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8 Miscellaneous Conjugates—Acylation and Alkylation of Xenobiotics in Physiologically Active Systems JORG IWAN

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Schering AG, Berlin/Bergkamen, Plant Protection Division, D-1 Berlin 65, Postfach 650311, West Germany

"Conjugation", as used by the p e s t i c i d e chemist, represents a c o l l e c t i v e term f o r reactions u s u a l l y catalyzed by enzymes and in which endogenous substrates are l i n k e d to xenobiotics. Binding to the f o r e i g n compounds takes place at f u n c t i o n a l groups that may already be present in the parent molecules or added in the course of the metabolic process. Formation of conjugates can a f f e c t the b i o l o g i c a l a c t i v i t y of a p e s t i c i d e or drug in two ways: Through decrease of lipid s o l u b i l i t y and through the a l t e r a t i o n of molecu l a r structures e s s e n t i a l f o r the exertion of physiol o g i c a l e f f e c t s . Therefore, conjugation normally r e s u l t s in d e t o x i f i c a t i o n but, as f a r as a c y l a t i o n s and a l k y l a t i o n s are concerned, t h i s is not always the case. 1. ACYLATION 1.1. A c e t y l a t i o n . Transfer of acetate from acetyl-coenzyme A (whose biosynthesis, as a reminder, is shown in fig. 1)to an amino group of a xenobiotic, is c e r t a i n l y the best understood a c y l a t i o n r e a c t i o n observed in biotransformations of f o r e i g n compounds. This r e a c t i o n , which is catalyzed by arylamine N — a c e t y l t r a n s f e r a s e s , represents a general metabolic pathway of aromatic amines, sulfonamides and hydrazino compounds as well as nonaromatic amines in mammals (1 - 4). There are strong i n d i c a t i o n s that generation of the N-acetyl d e r i v a t i v e s proceeds v i a a simple "ping-pong" mechanism i n v o l v i n g two consecutive steps: Formation of a c e t y l a c e t y l t r a n s f e r a s e through a r e a c t i o n between acetyl-CoA and a c e t y l t r a n s f e r a s e and, secondly, r e a c t i o n of the enzyme complex with a s u i t a ble substrate to produce the N-acetate and a c e t y l transferase (5 - 7). 132

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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Studies on d e t o x i f i c a t i o n of k,6-dinitro-oc r e s o l i n the r a b b i t , conducted by Smith, Smithies and Williams in 1952 (8), belong to the early investigat i o n r e v e a l i n g N - a c e t y l a t i o n of a p e s t i c i d e or or one of i t s degradation products ( f i g » 2). Among the DNOC metabolites extracted from u r i n e these same workers found 6 - a m i n o - 4 - n i t r o - o - c r e s o l and 6 - a c e t a m i d o - 4 - n i t r o - o - c r e s o l , the N - a c e t y l compound (as an O-glucuronide) being the most abundant convers i o n product. Detection of these substances, whose i d e n t i f i c a t i o n was based on paper chromatography as w e l l as on UV spectrophotometry, and comparison with authentic standards demonstrated that i n the r a b b i t , r e d u c t i o n and subsequent a c e t y l a t i o n of d i n i t r o - o c r e s o l c o n s t i t u t e d the predominant mechanism of inactivation. Intensive research on m i c r o b i a l decomposition of a n i l i n e d e r i v a t i v e s o c c u r r i n g as degradation products from a v a r i e t y of p e s t i c i d e s i n the s o i l environment s t a r t e d approximately i n the middle s i x t i e s . In the course of these studies which r e c e i v e d a s p e c i a l momentum by the discovery of m i c r o b i o l o g i c a l azobenzene formation, N - a c e t y l a t i o n of a n i l i n e s was recognized as a metabolic conversion common i n a v a r i e t y of f u n g i , b a c t e r i a and algae (9 - 15)* Some of the compounds studied are shown i n f i g . 3» During t h e i r i n v e s t i g a t i o n s on the metabolism of metobromuron by s e l e c t e d s o i l microorganisms Tweedy and coworkers detected the r a p i d and q u a n t i t a t i v e a c e t y l a t i o n of p-bromoaniline (9) {fig* *0 • These authors suggested that conversion of a n i l i n e s to t h e i r r e s p e c t i v e N - a c e t y l d e r i v a t i v e s may be competitive with the concentration dependent o x i d a t i v e coupling to azobenzenes i n s o i l . I d e n t i f i c a t i o n of the a c e t a n i l i d e s was accomp l i s h e d through cochromatography of s o i l and c u l t u r e extracts with known standard compounds or t h i n l a y e r and gas chromatography combined with mass spectrometry, A c h a r a c t e r i s t i c of the mass spectra obtained using e l e c t r o n impact i o n i z a t i o n was the loss of ketene from the molecular ions thus g i v i n g r i s e to the appearance of the corresponding a n i l i n e r a d i c a l ions i n high r e l a t i v e abundance. Needless to say these modern studies were c a r r i e d out employing carbon 1^ l a b e l e d parent compounds. Highly i n t e r e s t i n g as to the way of i t s format i o n , i t s mass spectrometric fragmentation pattern and i t s ultimate metabolic f a t e was the d e t e c t i o n of 4 - c h l o r o - 2 - h y d r o x y - a c e t a n i l i d e i n c u l t u r e s of Fusarium oxysporum fed with p - c h l o r o a n i l i n e (13) ( f i g . 5)•

