Xenobiotic Conjugation Chemistry - ACS Publications - American

8 Identification of Xenobiotic Conjugates by Nuclear Magnetic Resonance Spectrometry V. J. Feil

Downloaded by UNIV LAVAL on July 14, 2016 | http://pubs.acs.org Publication Date: January 24, 1986 | doi: 10.1021/bk-1986-0299.ch008

Metabolism and Radiation Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Fargo, ND 58105 A review of the l i t e r a t u r e that contains 120 references on the application of NMR spectrometry for the i d e n t i f i c a t i o n of xenobiotic conjugates. The references (primarily 1980 to 1984) provide structures and spectral summaries for sulfate, carbohydrate, amino acid, and miscellaneous conjugates. This review i s primarily a compilation of examples where nuclear magnetic resonance (NMR) has been used i n some capacity for the characterization of xenobiotic conjugates. Most references are taken from the period 1980-1984$ some e a r l i e r references are included because they provide examples of metabolites that were not available i n more recent references. The following information, when available, i s included i n the compilations! conjugate structure, source of the conjugate, NMR characteristics of the conjugate, effect of the conjugating group on the spectrum of the xenobiotic, MIR frequency, solvent, and l i t e r a t u r e reference. Related reviews have been published on the use of NMR i n pesticide analysis U ) , metabolic studies (2,3), drug metabolism (4,5), and medicinal chemistry (6,7). Since most of these reviews as well as many texts contain introductions to NMR, none w i l l be presented here. Furthermore, an audio course on Fourier Transform NMR Spectroscopy i s also available (8). Since structural characterization i s often based on empirical correlations, we have found comp i l a t i o n s of proton spectra by Aldrich, Sadtler, and Varian (9-11) and carbon spectra by Breitmaier et a l . and Bruker (12,13) to be useful. Becker (14) has presented extensive l i s t s o7~reFerences that contain useful compilations of NMR data (pp 77 and 106). Techniques The use of NMR i n the i d e n t i f i c a t i o n of xenobiotic conjugates i s quite limited. Many recent publications that include NMR i n struct u r a l characterizations used high f i e l d instruments (200 MHz or higher). Thus, the use of NMR i n metabolite characterization may increase with increased a v a i l a b i l i t y of high f i e l d instruments This chapter not subject to U.S. copyright. Published 1986, American Chemical Society

Paulson et al.; Xenobiotic Conjugation Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 1986.


178

XENOBIOTIC CONJUGATION CHEMISTRY

because the p o t e n t i a l for obtaining s t r u c t u r a l information from high f i e l d spectra i s so great. No published report on the use of two-dimensional NMR (15), 2-D NMR, i n xenobiotic conjugate i d e n t i f i c a t i o n was found, presumably because large samples are required. 2-D NMR should prove useful i n the assignment of peaks i n the spectrum of the parent xenobiotic such as i n the i d e n t i f i c a t i o n of hernandulcin (16). The accurate assignment of a spectrum i s often a p r e r e q u i s i t e To the determination of the conjugate by NMR. 2-D NMR was used i n conjunction with l^C enrichment i n a study of glycine metabolism i n tobacco suspension c e l l s (17). Sample requirements usually pose no problem i n lfH NMR, but may be a problem with other n u c l e i . Even sub microgram samples are f e a s i b l e with high f i e l d instruments! however, sample and solvent i m p u r i t i e s may prevent good spectroscopy at that l e v e l . Micro c e l l s , exhaustive exchange with a deuterium solvent (D2O or CD3OD), multiple pulse sequences , and presaturation pulses for solvent peak suppression have a l l been reported i n xenobiotic conjugate i d e n t i f i c a t i o n . High f i e l d instruments, u n l i k e i r o n magnet instruments, have not been a v a i l a b l e with micro c e l l s j however, an unsupported 1.8 mm tube placed i n s i d e a 5 mm tube has provided spectra of adequate r e s o l u t i o n with a 470 MHz spectrometer (18). Nuclear Overhauser Enhancement Nuclear Overhauser enhancement (NOE) was used i n the assignment of structures I - I I I . I r r a d i a t i o n of I at * produced a 27% increase of the integrated i n t e n s i t y for the proton at + but had no e f f e c t on the i n t e n s i t y of the remaining protons (19). I r r a d i a t i o n of the H at * of I I produced larger increases (22 and 41%) i n i n t e n s i t i e s of the two protons designated + than did i r r a d i a t i o n of the CH3 at * (17 and 27%) (20). I r r a d i a t i o n of I I I a t * caused a 12% increase i n the i n t e g r a t e d T n t e n s i t y for the benzylic proton designated +, but no increase i n the i n t e n s i t y f o r the proton associated with the hydroxyl group (21).

