Amino Acid Conjugates

5 Amino A c i d Conjugates RALPH O. MUMMA and ROBERT H. HAMILTON

Downloaded by MICHIGAN STATE UNIV on February 18, 2015 | http://pubs.acs.org Publication Date: June 1, 1976 | doi: 10.1021/bk-1976-0029.ch005

Departments of Entomology and Biology, Pesticide Research Laboratory and Graduate Study Center, The Pennsylvania State University, University Park, Pa. 16802

Amino acid conjugates of pesticides have been reported in both plants and animals. Perhaps the most well known conjugates are the glycine conjugates of acidic materials which are commonly found in animals (1-3). The glycine conjugate of benzoic acid, hippuric acid, was first isolated by Liebig in horse urine in 1829 (4). Knoop found in 1904 that long chain phenylalkyl acids fed to dogs were excreted as glycine conjugates of either phenylacetic acid or benzoic acid suggesting the existence of the β-oxidation pathway (5). Nicotinic acid also is excreted in urine of man as the glycine conjugate (nicotinuric) while phenylacetic acid is excreted as the glutamine conjugate (5). Birds excrete both these substances as the diornithine conjugates (5). Most of the pesticides that are recognized to form amino acid conjugates in plants are acidic insecticides, fungicides and herbicides but primarily the latter. The aspartic acid conjugate of indole-3-acetic acid, phenoxy herbicides and auxin-like plant growth regulators has been reported in plants by numerous laboratories (6-25). To further complicate the picture Feung et al. (26, 27) identified six additional amino acid conjugates (glutamic acid, alanine, valine, leucine, phenylalanine and tryptophan) of 2,4-D in soybean callus tissue. Figure 1 shows some examples of simple amino acid conjugates a l l involving con jugation through anα-amidebond. More complex amino acid conju gates have been reported such as the glutathione conjugate of triazines and diphenylether herbicides (28-33). However, in this case the glutathione is conjugated by means of a sulfur-carbon bond and its biochemical origin is different from amide linked amino acid conjugates. The glutathione conjugates are covered in more detail in another chapter (see Chapter by D. H. Hutson). Some other amino acid conjugates not linked through amide linkages with theα-aminogroup have been reported. For example 3-amino-1,2,3-triazole has been reported conjugated with alanine, glycine or serine (34) and alanine conjugates of N,N-dialkyldithio carbamates have also been reported (35).

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


5.

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69

Amino Acid Conjugates

Although amino a c i d conjugates o f a u x i n type h e r b i c i d e s would now appear t o be commonly found I n p l a n t s ; t h e i r l e v e l s , mechanism of formation and b i o l o g i c a l s i g n i f i c a n c e remain t o be evaluated. With regard t o l e v e l s o f s p e c i f i c conjugates, Feung e t a l . (36) have shown that w h i l e 2,4-D amino a c i d conjugation was common t o f i v e p l a n t c a l l u s t i s s u e s , the k i n d and percentage o f each conjugate was species s p e c i f i c . I n a d d i t i o n the l e v e l s and amounts of conjugates a l s o v a r i e d w i t h time, thus when soybean c a l l u s was incubated w i t h 2,4-D the e t h e r - s o l u b l e metabolites (amino a c i d conjugates) a r e very r a p i d l y formed (Figure 2) but degrade w i t h time (8). This was p a r t i c u l a r l y s i g n i f i c a n t i n the case o f the glutamic and a s p a r t i c a c i d conjugates (Figure 3 ) . Other metabol i t e s accumulated i n longer exposure times a t the expense o f t h e glutamic and a s p a r t i c conjugates. Although no enzymatic conjug a t i n g system has y e t been demonstrated there i s evidence that the a s p a r t i c conjugation system i s i n d u c i b l e (37). The b i o l o g i c a l s i g n i f i c a n c e of these conjugates a l s o demands greater a t t e n t i o n s i n c e the 2,4-D amino a c i d conjugates a r e b i o l o g i c a l l y a c t i v e , they s t i m u l a t e p l a n t c e l l d i v i s i o n and c e l l e l o n g a t i o n a t concent r a t i o n s t y p i c a l of auxins (38). The chemical, p h y s i c a l and b i o l o g i c a l p r o p e r t i e s , the i s o l a t i o n , i d e n t i f i c a t i o n and a n a l y t i c a l methods f o r amino a c i d conjugates w i l l be d i s c u s s e d . Since t h i s r e p o r t i s t o r e f l e c t the s t a t e of the a r t of work w i t h amino a c i d conjugates, most o f the examples w i l l be taken from our own i n v e s t i g a t i o n s of amino a c i d conjugates o f 2,4-D. These data represent the most extensive i n v e s t i g a t i o n o f amino a c i d conjugates. Chemical and P h y s i c a l P r o p e r t i e s Most amino a c i d conjugates behave as weak a c i d s . They a r e s o l u b l e i n water under b a s i c c o n d i t i o n s and i n s o l u b l e under a c i d i c c o n d i t i o n s . A t pH 7 they a r e u s u a l l y s o l u b l e i n p o l a r organic s o l vents such as methanol, e t h a n o l , 1-butanol and acetone. At pH 3 or lower most amino a c i d conjugates a r e nonionized and e x t r a c t a b l e i n t o e t h y l ether. A l s o 1-butanol e x t r a c t s the conjugates out of water a t a l l pH's. Some amino a c i d conjugates complicate the e x t r a c t i o n procedure because o f being d i f u n c t i o n a l such as t h e d i c a r b o x y l i c amino a c i d s , glutamic and a s p a r t i c a c i d s , and t h e b a s i c amino a c i d s such as a r g i n i n e , l y s i n e , and h i s t i d i n e . The l a t t e r three are i o n i z e d o r z w i t t e r ions a t a l l pH s and do not e x t r a c t w i t h e t h y l ether. A t pH 3 four e x t r a c t i o n s a r e u s u a l l y necessary t o e f f e c t i v e l y e x t r a c t the amino a c i d conjugates out o f water w i t h e t h y l ether. The amino a c i d conjugates o f 2,4-D can be e a s i l y c r y s t a l l i z e d from aqueous-alcoholic s o l v e n t s under a c i d i c c o n d i t i o n s . A l l possess low v o l a t i l i t y and high m e l t i n g p o i n t s . Most o f these conjugates a r e r e l a t i v e l y s t a b l e i n a c i d and b a s i c s o l u t i o n s a t room temperatures. The amino a c i d conjugates a r e r e a d i l y hydrolyzed i n f


