6 Synthesis of Xenobiotic Conjugates Xenobiotic Conjugation Chemistry Downloaded from pubs.acs.org by UNIV OF CALIFORNIA SANTA BARBARA on 07/12/18. For personal use only.
Ake Bergman Section of Organic Chemistry, Wallenberg Laboratory, University of Stockholm, S-106 91 Stockholm, Sweden Chemical methods for the synthesis of the major types of conjugates known to be formed as metabolites of xenobiotics are reviewed. The synthesis of glucuronic acid, sulfate, glutathione, cysteine, N-acetyl-cysteine, glucose, amino acid, bile acid, fatty acid and methylthiolated conjugates can be accomplished by a variety of reactions. These synthetic methods are described, exemplified and the u t i l i t y of these in macro and micro scale preparations is discussed in relation to synthesis of radiolabeled and unlabeled conjugates. Preparative scale enzymatic systems that have been used for the synthesis of conjugates are included. Most xenobiotic conjugation reactions result in products that have very different physical and chemical properties than the unconjugated xenobiotic. Many xenobiotic conjugates have less biological activity than the parent compound; however some conjugates are reactive species with greater biological activity than the xenobiotic from which they were derived (1). Some xenobiotic conjugates have lipophilic characteristics quite similar to the parent xenobiotic C?,3) and may be bioaccumulated in animal tissues with or without enterohepatic circulation. Xenobiotic conjugates may be further metabolized to either more or less polar products through degradation or additional conjugation reactions. Xenobiotic conjugates must be synthesized or isolated from biological matter in a preparative scale in order to prove the structure of these conjugates, and to describe their physical and chemical characteristics, their biological activity and bioaccumulation properties. The chemical synthesis of xenobiotic conjugates may also elucidate the often complex sterochemistry involved in the formation of these compounds. The present review on synthesis of xenobiotic conjugates will concentrate on methods for synthesis of the carbohydrate (glucuronic acid, glucose), sulfate, and mercapturic acid pathway conjugates, the latter including glutathione, cysteine, N-acetyl-cysteine, methyl sulfide and sulfone containing compounds. 0097-6156/86/0299-0124$09.50/0 © 1986 American Chemical Society
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The s y n t h e s i s o f model compounds t o v e r i f y t h e s t r u c t u r e s o f x e n o b i o t i c amino a c i d a d d u c t s ( p r o d u c t s o f nonenzymatic r e a c t i o n s of e l e c t r o p h i l i c x e n o b i o t i c s w i t h amino a c i d s i n p r o t e i n s ) a r e i n c l u d e d . Methods f o r s y n t h e s i s o f some r a r e l y o b s e r v e d c o n j u g a t e types w i l l be b r i e f l y i n t r o d u c e d . The major o r g a n i c methods f o r s y n t h e s i s a r e e x e m p l i f i e d and x e n o b i o t i c c o n j u g a t e s p r e p a r e d v i a these r e a c t i o n s a r e t a b u l a t e d w i t h r e f e r e n c e s t o t h e o r i g i n a l publications. Synthesis