H I

II

III

S h i f t Reagents S h i f t reagents have not been used extensively i n xenobiotic metabolism studies; however, they may f i n d increased use for determination of isomer composition i n studies on metabolic mechanisms because metabolic rates are often d i f f e r e n t for geometric and stereoisomers. Mizugaki et a l . used *H NMR with Eu(fod)3 to determine structures of c i s - and trans-3-alkenoic acids (22). Moser et a l . used *H NMR and a c h i r a l s h i f t reagent for a n a l y s i s of the


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Identification of Xenobiotic Conjugates by NMR

179

stereoisomers of Metolachlor (23). Wainer et a l . used *H NMR and a europium c h i r a l s h i f t reagent for a n a l y s i s of d- and 1-amphetamine (24). Stec et a l . used 3Ip NMR and a praseodymium c h i r a l s h i f t reagent f o r the determination of enantiomeric p u r i t y of ifosfamide and two of i t s urinary metabolites (25). Carbon-13 Enrichment 1 3

The use of compounds enriched with C i s common i n biosynthetic studies (26), but quite rare i n xenobiotic metabolism studies. Hernandez et a l . (27) studied the enzymic and non-enzymic reaction of glutathione with benzo(a)pyrene 4,5-oxide enriched with C at the 4 and 5 p o s i t i o n s . The oxide was prepared with an enrichment of 70 atom % a t p o s i t i o n 4 and 97 atom % at p o s i t i o n 5. The i s o meric compositions of the reaction mixtures could be determined because of the d i f f e r e n t enrichment l e v e l s . C NMR spectra showed two c l u s t e r s containing f i v e peaks each. Each c l u s t e r contained one intense peak corresponding to e i t h e r a hydroxy substituent or a g l u tathione substituent for the carbon at p o s i t i o n 5 (97 atom % enrichment). Each c l u s t e r also contained two doublets with appropriate 13rj-13c coupling constants corresponding to the doubly labeled isomers a t p o s i t i o n s 4 and 5. F e l l et a l . (28) i s o l a t e d a Propachlor metabolite from germfree r a t s that had e i t h e r structure IV or V (reaction with butanol/HCl had yielded the butyl ether-butyl ester d e r i v a t i v e of IV). The proton decoupled C NMR spectrum of the metabolite i s o lated from r a t s dosed with 2-chloro-N-isopropyl-acetanilide-2-L CJ showed i n CD3OO a broadened t r i p l e t a t 658.1 rather than the expected large s i n g l e t . This r e s u l t suggested isotope exchange was occurring t o a f f o r d a structure that contained only one deuterium on the C enriched carbon. A model compound, 4*-Methoxy-2-(methyls u l f o n y l ) a c e t a n i l i d e , i n CD3OD i n i t i a l l y yielded a s i n g l e t at 659.2 for the comparable carbon, but a t r i p l e t at 658.8 on standing overnight. Structure VI i s one of several that can be drawn showing either carbanion or s p character. The observed chemical s h i f t i s s i m i l a r t o those obtained by Matsuyama et a l . (29) f o r some carbonyl substituted sulfonium y l i d e s .


1 5

1 5

i 5

13

1 5

2

HNAc R

2

= CH2CHCOOH

1 3

Russo et a l . followed i h e metabolism of C labeled hordenine (£-hydroxy-N,N-dimethyl[ CJphenethylamine) by root homogenates with C NMR. They observed a decrease i n the signal due t o the dimethylamino group with a concomitant increase i n a s i g n a l due to the methylamino group, but observed no s i g n a l due t o the metabo l i z e d methyl group (30). 15

1 3

Other Nuclei 1

1 3

Few a p p l i c a t i o n s of NMR with n u c l e i other than H and C have been reported for xenobiotic metabolism studies. Some studies i n


