i t r i l e and e t h y l a c r y l a t e . A l t h o u g h t h e s e were not f u l l y c h a r a c t e r i z e d , they were observed by HPLC a n a l y s i s .


210



UNK

R«-H

I R -C3H -CH(CH ) 3

IS

5

2

10

S

0

TIME

F i g u r e 1 . Chromatogram showing s e p a r a t i o n o f a l k y l bromide d e r i v a t i v e s o f Reagent I I I . C o n d i t i o n s as d e s c r i b e d i n the t e x t under "HPLC S e p a r a t i o n C o n d i t i o n s . "



NELSON ET AL.

Fluorescent 1,3-Disubstituted 2(lR)Pyridones

a

C0NH +

N a

*

2

CH =CHCN —> 2

(j

^ ^ C 0 N H T

2

CH CH CN 2

2

Figure 2. Formation of d e r i v a t i v e s of heptachlor acrylonitrile.

and


212



2. Pyridone Acid Chlorides as Fluorescent Derivatizing Reagents. A second d e r i v a t i z i n g r e a g e n t , a f l u o r e s c e n t a c i d c h l o r i d e , was s y n t h e s i z e d from the sodium s a l t o f 3 - p h e n y l - 2 ( l H ) p y r i d o n e ( I V ) . The s a l t o f IV can be c o n v e r t e d i n t o Reagent V by the r e a c t i o n shown i n Scheme I I .

COOMe Scheme I I T h i s f l u o r e s c e n t a c i d c h l o r i d e can be used t o form d e r i v a t i v e s o f a l c o h o l s , amines, and p h e n o l s . Using these fluorescent d e r i v a t i v e s , an a n a l y s i s o f a s e r i e s o f η - a l c o h o l s from C ! t o C was d e v e l o p e d . A chromatogram produced by t h i s t e c h n i q u e i s shown i n F i g u r e 3 . D e r i v a t i v e s were a l s o formed from ammonia, d i m e t h y l a m i n e , and p h e n o l . A d e r i v a t i v e was formed from p e n t a c h l o r o p h e n o l but was not f u l l y characterized. The quantum y i e l d s o f f l u o r e s c e n c e o f the a l c o h o l d e r i v a t i v e s o f V were lower t h a n those o f the a l k y l h a l i d e d e r i v a t i v e s of I I I . 4

3. Water-Tracing Compounds. Another a p p l i c a t i o n o f t h e s e f l u o r e s c e n t p y r i d o n e s has been the development o f a s e r i e s o f compounds u s e f u l as ground-water t r a c e r s . Smart and L a i d l a w have o u t l i n e d s e v e r a l q u a l i t i e s d e s i r a b l e i n f l u o r e s c e n t w a t e r - t r a c i n g compounds (is). The p r o p e r t i e s o f an o r g a n i c t r a c i n g compound s h o u l d m i n i m i z e loss while i n t r a n s i t . There are two main sources o f dye l o s s , nona d s o r p t i v e l o s s and a d s o r p t i v e l o s s . N o n a d s o r p t i v e l o s s e s can be due, among o t h e r r e a s o n s , t o p h o t o c h e m i c a l d e c o m p o s i t i o n , c h e m i c a l d e c a y , pH e f f e c t s , and b i o d é g r a d a t i o n o f the compound by m i c r o o r g a n i s m s . A d s o r p t i o n o f the t r a c e r onto b o t h o r g a n i c and i n o r g a n i c s u b s t r a t e s i s o f t e n i r r e v e r s i b l e and can be a source o f much l o s s . In t e s t s conducted by Smart and L a i d l a w , a d s o r p t i o n was the l e a s t p r e v a l e n t i n compounds c o n t a i n i n g s u l f o n i c a c i d g r o u p s . T h i s seemed t o be due t o t h e i r low pK , w h i c h g i v e s an a n i o n i c group t h a t r e p e l s the u s u a l a n i o n i c charges o f the a d s o r b e n t s . Many o f the p r o p e r t i e s o f the 3 - p h e n y l - and 3 - c a r b a m o y l - 2 ( l H ) p y r i d o n e s i n d i c a t e t h a t they may be u s e f u l as water t r a c e r s . As a l r e a d y shown t h e s e compounds are i n t e n s e l y f l u o r e s c e n t . They show



12.

NELSON ET AL.

Fluorescent 1,3-Disubstituted 2(lYL)Pyridones

213

F i g u r e 3. Chromatogram showing s e p a r a t i o n o f a l c o h o l d e r i v a t i v e s of Reagent V . C o n d i t i o n s as d e s c r i b e d i n the t e x t under "HPLC Separation Conditions."


214


s t a b i l i t y toward p h o t o c h e m i c a l decay, s i n c e t h e i r maximum absorbance, 315-324 nm, i s o u t s i d e the normal range o f s u n l i g h t . T h e i r f l u o r e s cence i n t e n s i t y , as mentioned above, i s e s s e n t i a l l y c o n s t a n t o v e r a range o f pH from 2 t o 12. A p r e l i m i n a r y t e s t f o r the b i o d e g r a d a b i l i t y o f the 3 - p h e n y l - and 3 - c a r b a m o y l - 2 ( l H ) p y r i d o n e s was conducted i n a b a r n y a r d humus suspen sion. The a n a l y s i s by HPLC showed some l o s s , and the f l u o r e s c e n t compounds seemed t o be adsorbed onto the s o l i d . The 3 - c a r b a m o y l 2(lH)pyridone ( I I ) also hydrolyzed to 3 - c a r b o x y l i c a c i d - 2 ( l H ) p y r i d o n e b o t h i n the s l u r r y t e s t and i n water s o l u t i o n s t h a t had been l e f t s t a n d i n g 1-2 weeks. I n p r e l i m i n a r y t e s t s b o t h the 3 - p h e n y l - and the 3 - c a r b a m o y l - 2 ( l H ) p y r i d o n e s a p p a r e n t l y adsorbed t o some e x t e n t on s i l i c a sand columns. I n a d d i t i o n , the s o l u b i l i t y o f b o t h 1-H compounds was somewhat l o w , 1.3 x 1 0 ~ M f o r I I , and 1.0 x 1 0 ~ Jlf f o r I V . S i n c e s u l f o n a t e groups have been used i n o t h e r ground-water t r a c e r s , the g o a l o f t h i s work was t o s y n t h e s i z e s e v e r a l J V - s u b s t i t u t e d p y r i d o n e a l k y l s u l f o n a t e s which might be l e s s s u s c e p t i b l e t o a d s o r p t i o n as w e l l as more s o l u b l e . A l k y l s u l f o n a t e s o f v a r y i n g c h a i n l e n g t h s would be unique i n a water system and s e p a r a t e l y i d e n t i f i a b l e by HPLC a n a l y s i s . Two s e r i e s o f compounds were s y n t h e s i z e d ( V l a - c and V I I a - c ) . These compounds c o u l d be p r e p a r e d by t r e a t i n g compounds I I I and IV w i t h the a p p r o p r i a t e u>-bromoalkyl sulfonate. A s e r i e s o f b r o m o a l k y l s u l f o n a t e s was t h e r e f o r e needed t o form the J V - a l k y l s u l f o n a t e d 2 - p y r i d o n e s . F o r m a t i o n o f b r o m o a l k y l s u l f o nates has not been d e s c r i b e d e x t e n s i v e l y i n the l i t e r a t u r e ( 1 9 ) . A l k y l h a l i d e s r e a c t w i t h s u l f i t e i o n t o form a l k y l s u l f o n a t e s , a r e a c t i o n known as the S t r e c k e r r e a c t i o n ( 2 0 ) . "Organic Syntheses" (21) g i v e s a b r i e f d e s c r i p t i o n o f the s y n t h e s i s o f 2-bromoethane w i t h sodium s u l f i t e . A more g e n e r a l s y n t h e t i c method was developed u s i n g a l k y l d i h a l i d e s t o form α , ω - b r o m o a l k y l s u l f o n a t e s . The f o r m a t i o n o f 2 - b r o m o e t h y l s u l f o n a t e f o l l o w e d the method described i n "Organic Syntheses" (21). I n the attempt t o g e n e r a l i z e t h i s r e a c t i o n , we noted t h a t n e i t h e r 1,3-dibromopropane nor 1 , 4 - d i bromobutane was m i s c i b l e i n the e t h a n o l - w a t e r r e a c t i o n s o l v e n t . D i r e c t l y f o l l o w i n g the d e s c r i b e d p r o c e d u r e d i d produce b o t h the 3 - b r o m o p r o p y l s u l f o n a t e and 4 - b r o m o b u t y l s u l f o n a t e , but i n low y i e l d s o f r o u g h l y 20%. Improved p r o c e d u r e s f o r a l k y l a t i o n were developed u s i n g a c e t o n i t r i l e as the s o l v e n t . The y i e l d s o f the r e a c t i o n s f o r b o t h I I I and IV w i t h the e t h y l , p r o p y l , and b u t y l b r o m o s u l f o n a t e s are g i v e n i n the " E x p e r i m e n t a l " s e c t i o n . These r e a c t i o n s are u s e f u l because t h e y run under m i l d c o n d i t i o n s , use i n e x p e n s i v e or e a s i l y r e c o v e r a b l e s t a r t i n g m a t e r i a l s , and have s h o r t r e a c t i o n t i m e s . The major problem i n p u r i f i c a t i o n i s the s e p a r a t i o n o f the sodium p y r i d o n e s u l f o n a t e from excess sodium s u l f i t e , sodium b r o m i d e , and sodium b r o m o a l k y l s u l f o n a t e . However, t h e s e l a t t e r compounds u s u a l l y would not i n t e r f e r e w i t h the use o f the p y r i d o n e s u l f o n a t e as a water t r a c e r . From a p r a c t i c a l p o i n t o f v i e w , the p y r i d o n e s u l f o n a t e s need not be p u r i f i e d , b u t can be used directly. A m o d i f i e d s y n t h e t i c procedure i n v o l v e s the t r e a t m e n t o f the p y r i d o n e sodium s a l t w i t h a t e n f o l d excess o f α , ω - d i b r o m o a l k a n e i n a c e t o n i t r i l e , f o l l o w e d by removal o f the excess d i b r o m i d e by vacuum distillation. The r e s u l t i n g p r o d u c t i s t r e a t e d w i t h an excess o f sodium s u l f i t e i n aqueous e t h a n o l . E v a p o r a t i o n o f the s o l v e n t y i e l d s a useful tracer. Procedures g i v e n i n the e x p e r i m e n t a l s e c t i o n were