70

BOUND AND CONJUGATED PESTICIDE RESIDUES

« Cl-V

COOH

j~X.O C H . C - N H CIH

-

00H

V0CH C-NHCH CH

Cl-V

2

V

CH_

2


2,4-0-Asp

2,4-0-Gly

2,4-D-Phe

OOH

COOH Hippufic Acid

Figure 1.

IAA-ASP

Typical amino acid conjugates

O-

-i 5

1 10

1 15

INCUBATION

ETHER SOLUBLE FRACTION

1 20

1 25

1 30

I 35

TIME (DAYS)

Figure 2. Distribution of the radioactivity taken up by the soybean callus in water-soluble, ether-soluble (amino acid conjugates), and residue fraction



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AND HAMILTON


71

6 N HC1 a t 70°C i n 24 hours t o f r e e amino a c i d s and the parent acidic pesticide. Amino a c i d conjugates r e a d i l y form e s t e r s w i t h a l c o h o l s and t h i s sometimes causes problems i n the p u r i f i c a t i o n o f the i s o l a t e d m e t a b o l i t e s . Since methanol, e t h a n o l , and 1-butanol are used i n our i s o l a t i o n procedure and i n the chromatographic s o l v e n t s , s i g n i f i c a n t amounts o f these e s t e r s were i s o l a t e d and r e a d i l y detected by mass spectroscopy. A l l the amino a c i d conjugates o f 2,4-D that were t e s t e d were p a r t i a l l y hydrolyzed by Emulsin (27, 39) which i s a crude enzyme p r e p a r a t i o n used t o hydrolyze 3-glucosides. With these r e s u l t s one could assume the i s o l a t e d amino a c i d conjugates a c t u a l l y were B-glucosides, however, Emulsin hydrolyzed the s y n t h e t i c amino a c i d conjugates a l s o . E v i d e n t l y the enzymatic p r e p a r a t i o n contains s u f f i c i e n t peptidase t o e f f e c t the h y d r o l y s i s . Nineteen amino a c i d conjugates of 2,4-D were prepared by the r e a c t i o n o f 2 , 4 - d i c h l o r o p h e n o x y a c e t y l c h l o r i d e w i t h the c o r r e s ponding L-amino a c i d i n aqueous sodium hydroxide (40) as i s shown i n F i g u r e 4. The N - l y s i n e conjugate was prepared i n a s l i g h t l y d i f f e r e n t manner. The b a s i c e-amino group o f l y s i n e was d e r l v a t i z e d t o a carbobenzoxy group which was e v e n t u a l l y removed by hydrogenation (42). A s l i g h t l y modified r e a c t i o n has been used t o prepare the amino a c i d conjugates of i n d o l e - 3 - a c e t i c a c i d (43). Since both paper and t h i n - l a y e r chromatography are so important i n the i s o l a t i o n , p u r i f i c a t i o n , and i d e n t i f i c a t i o n of amino a c i d conjugates, s p e c i a l emphasis must be placed on the proper s e l e c t i o n of good s o l v e n t systems. Since t h e o r e t i c a l l y n e a r l y twenty amino a c i d conjugates are p o s s i b l e , the chromatographic s o l v e n t system must be a b l e t o separate most o f the conjugates. Table I shows the m o b i l i t y of t e n s e l e c t e d amino a c i d conjugates of 2,4-D i n t h i n - l a y e r (TLC) and paper chromatographic (PC) s o l vent systems (42). V a l i n e , l e u c i n e and i s o l e u c i n e conjugates have s i m i l a r chromatographic p r o p e r t i e s and are d i f f i c u l t t o separate. Chromatographic techniques must be used f o r i d e n t i f i c a t i o n purposes when only t r a c e amounts (0.01 yg) mass s p e c t r o m e t r i c a n a l y s i s can be extremely u s e f u l . For example, n e a r l y a l l of the amino a c i d conjugates of 2,4-D o r of i n d o l e - 3 - a c e t i c a c i d gave molecular ions and c h a r a c t e r i s t i c fragmentation p a t t e r n s t y p i c a l o f both the amino a c i d and of the parent p e s t i c i d e (42, 43). The upper r e g i o n of the s p e c t r a (>m/e 219) i s c h a r a c t e r i s t i c of the s p e c i f i c conjugate and p a r t i c u l a r l y u s e f u l f o r i d e n t i f i c a t i o n purposes. F i g u r e 5 i l l u s t r a t e s the 01



72

BOUND

A N D CONJUGATED

PESTICIDE

RESIDUES

8 16 INCUBATION TIME (DAYS) Figure 3. Relative amounts of the ether-solubles isolated from soybean callus tissues grown for different times in 2,4-D-l- C. E * =» glutamic acid conjugate, E =- aspartic acid conjugate. 14

CI

Figure 4.