of Glucuronic
a c i d conjugates
I n v i v o D - g l u c u r o n i c a c i d c o n j u g a t e s o f x e n o b i o t i c s a r e formed from U D P - g l u c u r o n i c a c i d and x e n o b i o t i c s . The r e a c t i o n s a r e c a t a l y z e d by U D P - g l u c u r o n y l t r a n s f e r a s e enzymes; 0-, S-, N- and C - g l u c u r o n i d e s c a n be formed i n t h i s manner. The b i o c h e m i c a l f o r m a t i o n o f g l u c u r o n i c a c i d c o n j u g a t e s and t h e c h e m i c a l p r o p e r t i e s o f both enzyme and c o n j u g a t e s have been t h o r o u g h l y s t u d i e d (4,5,6). C h e m i c a l methods f o r t h e s y n t h e s i s o f g l u c u r o n i c a c i d c o n j u g a t e s o f 0-, S-, N- and C - a g l y c o n e s were most r e c e n t l y reviewed by K e g l e v i c ( 7 ) . Both d i r e c t and i n d i r e c t methods a r e a v a i l a b l e f o r synthesis of glucuronic a c i d conjugates. K o e n i g s - K n o r r s y n t h e s i s . T h i s method, based on the method d e s c r i b e d by K o e n i g s and K n o r r ( 8 ) f o r t h e s y n t h e s i s o f g l y c o p y r a n o s i d e s i s t h e p r o c e d u r e most o f t e n used f o r s y n t h e s i s o f g l u c u r o n i c a c i d c o n j u g a t e s . The K o e n i g s - K n o r r r e a c t i o n w i t h i t s m o d i f i c a t i o n s ( c f . s y n t h e s i s o f g l u c o s e c o n j u g a t e s ) has been reviewed by I g a r a s h i ( 9 ) . The r e a c t i o n i s t y p i c a l l y c a r r i e d o u t a s d e s c r i b e d by B o l l e n b a c k ( 1 0 ) : m e t h y l ( t r i - O - a c e t y l - c t - D - g l u c o p y r a n o s y l bromide) u r o n a t e i s s t i r r e d w i t h a t e n t i m e s m o l a r e x c e s s o f a phenol i n the presence o f s i l v e r carbonate ( F i g u r e 1). Solvents, such a s d i e t h y l e t h e r , m e t h y l e n e c h l o r i d e , a c e t o n e , benzene, toluene, 1,2-dichloroethane, methanol and dimethylformamide (DMF), s u i t a b l e t o d i s s o l v e the a g l y c o n e , may be u s e d . However, t h e y i e l d s a r e improved by d r y r e a c t i o n c o n d i t i o n s ; the water c a n be t r a p p e d by magnesium s u l f a t e , c a l c i u m s u l f a t e , o r m o l e c u l a r s i e v e s (3&) added t o t h e r e a c t i o n m i x t u r e o r c a n be removed by c o n t i n o u s a z e o t r o p i c d e s t i n a t i o n o f t h e s o l v e n t . The r e a c t i o n p r o c e e d s w i t h inversion of configuration a t C - l of the glucuronic a c i d . The K o e n i g s - K n o r r r e a c t i o n , i n i t s o r i g i n a l form, r e q u i r e s r e a c t i o n a t room temperature o v e r n i g h t . The a p p r o p r i a t e molar r a t i o o f the r e a c t a n t s c a n f u r t h e r improve t h e y i e l d s a s shown i n the s y n t h e s i s o f c e r t a i n s t e r o i d g l u c u r o n i d e s (11,12). Promotors o t h e r than s i l v e r s a l t s have been used a s shown i n T a b l e I . Cadmium c a r b o n a t e may be e s p e c i a l l y u s e f u l i n t h i s r e g a r d ( 1 2 ) ; the cadmium bromide formed i n t h i s r e a c t i o n l i k e l