X E N O B I O T I C CONJUGATION CHEMISTRY


180

related f i e l d s are included here as they may serve as models for xenobiotic metabolism studies. No examples of the use of deuterium and t r i t i u m WÎR i n xenob i o t i c metabolism were found. Their use i n biosynthetic studies has been reviewed by Garson and Staunton (31). S e n s i t i v i t y problems e x i s t with deuterium, but should not be a problem with t r i t i u m since i t i s the most s e n s i t i v e nucleus a v a i l a b l e ( 1 . 2 1 x proton) and because of n e g l i g i b l e t r i t i u m backgrounds. Tritium NMR may be useful i n the studies of xenobiotic-enzyme i n t e r a c t i o n s as shown by Scott et a l . (32). Hazards due to the use of radioact i v i t y should be minimal because 1 mCi of a c t i v i t y should provide s u f f i c i e n t material for many experiments. However, isotope e f f e c t s may be a problem i f the metabolic reaction d i r e c t l y involves the t r i t i u m (or deuterium) atom because isotopes of hydrogen can greatly a f f e c t enzymic reaction rates. Also, l a b i l i t y may be a problem as Bakke and F e l l have found with CD3SO compounds, where exchange was too rapid to permit metabolism studies (33). Tsoupras et a l . used 31p NMR to detect the presence of phosphate groups i n the 22-adenosinemonophosphoric ester of 2-deoxyecdysone and 22-phospho-2-deoxyecdysone (34). Quantitative P NMR has been used i n several studies. Wayne et a l . used 31p NMR for q u a n t i t a t i v e analysis of organophosphorous p e s t i c i d e s (35). Zon et a l . used 31p NNR to study chemical and microsomal oxidation of cyclophosphamide (36), and to determine the h a l f - l i f e of a cyclophosphamide analogue (37). Mazzola et a l . determined p e s t i c i d e residues i n foods by NMR (38). Wyrwicz et a l . made unsuccessful attempts at detecting metabolites of methoxyflurane i n c i r c u l a t i n g blood during 2 hours of continuous anesthesia (39). The compound had previously been reported to be highly metabolized by the l i v e r . Spratt and Dorn (40) have prepared a large number of £-fluorobenzoyl d e r i v a t i v e s of alcohols, phenols, carboxylic acids, amines and t h i o l s . These compounds generally are formed i n high y i e l d and may be useful for i s o l a t i o n and analysis of metabolites. Chemical s h i f t s vary over a 10 ppm range, but model compounds would l i k e l y be required, for example, to d i s t i n g u i s h between compounds containing SH and OCH3 groups from compounds containing OH and SCH3 groups. This i s indicated by the following s h i f t s r e l a t i v e to 1,2-difluor0-tetrachloroethanet PhS, -36.89} PhO, -37.23} PhN, -40.38} PhCh 0, -38.12. 3 1

2

Amino a c i d conjugates Examples of the use of NMR i n the i d e n t i f i c a t i o n of amino acid conjugates are l i s t e d i n Table I . R e l a t i v e l y few examples of the use of l^C NMR were found, presumably because of the large amounts of sample required. Carbon isWR spectra obtained on an i r o n magnet instrument (15-25 MHz) can provide useful information for i d e n t i f i c a t i o n of amino acid conjugates (27,28,42,45,48,50,63,64)} however, proton spectra obtained on these instruments (60-100 MHz) are often of l i m i t e d value because of inadequate s p e c t r a l dispersion. Many of the examples i n Table I are, therefore, of proton spectra taken at 200 MHz or higher. Figure 1 l i s t s approximate proton and carbon s h i f t values of glutathione-type conjugates (proton and carbon s h i f t values are generally found within 0.4 and


8.

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181


1.9 ppm respectively of these values). Mercapturate and cysteine conjugates show s i m i l a r values for the cysteine absorbances. Because of a c h i r a l center i n cysteine, the protons on the B carbon are magnetically non equivalent% therefore, the protons of cysteine (also of the glutamyl group) y i e l d an ABC coupling pattern. Chemical s h i f t and coupling constant v a r i a t i o n s often render the three expected sets of doublet of doublets unrecognizable. Computer programs are a v a i l a b l e (often as part of FT NMR programs) f o r c a l c u l a t i o n of spectra from suspected s h i f t values and coupling constants to determine the accuracy of assignments, and f o r conversion of spectra from one frequency to another. A u s e f u l hard copy source that may a i d pattern recognition a l s o e x i s t s (68).


Cysteine a 4.4 (53.8), 0 3.2, 3.5 (34.1) a 0 a 0 II II R-S-CH2-CH-C-N-CH2-C-OH H 1 Glycine a 3.7 (43.7) C=0 r

8

1

1

y CH2 Glutamic a 3.6 (54.2) \ B CH B 2.1 (27.6) a CH-NH Y 2.5 (32.8) C=0 OH 2

2

L

Figure 1. Approximate chemical s h i f t s of glutathione-type conjugates. Table I . Examples of NMR usage i n i d e n t i f i c a t i o n of amino a c i d conjugates. Cysteine, mercapturate (N-acetylcysteine), g l u t a thione, c y s t e i n y l g l y c i n e , and glutamylcysteine are a l l conjugated through s u l f u r . Compound

Spectroscopic Summary

Ref.