7

t

t

CI

General scheme for the synthesis of amino acid conjugates of 2,4-D


5.

MUMMA

Table I .

AND HAMILTON

R


Values of Amino A c i d Conjugates of 2,4-D. Solvent System


73

Compound

I

II

Gly

0.00

0.27

Ala

0.35

Ser

5

TLC IV

V

VI

PC VII

0.00

0.17

0.00

0.80

0.68

0.33

0.48

0.20

0.54

0.77

0.74

0.14

0.17

0.12

0.11

0.35

0.73

0.65

Val

0.36

0.39

0.52

0.27

0.50

0.74

0.79

Leu

0.40

0.42

0.56

0.29

0.58

0.75

0.82

He

0.42

0.43

0.56

0.27

0.58

0.74

0.82

Asp

III

0.17

0.01

0.26

0.03

0.31

0.71

0.36

Glu

0.13

0.02

0.21

0.03

0.30

0.71

0.43

Phe

0.33

0.37

0.49

0.25

0.49

0.74

0.80

Trp

0.27

0.28

0.32

0.18

0.42

0.80

0.80

I, benzene-dioxane-formic a c i d (90:25:2, v / v / v ) ; I I , c h l o r o form-methanol-concentrated ammonium hydroxide (70:35:2, v / v / v ) ; I I I , d i e t h y l ether-petroleum ether (60-70°)-formic a c i d (70:30:2, v / v / v ) ; IV, benzene-triethylamine-methanol-concentrated ammonium hydroxide (85:15:20:2, by v o l ) ; V, benzene-methanol-cyclohexaneformic a c i d (80:10:20:2, by v o l ) ; V I , 1-butanol-acetic a c i d water (90:20:10, v / v / v ) ; and V I I , l-butanol-95% ethanol-3 N ammonium hydroxide (4:1:5, v / v / v ) .


74

BOUND

AND CONJUGATED

PESTICIDE

RESIDUES

mass fragmentation (>m/e 219) of 2,4-D-Ile. I t gives a strong molecular i o n (53%, m/e 333) and fragments t y p i c a l of the amino a c i d . The main fragments >m/e 219 can be grouped i n t o four types as f o l l o w s : (a) p a r e n t - C l (P-35); (b) P-COOH (P-45); (c) P-H 0 (P-18); and (d) P-side chain fragmentation. The s i d e chain fragmentation i s s i m i l a r to the s i d e chain fragmentation p r e v i ously reported f o r peptides and d e r i v a t i v e s and are c h a r a c t e r i s t i c of the amino a c i d (44).


2

Figure 6 shows the prominent mass s p e c t r a l ions a r i s i n g from the fragmentation of the 2,4-D p o r t i o n of the molecule and are t y p i c a l f o r a l l the amino a c i d conjugates as w e l l as 2,4-D (42). The presence of the c h a r a c t e r i s t i c c h l o r i n e isotope peaks permits i d e n t i f i c a t i o n of metabolites even when s i g n i f i c a n t i m p u r i t i e s are present. I s o l a t i o n , P u r i f i c a t i o n and I d e n t i f i c a t i o n In our hands the procedure of the e x t r a c t i o n of the p l a n t t i s s u e and the time involved i n t h i s procedure depends upon the t i s s u e being examined. P e s t i c i d e metabolism s t u d i e s with p l a n t c a l l u s t i s s u e s o f f e r s many advantages over using the whole p l a n t . C a l l u s t i s s u e does not r e q u i r e a l i g h t e d growth chamber. I t i s s t e r i l e , uses inexpensive equipment, r e q u i r e s l i t t l e space, u s u a l l y does not c o n t a i n many i n t e r f e r i n g substances and o f f e r s v e r s a t i l i t y i n comparing metabolism i n d i f f e r e n t p l a n t s a t the same time by using d i f f e r e n t p l a n t c a l l u s t i s s u e . Whole p l a n t s , on the other hand, do r e q u i r e c o n t r o l l e d environmental growth chambers or greenhouse space and c o n t a i n s i g n i f i c a n t i n t e r f e r i n g phenolic substances and pigments. Obtaining s t e r i l e i n t a c t p l a n t s i s a l s o not u s u a l l y f e a s i b l e and r e s t r i c t s the method of treatment i f m i c r o b i a l metabolism i s to be avoided. U s u a l l y we can i s o l a t e and i d e n t i f y a metabolite from p l a n t c a l l u s t i s s u e i n 1/5 to 1/15 the time i t takes to work with the whole p l a n t . The l a r g e amount of p l a n t pigments and sugars o f t e n causes s t r e a k i n g of chromatograms and thus does not give good separations as i s t y p i c a l of c a l l u s t i s s u e . Therefore, most of our i d e n t i f i c a t i o n of metabolites have been performed with p l a n t c a l l u s t i s s u e s . However, once the metabolites have been i d e n t i f i e d the r e l a t i v e amount and types of metabolites must be


5.

MUMMA

AND

0 Cl-^

HAMILTON

Acid

75

Conjugates

COOH

VoCH,C-NHCH

m/e 298 100 %(base)

/=\

Cl-f'

° NHCH

HCH Cl-(^J>-OCH C-NHCf CH, m/e 252 14% CH.

-CI


Amino

2

VOCH^C-NHCH

m/e 3 3 3 53% -C H 4

0

C l

CI I—

8

COOH

OCHXNHCH l H m/e 2 7 7 14%

Figure 5.

Figure 6.