y a c t s a s a c a t a l y s t . S i l v e r p e r c h l o r a t e (0.2 mol/mol a g l y c o n e ) has been r e p o r t e d t o be a n e f f e c t i v e c a t a l y s t when used t o g e t h e r w i t h s i l v e r c a r b o n a t e ( 1 3 ) . Phase t r a n s f e r c a t a l y s i s (PTC) c o n d i t i o n s were used f o r s y n t h e s i s o f m e t h y l ( 2 - b e n z y l o x y - 4 - f o r m y l p h e n y l 2 , 3 , 4 - t r i - 0 - a c e t y l - B - D - g l u c o p y r a n o 8 i d e ) u r o n a t e ( F i g u r e 2) but o n l y a moderate y i e l d (28%) was o b t a i n e d ( 1 4 ) . O t h e r p r o t e c t i v e groups f o r t h e c a r b o h y d r a t e h y d r o x y l s c a n a l s o be used, thus methyl ( 2 , 3 , 4 - t r i - O - p i v a l o y l - a - D - g l u c o p y r a n o s y l bromide) u r o n a t e
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was s u c e s s f u l l y s y n t h e s i z e d and f u r t h e r used f o r t h e p r e p a r a t i o n o f p r i m a r y , secondary and t e r t i a r y a l c o h o l g l u c u r o n i d e s i n h i g h y i e l d s (15). S y n t h e s i s o f S-, N-, N-hydroxy- and C - g l u c u r o n i d e s can a l s o be performed by r e a c t i o n s a n a l o g o u s t o the one shown i n F i g u r e 1. S - N u c l e o p h i l e s may i n d u c e p a r t i a l h y d r o l y s i s o f the a c e t y l p r o t e c t i v e groups but a d d i t i o n o f an a c e t y l a t i n g agent p r i o r t o i s o l a t i o n o f the S - g l u c u r o n i d e d e r i v a t i v e s w i l l improve the y i e l d s . Some N - g l u c u r o n i d e s can be formed by rearrangement o f c e r t a i n O - g l u c u r o n i d e s as i l l u s t r a t e d i n F i g u r e 3 ( 1 6 ) . A C - g l u c u r o n i d e o f e q u i l e n i n e was o b t a i n e d as a b y p r o d u c t from t h e s y n t h e s i s o f methylequilenine ( t r i - O - a c e t y l - B - D - g l u c o p y r a n o s i d e ) uronate (12). The methyl ( t r i - O - a c e t y l - c t - D - g l u c o p y r a n o s y l bromide) u r o n a t e i s r e a d i l y p r e p a r e d (10,17) but i t i s a l s o c o m m e r c i a l l y a v a i l a b l e . The d e r i v a t l z e d c o n j u g a t e i s commonly p u r i f i e d by c h r o m a t o g r a p h i c p r o c e d u r e s b e f o r e i t i s d e p r o t e c t e d by a l k a l i n e h y d r o l y s i s a t +4 C o r room temperature (4,7,10,12). The s y n t h e s i s o f a s e l e c t e d number o f d e r i v a t l z e d and d e p r o t e c t e d g l u c u r o n i c a c i d c o n j u g a t e s o f x e n o b i o t i c s a r e summarized i n T a b l e I . F u s i o n type s y n t h e s i s . M e t h y l ( t e t r a - O - a c e t y l - f H D - g l u c o p y r a n o s e ) u r o n a t e r e a c t e d w i t h an e x c e s s o f p h e n o l i n t h e p r e s e n c e o f z i n c c h l o r i d e a t reduced p r e s s u r e and 110-120°C ( 1 0 ) . The p r o d u c t , m e t h y l ( p h e n y l - t r i - 0 - a c e t y l - 3 - D - g l u c u p y r a n o s i d e ) u r o n a t e , was i s o l a t e d i n 8% y i e l d . I f p - t o l u e n e s u l f o n i c a c i d was used i n s t e a d o f z i n c c h l o r i d e a 55% y i