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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BOUND AND CONJUGATED PESTICIDE RESIDUES

Figure 1.

O

OH

°

2

N

i0r N0

C

2

H



HoN

OH

it

— N0

2

N

i^r N0

C H 3

2

Figure 2.

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

IWAN

Acylation and Alkylation of Xenobiotics

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8.

Figure 5.

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

135

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136

B O U N D A N D C O N J U G A T E D PESTICIDE

RESIDUES

Upon e l e c t r o n impact, generation of a benzoxazole r a d i c a l i o n by o r t h o - e l i m i n a t i o n of water was observed ( f i g . 6). Discovery of such a fragment may be a u s e f u l diagnostic t o o l f o r the i n t e r p r e t a t i o n of respective mass spectra; however, we must bear i n mind that ohydroxy-acetanilides w i l l undergo the same c y c l i z a t i o n on heating thus forming benzoxazoles as a r t e f a c t s . This behaviour could lead to misinterpretations i f a glc-ms combination i s used f o r i d e n t i f i c a t i o n of metabolic products. As f a r as f u r t h e r degradation i s concerned, ohydroxylation may be one of the f i r s t steps toward m i c r o b i a l r i n g f i s s i o n of a n i l i n e s . Conversion of m-arainophenol to m-hydroxy-aceta n i l i d e on sugar beet leaves a f t e r spraying with desmedipham provides an example of the p a r t i c i p a t i o n of higher plants i n the a c e t y l a t i o n of a f o r e i g n compound (~16) (fig. 7). However, the N - a c e t y l d e r i v a t i v e was detected among the chloroform soluble materials recovered from the l e a f r i n s e of treated beets. M i c r o b i a l i n t e r a c t i o n at the plant surface thus e f f e c t i n g N - a c e t y l a t i o n , t h e r e f o r e , cannot be excluded. A c e t y l t r a n s f e r from ^-(N-hydroxyacetaraido)biphenyl to 4-aminoazobenzene, a r e a c t i o n catalyzed by a s p e c i a l acetyltransferase occurring i n rat l i v e r , may i l l u s t r a t e that transacylations do not n e c e s s a r i l y b r i n g about d e t o x i f i c a t i o n (17). In t h i s case a c e t y l a t i o n and deacetylation apparently play an important r o l e i n the carcinogenic act i v i t y of arylamlnes. By way of a c e t y l t r a n s f e r from the hydroxamic a c i d to another arylamine - the metabo l i c step shown i n f i g . 8 - an arylhydroxylamine i s released that can be o x i d i z e d to the corresponding n i t r o s o d e r i v a t i v e . T h i s , i n t u r n , i s more carcinogeni c than any of i t s precursors. 1.2. Formylation. Besides a c e t y l a t i o n , conjugat i o n with other carboxylic acids i s also p o s s i b l e . The simplest representative of the f a t t y a c i d homologues, formic a c i d , has been found i n a v a r i e t y of a c t i v a t e d forms i n l i v i n g c e l l s . Formyltetrahydrofolic a c i d occuring as N-10-formyl and N-5-N-1O-methenyl-tetrahydrofolate ( f i g . 9) i s c e r t a i n l y the most important formate c a r r i e r . F s s e n t i a l metabolic steps i n the biosynthesis of purines ( f i g . 10) depend on the a v a i l a b i l i t y of t h i s formyl source. Other b i o l o g i c a l l y a c t i v e compounds may also be considered as formate donors. Formyl-CoA, f o r i n -

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

Acylation and Alkylation of Xenobiotics H c

,A^ogC.