2

Prominent mass spectral ions arising from fragmentation of 2,4-D-Ile >219)

(m/e

Prominent mass spectral ions arising from the fragmentation of the 2,4-D portion of the molecule of amino acid conjugates of 2,4-D



76

BOUND

A N D CONJUGATED

PESTICIDE RESIDUES

determined i n the whole p l a n t i n order to determine the s i g n i f i cance of the metabolites. Figure 7 shows the i s o l a t i o n scheme of amino a c i d conjugates of 2,4-D from p l a n t c a l l u s t i s s u e . The f r o z e n t i s s u e was homogenized i n 95% ethanol i n a Waring Blendor. The homogenate was f i l t e r e d with s u c t i o n , and the r e s i d u e r i n s e d thoroughly with 80% ethanol. The pooled f i l t r a t e was evaporated and the aqueous concentrate (adjusted to pH 3.0) was e x t r a c t e d four times with e t h y l ether. The aqueous l a y e r contains the g l u c o s i d i c conjugates i n c l u d i n g the hydroxylated m e t a b o l i t e s . The water f r a c t i o n was then u s u a l l y extracted four times with equal volumes of 1-butanol (water s a t u r a t e d ) . The e t h y l ether f r a c t i o n contains the amino a c i d conjugates of 2,4-D as w e l l as any f r e e 2,4-D. The e t h y l ether e x t r a c t was concentrated and the components separated on paper chromatography. The r a d i o a c t i v e compounds were e l u t e d from the paper chromatograms and p u r i f i e d by t h i n - l a y e r chromatography. The solvent systems that we found best s u i t e d f o r our work are given i n Table I . The t h i n - l a y e r tanks always contained a paper l i n e r which does a f f e c t the m o b i l i t y of the s o l v e n t system and the s i l i c a g e l l a y e r was a c t i v a t e d (135°C f o r 4-8 h r ) . Figure 8 shows a t y p i c a l s e p a r a t i o n on paper chromatograms of whole p l a n t and c a l l u s t i s s u e e x t r a c t s . As i n d i c a t e d the c a l l u s t i s s u e e x t r a c t s give much b e t t e r r e s o l u t i o n . U s u a l l y only one a d d i t i o n a l t h i n l a y e r chromatographic separation of each eluted band i s necessary to o b t a i n s u f f i c i e n t p u r i t y f o r mass s p e c t r a l a n a l y s i s . A l l compounds e l u t e d from r a d i o a c t i v e bands are subjected to a c i d hyd r o l y s i s (6 N HC1, 70°C, 24 h r ) , enzymatic h y d r o l y s i s (Emulsin, N u t r i t i o n a l Biochemical Company) and chromatographic c h a r a c t e r i z a t i o n i n a l l the l i s t e d t h i n - l a y e r and paper chromatographic s o l v e n t s , i n c l u d i n g comparison with standard s y n t h e t i c compounds. Following t h i s procedure the sample i s analyzed i n a mass s p e c t r o meter v i a a s o l i d probe i n l e t . Unfortunately mass spectrometric a n a l y s i s destroys the sample and r e q u i r e s a r e l a t i v e l y pure f i n i t e (>0.01 yg) amount of compound. T h i s amount of m a t e r i a l i s sometimes hard to o b t a i n when metabolites are present i n small q u a n t i t i e s . When i n s u f f i c i e n t amounts of sample are a v a i l a b l e f o r mass spectrometric a n a l y s i s , i d e n t i f i c a t i o n must be based p u r e l y upon chromatographic data. Since the c o n c e n t r a t i o n of amino a c i d conjugates i n the c a l l u s t i s s u e v a r i e s g r e a t l y with time of exposure (8) i t i s des i r a b l e to determine the c o n c e n t r a t i o n of metabolites a f t e r s e v e r a l time i n t e r v a l s as i s i l l u s t r a t e d i n Figure 2 f o r soybean c a l l u s t r e a t e d with 2,4-D. The e t h e r - s o l u b l e f r a c t i o n (conjugates) decreases with time while the water-soluble metabolites i n c r e a s e . In f a c t the major conjugates (glutamate and aspartate) both i n crease and l a t e r decrease over d e f i n i t e time i n t e r v a l s , f i r s t the glutamic and then the a s p a r t i c (Figure 3). A comparison of the metabolism of 2,4-D (Table II) shows the r e l a t i v e amount of 2,4-D-Asp and 2,4-D-Glu i n the e t h y l ether e x t r a c t of s i x d i f f e r e n t p l a n t c a l l u s t i s s u e s ( c a r r o t , jackbean,


5.

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AND

HAMILTON

77


C a l l u s Tissue (2,4-D) 1.

Ethanol homogenization

RESIDUE

ETHANOL EXTRACT


1. 2.

Concentrate E t h y l ether, pH 3

ETHER EXTRACT (2,4-D and amino a c i d conjugates)

WATER SOLUBLE

l-Butanol Extract (mostly glycosides)

water saturated 1-butanol

Water soluble

Figure 7. Scheme of isolation of amino acid conjugates of 2,4-D from plant callus tissue

Figure 8. Radioautographu of decending paper chromatograms of ether-soluble (pH 3.0) metabolites of 2,4-D-l* C isolated from soybean plant (A) and soybean callus tissue (B). Solvent system: 1-butanol-ethanol (95% )-ammonium hydroxide (3N) (4:1.25:1, v/v/v); Whatman No. 1 paper. 4


78


soybean, sunflower, tobacco and corn) (36). Once the m e t a b o l i t e s have been thoroughly c h a r a c t e r i z e d i n the c a l l u s t i s s u e , i t i s e a s i e r t o recognize and q u a n t i f y them i n the whole p l a n t e x t r a c t s . A l l whole p l a n t s and p l a n t c a l l u s t i s s u e examined i n our l a b o r a t o r i e s to date, contained some amino a c i d conjugates but i n v a r y i n g amounts.