8

200

C4i)

,C-CH CH 2

2

0^C6 so -

Continued

3

40.1 75.5 75.5 25.1 45.4 78.8 39.2

25 MHz CO3OO

(40.4) (69.3) (69.9) (25.3) (45.8) (70.4) (41.6)


Okinawan sponge. Spectrum s i m i l a r t o t h a t o f v i t a m i n 03.

270 M H Z CO3OO

3a 4 . 7 ( 3 . 9 5 ) 4^N=CH -0390l»»LJ 3 2 HO

3

2

Synthesis.

2

( 78)

-0 S0-v^^-CH2CH NH3 3

2

5 6 R e f . t o a n i m a l metab.

Synth.

2 6.73 5 7.07 6 6.63

250 MHz DMS0-d

6

1st o r d e r s p l i t t i n g p a t t e r n . 250 M H Z DMSO-ds TMS

(78) -CfrSO^

2 7.06 5 6.75 6 6.82

2

HOU^\oCH CH NH 2

2

3

5

6 Synth. R e f . t o a n i m a l metab. 3 2 (79) 4 /—y 1 OH CH —

-0 S0/Q\0CH2vWl2N-OH

3

3

H CH 5 6 * R a t , dog, man ( u r i n e ) . 3

s o ^ ^ A ^ k

0

4 ^ ^ 2 OH

(

~

3

NH . A . Man ( u r i n e ) . (73)

3

2

4-0S033

5 3

Synthesis.

*H NMR and IR a l s o r e p o r t e d .

Synthesis.

1 153.2 2 , 6 126.2 3.5 135.4 4 121.7

2

5

6 3

2

(74)

02N-^^-0S0 3

5

6

3

2

Synthesis.

5

3

6

Synthesis.

2 3

6

22.6 MHz CÔ/MaOD

1 159.2 2,6 124.3 3,5 128.4 4 147.5 1 151.7 2,6 124.0 3,5 132.9 4 139.1

22.6 MHz D20/te00 7 22.7

(74)

4Q)-0S0 5

22.6 MHz CÔ/NaOD

(74)

W 3 ^ K J S 0

3

25 MHz D2O

1,4 149.0 2 , 3 , 5 , 6 123.0

6

Br-(Q)-0S03-

3

1 152.9 2,6 101.2 3,5 158.4

Summary

1,2 143.4 3,6 123.5 4 , 5 127.4

(73)

CH

NMR

(73)

3

-OjSO^

Continued

Ref,

HO £ 2

NMR

Synthesis.

1 2 3 4

153.9 124.7 143.2 129.6

22.6 MHz D20/NaOD 5 132.3 6 121.0 7 23.1 Continued on next page


190


Table II. Ref. (74)

Compound 3 2 CH30^)0S0 3

5 3

6 2

5

6

CI 3

2

3

Synthesis.

1 146.2 2,6 124.0 3,5 116.1 4 158.0

7 57.0

22.6 MHz D20/Ma0D

22.6 mz D20/Wa0D

1 152.6 2,6 125.8 3,5 133.4 4 133.6

(74) Synthesis.

5

NMR Surnmarv

(74)

c i < ^ o s o


Synthesis.

Continued

1 154.5 2 124.7 3 136.9

4 129.1 5 133.6 6 122.9

22.6 MHz D20/Na00

6 0S0 -

(81)

3

^ ^ ^ ^ ^ Carbohydrate

Rat ( u r i n e ) .

80 MHz Protons of carbons Acetone/D20 2 and 3 s h i f t e d d o w n f i e l d 0.28 and 0.15 ppm r e s p e c t i v e l y .