e l d o f t h e g l u c u r o n i d e was o b t a i n e d . P e n t a - O - a c e t y l - c t - L - i d o p y r a n o s e was f u s e d under s i m i l a r c o n d i t i o n s w i t h p h e n o l t o g i v e the anomeric p h e n y l t e t r a - O - a c e t y l - L - i d o p y r a n o s i d e s ( 1 8 ) . These compounds were o x i d i z e d t o t h e c o r r e sponding g l u c u r o n i c a c i d c o n j u g a t e s by oxygen o v e r a p l a t l n l u m o x i d e c a t a l y s t . D i e t h y l s t i l b o e s t r o l - 3 - D - g l u c u r o n i c a c i d was o b t a i n e d i n 40% y i e l d a f t e r c o n d e n s a t i o n u s i n g a c a t a l y t i c amount o f p - t o l u e n e s u l f o n i c a c i d and subsequent a l k a l i n e h y d r o l y s i s ( 1 9 ) . A n o t h e r c a t a l y s t , s t a n n i c c h l o r i d e , has shown advantageous p r o p e r t i e s f o r c a r r y i n g out t h i s type o f c o n d e n s a t i o n r e a c t i o n under m i l d c o n d i t i o n s ( 2 0 ) . T h e r e i s o n l y l i m i t e d i n f o r m a t i o n on the s y n t h e s i s o f C - g l u c u r o n i d e s o f x e n o b i o t i c s p r o b a b l y due t o t h e f a c t t h a t r e l a t i v e l y few c o n j u g a t e s o f t h i s type have been r e p o r t e d . The C - g l u c u r o n i d e o f A " - t e t r a h y d r o c a n n a b i n o l (A -THC) was p r e p a r e d from A -THC and m e t h y l ( t e t r a - O - a c e t y l - 3 - D - g l u c o p y r a n o s e ) u r o n a t e u s i n g boron t r i f l u o r i d e e t h e r a t e as a c a t a l y s t a t 0°C f o r 4 h ( 2 1 ) . I f t h e r e a c t i o n was c a t a l y z e d by p - t o l u e n e s u l f o n i c a c i d under r e f l u x an e q u a l amount o f t h e A6-THC 4 ' - C - g l u c u r o n i d e and A -THC 0 - g l u c u r o n i d e and a m i n o r amount o f A6-THC 6'-C-glucu r o n i d e were o b t a i n e d . 6
6
6
O x i d a t i v e r e a c t i o n s . O x i d a t i o n o f the p r i m a r y a l c o h o l group o f p a r t i a l l y p r o t e c t e d g l u c o p y r a n o s i d e s can be performed w i t h o x i d i z i n g agents such as p o t a s s i u m permanganate, hydrogen p e r o x i d e o r h y p o h a l o g e n i t e (7). Ruthenium t e t r a o x i d e , p r e s e n t i n c a t a l y t i c amount t o g e t h e r w i t h molar amounts of p e r i o d a t e e f f e c t i v e l y o x i d i z e s a l c o h o l s such as the p r i m a r y h y d r o x y l group o f g l u c o s i d e s i f the r e m a i n i n g h y d r o x y l groups a r e p r o t e c t e d ( 3 5 ) . O x i d a t i o n o f g l u c o s i d e s w i t h oxygen and a p l a t i n u m c a t a l y s t i s an i m p o r t a n t
BERGMAN
Synthesis of Xenobiotic Conjugates
COOMe
COOMe
OAc
CAc
F i g u r e 1. S y n t h e s i s o f m e t h y l ( p h e n y l - 2 , 3 , 4 - t r i - 0 - a c e t y l 3^D-glucopyranoside) u r o n a t e by t h e K o e n i g s - K n o r r method.
COOMe
Q
OAC F i g u r e 2. S t r u c t u r e o f m e t h y l ( 2 - b e n z y l o x y - 4 - f o r m y l p h e n y l 2,3,4-tri-O-acetyl-B-D-glucopyranoside) uronate.
COOMe
F i g u r e 3. 0-N rearrangement o f m e t h y l -acety1-8-D-glucopyranosid) uronate.