Table I I . R e l a t i v e Percentage of 2,4.-D-Asp and 2,4-D-Glu i n S i x C a l l u s Tissues Incubated w i t h 2,4-D f o r 8 Days Callus Tissue

2,4-D-Asp

2 4-D-Glu f

Carrot

0

23.8

Jackbean

1.2

32.5

Soybean

3.7

12.9

Sunflower

0

5.0

Tobacco

0.8

6.7

Corn

1.6

1.3

Biological

P r o p e r t i e s and Metabolism

U n f o r t u n a t e l y the l i t e r a t u r e does not c o n t a i n many examples where the b i o l o g i c a l a c t i v i t y of amino a c i d conjugates has been determined. Twenty amino a c i d conjugates of 2,4-D and s e v e r a l amino a c i d conjugates of i n d o l e - 3 - a c e t i c a c i d have been r e p o r t e d to possess b i o l o g i c a l a c t i v i t y (38, 39). They s t i m u l a t e p l a n t c e l l d i v i s i o n and c e l l e l o n g a t i o n (38, 39). Table I I I i n d i c a t e s the e l o n g a t i o n of Avena c o l e o p t i l e s e c t i o n s and Table IV the s t i m u l a t i o n of soybean cotyledon c a l l u s t i s s u e induced by s e l e c t e d amino a c i d conjugates of 2,4-D. As i n d i c a t e d i n these Tables the amino a c i d conjugates of 2,4-D are b i o l o g i c a l l y a c t i v e a t p h y s i o l o g i c a l c o n c e n t r a t i o n s ( 1 0 ~ - 1 0 ~ M) and i n some cases considerably more a c t i v e than 2,4-D. Their p h y s i o l o g i c a l e f f e c t i s theref o r e t y p i c a l of the e f f e c t of the parent h e r b i c i d e . At higher than p h y s i o l o g i c a l c o n c e n t r a t i o n these amino a c i d conjugates possess h e r b i c i d a l p r o p e r t i e s and the D-amino a c i d conjugates of 2,4-D have been observed t o s t i m u l a t e f r u i t growth (41). I n a d d i t i o n we have determined the t o x i c o l o g y of a number of L-amino a c i d conjugates of 2,4-D i n r a t s and showed t h e i r L D 5 0 t o be s i m i l a r to that of 2,4-D. 6

7


5.

MUMMA

AND HAMILTON

Table I I I .

79


Growth of Soybean Cotyledon C a l l u s T i s s u e Induced by Amino A c i d Conjugates of 2,4-D R e l a t i v e Percent Greater or• Less than

2,4-D or Conjugate

5

10" M

C o n t r o l (no a d d i t i v e ) Downloaded by MICHIGAN STATE UNIV on February 18, 2015 | http://pubs.acs.org Publication Date: June 1, 1976 | doi: 10.1021/bk-1976-0029.ch005

7

10" M

6

10" M

2,4-D 8

10- M

A l l Died

2,4-D-Gly

+13

- 4

-14

- 35

2,4-D-Glu

+56

+29

+53

+ 85

2,4-D-Leu

+26

+28

+35

+155

2,4-D-Phe

+92

+93

+ 9

+110

Table IV.

E l o n g a t i o n of Avena c o l e o p t i l e Sections Induced by Amino A c i d Conjugates of 2,4-D

2,4-D or Conjugate

% Elongation 8

10" M

10- M

10- M

10" M

2,4-D

39

74

45

39

2,4-D-Asp

57

35

26

22

2,4-D-Ile

45

55

47

24

2,4-D-Phe

49

59

32

26

2,4-D-Try

66

41

24

22

5

6

7

The amino a c i d conjugates are capable of being metabolized to other b i o l o g i c a l l y a c t i v e compounds (8). 2,4-D-Glu i s metabolized by soybean c a l l u s t i s s u e to 2,4-D, 2,4-D-Asp and to the hydroxy1ated metabolites; 4-hydroxy-2,5-dichlorophenoxyacetic a c i d and 4-hydroxy-2,3-dichlorophenoxyacetic a c i d . I n t e r e s t i n g l y 2,4-D-Glu i s more r a p i d l y metabolized by soybean c a l l u s t i s s u e than i s 2,4-D, (Table V) e s p e c i a l l y to the hydroxylated m e t a b o l i t e s . Of s p e c i a l note i s that 2,4-D-Glu i s metabolized to other amino a c i d conjugates, p a r t i c u l a r l y the a s p a r t i c conjugate.


80

BOUND

Table V.