3

2


12.

NELSON ET AL.

Fluorescent 1,3-Disubstituted 2(lB)Pyridones

215

t h o s e used f o r i s o l a t i o n o f pure compounds. The molar a b s o r p t i v i t i e s and f l u o r e s c e n c e quantum y i e l d s o f t h e l - a l k y l - 3 - p h e n y l - and 3 - c a r b a m o y l - 2 ( l H ) p y r i d o n e s u l f o n a t e s a r e shown i n T a b l e I I .


Table I I

R e l a t i v e F l u o r e s c e n c e and S p e c t r o s c o p i c Data for Pyridone Sulfonates

I

-

(CH ) S0 2

R

η

Compound Number

n

3

+

Na

Molar Absorptivity, ε x 10

Fluorescence Quantum Y i e l d , Φ* »

Wavelength o f Maximum A b s o r b a n c e , λ (nm) may

3

C

6 5

H

2

Vila

8.6

0..29

315

C

H

6 5

3

Vllb

5.9

0..34

315

C

H

6 5

4

VIIc

2.3

0..38

315

C0NH

2

2

Via

6.1

0..95

325

C0NH

2

3

VIb

6.4

0..95

325

C0NH

2

4

Vic

2.3

0..98

325

R e l a t i v e to compound I .

A n a l y t i c a l HPLC s e p a r a t i o n s o f t h e homologous a l k y l s u l f o n a t e s e r i e s o f t h e 3 - p h e n y l - and 3 - c a r b a m o y l - 2 ( l H ) p y r i d o n e s were developed u s i n g i o n - p a i r i n g r e a g e n t s . These s e p a r a t i o n s , shown i n F i g u r e s 4 and 5 , demonstrate t h a t t h e s e compounds would be s e p a r a t e l y i d e n t i f i a b l e from t h e same sample i f they were t o be used as s i t e - s p e c i f i c water t r a c e r s . F o r example, s e v e r a l l o c a t i o n s a t t h e same waste d i s p o s a l s i t e c o u l d be marked. S i n c e t h e s y n t h e s e s we have developed are g e n e r a l , a d d i t i o n a l members o f a s e r i e s c o u l d be s y n t h e s i z e d i f needed. L e v e l s o f d e t e c t i o n o f the compounds a r e i n t h e p a r t s - p e r t r i l l i o n range. A d s o r p t i o n s t u d i e s u s i n g s a n d , sawdust, p e a t moss, b a r n y a r d s o i l , k a o l i n i t e , and b e n t o n i t e showed e s s e n t i a l l y no a d s o r p t i o n from 1 0 0 - p g / L s o l u t i o n o f p y r i d o n e V I b o v e r a p e r i o d o f 24 h r ( 2 2 ) . These compounds a r e c u r r e n t l y u n d e r g o i n g some f i e l d t e s t s . Quantum Yields of Fluorescence. T a b l e I I I l i s t s t h e r e l a t i v e quantum y i e l d s o f f l u o r e s c e n c e o f 24 3 - s u b s t i t u t e d 2 ( l H ) - p y r i d o n e s . P y r i d o n e I has the h i g h e s t y i e l d measured, w h i c h i s s e t a t 1.00. An attempt was made t o measure the ' a b s o l u t e ' quantum y i e l d Φ _ o f I r e l a t i v e t o rhodamine Β u s i n g f e r r i o x a l a t e a c t i n o m e t r y . Α value of 0 . 9 8 ± 0 . 0 2 was o b t a i n e d . However, t h e d e t e r m i n a t i o n o f ' a b s o l u t e '



216


F i g u r e 4. Chromatogram showing s e p a r a t i o n o f the 3 - p h e n y l 2 ( l H ) p y r i d o n e a l k y l s u l f o n a t e sodium s a l t s . C o n d i t i o n s as d e s c r i b e d i n the t e x t under "HPLC S e p a r a t i o n C o n d i t i o n s . "



12.

NELSON ET AL.