COOMe
(2-pyridyl-2,3,4-tri-0-
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XENOBIOTIC CONJUGATION CHEMISTRY
T a b l e I . X e n o b i o t i c s o r x e n o b i o t i c m e t a b o l i t e s ( a g l y c o n e s ) used i n K o e n i g s - K n o r r type s y n t h e s i s o r f u s i o n r e a c t i o n . Aglycone Phenol* 2- and 4 - H a l o p h e n o l s * 2-,3- and 4 - M e t h y l p h e n o l * Catechol* 1- and 2-Naphthol* Methyl g e n t i s a t e * N-Acetyl-N-phenylhydroxylamine 4-Methoxy-2-methyl-6sulfanilamidopyrimidine 2-Pyridinol Cortisone Cortisol Corticosterone P o t a s s i u m m e t h y l and e t h y l x a n t h a t e ^ Potassium benzyl xanthate^ 188-Glycyrrhetic acid Estrone 173-Estradiol Equilin Equilenin* o,o -Diethyl-4,4 -stilbenediol* 2-Benzyloxy-4-hydroxybenzaldehyde Phenolphthalein methyl e s t e r Hexoestrol Procaterol Methaqualone 1-Phenylethanol N2-Methyl-9-hydroxyellipticinium acetate 4-Chloro-3-hydroxybenzotrifluoride tert-Butanol 2,2,2-Trichloroethanol 2-Adamantano1 5-Ethoxy-2-nitrophenol 1
2
1
1
Promotor Ag2C03 Ag2C03 Ag2C03 Ag2C03 Ag2C03 Ag2C03 Ag2C03
Ref. 10 10 10 10 10 10 22
-
23 16 24 24 24 25 25 11 12 12 12 12 26/27
-
Ag2C3 Ag2C03 Ag2C03
-Ag2C03 CdC03 CdC0 CdC03 CdC0 -/CdC0 3
3
-C d C 0
3
T2T 3
CdC03
-
Ag C0 Ag2C03 2
3
Ag2C03 Ag2C03 Ag2C03
1. * - Aglycone8 used a l s o i n s y n t h e s i s o f g l u c u r o n i d e s f u s i o n r e a c t i o n ; 2. N - g l u c u r o n i d e ; 3. S - g l u c u r o n i d e ; 4. C - g l u c u r o n i d e .
28 27 29 30 31
32 33 15 15 15 34 v i a the
method f o r t h e p r e p a r a t i o n o f g l u c u r o n i c a c i d c o n j u g a t e s ( 3 6 ) . F o r example, ( 2 - e t h o x y - 5 - n i t r o p h e n y l ) - $ - D - g l u c o p y r a n o s i d e uronic acid was o b t a i n e d by a P t c a t a l y z e d o x i d a t i o n o f t h e c o r r e s p o n d i n g g l u c o s i d e a t 80°C ( 3 7 ) . S i n c e t h e s t a r t i n g m a t e r i a l f o r t h e o x i d a t i o n must be t h e g l u c o s e c o n j u g a t e , o r a d e r i v a t i v e t h e r e o f , t h e method i s v a l u a b l e i f both g l u c o s e and g l u c u r o n i c a c i d c o n j u g a t e s a r e needed. The s y n t h e s i s o f g l u c o s e c o n j u g a t e s w i l l be d i s c u s s e d below. However, o x i d a t i o n p r o c e d u r e s c a n n o t always be used because o t h e r f u n c t i o n a l groups i n t h e a g l y c o n e m o i e t y may a l s o be oxidized.