A N D CONJUGATED

R e l a t i v e Percentage Metabolites of 2,4-D Incubated with Soybean C a l l u s Tissue

Ether Soluble Metabolite


RESIDUES

and 2,4-D-Glu

Water Soluble

% In T i s s u e * 2,4-D

Metabolite

2,4-D-Glu

2,4-D-Asp

3.7

11.7

2,4-D-Glu

12.9

6.7

2,4-D

33.7

6.0

Others

11.9

1.9

62.2

26.1

Total

PESTICIDE

% In T i s s u e * 2,4-D

(4-OH-2,5-D, 4-0H-2,3-D)

2,4-D-Glu

26.3

54.9

2,4-D

0.8

4.2

Others

6.7

11.7

33.8

70.8

Total

*2,4-D Incubated 12 days, 2,4-D-Glu Incubated 8 days. A n a l y t i c a l Methods Although amino a c i d conjugates of p e s t i c i d e s have been i s o l a t e d f o r many years no comprehensive i n v e s t i g a t i o n has been reported concerning the development of a n a l y t i c a l methods f o r these compounds. Recently, i n t h i s l a b o r a t o r y Arjmand (45) developed an a n a l y t i c a l method f o r the a n a l y s i s of nineteen metabolites of 2,4-D i n c l u d i n g the amino a c i d conjugates. T h i s technique i n volved the gas chromatographic a n a l y s i s of the t r i m e t h y l s i l y l (TMS) d e r i v a t i v e s . He showed that s i x t e e n amino a c i d conjugates could be separated and q u a n t i f i e d when analyses were performed i n two separate columns (OV-1 and OV-17 s t a t i o n a r y phases) with temperature programming c o n d i t i o n s . A t y p i c a l separation i s shown i n Figure 9. The proper d e r i v a t i z a t i o n reagent and c o n d i t i o n s were found to be important. Hexamethyldisilazane gave monosilyl a t e d amino a c i d conjugates while more stronger s i l y l a t i n g r e a c t i o n c o n d i t i o n s always r e s u l t e d i n a mixture of mono- and d i s i l y l a t e d products. A l l the TMS d e r i v a t i v e s of the amino a c i d conjugates of 2,4-D were s t a b l e and gave a l i n e a r response with a flame i o n i z a t i o n detector i n the range of 1-10 yg. Unfortunately e l e c t r o n capture detectors were not a p p l i c a b l e s i n c e temperature program c o n d i t i o n s were employed and the TMS d e r i v a t i v e s do not work w e l l with t h i s d e t e c t o r . Figure 10 shows a t y p i c a l GLC s e p a r a t i o n of the ether e x t r a c t of soybean c a l l u s t i s s u e f o r t i f i e d with 30 ppm amino a c i d conjugates and hydroxylated 2,4-D metabolites. Unfortunately the percentage recovery of the amino a c i d conjugates from the f o r t i f i e d c a l l u s t i s s u e v a r i e d g r e a t l y as evidenced i n Table VI.



5.

MUMMA

i 0

•

1 2

1

1 4

1

81


AND HAMILTON

1 6

1

1

•

1

8

1

1

10

1

1

12

' 14

r 16

T I M E (min) Figure 9. GLC separation of TMS derivatives of 2,4-D metabolites and amino acid conjugates. Column: 1% OV-17 on 80/100 mesh Supelcoport, 6' X 4 mm i.d. glass. Temperature programmed at 5°/min up to 280°C, initial temperature 180°C.

1 0

1

1 2

1

1 4

'

1 6

1

1 8

'

1 10

1

1

' 12

1 14

1

1

'

1

16

r

18

TIME (min) Figure 10. GLC of ether extract of soybean callus tissue fortified with 2,4-D metabolites and amino acid conjugates. Lower tracing is control tissue extract without fortification. Column: 2% OV-1 on 100/120 mesh Supelcoport, 6' X 4 mm i.d. glass. Temperature programmed at 5°/min up to 280°C, initial temperature 180°C.



82

Recovery ranged from 18.5 t o 91.4%. No i n v e s t i g a t i o n was conducted on ways t o improve the recovery which o b v i o u s l y needs f u r t h e r study.

Table VI.

Percentage Recovery o f 2,4-D-Conjugates from Soybean C a l l u s Tissue F o r t i f i e d w i t h 30 ppm each


Compound

ppm Recovered

% Recovery

2,4-D

25.6

85.49

2,4-D-Ala

21.7

72.33

2,4-D-Val

27.4

91.40

2,4-D-Leu

24.5

81.77

2,4-D-Asp 2,4-D-Phe

5.6 21.6

18.53 71.88

Discussion Amino a c i d conjugates a r e o b v i o u s l y more wide spread i n p l a n t t i s s u e than once e n v i s i o n e d . A s p a r t i c a c i d conjugates have been found t o be most abundant, however, conjugates w i t h glutamic a c i d , a l a n i n e , v a l i n e , l e u c i n e , phenylalanine and tryptophan have been i d e n t i f i e d . Probably as more t i s s u e s and p l a n t s a r e examined conjugates w i t h a d d i t i o n a l amino a c i d s w i l l be found. Since d i f f e r e n t p l a n t t i s s u e s c o n t a i n d i f f e r e n t concentrations of amino a c i d conjugates, perhaps the c o n c e n t r a t i o n o f the conjugate i n the t i s s u e r e f l e c t s a f r e e amino a c i d p o o l s i z e and should be examined f u r t h e r . Since i t i s now c l e a r t h a t the glutamic a c i d conjugate i s a major m e t a b o l i t e , a number of r e p o r t s of the a s p a r t i c a c i d conjugates must be examined c r i t i c a l l y e s p e c i a l l y s i n c e i t i s d i f f i c u l t t o separate the glutamic and a s p a r t i c conjugates by chromatography. Although i t seems c l e a r that amino a c i d conjugates a r e important i n p l a n t c a l l u s t i s s u e , j u s t how s i g n i f i c a n t these compounds are i n the whole p l a n t remains t o be proved. U n f o r t u n a t e l y , almost a l l of the 2,4-D metabolism s t u d i e s w i t h c a l l u s t i s s u e has been performed a t p h y s i o l o g i c a l concentrations and metabolism of 2,4-D a t h e r b i c i d a l concentrations might be s i g n i f i c a n t l y d i f f e r e n t . The c o n c e n t r a t i o n o f amino a c i d conjugates found i n soybean c a l l u s t i s s u e e x h i b i t e d a temporal r e l a t i o n s h i p . The highest c o n c e n t r a t i o n was found the f i r s t day of exposure of the p l a n t t o 2,4-D and the amino a c i d conjugates s t e a d i l y decreased w i t h a concomltment i n c r e a s e i n the c o n c e n t r a t i o n of the n o n b i o l o g i c a l l y



5.