-CH -C« O / / 2

C H 3

\ -H20 \

m/e 185 185 13 r.a. 13

OH NH; O^CH

CI

m/e 167 r.a. 68

m/e 143 r.a. 100

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3

Figure 6.

I

H-N-C-CH3

NHo

OH O

OH

N

N-C-CH

NH 3

N

acetyl

N

transferase

^

Ox.

Figure 8.

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

BOUND AND CONJUGATED PESTICIDE RESIDUES

H

H

N -Formyl-tetrahydrofolate

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10

N5-Nio-Methenyl-tetrahydrofolate Figure 9.

„THF"

Fumarate Figure 10.

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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Acylation and Alkylation of Xenobiotics

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stance has been demonstrated i n microorganisms and higher plants as shown i n f i g . 11 which gives a part i a l view of the glyoxylate c y c l e . N-Formyl-L-kynurenine an intermediate i n t r y p t o phane metabolism, was found to be capable of transf e r r i n g formate to a n i l i n e , naphthylamine and ant h r a n i l i c a c i d i n v i t r o (18) ( f i g . 12). The transformylation was catalyzed by kynurenine formamidase from guinea-pig l i v e r . 2-Formamido-1naphtyl hydrogen sulphate, detected as a metabolite of 2-naphthylamine i n the urine of dogs and r a t s , may have received i t s formyl group by r e a c t i o n with t h i s enzyme system (19)• M i c r o b i a l transformation of a n i l i n e s to formanilides i n s o i l and pure fungal cultures was observed by Kaufman, Kearney and Plimmer (12, 13, 20) ( f i g . 13). 3,^-Dichloroformanilide, as a metabolite of 3t^d i c h l o r o a n i l i n e i n s o i l , could be i d e n t i f i e d a f t e r p u r i f i c a t i o n by column and preparative t h i n l a y e r chromatography using i n f r a r e d and mass spectrometry. Loss of 28 mass u n i t s from the parent i o n was i n d i c a t i v e of the e l i m i n a t i o n of a formyl group as carbon monoxide, a fragmentation analogous to the loss of ketene from a c e t a n i l i d e s • I n t e r p r e t a t i o n of the spect r a was confirmed by synthesis of authentic 3t^dichloroforraanilide obtained from 3,4-dichloroaniline through r e a c t i o n with a c e t i c formic anhydride or r e f l u x i n g i n e t h y l formate under atmospheric pressure. 9

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f

1.3* Malonic a c i d conjugation. Conjugation with malonic a c i d has been observed as a mechanism f o r d e t o x i f i c a t i o n of D-amino acids i n higher plants (21, 22). Malonyl-CoA which plays a c e n t r a l r o l e i n the biosynthesis of f a t t y acids ( f i g * 1*0 presumably mediates t h i s r e a c t i o n . Malonyl t r a n s f e r to a f o r e i g n compound by microorganisms was reported by Ross and Tweedy (2^)• Studi e s on the fate of chlordimeform i n mixed m i c r o b i a l cultures revealed that ^ - c h l o r o - o - t o l u i d i n e , occurring through stepwise h y d r o l y t i c a l cleavage of the parent compound, was transformed to the respective malonanilic a c i d d e r i v a t i v e ( f i g . 15)• In some experiments t h i s metabolite amounted to as much as k6 $ of the t o t a l administered r a d i o a c t i v i t y thus suggesting that malonic a c i d conjugation may be an important mechanism f o r d e t o x i f i c a t i o n of aromatic amines. I d e n t i f i c a t i o n of the conjugate was accomplished using t h i n l a y e r chromatographic p u r i f i c a t i o n and mass

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

BOUND AND CONJUGATED PESTICIDE RESIDUES

I I

Formyl CoA

I i

! I

Formate 0 0H V

H H 0 CoA-SH o

if

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CO.

II II

Ml

1V C0

o

i!