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Enzymatic methods. I n v i t r o s y n t h e s i s o f g l u c u r o n i d e s by u s e o f i s o l a t e d U D P - g l u c u r o n y l t r a n s f e r a s e and a n a g l y c o n e may be p e r formed ( e . g . 38) b u t o n l y v e r y s m a l l amounts o f a g l y c o n e s c a n be p r o c e s s e d by t h i s method. Immobilized UDP-glucuronyltransferase, c o v a l e n t y bound t o cyanogen bromide a c t i v a t e d a g a r o s e (39) o r Sepharose ( 4 0 ) , has been used f o r t h e p r e p a r a t i o n o f a v a r i e t y o f g l u c u r o n i d e c o n j u g a t e s . The p u r i f i e d and i m m o b i l i z e d enzyme from r a b b i t l i v e r was more s t a b l e than t h e enzyme o b t a i n e d from r a t o r c a l f l i v e r . I m m o b i l i z a t i o n s t a b i l i z e s the enzyme which may be used r e p e a t e d l y . S m a l l amounts o f e t h a n o l , d i m e t h y l s u l f o x i d e (DMSO), and p r o p y l e n e g l y c o l c a n be added t o t h e aqueous r e a c t i o n medium w i t h o u t a major l o s s o f enzymatic a c t i v i t y . However, t h e a d d i t o n of o n l y 2% o f dioxane r e s u l t e d i n 100% i n h i b i t i o n (39,40). The o r i g i n a l l y r e p o r t e d 50-70% v i e l d o f 4 - n i t r o p h e n o l g l u c u r o n i d e was improved by a d d i t i o n s o f M g ( s 3 . 0 mM) and C a ( 0 . 8 - 3 . 0 mM) s a l t s ( 4 1 ) , but h i g h e r c o n c e n t r a t i o n s o f t h e s e i o n s were i n h i b i t o r y . The r e a c t i o n h a s been c a r r i e d o u t i n a 5-30 mg s c a l e o f a g l y c o n e and t h e p r o d u c t s were p u r i f i e d by TLC methods (39,41). I f l a r g e r amounts o f c o s o l v e n t s must be added i n o r d e r t o d i s s o l v e more n o n p o l a r x e n o b i o t i c s lower y i e l d s a r e o b t a i n e d . Sometimes t h i s problem c a n be overcome by e x t e n d i n g t h e i n c u b a t i o n t i m e . Several glucuronide conjugates o f x e n o b i o t i c s , exemplified i n T a b l e I I , have been s y n t h e s i z e d by t h e i m m o b i l i z e d UDP-glucuronylt r a n s f e r a s e enzyme method (40-47). i +
2+
G l u c u r o n i d e e s t e r s y n t h e s i s . The c h e m i c a l s y n t h e s i s o f g l u c u r o n i c a c i d c o n j u g a t e s o f x e n o b i o t i c c o n t a i n i n g a c a r b o x y l group c a n be a c c o m p l i s h e d by r e a c t i n g t h e x e n o b i o t i c and a p r o t e c t e d g l u c u r o n i c a c i d i n t h e presence o f molar amounts o f e.g. d i c y c l o h e x y l c a r b o d i i m i d e (DCC) (48,49), o r l j l ' - c a r b o n y l d i i m i d a z o l e a s i l l u s t r a t e d i n F i g u r e 4A ( 5 0 ) . The d i i m i d e a c t i v a t e d x e n o b i o t i c a c i d r e a c t s with the protected g l u c u r o n i c a c i d t o give e s t e r conjugates i n f a i r y i e l d (-50%). The r e a c t i o n s may be c a r r i e d o u t i n o r g a n i c s o l v e n t s such a s DMF, methylene c h l o r i d e and t e t r a h y d r o f u r a n . The DCC mediated procedure cannot be used t o p r e p a r e t h e g l u c u r o n i c a c i d conjugates of a r o y l a c i d s . Instead the corrsponding a r o y l a c i d c h l o r i d e s c a n be used a s shown i n F i g u r e 4B ( 5 1 ) . The K o e n i g s - K n o r r r e a c t i o n may a l s o be used f o r t h e s y n t h e s i s o f g l u c u r o n i c a c i d c o n j u g a t e s . A more r e a d i l y d e