MUMMA

AND HAMILTON


83

a c t i v e hydroxylated m e t a b o l i t e s . Whether a s i m i l a r temporal r e l a t i o n s h i p e x i s t s i n the whole p l a n t remains t o be determined. Although a l l the amino a c i d conjugates of 2,4-D possess a u x i n - l i k e p r o p e r t i e s , whether they express these p r o p e r t i e s as the amino a c i d conjugate o r some d e r i v a t i v e o r hydrolyzed product i s not known. The evidence would suggest that perhaps the conjugates may have i n v i v o b i o l o g i c a l a c t i v i t y . The f a c t that they are so r a p i d l y formed and s t i m u l a t e p l a n t c e l l d i v i s i o n (at p h y s i o l o g i c a l c o n c e n t r a t i o n s ) i n excess o f that of 2,4-D i s very suggestive. Venis (37) has shown the amino a c i d c o n j u g a t i o n o f i n d o l e - 3 - a c e t i c a c i d and other aromatic a c i d s i s c a t a l y z e d by an auxin (2,4-D, naphthyl a c e t i c a c i d , i n d o l e - 3 - a c e t i c a c i d ) i n d u c i b l e enzyme. I t i s i n t e r e s t i n g that the s t r u c t u r a l r e q u i r e ments f o r i n d u c t i o n are more s p e c i f i c than the s u b s t r a t e r e q u i r e ments. Since 2,4-D-Glu can be converted t o 2,4-D-Asp i n higher c o n c e n t r a t i o n s than 2,4-D i t s e l f i t suggests that the p l a n t t i s s u e may possess an enzyme capable of c a t a l y z i n g the d i r e c t conversion of 2,4-D-Glu t o 2,4-D-Asp. The hydroxylated m e t a b o l i t e s are more r a p i d l y formed from 2,4-D-Glu than from 2,4-D thus r a i s i n g the q u e s t i o n , can the amino a c i d conjugates be d i r e c t l y hydroxylated and i f so are they r e q u i r e d f o r h y d r o x y l a t i o n ? To our knowledge the i n v i t r o h y d r o x y l a t i o n o f 2,4-D has not been demonstrated i n a c e l l f r e e system and warrants f u r t h e r i n v e s t i g a t i o n s employing amino a c i d conjugates as s u b s t r a t e s . U s u a l l y the hydroxylated 2,4-D m e t a b o l i t e s are present as the g l u c o s i d e s , however, s m a l l amounts of the f r e e aglycone were reported i n bean p l a n t s ( 8 ) . Amino a c i d conjugates of hydroxylated 2,4-D m e t a b o l i t e s have not been r e p o r t e d , however, we do have p r e l i m i n a r y evidence f o r t h e i r occurrence. A s i g n i f i c a n t q u a n t i t y o f 2,4-D i s e v i d e n t l y present as a glucose e s t e r ( 7 ) . The glucose e s t e r s of the amino a c i d conjugates have not y e t been r e p o r t e d , however, i t i s q u i t e p o s s i b l e that they may e x i s t . Small amounts o f the amino a c i d conjugates are o f t e n found i n the water s o l u b l e f r a c t i o n a f t e r Emulsin treatment. The amino a c i d conjugates would undoubtedly possess d i f f e r e n t p e r m e a b i l i t i e s than the parent p e s t i c i d e t o cytoplasmic and subc e l l u l a r membranes. Thus, the b i o l o g i c a l a c t i v i t y and r a p i d metabolism of the amino a c i d conjugates might be owing t o t h e i r more r a p i d p e n e t r a t i o n o f the c e l l than the parent p e s t i c i d e , which r e s u l t s i n an accumulation a t the t a r g e t s i t e s o f b i o l o g i c a l a c t i v i t y and metabolism. Although o n l y the amino a c i d conjugates o f 2,4-D and i n d o l e 3 - a c e t i c a c i d have so f a r been s t u d i e d i n depth, probably other amino a c i d conjugates are e q u a l l y important. P o s s i b l y a l l the a c i d i c a u x i n - l i k e h e r b i c i d e s form amino a c i d conjugates and need to be reexamined i n l i g h t of c u r r e n t t h i n k i n g . A d d i t i o n a l s t u d i e s a r e needed t o determine i f the d i f f e r e n t h e r b i c i d a l d e r i v a t i v e s o f 2,4-D, such as the amine s a l t s , the b u t y l e s t e r and the butoxyethanol e s t e r , a l s o g i v e r i s e t o the


84

BOUND AND CONJUGATED PESTICIDE RESEDUES


amino a c i d conjugates when a p p l i e d to p l a n t s . The metabolism of 2,4-D has been examined i n only a few p l a n t s and a d d i t i o n a l p l a n t s should be i n v e s t i g a t e d . These data c o l l e c t i v e l y demonstrate the wide d i s t r i b u t i o n of amino a c i d conjugates, t h e i r b i o l o g i c a l s i g n i f i c a n c e and p l a n t species s p e c i f i c i t y . Hopefully these r e s u l t s w i l l stimulate other i n v e s t i g a t o r s to be more aware of amino a c i d conjugates. A n a l y t i c a l methods should be modified so that amino a c i d and e s t e r conjugates can be determined i n residue work. F i n a l l y the use of s t e r i l e p l a n t t i s s u e c u l t u r e s o f f e r s many advantages f o r metabolism s t u d i e s .