Conjugates

P r o t o n NMR has been used f o r t h e d e t e r m i n a t i o n o f c o n f i g u r a t i o n about t h e anomeric carbon and t h e c o n j u g a t i o n s i t e o f c a r b o h y d r a t e s w i t h x e n o b i o t i c s . Most x e n o b i o t i c c o n j u g a t e s appear t o have t h e B c o n f i g u r a t i o n ! however, i n many c a s e s t h i s has been e s t a b l i s h e d o n l y by e n z y m a t i c h y d r o l y s i s . Enzyme h y d r o l y s i s may g i v e erroneous r e s u l t s because o f impure enzyme o r l a c k o f s p e c i f i c i t y , a s demonstrated by t h e h y d r o l y s i s o f an a - g l u c o s i d e by e i t h e r a - o r 8 g l u c o s i d a s e ( 8 2 ) . The d e t e r m i n a t i o n o f t h e c o n j u g a t i o n s i t e and t h e establishment of configuration are e s s e n t i a l t o the determination of m e t a b o l i c mechanisms, b i o l o g i c a l a c t i v i t y and c a t a b o l i s m o f x e n o b i o t i c c o n j u g a t e s . T a b l e I I I l i s t s examples i n which NMR has been used i n a v a r i e t y o f ways. F o r thorough a n a l y s i s , t h e NMR o f t h e c o n j u g a t e s h o u l d be o b t a i n e d , t h e c o n j u g a t i n g group s h o u l d be removed and i d e n t i f i e d ( i . e . g l u c o s e o x i d a s e , d e r i v a t i z a t i o n , GC, QC/MS), and t h e NMR o f t h e unconjugated m e t a b o l i t e s h o u l d be d e t e r mined. Comparison o f s p e c t r a o f t h e c o n j u g a t e d and unconjugated m e t a b o l i t e may y i e l d i n f o r m a t i o n on t h e s i t e o f c o n j u g a t i o n . F o r example, g l u c u r o n i d a t i o n o f a phenol s h i f t s t h e o r t h o p r o t o n s downf i e l d a p p r o x i m a t e l y 0.15 ppm ( 8 3 ) , and s h i f t s t h e p r o t o n o f a s e c o n dary a l c o h o l d o w n f i e l d 0.1 - 0.3 ppm ( 8 4 ) . Reviews on t h e u s e o f *H NMR i n t h e i d e n t i f i c a t i o n o f c a r b o h y d r a t e s i n d i c a t e t h a t c a u t i o n s h o u l d be e x e r c i z e d i n assignment o f c o n f i g u r a t i o n ( 8 5 - 8 7 ) . The e s t a b l i s h m e n t o f t h e anomeric c o n f i g u r a t i o n by NMR has u s u a l l y been done by d e t e r m i n i n g t h e c o u p l i n g c o n s t a n t between t h e anomeric p r o t o n and t h e p r o t o n on C-2 o f t h e c a r b o h y d r a t e m o i e t y . C h e m i c a l s h i f t may a l s o be i n d i c a t i v e o f c o n f i g u r a t i o n a s p r o t o n s i n t h e a c o n f i g u r a t i o n u s u a l l y absorb f a r t h e r d o w n f i e l d than 8 p r o t o n s , ( e . g . t h e s h i f t s f o r t h e a and 6 anomeric p r o t o n s f o r £ - n i t r o p h e n y l g l u c o s i d e a r e 6 5.9 and 5.2 r e s p e c t i v e l y ) ! however, c h e m i c a l s h i f t s can Paulson et al.; Xenobiotic Conjugation Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

8. FEIL

191


o n l y p r o v i d e s u p p o r t i n g e v i d e n c e i n c o n f i g u r a t i o n a l assignments because o f s u b s t i t u e n t and s o l v e n t e f f e c t s . The magnitude o f t h e c o u p l i n g c o n s t a n t between t h e p r o t o n s o f C - l and C - 2 , ^ 1 , 2 . f r e q u e n t l y been used t o e s t a b l i s h anomeric c o n f i g u r a t i o n to8a). V i c i n a l protons i n a glucose o r glucuronic a c i d conjugate i n the 8 c o n f i g u r a t i o n ( t r a n s - d i a x i a l ) g e n e r a l l y have c o u p l i n g c o n s t a n t s o f 7 t o 12 Hz w h i l e v i c i n a l p r o t o n s i n t h e a c o n f i g u r a t i o n ( a x i a l e q u a t o r i a l ) have c o u p l i n g c o n s t a n t s o f 2 t o A H z . D e v i a t i o n s from t h e normal c o n f i g u r a t i o n s may be generated by i n t r a m o l e c u l a r h y d r o gen b o n d i n g , i n t e r a c t i o n between t h e a c i d group o f a g l u c u r o n i c a c i d and an amino group, and by t h e anomeric e f f e c t ( h i g h l y e l e c t r o n e g a t i v e s u b s t i t u e n t s on t h e anomeric carbon o f a c a r b o h y d r a t e i n t h e 8 c o n f i g u r a t i o n p r e f e r an a x i a l p o s i t i o n ) (89-90). These e f f e c t s can be suppressed by u s i n g h y d r o x y l i e s o l v e n t s (DoO and CD3OD) and by m e t h y l a t i o n o f g l u c u r o n i d e s . D e r i v a t i z a t i o n ( a c e t y l , m e t h y l , and t r i m e t h y l s i l y l ) o f the conjugates t o suppress i n t r a m o l e c u l a r i n t e r a c t i o n s i n s o l v e n t s such a s c h l o r o f o r m and acetone may support the assignments. D e r i v a t i z a t i o n may a l s o be necessary because o f i n t e r f e r e n c e o f t h e HOD peak ( c a 64.8) w i t h t h e anomeric p r o t o n resonance.