2

Fluorescent 1,3-Disubstituted 2(lYL)Pyridanes

4

t

S

10

12

MIN

Figure 5. Chromatogram showing s e p a r a t i o n o f the 3 - c a r b a m o y l 2 ( l H ) p y r i d o n e a l k y l s u l f o n a t e sodium s a l t s . C o n d i t i o n s as d e s c r i b e d i n the t e x t under "HPLC S e p a r a t i o n C o n d i t i o n s . "


217

218


quantum y i e l d s o f f l u o r e s c e n c e p r e s e n t s many p r o b l e m s . F o r t h i s r e a s o n , a l l v a l u e s o f quantum y i e l d s r e p o r t e d here are v a l u e s r e l a t i v e to I . The 3 - C 0 N H , COOH, C 0 0 C H , and CN p y r i d o n e s i n T a b l e I I I A are a l l h i g h l y f l u o r e s c e n t . The 3 - p h e n y l p y r i d o n e s have r e l a t i v e f l u o r e s c e n c e i n the range 0 . 2 - 0 . 3 . Some l e s s f l u o r e s c e n t p y r i d o n e s are i n c l u d e d i n T a b l e I I I B . In general, s u b s t i t u t i o n at the 1 - p o s i t i o n does not have a s i g n i f i c a n t e f f e c t on the f l u o r e s c e n c e yield. The a l c o h o l d e r i v a t i v e s o f compound V are u n u s u a l i n t h i s r e g a r d , g i v i n g f l u o r e s c e n c e y i e l d s an o r d e r o f magnitude l e s s t h a n t h a t o f the c o r r e s p o n d i n g a c i d . 2

3

Limits of Detection. A s i g n a l - t o - n o i s e r a t i o o f 2 c o u l d be o b t a i n e d from 5pL o f a 1 0 ~ M s o l u t i o n o f p y r i d o n e I , under the c o n d i t i o n s d e s c r i b e d i n the " E x p e r i m e n t a l " s e c t i o n . T h i s amount o f the s o l u t i o n Downloaded by UNIV OF SYDNEY on May 3, 2015 | http://pubs.acs.org Publication Date: December 30, 1989 | doi: 10.1021/bk-1989-0383.ch012

8

Table I I I

R e l a t i v e F l u o r e s c e n c e and S p e c t r o s c o p i c Data f o r 3 - S u b s t i t u t e d 2(1H)Pyridones

Table I I I A

I R R

CH H CH2CH3 CH2CH2CH3 3

CH(CH ) 0^2(^2)2^3 C10H5CÏ-6 H 3

CH H CH CH

2

2

2

3

4

3 3

(I) (II)

2

2

C0NH C0NH C0NH C H (IV) 2

2

2

6

5

H

6 5 CI CI CN

3

3

6

( )

C0NH C0NH C0NH C0NH

C 3

CH CH C H C00CH3 CH CH 2

a

R'

COOH C0NH COOCH3 N0 2

2

Fluoréscence Quantum, Y i e l d , |

Molar Absorptivity, ε x 10

3

Wavelength (nm) Emission Maximum λ Absorbance, em λ max

1.00 0.,93 0..93 0.,92

8.77 7.50 8.59 8.44

324 321 324 325

384 382 382 382

0..83 0.,93 0.,68 0. 24

8.57 8.56 10.3 9.95

324 326 331 311

384 390 386 392

0.,33 0.,012 0..0083 0..88

9.83 7.79 6.83 8.84

313 304 308 327

394 369 368 380

0..65 0..68 0..58 0..0

6.50 5.11 9.95 6.68

308 326 327 365

380 384 389

Roman numerals i n p a r e n t h e s e s denote compounds d i s c u s s e d i n t e x t . R e l a t i v e t o compound I .


12.

NELSON ET AI»

219

Fluorescent 1,3-Disubstituted 2(IHl)Pyridones


Table I I I B

COR Fluorescence Quantum. Y i e l d , |

a

R

( )

Wavelength (nm) Maximum Emission, Absorbance, λ em λ max

Molar Absorptivity, ε x ΙΟ 3

OH (Va) 0CH 0C H OCH2CH2CH3

0.28 0.023 0.016 0.015

10.1 10.5 9.69 10.6

317 317 318 325

400 390 390 390

0(CH ) CH NH N(CH ) 0C H

0.014 0.066 0.31 0.088

9.85 9.54 9.41 9.27

325 319 319 319

392 398 398 400

3

2

5

2

3

3

2

3

6

2

5

^Roman numerals i n p a r e n t h e s e s denote compounds d i s c u s s e d i n t e x t . R e l a t i v e t o compound I .

1 4

1 2

contains 5 x 1 0 " m o l e s , o r 6.9 x 1 0 ~ g, of I . S i n c e the r e g i o n s o f a b s o r p t i o n and e m i s s i o n o f the o t h e r p y r i d o n e s r e p o r t e d here are e s s e n t i a l l y the same as f o r I , o t h e r d e t e c t i o n l i m i t s can be e s t i m a t e d by d i v i d i n g the v a l u e s f o r I by the r e l a t i v e quantum y i e l d s , assuming the same chromatographic k ' v a l u e o f 2 . I n an attempt t o lower the d e t e c t i o n l i m i t s , the S c h o e f f e l d e t e c t o r was m o d i f i e d t o use a z i n c m e t a l lamp, w h i c h has a resonance l i n e o f 308 nm, and a cadmium resonance lamp, w h i c h has an i n t e n s e l i n e a t 326 nm. The l a t t e r was v e r y c l o s e t o the w a v e l e n g t h o f maximum absorbance ( λ ) o f the p y r i d o n e s . Each change lowered the d e t e c t i o n l i m i t an orSer o f magnitude. Thus, u s i n g the cadmium lamp, 5 μ ΐ o f a 1 0 " if s o l u t i o n o f I was d e t e c t e d . This i s 5 x 1 0 " moles ( 0 . 5 femtomoles) or 7 x 1 0 ~ g (70 femtograms). 1 0

1 6

1 4

Metal Ion Analysis. We have r e p o r t e d a s e n s i t i v e t r a c e - m e t a l a n a l y s i s based upon HPLC s e p a r a t i o n o f p-aminophenyl EDTA c h e l a t e s and f l u o r e s c e n c e d e t e c t i o n by postcolumn r e a c t i o n w i t h f l u o r e s c a m i n e ( 2 3 ) . An a p p l i c a t i o n o f the p y r i d o n e c h e m i s t r y a l r e a d y d i s c u s s e d l e a d s t o a f l u o r e s c e n t - l a b e l e d EDTA ( V I I I ) .


220


^xC0NH

Br

CH

Na®

2

(HOOCCHJNCHCH N(CH COOH) S

2

t

2

2

III 1

(HOOCCHJNCHCH,N(CH COOH) 1

I

VIII


Scheme I I I We have developed r e v e r s e - p h a s e i o n - p a i r i n g HPLC s e p a r a t i o n s o f s u b s t i t u t e d EDTA m e t a l c h e l a t e s o f s e v e r a l t r a n s i t i o n m e t a l s ( i n c l u d i n g C d , Z n , P b , and Hg) and s e v e r a l l a n t h a n i d e s ( L a , Ce, E u , Dy, E r , Yb, L u ) . D e t e c t i o n l e v e l s o f t h e s e c h e l a t e s are c u r r e n t l y b e i n g a s s e s s e d . A s e n s i t i v e m e t a l i o n a n a l y s i s employing an i n h e r e n t l y f l u o r e s c e n t EDTA seems f e a s i b l e . Experimental Apparatus. HPLC s e p a r a t i o n s were a c c o m p l i s h e d u s i n g a system c o n s i s t i n g o f two Waters A s s o c i a t e s Model 6000-A L i q u i d Chromatography pumps, a Model 660 s o l v e n t programmer, a Model 440 absorbance d e t e c t o r s e t a t 313 nm, and a S c h o e f f e l FS 970 f l u o r o m e t e r w i t h e x c i t a t i o n a t 320 nm u s i n g a S c h o e f f e l 7-54 e x c i t a t i o n f i l t e r and a S c h o e f f e l KV 370 e m i s s i o n f i l t e r . NMR s p e c t r a were o b t a i n e d on a JEOL FX-270 s p e c t r o m e t e r and are r e f e r e n c e d e i t h e r t o t e t r a m e t h y l s i l a n e i n chloroform-d, to t - b u t y l - O D i n deuterium o x i d e , or to d i m e t h y l - d s u l f o x i d e as an i n t e r n a l s t a n d a r d . U l t r a v i o l e t absorbance data were o b t a i n e d on a H i t a c h i 100-80 s p e c t r o p h o t o m e t e r . Fluorescence spectra and quantum y i e l d s were determined u s i n g a P e r k i n - E l m e r H i t a c h i MPF-2A s p e c t r o p h o t o m e t e r . M e l t i n g p o i n t s were determined u s i n g a Thomas-Hoover U n i m e l t , and are u n c o r r e c t e d . E l e m e n t a l a n a l y s e s were o b t a i n e d from A t l a n t i c M i c r o l a b s , A t l a n t a , GA, o r Huffman L a b o r a t o r i e s , Wheat R i d g e , CO. 6