Literature Cited 1. Knaak, J. B., Sullivan, L. J., J. Agr. Food Chem. (1968) 16, 454. 2. Wit, J. G., Van Genderen, H., Biochem. J. (1966) 101, 698. 3. Lethco, E. J., Brouwer, E. A., J. Agr. Food Chem. (1966) 14, 532. 4. Fieser, L. F., Fieser, M., "Organic Chemistry", p. 2, Reinhold Publishing Corp., New York, 1956. 5. Fruton, J. S., Simmonds, S., "General Biochemistry", John Wiley & Sons, New York, 1953. 6. Andrea, W. A., Good, N. E., Plant Physiol. (1961) 32, 566. 7. Klämbt, H. D., Planta (1961) 57, 339. 8. Feung, C. S., Hamilton, R. H., Witham, F. H., Mumma, R. O., Plant Physiol. (1972) 50, 80. 9. Andrea, W. A., Good, N. E., Plant Physiol. (1955) 30, 380. 10. Andrea, W. A., van Ysselstein, M. W. H., Plant Physiol. (1956) 31, 235. 11. Andrea, W. A., van Ysselstein, N. W. H., Plant Physiol. (1960) 35, 225. 12. Good, N. E., Andrea, W. A., van Ysselstein, N. W. H., Plant Physiol. (1956) 31, 321. 13. Fang, S. C., Theisen, P., Butts, J. S., Plant Physiol. (1959) 34, 26. 14. Bennet-Clark, T. A., Wheeler, A. W., J. Exp. Botany (1959) 10, 468. 15. Thurman, D. A., Street, H. E., J. Exp. Botany (1962) 13, 369. 16. Wightman, F., Can. J. Botany (1962) 40, 689. 17. Zenk, M. H., Collow. Intern. Centre, Nat. Recherche Sci., Paris (1963) 123, 241. 18. Sudi, J., Nature (1964) 201, 1009. 19. Sudi, J., N. Phytotologist (1966) 65, 9. 20. Winter, A., Thimann, K. V., Plant Physiol. (1966) 41, 335. 21. Olney, H. O., Plant Physiol. (1968) 43, 293. 22. Robinson, B. J., Forman, M., Addicott, F. T., Plant Physiol. (1968) 43, 1321. 23. Morris, D. A., Briant, R. E., Thomson, P. G., Planta (1969) 89, 178.



5. M U M M A AND HAMILTON


85

24. Beyer, E. M., Morgan, P. W., Plant Physiol. (1970) 46, 157. 25. Lau, O. L., Murr, D. P., Yang, S. F., Plant Physiol. (1974) 54, 182. 26. Feung, C. S., Hamilton, R. H., Witham, F. H., J. Agr. Food Chem. (1971) 19, 475. 27. Feung, C. S., Hamilton, R. H., Mumma, R. O., J. Agr. Food Chem. (1973) 21, 637. 28. Frear, D. S., Swanson, H. R., Pesticide Biochem. and Physiol. (1973) 3, 473. 29. Shimabukuro, R. H., Lamoureux, G. L., Swanson, H. R., Walsh, W. C., Stafford, L. E., Frear, D. S., Pesticide Biochem. and Physiol. (1973) 3, 483. 30. Shimabukuro, R. H., Swanson, H. R., Walsh, W. C., Plant Physiol. (1970) 46, 103. 31. Shimabukuro, R. H., Frear, D. S., Swanson, H. R., Walsh, W. C., Plant Physiol. (1971) 47, 10. 32. Lamoureux, G. L., Stafford, L. E., Shimbukuro, R. H., Zaylskie, R. G., J. Agr. Food Chem. (1973) 21, 1020. 33. Shimabukuro, R. H., Walsh, W. C., Lamoureux, G. L., Stafford, L. E., J. Agr. Food Chem. (1973) 21, 1031. 34. Carter, M. C., Physiol. Plant (1965) 18, 1054. 35. Kaslander, J., Sijpesteijn, A. K., Van Der Kerk, G. J. M., Biochim. Biophys. Acta (1962) 60, 417. 36. Feung, C. S., Hamilton, R. H., Mumma, R. O., J. Agr. Food Chem. (1975) 23, 373. 37. Venis, M. A., Plant Physiol. (1972) 49, 24. 38. Feung, C. S., Mumma, R. O., Hamilton, R. H., J. Agr. Food Chem. (1974) 22, 307. 39. Hamilton, R. H., Feung, C. S., Myer, H. E., Mumma, R. O., Fifth Annual Meeting American Society Plant Physiologists, June 20, Cornell University, Ithaca, New York, 1974. 40. Wood, J. W., Fontaine, T. D., J. Org. Chem. (1952) 17, 891. 41. Wood, J. W., Fontaine, T. D., Mitchell, J. W., U. S. Patent No. 2,734,816 (1956). 42. Feung, C. S., Hamilton, R. H., Mumma, R. O., J. Agr. Food Chem. (1973) 21, 632. 43. Feung, C. S., Hamilton, R. H., Mumma, R. O., J. Agr. Food Chem. (1975) in press 44. Biemann, K., Cone, C., Webster, B. R., J. Amer. Chem. Soc. (1966) 88, 2597. 45. Arjmand, M., "Quantitative Gas-Liquid Chromatographic Analysis of 2,4-D Metabolites", Masters Thesis, 1975, The Pennsylvania State University, University Park, Pa.


Amino Acid Conjugates

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