h

a

s

m

o

s

t

Carbon NMR has r a r e l y been used i n t h e i d e n t i f i c a t i o n o f xenob i o t i c c o n j u g a t e s ! t h e r e f o r e , a p p r o p r i a t e model compounds must o f t e n be t a k e n from t h e c a r b o h y d r a t e o r n a t u r a l p r o d u c t l i t e r a t u r e . G l u c o s e and g l u c u r o n i c a c i d a r e t h e most common c a r b o h y d r a t e c o n j u g a t e s . The a anomers o f g l u c o s e , g l u c u r o n i c a c i d , and t h e i r g l y c o n e d e r i v a t i v e s absorb a t h i g h e r f i e l d t h a n t h e 8 anomers; however, t h e d i f f e r e n c e s a r e n o t s o g r e a t t h a t assignments can be c o n f i d e n t l y made w i t h o u t a d d i t i o n a l i n f o r m a t i o n . T h i s i s shown by t h e f o l l o w i n g compounds! g l u c o s e a 9 2 . 8 , 8 9 6 . 7 j methyl g l u c o s i d e a 9 9 . 6 , 8 103.4i £ - n i t r o p h e n y l g l u c o s i d e a 100.4, 8 102.7 ( 1 2 ) . S i n g l e bond &C^-H c o u p l i n g c o n s t a n t s a t t h e anomeric carbon v a r y w i t h c o n f i g u r a t i o n , b e i n g a p p r o x i m a t e l y 160 Hz f o r 8 anomers and 170 Hz f o r a anomers ( 8 8 b ) j however, t o t h e a u t h o r ' s knowledge, t h i s t e c h n i q u e has not been a p p l i e d t o x e n o b i o t i c c o n j u g a t e c h a r a c t e r ization. The a p p l i c a t i o n o f both carbon and p r o t o n NMR t o t h e e s t a b l i s h m e n t o f anomeric c o n f i g u r a t i o n s i n x e n o b i o t i c c o n j u g a t e s has been q u i t e l i m i t e d , and v e r i f i c a t i o n by s y n t h e s i s has been even more l i m i t e d . The NMR method appears t o be i n g e n e r a l agreement w i t h s y n t h e s i s a s no c l e a r l y c o n t r a d i c t o r y r e p o r t s were f o u n d . U n f o r t u n a t e l y , t h e u s u a l method o f s y n t h e s i s , t h e K o e n i g s - K n o r r r e a c t i o n , may g i v e m i x t u r e s ( 9 1 ) , and i t i s c o n c e i v a b l e t h a t t h e i n c o r r e c t isomer may have been i s o l a t e d i n some c a s e s . A p o s s i b l e d i s c r e p e n c y e x i s t s w i t h t h e g l u c u r o n i d e o f f l u o r e s e i n . The r e p o r t e d p r o t o n NMR absorbance f o r t h e anomeric carbon a t 65.09 Oi 2=2.6 ) s u g g e s t s an a c o n f i g u r a t i o n w h i l e s y n t h e s i s from t h e a bromo compound and h y d r o l y s i s by 8 - g l u c u r o n i d a s e suggest a 8 c o n f i g u r a t i o n (98). Hz

9


192


Table I I I . Examples o f NMR usage i n i d e n t i f i c a t i o n o f c a r b o hydrate conjugates. Compound B-galactose B-glucose -OCH2 B-cellobiose |= 0 HO jL0H

(Ref.) (92)

NMR Summary

200 MHz Anomeric H 6 ( J i i n Hz)i DMSO G l u c o s i d e s 4.18 ± 0.01 ( 7 . 6 ) p e r a c e t y l 4.22 ± 0.02 (7.6 ± 0.1) 2