Chemicals. A l l c h e m i c a l s were o b t a i n e d from A l d r i c h Chemical Company u n l e s s o t h e r w i s e i n d i c a t e d . A l l o t h e r reagents were ACS c e r t i f i e d o r equivalent. HPLC Separation Conditions. S e p a r a t i o n s o f a l k y l bromide and a l c o h o l d e r i v a t i v e s were performed u s i n g an A l t e x U l t r a s p h e r e C 5-μπι, 4 . 6 x 150 mm column. The d e r i v a t i v e s o f m e t h a n o l , e t h a n o l , 1 - p r o p a n o l , and 1-butanol were s e p a r a t e d u s i n g a m o b i l e phase o f 65% a c e t o n i t r i l e and 35% w a t e r , w i t h a f l o w r a t e o f 1 mL/min. The d e r i v a t i v e s o f the a l k y l bromides were s e p a r a t e d u s i n g a l i n e a r program from 10% a c e t o n i t r i l e and 90% water t o 50% a c e t o n i t r i l e and 50% water i n 20 m i n . The f l o w r a t e was 1 mL/min. S e p a r a t i o n s o f p y r i d o n e s u l f o n a t e s were performed u s i n g an A l t e x Ultrasphere I . P . C 5-μιη, 4 . 6 x 150 mm column. The 3 - c a r b a m o y l 2 ( l H ) p y r i d o n e s e r i e s was s e p a r a t e d u s i n g a m o b i l e phase o f 89% 0.005-flf tetrabutylammonium phosphate (Waters A s s o c i a t e s P I C - A 1 8

1 8


12.

NELSON ET AL.


221

i o n - p a i r i n g r e a g e n t ) and 11% a c e t o n i t r i l e w i t h a f l o w r a t e o f 1 mL/min. The 3 - p h e n y l - 2 ( l H ) p y r i d o n e s e r i e s was s e p a r a t e d u s i n g a m o b i l e phase o f 75% 0.005-W tetrabutylammonium phosphate and 25% a c e t o n i t r i l e w i t h a flow r a t e o f 1 mL/min. Quantum Yields of Fluorescence Measurements. A l l o f the quantum y i e l d s o f f l u o r e s c e n c e were measured by the r e l a t i v e f l u o r e s c e n c e measurement t e c h n i q u e o f P a r k e r and Rees ( 2 4 ) . T h i s method compares the f l u o r e s c e n c e o f the compound o f i n t e r e s t t o the f l u o r e s c e n c e o f some known compound. A l l o f the f l u o r e s c e n c e quantum y i e l d s were measured u s i n g I as a r e f e r e n c e . Compound I had p r e v i o u s l y been measured by t h i s same method u s i n g rhodamine Β as a s t a n d a r d . (I ) SiCi Downloaded by UNIV OF SYDNEY on May 3, 2015 | http://pubs.acs.org Publication Date: December 30, 1989 | doi: 10.1021/bk-1989-0383.ch012

£

*

2

=

d

f

2

) i e

2

c

2

φ

ι

In the e q u a t i o n , the s u b s c r i p t s 1 and 2 r e f e r to the r e f e r e n c e compound and the compound o f i n t e r e s t , r e s p e c t i v e l y , 1^ i s the i n t e n s i t y o f the f l u o r e s c e n t s i g n a l o f each compound measured as peak h e i g h t i n c e n t i m e t e r s , ε i s the molar a b s o r p t i v i t y , c i s the concen t r a t i o n i n moles p e r l i t e r , and Φ i s the f l u o r e s c e n c e quantum y i e l d . In t h i s a p p l i c a t i o n , i s s e t a t 1.00. The c o n c e n t r a t i o n s o f the s o l u t i o n s t h a t were t e s t e d ranged from 1 0 ~ t o 1 0 " M. The s o l u t i o n s run a t the h i g h e r c o n c e n t r a t i o n s were a l l checked f o r s e l f - q u e n c h i n g , but none was f o u n d . A l l measurements, e x c e p t the f l u o r e s c e n c e v e r s u s - s o l v e n t s t u d y , were made i n 0.1-W phosphate b u f f e r , pH 7 . 4 . S l i t s e t t i n g s on the P e r k i n - E l m e r MPF-2A were 10 πψ (nm) f o r b o t h e m i s s i o n and e x c i t a t i o n monochromators. 4

7

Limits of Detection Study. The l i m i t s o f d e t e c t i o n were measured by i n j e c t i n g s u c c e s s i v e l y s m a l l e r samples ( d e f i n e d as volume times c o n c e n t r a t i o n ) o f compound I , a t the most s e n s i t i v e s e t t i n g o f the S c h o e f f e l 970 u n t i l the peak h e i g h t o f the s i g n a l from the sample was t w i c e t h a t o f the n o i s e . The chromatographic c o n d i t i o n s were a d j u s t e d so t h a t k = 2 f o r the peak. The s t a n d a r d d e u t e r i u m lamp was used f o r i n i t i a l measurements. A m o d i f i e d lamp h o u s i n g was c o n s t r u c t e d t o accommodate a z i n c lamp and a cadmium lamp, b o t h from U l t r a v i o l e t P r o d u c t s , San G a b r i e l , CA. The cadmium lamp was used w i t h an AL3261-1 i s o l a t i o n f i l t e r from C o r i o n C o r p . , H o l l i s t o n , MA. In u s i n g the m e t a l lamps, the monochromator s e t t i n g on the S c h o e f f e l 970 was a d j u s t e d t o g i v e a maximum s i g n a l when a 1 0 ~ M s o l u t i o n o f compound I was i n the d e t e c t o r c e l l . 1

6

In Situ Derivatization. To demonstrate t h a t a group o f a l c o h o l s c o u l d be d e r i v a t i z e d a t low a l c o h o l c o n c e n t r a t i o n , the f o l l o w i n g experiment was performed. F i r s t , 6 x 1 0 ~ mole o f each o f the a l c o h o l s used t o form d e r i v a t i v e s was d i s s o l v e d i n 100 mL o f a c e t o nitrile. Then 2 x 1 0 " mole o f compound V was added, and the s o l u t i o n was heated a t 70°C. T h i s s o l u t i o n was then d i l u t e d by t a k i n g 1.0 mL o f the r e a c t i o n m i x t u r e and d i l u t i n g t o 10 mL w i t h water and a c e t o n i t r i l e , and the d i l u t e d s o l u t i o n was a n a l y z e d . A f t e r 4 h the methanol and e t h a n o l were c o m p l e t e l y d e r i v a t i z e d , b u t i t took 6 h f o r the n - p r o p a n o l and n - b u t a n o l d e r i v a t i v e s t o form. To demonstrate t h a t a group o f a l k y l bromides c o u l d be d e r i v a t i z e d a t low a l k y l bromide c o n c e n t r a t i o n , a s i m i l a r experiment 5

4


222


x

6

was performed w i t h reagent I I I . In t h i s experiment, 5 1 0 " mole o f each a l k y l bromide was d i s s o l v e d i n dimethylformamide. To t h i s s o l u t i o n was added 1.8 x 1 0 ~ mole o f reagent I I I , and the m i x t u r e was heated a t 70°C. A f t e r 3 h the p r i m a r y a l k y l bromides were c o m p l e t e l y d e r i v a t i z e d , b u t the secondary a l k y l bromides took 40 h t o be c o m p l e t e l y d e r i v a t i z e d .