p r o t e c t e d g l u c u r o n i c acid derivative, 2,2,2-trichloroethyl (2,3,4-tri-0-(2,2,2-tric h l o r o - e t h y l o x y c a r b o n y l ) - D - g l u c o p y r a n o s i d e ) u r o n a t e , h a s been used i n glucuronide e s t e r s y n t h e s i s i n order t o minimize h y d r o l y s i s o f the 1 - e s t e r bond ( 4 9 ) . M i s c e l l a n e o u s methods and comments. The N ^ - g l u c u r o n i c a c i d c o n j u g a t e o f metoelopramide was o b t a i n e d i n low y i e l d by s i m p l y r e a c t i n g t h i s compound w i t h a molar amount o f g l u c u r o n i c a c i d a t room temperature o v e r n i g h t ( 5 2 ) . T r i m e t h y l s i l y l t r i f l u o r o m e t h a n e s u l f o n a t e h a s been used t o a c t i v a t e m e t h y l ( 2 , 3 , 4 - t r i - O - a c e t y l glucopyranoside) uronate f o r r e a c t i o n with hydroxy-containing a r o m a t i c and a l i p h a t i c a g l y c o n s a t low temperatures ( 5 3 ) . Both a - and B-anomeric p r o d u c t s were formed. Chemical s y n t h e s i s o f g l u c u r o n i c a c i d conjugates o f p o l y f u n c t i o n a l x e n o b i o t i c s may y i e l d u n d e s i r a b l e s i d e p r o d u c t s ; enzymatic methods may be p r e f e r a b l e f o r g l u c u r o n i c a c i d
130
XENOBIOTIC CONJUGATION CHEMISTRY
T a b l e I I . X e n o b i o t i c s o r x e n o b i o t i c m e t a b o l i t e s ( a g l y c o n e s ) used i n s y n t h e s i s o f g l u c u r o n i c a c i d c o n j u g a t e s by o x i d a t i v e ( A ) , enzymatic (B) and e s t e r i f i c a t i o n (C) methods. Aglycone Methanol Tr iphenylmethanol 2-Ethoxy-5-nitrophenol 4-Nitrophenol Borneol Meprobamate 4-Nitrothiophenol ^-Tetrahydrocannabinol Cannablnol Isoborneol N-Hydroxyphenacetin N-Hydroxy-4-chloroacetanilide N-Hyd roxy-2-ace t y l a m i n o n a p h t h a l e n e N-Hydroxy-2-acetylaminofluorene Flurbiprofen Gemfibrozil Prodolic acid Flufenamic a c i d Tripelennamine Cyproheptadine Indomethacin Ananilino acid^ 3-Phenoxybenzoic a c i d 4-Chlorobenzoic a c i d
Method A A A A,B B B B B B B B B B B B B B B B B C C C C
Ref. 54 35 37 38-41 41 41
TT 42 43 44 46 46 46 46 47 47 47 77 45
T5 49 55 48 56
1. F l u v a l i n a t e m e t a b o l i t e .
c o n j u g a t i o n of compounds o f t h i s type and i f o n l y l i m i t e d amounts of t h e a g l y c o n e i s a v a i l a b l e . However, the K o e n i g s - K n o r r r e a c t i o n can a l s o be used f o r m i c r o s c a l e s y n t h e s i s . O x i d a t i v e methods, on the o t h e r hand, r e q u i r e somewhat l a r g e r amounts o f r e a c t a n t s . The s y n t h e s i s o f r a d i o l a b e l e d x e n o b i o t i c g l u c u r o n i d e s seem t o be c a r r i e d out l e s s o f t e n . I t i s u s u a l l y p r e f e r a b l e t o l a b e l t h e x e n o b i o t i c moiety but t h e g l u c u r o n i c a c i d c a n c e r t a i n l y be l a b e l e d as w e l l . L a b e l e d m e t h y l ( 2 , 3 , 4 - t r i a c e t y l - a - D - g l u c o p y r a n o s y l bromide) u r o n a t e i s a t p r e s e n t n o t c o m m e r c i a l l y a v a i l a b l e . S y n t h e s i s of G l u c o s e
conjugates
G l u c o p y r a n o s i d e c o n j u g a t e s o f exogenous compounds a r e commonly found i n p l a n t s ( 5 7 ) and i n s e c t s ( 5 8 ) . S t e r o i d g l y c o s i d e s were a l s o r e c e n t l y c o n s i d e r e d as p o t e n t i a l prodrugs w i t h s p e c i f i c t i s s u e d i s t r i b u t i o n ( 5 9 ) . The s y n t h e s i s o f