0.02 (7.6 ± p e r a c e t y l 4.21 ± 0.04 ( 7 . 5 ±


G a l a c t o s i d e s 4.17 ± Hydrocortisone, prednisolone, dexamethasone, f l u d r o c o r t i s o n e . Also peracetyl derivatives. Synthesis. CH

(93) | O-B-glucuronide

3

)

^ O ^

H

(

0

H

)

0.3) 0.5)

C e l l o b i o s i d e 4.15 ( 7 . 6 ) p e r a c e t y l 4.28 ( 7 . 6 ) Other assignments a l s o made. 20 MHz Anomeric C 103.04 DMSO Other g l u c u r o n i d e assignments a l s o made. NMR o f n o r a n t i p y r i n e tautomers a i d e d i n assignment o f conjugation s i t e .

Man and r a t . (94) ^ H Metab. o f

No anomeric NMR d a t a . NMR used t o e s t a b l i s h MeO and c o n j u g a t e s i t e s .

O-glucuronide Man ( u r i n e ) . 5,6-dihydroxyindole.

F

F

(95)

0 0 B-glucoside B-glucoside Mixture - fungal metabolites. x

H ^N-CH

(84) — 3

I^JH X

^

@$©

360 MHz DMSO D2O

500 MHz NMR on m i x t u r e o f Acetone i s o m e r s as a c e t y l d e r i v a t i v e s . Conjugation s i t e s determined by NMR. Anomeric H 5.57 ( 7 . 7 ) 5.65 ( 7 . 7 )

Anomeric H 4.95 (J=8 Hz) Other assignments a l s o made.

100 MHz 360 MHz 400 MHz CD 0D

O-B-glucuronide

Man ( u r i n e ) . Metabolite of o x a p r o t a l i n e .


3

8.

FEIL

Identification of Xenobiotic Conjugates by NMR Table I I I .

Compound

(84)

N-CH

Continued

Ref.

H 3

/tri O - B - g l u c u r o n i d e

193

NMR Summary 100 KHz Anomeric H 360 MHz 4.45 (J=8 H z ) 400 MHz Location of hydroxyl CO3OO group based on a r o m a t i c s h i f t s .

Q ^ y © ^ " Man ( u r i n e ) . Metabolite of oxaprotaline. 0

(83)


CH3O-@H~-.CCl3 Olo-glucuronide(H) OH(glucuronide) (urine).

Chicken

(96) 0-8-glucuroniae

A l s o c o n j u g a t e s o f more h i g h l y o x i d i z e d compounds. Rat ( u r i n e ) .

, %

60 MHz B - G l u c u r o n i d a s e , NMR COCI3 on unconjugated cpds t o determine degree and s i t e o f o x i d a t i o n . No anomeric NMR data.

(2Z)

25 MHz Anomeric C 101.7 CO3OD Anomeric H J i 2 7 . 5 Hz l^C and ^H NMR used t o d e t e r m i n e c o n j u g a t i o n s i t e . *H NMR used t o establish original configuration of a g l y c o n e .

(97)

25 MHz Anomeric C 9 8 . 1 CO3OO Anomeric H J i 2 7 . 5 Hz !3c and H NMR used t o d e t e r m i n e c o n j u g a t i o n s i t e . H NMR used t o establish original configuration of aglycone.

^\O-B-glucuronide

JtrOH Rat

90 KHz No anomeric d a t a . NMR suggested a 4-methoxyphenyl group. C o n j u g a t i o n s i t e s d e t e r m i n e d by comp a r i s o n t o s y n t h e t i c cpds a f t e r f o l l o w i n g sequence1 OH t o 0 b e n z y l j B - g l u c u r o n i d a s e j CH2N2.

(urine). ,

X

O-B-glucuronide Rat

l

(urine).

°

m

C-0-a-glucoside(H) C l ^ c ^ - NH-H( B - g l u c o s i d e ) F o x t a i l and B a r l e y .

90 MHz S p e c t r a on a c e t y l d e r i v s . CDCI3 A c y l anomeric H 6.34 TMS (J 2=3.7 Hz) N- anomeric H 5.95 (0=8.0 H z ) Synthetic acyl B-glucoside, anomeric H 5.97 (J=8.0 H z ) lf

Continued on next page


194


Table I I I . ReTi

Compound

Continued NMR Summary 100 MHz H y d r o l y z e d by BCO3OO g l u c u r o n i d a s e but anomeric p r o t o n a t 5.09 ( J i 2 = 2 . 6 Hz) s u g g e s t s a. F

H O ^ ^ O ^3^0-glucuronide Synthesis - r e f . to rabbit and human m e t a b o l i t e s .