4

Synthesis of Derivatizing Reagent III. We p l a c e d 50 mL o f m e t h a n o l , w h i c h had been p r e v i o u s l y d r i e d over 4-Â m o l e c u l a r s i e v e s , i n a 100-mL round-bottom f l a s k and added 6.0 g o f 2 - h y d r o x y n i c o t i n i c a c i d and 3 mL o f boron t r i f l u o r i d e e t h e r a t e . The s o l u t i o n was heated t o r e f l u x f o r 24 h and the s o l v e n t was removed under reduced p r e s s u r e . The r e s i d u e was d i s s o l v e d i n 50 mL o f 0.1-JV sodium h y d r o x i d e and e x t r a c t e d w i t h 60 mL o f c h l o r o f o r m . The c h l o r o f o r m e x t r a c t was c o n c e n t r a t e d under reduced p r e s s u r e and the r e s i d u e c r y s t a l l i z e d from i s o p r o p y l a l c o h o l . The y i e l d o f 3 - c a r b o m e t h o x y - 2 ( l / f ) p y r i d o n e was 5 . 0 g; rap 1 5 2 . 5 - 1 5 4 ° C ; NMR (CDC1 ) δ 3.85 ( s , 3 , - C H ) , 6.34 ( t , 1, ^4,5 = J s , 6 = 7 H z , H - 5 ) , 7.74 ( d d , 1, J , = 2 H z , J , = 7 H z , H-6), 8.21 ( d d , J , e = 2 H z , J , s = 7 H z , H - 4 ) ; mass spectrum, m/e 153, M . T h i s p r o d u c t was p l a c e d i n a 100-mL round-bottom f l a s k , and 50 mL o f c o n c e n t r a t e d ammonium h y d r o x i d e was added. The m i x t u r e was s t i r r e d o v e r n i g h t a t room t e m p e r a t u r e . A f t e r 18 h the s o l u t i o n was c o o l e d and the c r y s t a l s o f compound I I were c o l l e c t e d . No r e c r y s t a l l i z a t i o n was r e q u i r e d . The y i e l d was 4.20 g (93%) o f I I , mp 2 6 8 - 2 6 9 ° C ; NMR (DMS0-d ) δ 6.33 ( t , 1, J , = J , = 7 H z , H - 5 ) , 7.41 ( s , 1, - N H ) , 7.59 ( d d , 1, J , = 7 H z , J , = 2.6 H z , H - 6 ) , 8.23 ( d d , 1, ^ 5 , 6 = 7 H z , J , = 2.5 H z , H - 4 ) , 9.00 ( s , 1, - N H ) , 12.30 ( s , 1, H - l ) ; mass spectrum, m/e 138, M . A n a l . C a l c d f o r C H N 0 : C, 5 2 . 1 5 ; H , 4 . 3 8 ; N , 2 0 . 2 9 . Found: C, 5 1 . 8 5 ; H , 4 . 4 6 ; N , 2 0 . 3 4 . Reagent I I I was formed by d i s s o l v i n g 0.50 g ( 3 . 5 mmol) o f I I i n 25 mL o f water i n a 50-mL round-bottom f l a s k . To t h i s s o l u t i o n was added 0.15 g (3.61 mmol) o f sodium h y d r o x i d e , and the m i x t u r e was heated on a steam b a t h f o r 30 m i n . The water was removed and the r e s i d u e suspended i n hot t o l u e n e . The t o l u e n e s u s p e n s i o n was f i l t e r e d and the r e m a i n i n g s o l i d d r i e d i n an Abderhalden d r y i n g p i s t o l w i t h phosphorus p e n t o x i d e . T h i s procedure gave 0.55 g (99%) y i e l d o f Reagent I I I . 3

3

4

4

5

5

6

4

+

6

5

4

6

4

5

4

6

5

6

6

+

6

6

2

2

General Method for Forming Derivatives with Reagent III. To make the d e r i v a t i v e s , I I I was d i s s o l v e d i n 5 mL dimethylformamide, and the a l k y l h a l i d e was added t o the s a l t s o l u t i o n . The m i x t u r e was a l l o w e d t o s t a n d a t room temperature f o r 24 h , and the dimethylformamide was removed under reduced p r e s s u r e . The r e s i d u e was d i s s o l v e d i n w a t e r , and the d e r i v a t i v e was e x t r a c t e d w i t h 25 mL o f c h l o r o f o r m . The d e r i v a t i v e was t h e n p r e c i p i t a t e d from the c h l o r o f o r m w i t h e t h e r hexane. Heptachlor Derivative of III. A d d i t i o n o f 0.11 g ( 0 . 6 8 mmol) o f I I I and 0.25 g (0.68 mmol) o f h e p t a c h l o r (1,4,5,6,7,8,8-heptachloro3 a , 4 , 7 , 7 a - t e t r a h y d r o - 4 , 7 - m e t h a n o i n d e n e ) t o dimethylformamide gave 0.17 g (50%) o f the d e r i v a t i v e : mp 2 8 0 - 2 8 1 ° C ( d e c ) ; NMR (CDC1 ) δ 3.19 ( d d , 1 ) , 3 . 9 6 - 4 . 1 8 (m, 1 ) , 5 . 6 2 - 6 . 8 1 (m, 3 ) , 6.39 ( t , 1, J , = J ,6 = 7 H z , H - 5 ) , 7.16 ( d d , 1, J , = 2 H z , J , = 7 H z , H - 6 ) , 8.38 ( d d , 1, J , = 2 H z , J , = 7 H z , H - 4 ) ; mass spectrum, m/e 472, M . 3

4

4

5

6

5

5

6

+

4

6

4

5


12.

NELSON ET A U

Anal. Calcd for C H C 1 N 2 0 2 : C, 4 0 . 6 7 ; H , 2 . 2 5 ; N , 5 . 6 9 . 1 6


223


1 0

6

C, 4 0 . 4 6 ; H , 2 . 1 2 ; N , 5 . 9 0 .

Found:

Synthesis of Derivatizing Reagent V. 2 5 . 3 g (163 mmol) o f 3 - p h e n y l p y r i d i n e was heated t o r e f l u x w i t h 60 mL o f 30% hydrogen p e r o x i d e for 5 h i n 100 mL o f g l a c i a l a c e t i c a c i d ( 2 5 ) . The g l a c i a l a c e t i c a c i d and excess hydrogen p e r o x i d e were removed under reduced p r e s sure. To the r e s i d u e 150 mL o f a c e t i c a n h y d r i d e was added, and the m i x t u r e was heated t o r e f l u x f o r 24 h . The a c e t i c a n h y d r i d e was removed under reduced p r e s s u r e , and 10% h y d r o c h l o r i c a c i d was added t o the dark r e s i d u e . T h i s m i x t u r e was a l l o w e d t o s t a n d f o r 10 h , and the a c i d was removed under reduced p r e s s u r e . The r e s i d u e was e x t r a c t e d w i t h hot benzene. The s o l i d t h a t formed on c o o l i n g was r e c r y s t a l l i z e d from benzene t o g i v e crude I V . P u r i f i c a t i o n o f the p r o d u c t was o b t a i n e d by column chromatography o f the crude p r o d u c t on s i l i c a g e l u s i n g c h l o r o f o r m as the m o b i l e phase. A t o t a l o f 13.2 g (47%) o f m a t e r i a l was c o l l e c t e d : mp 2 2 2 - 2 2 3 . 5 ° C ; NMR (CDC1 ) δ 6.18 Ù , 1, J , 5 = ^ 5 , 6 = 7 H z , H - 5 ) , 7.24 (m, 4, a r o m a t i c ) , 7.58 (m, 3 , H-4 + H-6 + a r o m a t i c ) ; mass spectrum, m/e 171, M . A n a l . C a l c d f o r CnH N0: C, 7 7 . 1 7 ; H , 5 . 3 0 ; N , 8 . 1 8 . Found: C, 7 6 . 9 2 ; H , 5 . 2 3 ; N , 8.27. P y r i d o n e IV was c o n v e r t e d t o i t s sodium s a l t by t r e a t m e n t w i t h an e q u i m o l a r amount o f sodium methoxide i n methanol (see procedure below under f o r m a t i o n o f the 3 - p h e n y l - 2 ( l / f ) p y r i d o n e s u l f o n a t e s ) . The sodium s a l t was n e x t t r e a t e d w i t h the m e t h y l e s t e r o f cr-bromo-pt o l u i c a c i d , o b t a i n e d by t r e a t m e n t o f the a c i d w i t h B F e t h e r a t e . For the f o r m a t i o n o f V , 3.67 g (16.0 mmol) o f the m e t h y l e s t e r o f α - b r o m o - p - t o l u i c a c i d and 20 mL o f dimethylformamide were p l a c e d i n a 50-mL round-bottom f l a s k . To t h i s was added 3.04 g (15.7 mmol) o f the sodium s a l t o f I V . The r e a c t i o n t o o k p l a c e r a p i d l y . A f t e r 30 min the s o l v e n t was removed under reduced p r e s s u r e . Crystallization from i s o p r o p y l a l c o h o l gave 3.71 g (74%) o f 1 - ( p - c a r b o x y m e t h y l p h e n y l ) m e t h y l - 3 - p h e n y l - 2 ( l H ) p y r i d o n e . To 0.43 g (1.36 mmol) o f t h i s e s t e r was added 0.055 g ( 1 . 3 8 mmol) o f sodium h y d r o x i d e i n 35 mL o f w a t e r , and the m i x t u r e was heated on a steam b a t h u n t i l a l l the e s t e r dissolved. C o n c e n t r a t e d h y d r o c h l o r i c a c i d was added t o p r o t o n a t e the c a r b o x y l a t e a n i o n , and the a c i d r e l a t e d t o V p r e c i p i t a t e d . Recryst a l l i z a t i o n from i s o p r o p y l a l c o h o l gave 3.42 g (94%) o f the p r o d u c t : mp 1 9 7 - 1 9 9 ° C ; NMR (DMS0-d ) δ 5.25 ( s , 2 , - C H - ) , 6.36 ( t , 1, J , = SJ6 7 H z , H - 5 ) , 7.20 t o 7.80 (m, 1 1 , a r o m a t i c ) ; mass spectrum, m/e 305, M . A n a l . C a l c d f o r C H N 0 : C, 7 4 . 7 5 ; H , 4 . 9 5 ; N , 4 . 5 9 . Found: C, 7 4 . 5 3 ; H , 4 . 9 9 ; N , 4 . 3 2 . To 25 mL o f dimethylformamide i n a 50-mL round-bottom f l a s k was added 0.25 mL ( 3 . 4 2 mmol) o f t h i o n y l c h l o r i d e . A f t e r s e v e r a l m i n u t e s , 1.00 g (3.28 mmol) o f the a c i d o f V was added. The r e a c t i o n m i x t u r e was a l l o w e d t o s t a n d a t room temperature f o r 18 h , and the s o l v e n t was removed under reduced p r e s s u r e . The r e s i d u e was t h e n c r y s t a l l i z e d from c h l o r o f o r m t o g i v e 0.97 g (91%) o f Reagent V : mp 1 5 0 - 1 5 1 ° C ; NMR (DMS0-d ) δ 5.19 ( s , 2 , - C H - ) , 6.23 ( t , 1, J , = 5»6 7 H z , H - 5 ) , 7 . 1 6 - 8 . 0 8 (m, 11, a r o m a t i c ) ; mass spectrum, m/e 339, M . 3

4

+

8

3

6

2

4

5

=

J

+

1 9

6

J

1 5

3

2

4

5

=

+

General Methods of Forming Derivatives with V. The p r e p a r a t i o n o f a l l o f the d e r i v a t i v e s o f V f o l l o w e d one o f t h r e e g e n e r a l methods:


224



Method A . I n t h i s p r o c e d u r e , V was d i s s o l v e d i n 5 mL o f the l i q u i d t o be d e r i v a t i z e d , and the m i x t u r e was heated f o r 30 min a t 6 0 ° C . The s o l v e n t was removed under reduced p r e s s u r e , and the r e s i d u e was c r y s t a l l i z e d from a methanol-water s o l u t i o n . Method B. I n t h i s p r o c e d u r e , V was d i s s o l v e d i n 20 mL o f a c e t o n i t r i l e , and the compound t o be d e r i v a t i z e d was bubbled i n as a gas. The s o l v e n t was removed under reduced p r e s s u r e , and the r e s i d u e was c r y s t a l l i z e d from a methanol-water s o l u t i o n . Method C . I n t h i s p r o c e d u r e , V was d i s s o l v e d i n 10 mL o f p y r i d i n e , and the compound t o be d e r i v a t i z e d was then added. The m i x t u r e was heated a t 60°C f o r 1 h , and the s o l v e n t was removed under reduced p r e s s u r e . The r e s i d u e was then c r y s t a l l i z e d from a methanol-water s o l u t i o n . Methanol Derivative of V. Method A . A d d i t i o n o f 0.10 g (0.31 mmol) o f V t o methanol gave 0.06 g (59%) o f XXV: mp 1 3 3 - 1 3 4 ° C ; NMR (CDC1 ) δ 3.79 ( s , 3 , - 0 C H ) , 5.10 ( s , 2 , - C H - ) 6.10 ( t , J , = J , = 7 H z , H - 5 ) , 7 . 1 0 - 7 . 9 2 (m, 1 1 , a r o m a t i c ) ; mass spectrum, m/e 319, M . A n a l . Calcd f o r C20H17NO3: C, 7 5 . 2 2 ; H , 5 . 3 7 ; N , 4 . 3 9 . Found: C, 7 4 . 9 4 ; H, 5.48; N , 4.22. 3

3

2

4

5

5

6

+

Ethanol Derivative of V. Method A . A d d i t i o n o f 0.10 g (0.31 mmol) of V t o e t h a n o l gave 0.07 g (64%) o f X X V I : mp 8 7 . 5 - 8 9 . 5 ° C ; NMR (CDCI3) δ 1.32 ( t , 3 , - C H ) , 4.26 ( q , 2 , - 0 C H - ) , 5.14 ( s , 2 , - C H - ) , 6.12 ( t , 1, J , = J , = 7 H z , H - 5 ) , 7 . 0 8 - 7 . 9 6 (m, 1 1 , a r o m a t i c ) ; mass spectrum, m/e 333, M . A n a l . C a l c d f o r C H N 0 : C, 7 5 . 6 6 ; H , 5 . 7 5 ; N , 4 . 2 0 . Found: C, 7 5 . 4 4 ; H , 5 . 6 3 ; N , 3 . 9 5 . 3

4

5

5

2

2

6

+

2 1

1 9

3

1-Propanol-Derivative of V. Method A . A d d i t i o n o f 0.30 g (0.98 mmol) o f V t o η - p r o p y l a l c o h o l gave 0.18 g (56%) o f X X V I I : mp 6 8 - 7 0 ° C ; NMR (CDC1 ) δ 0.97 ( t , 3 , - C H ) , 1.72, ( s e x . , 2 , - C H - ) , 4.21 ( t , 2 , - 0 C H - ) , 5.18 ( s , 2 , - C H - ) , 6.18 ( t , 1, J , = J , = 8 H z , H - 5 ) , 7 . 1 4 - 8 . 0 0 (m, 11, a r o m a t i c ) ; mass spectrum, m/e 347, M . Anal. Calcd for C H N 0 : C, 7 6 . 0 6 ; H , 6 . 0 9 ; N , 4 . 0 3 . Found: C, 75.73; H, 6.11; N , 4.01. 3