II II II

I

!—!r

OxalylCoA O^^S-CoA

I Oxalate l G^OH i

O^-QH

O^OH ^•FADH E / J~°2

NADPH«i

9

\ \ NADP ^| \\CoA-SH4l

^A-FAD • E V - f H 0 2

Glyoxylate

V

H2^2 0 NH,j o

k

A"

4 COOH

1h II II II

2

3

I'

!!H OI !'|2 'I 5

—I

I

Pyr P

L-Glutamate

Figure 11.

Figure 12.

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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Acylation and Alkylation of Xenobiotics

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HS R

Cx

e

ooc

c

o HaC

CH

s

2

Bk*ln~COO

e

HS

e

R A c CH

O il OOC sC CH SCoA N

X

S

2

x

2

Makmyl-CoA NADP®

0

HC

II /Cv

3

O

-

11

SCoA

°OOC\ / \ CH S C

0

2

o CH H3C

M CH S

- - 6 C0

o

O

II

11

HsC H 0* 2

CH

2

H3C

S * ^ NADPH NADP* + H «

2

CH S 2

HS

Figure 14.

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

v

BOUND AND CONJUGATED PESTICIDE RESIDUES

142

spectrometry. C h a r a c t e r i s t i c of the mass spectrum was a loss of kk mass u n i t s from the parent i o n to 183/ 185 which i n d i c a t e d the e l i m i n a t i o n of carbon d i o x i d e . From t h i s point downward i n the d i r e c t i o n of lower mass numbers the spectrum became i d e n t i c a l to that of 4-chloro-o-acetotoluidide the base peak representing the t o l u i d i n e r a d i c a l i o n at § 1^1/143. Synthesis of authentic k -chloro-2 -methylmalonanilic a c i d confirmed these r e s u l t s . Evidence f o r the capacity of higher plants to conjugate a n i l i n e derivates with malonic a c i d was r e cently obtained i n studies on the metabolism of 2,6d i c h l o r o - 4 - n i t r o a n i l i n e i n soybeans (2k) ( f i g » 16). The metabolic pathway involved reduction of DCNA to the corresponding p-phenylenediamine and subsequent malonyl t r a n s f e r to y i e l d N-(4-amino-3$5-dichlorophenyl)-malonamic a c i d as the major conversion produ c t . Spectrometric i d e n t i f i c a t i o n could be confirmed by synthesis from 2,6-dichloro-p-phenylenediamine through r e a c t i o n with e t h y l chloroformylacetate f o l lowed by mild h y d r o l y s i s .

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1

1

*\,k. Miscellaneous a c y l a t i o n s . Various other carboxylic acids are known to e x i s t as a c t i v a t e d subs t r a t e s i n l i v i n g c e l l s . Assuming the a v a i l a b i l i t y of a s u i t a b l e t r a n s f e r a s e , a l l of them might be regarded as p o t e n t i a l a c y l a t i o n reagents f o r xenobiotics i f these f o r e i g n compounds can penetrate to the s i t e of enzymatic a c t i o n . The studies of Smith showing that Streptomyces venezuelae can produce a c e t y l , propionyl and b u t y r y l as w e l l as probably pentanoyl and hexanoyl analogues of chloramphenicol, i f the c h l o r i d e i o n conc e n t r a t i o n i n the growth medium i s l i m i t e d , may i l l u s t r a t e t h i s point (25). 2. ALKYLATION 2.1. Methylation. As early as i n 189^ i t was suggested by Hofmeister that methylation of organic and inorganic compounds i n animal t i s s u e s may occur v i a t r a n s f e r of an i n t a c t methyl group (26). A particu l a r compound a c t i n g as a p o s s i b l e methyl source, however, was not mentioned. In 1933t 39 years l a t e r , Challenger, Higginbottom and E l l i s succeeded i n e l u c i dating the nature of the s o - c a l l e d Gosio-gas, a v o l a t i l e arsenic compound produced by several fungi growi n g on arsenic containing media (27)• These authors demonstrated that Gosio-gas e n t i r e l y consisted of t r i m e t h y l a r s i n e , and they proposed a mechanism i n v o l v i n g stepwise a d d i t i o n of formaldehyde to arsenious

In Bound and Conjugated Pesticide Residues; Kaufman, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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CI

Figure 15.

OH

9

o N0

NH

2

2

NH

2

HN-C-CH -COOH 2

HN-C-CH

3

-C^OH NH NH v>H 0 2 NHo ? ? HN-C-CH -C-OC2H NM CI-C-CH -C-OC2H ?

2

2

,,