270 MHz Acetone

/X^VOH 1 /B-glucoside 0 0=C-0 Xanthium and s o i n a c h .

Anomeric H ( a c e t y l d e r i v ) 5.52 ( J i 2 8 . 5 ) H Z

F

100 MHz

O=C-0CH

JT B-glucoside Tomato

COCI3 Anomeric H 4.54 ( 7 . 0 Hz) f o r p e r - O - a c e t y l methyl e s t e r . H-4 s h i f t e d d o w n f i e l d 0.2 ppm from OH c p d .

3

(shoots). CH

0

(101)

3

80 MHz

Anomeric H 5.57 ( J l , 2 = 7 . 3 H z )

CO3OO

i

CH3 Man ( u r i n e ) .

B-glucuronide (1Q2)

Pr 0 ^-v 9 ^N-S -iCjy CO-B-glucur o n i d e Pr n Man ( u r i n e ) . x

Anomeric C

95.4

w

(103)

glucuronide

R a t s , dogs, and monkeys (urine). (104)

6-0-malonyl-B-glucoside \

80 MHz No anomeric NMR d a t a . CO3OO Spectrum s i m i l a r t o p a r e n t cpd except t h a t p r o t o n s on benzene r i n g o f b e n z i m i d a z o l e were s h i f t e d . A c y l g l u c u r o n i d e a l s o r e p o r t e d - no anomeric NMR d a t a .

180 MHz Anomeric H 4.87 (J=7.7 Hz) COCI3 Anomeric C 104.3 M a l o n y l C H , V 4 1 . 2 , H 3.38 S p e c t r a on p e r a c e t y l d e r i v . Other assignments a l s o made. 2

Spinach

leaves.


8.

FEIL


Table I I I .

Compound

Continued

Ref.

NMR Summary

(105)

90 MHz Anomeric H 4.92 (J=7.2Hz) DMSO A m i x t u r e o f two i s o m e r i c tris-t-butyldimethylsilyl derivatives yielded doublets at 4.34 and 4.50 (0=7.0 H z ) .

r^>) do6) l - S - B - g l u c o s i d e +N (glucuronide) .0 Synthesis. Rabbit, monkey, r a t , and dog ( u r i n e ) .

Anomeric C 83.1 f o r 25 MHz glucoside DMSO Anomeric C 82.6 f o r g l u c u r o n i d e S i t e of conjugation e s t a b l i s h e d by comparison t o C H S , C H S 0 , and CH S02 cpds o f p y r B p y r - N o x i d e .

Synthesis.

Anomeric C 106.7 f o r g l u c o s i d e .

H B-glucoside-N—N

0 II

CH si^ * N

>

3

Tomato.


195

N

(107) CI

3

100 MHz D2O

(108)

HC w CH i

3

3

Anomeric H 4 . 7 (J=7.7 Hz)

B - g l u c u r o n i d e - C = C-C0H R aHb b i t C 3

(urine).

(109)

CH B-glucuronide Swine ( l i v e r and m u s c l e ) . 3

Miscellaneous

360 MHz DMSO Anomeric H 4.40 ( J i , 2 = 9 . 0 Hz) NMR i n d i c a t e d unchanged p y r i m i d i n e r i n g and s h i f t e d £-aminophenyl p r o t o n s .

Conjugates

T a b l e IV l i s t s s t r u c t u r e s o f some uncommon c o n j u g a t e s t o which NMR has been a p p l i e d i n c h a r a c t e r i z a t i o n s . Both t h e x e n o b i o t i c s and t h e c o n j u g a t e s a r e o f d i v e r s e s t r u c t u r a l t y p e s . Ecdysonet y p e compounds, a l t h o u g h not x e n o b i o t i c s i n t h e s t r i c t d e f i n i t i o n o f t h e t e r m , a r e i n c l u d e d because they r e p r e s e n t c o n j u g a t i o n w i t h a phosphate group.


196


T a b l e I V . Examples o f NMR usage i n i d e n t i f i c a t i o n o f m i s c e l l a n e o u s conjugates. Compound

Ref. CHOJ

QH

M f t Summary

100 MHz C0 00 3

P r o t o n s on C-2 and C-3 s h i f t e d d o w n f i e l d 0.52 and 0.22 ppm from r e s p e c t i v e ecdysone values.

H0-

Xenobiotic Conjugation Chemistry - ACS Publications - American

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