3

2

2

2

4

5

5

6

+

2 2

2 1

3

1-Butanol Derivative of V. Method A . A d d i t i o n o f 0.30 g (0.98 mmol) o f V i n η - b u t y l a l c o h o l gave 0.20 g (60%) o f X V I I I : mp 5 8 6 0 ° C ; NMR (CDC1 ) δ 0.94 ( t , 3 , - C H ) , 1.22-1.79 (m, 4 , - ( C H ) - ) , 4.25 ( t , 2 , - 0 C H - ) , 6.18 ( t , 1, J , = J , = 7 H z ) , 7 . 1 7 - 8 . 0 2 (m, 11, a r o m a t i c ) ; mass spectrum, m/e 3 6 1 , M . A n a l . C a l c d f o r C 3H N0 : C, 7 6 . 4 3 ; H , 6 . 4 1 ; N , 3 . 8 8 . Found: C, 7 6 . 5 8 ; H , 6 . 1 6 ; N, 4.25. 3

3

2

4

2

5

5

2

6

+

2

2 3

3

Ammonia Derivative of V. Method B . A d d i t i o n o f 0.30 g (0.98 mmol) of V t o a c e t o n i t r i l e and a d d i t i o n o f ammonia gave 0.20 g (71%) o f X X I X : mp 2 1 1 - 2 1 2 ° C ; NMR ( D M S 0 - d , t-BuOD l o c k ) δ 5.16 ( s , 2 , - C H - ) , 6.27 ( t , 1, J , = J , = 7 H z , H - 5 ) , 7 . 0 5 - 7 . 8 0 (m, 1 1 , a r o m a t i c ) ; mass spectrum, m/e 304, M . A n a l . C a l c d f o r C H N 0 : C, 7 4 . 9 8 ; H , 5 . 3 0 ; N , 9 . 2 0 . Found: C, 7 4 . 7 9 ; H , 5 . 1 2 ; N , 9 . 1 9 . 6

4

5

5

2

6

+

1 9

1 6

2

2

Dimethyl amine Derivative of V. Method B . A d d i t i o n o f 0.30 g (0.98 mmol) o f V t o a c e t o n i t r i l e and a d d i t i o n o f dimethylamine gave 0.19 g of XXX: mp 1 1 9 - 1 2 0 ° C ; NMR (CDC1 ) δ 2.98 ( s , 6, N ( C H ) ) , 5.16 ( s , 2, - C H - ) , 6.19 ( t , 1, J , = J , = 7 H z , H - 5 ) , 7 . 1 7 - 7 . 6 6 (m, 11, 3

2

4

5

5

3

2

6


12.

NELSON ET AL.

225

Fluorescent 1,3-Disubstituted 2(m)Pyridones

+

a r o m a t i c ) ; mass spectrum, m/e 332, M . A n a l . C a l c d f o r C21H20N2O2: C, 7 5 . 8 8 ; H , 6 . 0 6 ; N , 1 0 . 2 5 . Found: C, 7 5 . 7 1 ; H , 5 . 9 9 ; N , 8 . 2 8 . Phenol Derivative of V. Method C. A d d i t i o n o f 0.49 g ( 1 . 4 4 mmol) of V and 0.14 g (1.49 mmol) o f p h e n o l t o 10 mL o f p y r i d i n e gave 0.33 g (58%) o f X X X I : mp 1 2 6 - 1 2 8 ° C ; NMR (CDC1 ) δ 5.22 ( s , 2 , - C H - ) , 6.21 ( t , 1, J , = J , = 7 H z , H - 5 ) , 7 . 0 4 - 8 . 0 2 (m, 16, a r o m a t i c ) ; mass spectrum, m/e 3 8 1 , M . A n a l . C a l c d f o r C 5 H N 0 : C, 78.72; H, 5.02; N , 3.67. Found: C, 7 8 . 8 6 ; H , 5 . 0 3 ; N , 3 . 9 2 . 3

2

4

5

5

6

+


2

1 9

3

2- Bromoethylsulfonate Sodium Salt. A s o l u t i o n o f 1,2-dibromoethane ( C a u t i o n : t o x i c ) (307.5 g , 1.65 mol) i n water (225 mL) and 95% e t h a n o l (625 mL) was heated t o r e f l u x and s t i r r e d w h i l e a s o l u t i o n of sodium s u l f a t e (62.5 g , 0.496 mol) i n w a t e r (225 mL) was s l o w l y added over a p e r i o d o f 2 h and t h e n r e f l u x e d f o r an a d d i t i o n a l 2 h . The e x c e s s 1,2-dibromoethane was t h e n d i s t i l l e d o f f w i t h the e t h a n o l and the r e s u l t a n t aqueous s o l u t i o n was a l l o w e d t o d r y . The r e s i d u e was r e c r y s t a l l i z e d from 95% e t h a n o l (2 L ) and d r i e d i n an A b d e r h a l d e n d r y i n g a p p a r a t u s under vacuum because i t was h y g r o s c o p i c . The y i e l d was 67.7 g (68%). *H NMR ( D 0 ) δ 3.43 ( t , 2 , H - l ) , 3.67 ( t , 2 , H-2); C NMR ( D 0 ) δ 2 5 . 6 1 , 5 4 . 6 6 . 2

1 3

2

3- Bromopropylsulfonate Sodium Salt. A s o l u t i o n o f 1,3-dibromopropane ( C a u t i o n : i r r i t a n t ) (10.9 mL, 20 g , 99 mmol) i n 95% e t h a n o l (37.5 mL) and water (13.5 mL) was s t i r r e d v i g o r o u s l y and brought t o r e f l u x . A s o l u t i o n o f sodium s u l f i t e (3.75 g , 29 mmol) d i s s o l v e d i n water (13.5 mL) was s l o w l y added t o the r e f l u x i n g m i x t u r e o v e r a p e r i o d o f 2 h and t h e n r e f l u x e d f o r an a d d i t i o n a l 2 h . The m i x t u r e was poured i n t o a s e p a r a t o r y f u n n e l and a l l o w e d t o c o o l , whereupon i t s e p a r a t e d i n t o a two-phase system. The excess 1,3-dibromopropane was drawn off, and the r e m a i n i n g s o l u t i o n was e v a p o r a t e d t o d r y n e s s . The r e s i d u e was r e c r y s t a l l i z e d from 95% e t h a n o l 3 t i m e s . The y i e l d was 1.15 g (17.7%): mp 2 7 9 - 2 8 5 ° C ( - H B r ) , 306°C ( d e c ) ; *H NMR ( D 0 ) δ 2.29 ( ρ , 2 , H - 2 ) , 3.07 ( t , 2 , H - l ) , 3.60 ( t , 2 , H - 3 ) ; C NMR ( D 0 ) δ 29.10, 33.20, 51.02. 2

1 3

2

4- Bromobutylsulfonate Sodium Salt. A s o l u t i o n o f 1,4-dibromobutane (Caution: l a c h r y m a t o r , i r r i t a n t ) ( 1 2 . 0 mL, 2 1 . 4 g , 99 mmol) i n 95% e t h a n o l (37.5 mL) and w a t e r ( 1 3 . 5 mL) was v i g o r o u s l y s t i r r e d and b r o u g h t to r e f l u x . A s o l u t i o n o f sodium s u l f i t e (3.75 g , 29 mmol) i n w a t e r (13.5 mL) was s l o w l y added t o the r e f l u x i n g m i x t u r e over a p e r i o d o f 2 h and t h e n r e f l u x e d f o r an a d d i t i o n a l 2 h . The m i x t u r e was poured i n a s e p a r a t o r y f u n n e l and a l l o w e d t o c o o l and s e p a r a t e i n t o a two-phase system. The excess 1,4-dibromobutane was drawn off, and the r e m a i n i n g s o l u t i o n was evaporated t o d r y n e s s . The r e s i d u e was r e c r y s t a l l i z e d from 95% e t h a n o l 3 t i m e s . The y i e l d was 2.08 g (29.3%). H NMR ( D 0 ) δ 1.88 (m, 2 ) , 1.97 (m, 2 ) , 2.89 (m, 2 , H - l ) , 3.45 (m, 2 , H - 4 ) . X

2

General Procedures for the Synthesis of the Pyridone Sulfonates VIa,b,c and VIIa,b,c (25). A s o l u t i o n o f 1.25 mmol o f the sodium s a l t o f 3 - c a r b a m o y l - 2 ( l H ) p y r i d o n e ( I I I ) o r 3 - p h e n y l - 2 ( l H ) p y r i d o n e and 1.25 mmol o f the c o r r e s p o n d i n g bromosulfonate sodium s a l t i n 50 mL o f a c e t o n i t r i l e was brought t o r e f l u x and s t i r r e d f o r 24 h . The r e a c t i o n m i x t u r e was c o o l e d and f i l t e r e d t h r o u g h a s i n t e r e d g l a s s


226



f u n n e l . The r e m a i n i n g s o l i d was t r i t u r a t e d w i t h t h r e e p o r t i o n s o f h o t a c e t o n i t r i l e , a d d i n g t h e f i l t r a t e s t o t h e o r i g i n a l f i l t r a t e . The a c e t o n i t r i l e was e v a p o r a t e d . Some o f t h e p r o d u c t s were contaminated w i t h sodium b r o m i d e . Good a n a l y s e s were d i f f i c u l t t o o b t a i n because o f t h i s c o n t a m i n a t i o n and because the s u l f o n a t e s were h y g r o s c o p i c . S t r u c t u r e s were supported by NMR d a t a . I n one c a s e , a good a n a l y s i s was o b t a i n e d . An example i s g i v e n b e l o w . 3-Phenyl-2(lH)Pyridone Sodium Salt. A s o l u t i o n o f sodium m e t a l (0.25 g , 1 1 . 3 mmol) and anhydrous methanol (10 mL) was a l l o w e d t o r e a c t t o form sodium methoxide. P y r i d o n e IV ( 1 . 8 8 g , 11 mmol) was added t o t h e s o l u t i o n and a l l o w e d t o s t a n d a t room temperature f o r 24 h . The methanol was removed under reduced p r e s s u r e and the r e s i d u e was e x t r a c t e d w i t h h o t benzene 3 times and d r i e d i n the A b d e r h a l d e n d r y i n g p i s t o l f o r 30 m i n . The y i e l d was 1.7 g (82%). 3-Phenyl-2(lH)Pyridone-Propylsulfonate Sodium Salt Vllb. A solution o f 3 - p h e n y l - 2 ( l H ) p y r i d o n e sodium s a l t (0.264 g , 1.25 mmol) and 3 b r o m o p r o p y l s u l f o n a t e sodium s a l t (0.241 g , 1.25 mmol) i n a c e t o n i t r i l e (650 mL) was brought t o r e f l u x w i t h s t i r r i n g f o r 24 h . The y i e l d was 41%, as determined by HPLC. An a n a l y t i c a l sample was o b t a i n e d from the f i l t e r e d r e s i d u e o f r e a c t i o n by p r e p a r a t i v e HPLC. A n a l . C a l c d f o r C H N S 0 N a H 0 : C , 5 0 . 4 5 ; H , 4 . 8 ; N , 4 . 2 . Found: C, 5 0 . 3 8 ; H, 4 . 8 3 ; N , 4 . 1 9 . A n a l y t i c a l samples o f l - a l k y l - 3 - s u b s t i t u t e d 2 ( l H ) p y r i d o n e s u l f o n a t e s were o b t a i n e d by u s i n g a Whatman P a r t i s i l M-9 10/50 0DS-2 C18 column. The 3-carbamoyl s e r i e s compounds were o b t a i n e d u s i n g a m o b i l e phase o f 90% water and 10% a c e t o n i t r i l e w i t h a f l o w r a t e o f 3.3 mL p e r m i n u t e . The 3 - p h e n y l s e r i e s compounds were o b t a i n e d u s i n g a m o b i l e phase o f 85% water and 15% a c e t o n i t r i l e w i t h a f l o w r a t e o f 3 . 3 mL p e r m i n u t e . Any 3 - s u b s t i t u t e d 2 ( l H ) p y r i d o n e was r e t a i n e d a t the head o f t h e column. 1 4

1 4

4

2

Literature Cited 1. Diebold, G. J.; Zare, R. N. Science 1977, 1439. 2. Furata, N.; Otsuki, A. Anal. Chem. 1983, 55, 2407. 3. Skoropinski, D. B.; Callis, J. B.; Danielson, J. D. S.; Christian, G. D. Anal. Chem. 1986, 58, 2831. 4. Pfeffer, W. D.; Yeung, E. S. Anal. Chem. 1986, 58, 2103. 5. Lawrence, J. F.; Frei, R. W. Chemical Derivatization in Liquid Chromatography; Elsevier Scientific: New York, 1976. 6. Grushka, E.; Lam, S.; Chassin, J. Anal. Chem. 1978, 50, 1398. 7. Frei, R. W.; Lawrence, J. F. J. Chromatogr. 1973, 83, 231. 8. Cassidy, R. M.; Le Gay, D. S.; Frei, R. W. J. Chromatogr. Sci. 1974, 12, 85. 9. Blau, K.; King, G. S., Eds. Handbook of Derivatives for Chromatography; Heyden: Philadelphia, PA, 1978, p 351-384. 10. Chen, R. F. Nature 1966, 209, 69. 11. Chen, R. F. Arch. Biochem. Biophy. 1967, 120, 609. 12. Seiler, N.; Wiechmann, M. Z. Anal. Chem. 1966, 220, 109. 13. Lawrence, J. F. J. Chromatogr. Sci. 1979, 17, 147. 14. Holman, W. I. M.; Weigand, C. Biochem. J. 1948, 43, 423. 15. Veda, K. J. Jpn. Biochem. Soc. 1950, 22, 196; Chem. Abstr. 1952, 46, 4541.



12. NELSON ET AL.

Fluorescent 1,3-Disubstituted 2(lVL)Pyridones

227

16. Robenson, T.; Cepurneck, C. Phytochemistry 1965, 4, 75. 17. Chung, N. M.; Tieckelmann, H. J. Org. Chem. 1970, 35, 2517. 18. Smart, P. L.; Laidlaw, I. M. S. Water Resour. Res. 1977, 13, 15. 19. Yoneda, G. S.; Griffin, M. T.; Carlyle, D. W. J. Org. Chem. 1975, 40, 375. 20. Gilbert, Ε. E. Sulfonation and Related Reactions; Robert E. Krieger Publishing: Huntington, NY, 1977, p 137. 21. Marvel, C. S.; Sparberg, M. S. Org. Synth., Coll. 1943, 2, 558. 22. Nelson, D. Α.; Lenz, D. M. Gov. Rep. Announce. Index (U.S.) 1984, 85(5), 130, NTIS No. PB84-132489. 23. Beckett, J. R.; Nelson, D. A. Anal. Chem. 1981, 53, 910. 24. Parker, C. Α.; Rees, W. T. Analyst (London) 1960, 85, 587. 25. Boekelheide, V.; Linn, W. J. J. Am. Chem. Soc. 1954, 76, 1286. RECEIVED September 26, 1988


Luminescence Applications - American Chemical Society

Recommend Documents