Citation
Analysis of 2,4-D metabolites in higher plants by gas chromatography

Material Information

Title:
Analysis of 2,4-D metabolites in higher plants by gas chromatography
Creator:
Glaze, Norman Cline, 1934- ( Dissertant )
Wilcox, M. ( Thesis advisor )
Humphreys, T. E. ( Reviewer )
West, S. H. ( Reviewer )
Place of Publication:
Gainesville, Fla.
Publisher:
University of Florida
Publication Date:
Copyright Date:
1966
Language:
English
Physical Description:
iv, 47 leaves : ill. ; 28 cm.

Subjects

Subjects / Keywords:
Agronomy thesis Ph. D
Dichlorophenoxyacetic acid ( lcsh )
Dissertations, Academic -- Agronomy -- UF
Herbicides ( lcsh )
Plants -- Metabolism ( lcsh )
Fatty acids ( jstor )
Soil science ( jstor )
Chlorine ( jstor )
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Abstract:
The use of herbicides is increasing rapidly year by year. 2,4-Dichlorophenoxyacetic acid (2,4-D) is the most widely used of the herbicides offered on the market. Although 2,4-D was one of the first selective herbicides discovered and has been investigated by many workers over the years, there are still many questions concerning its metabolism. Hydroxylated derivatives have been found in some microorganisms and plants . This might be expected as hydroxylation is a prime method of detoxication in biological systems. 2,4-D has been found to be hydroxylated predominantly in the 5-position. However, 4- hydroxy-2,3- and 4-hydroxy-2,5-dichlorophenoxyacetic acids have been identified in some cases. This is interesting as it involves much more elaborate mechanisms than hydroxylation in a vacant position. The formation of these hydroxy acids requires a chlorine shift to one of the meta positions and hydroxylation in the para position. The object of this work is to determine whether hydroxylated derivatives are formed by excised roots from 2,4-D or related herbicides. Some workers have also found muconic acids and gamma-lactones as succeeding stages in the metabolism of these herbicides. This would follow from an oxidative degradation of the ring structure. The lactones found are of interest from the public health standpoint. The gamma-lactones isolated contain double bonds in the even numbered positions and compounds of this type have been found to be carcinogenic. If these compounds appear in any appreciable amounts and are not very transitory in nature, the use of the herbicides involved might require reevaluation by regulating organizations.
Thesis:
Thesis--University of Florida, 1966.
Bibliography:
Includes bibliographical references (leaves 42-46).
Additional Physical Form:
Also available on World Wide Web
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Norman Cline Glaze.

Record Information

Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Resource Identifier:
029978523 ( AlephBibNum )
37498860 ( OCLC )
ACG1109 ( NOTIS )

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Full Text









ANALYSIS OF 2,4-D METABOLITES IN

HIGHER PLANTS BY GAS

CHROMATOGRAPHY




















By
NORMAN CLINE GLAZE 1, .

/








A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY









UNIVERSITY OF FLORIDA


April, 1966















ACKNOWLEDGEMENTS

The author wishes to express his sincere appreciation to Dr.

M. Wilcox, Dr. S. H. West, Dr. T. E. Humphreys, and Dr. H. C.

Harris for their advice and guidance during the course of these

studies.

Special acknowledgement is made to Professor Zygmunt Eckstein,

Katedra Technologii Organicznej II Politechniki, Warsaw, for

supplying reprints and translations of necessary passages from his

works, to Dr. G. R. Powell, Unit of Experimental Agronomy, Univer-

sity of Oxford, who supplied samples and physical properties of

4-hydroxy-2,3-dichloro- and 4-hydroxy-2,5-dichlorophenoxyacetic

acids and to Dr. R. H. Biggs for use of his gas chromatograph.

This work was made possible by a grant from'the American

Cancer Society.
















TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS. . . . . ... . . . ii

LIST OF TABLES. . . . . . . . . . .... iv

INTRODUCTION. . . . . . .... . ...... 1

LITERATURE REVIEW . . . .. . . . .. 3

MATERIALS AND METHODS .................. 17

EXPERIMENTAL RESULTS. ..... ...... 28

DISCUSSION. . . . . . . ... ... 34

SUMMARY ......... ........... . 40

LITERATURE CITED. . . ... . . . . . 42















LIST OF TABLES


Table Page

1 Operating Conditions for Gas Chromatography. . 29

2 Retention Times of Standard Solutions in
Methanol . . .. .. . . 30

3 Relative Peak Heights of Methyl and Propyl Esters
of the Propyl Ethers. .. . . . . . 32















INTRODUCTION

The use of herbicides is increasing rapidly year by year.

2,4-Dichlorophenoxyacetic acid (2,4-D) is the most widely used of

the herbicides offered on the market. Although 2,4-D was one of the

first selective herbicides discovered and has been investigated by

many workers over the years, there are still many questions con-

cerning its metabolism.

Hydroxylated derivatives have been found in some microorganisms

and plants. This might be expected as hydroxylation is a prime

method of detoxication in biological systems. 2,4-D has been found

to be hydroxylated predominantly in the 5-position. However, 4-

hydroxy-2,3- and 4-hydroxy-2,5-dichlorophenoxyacetic acids have been

identified in some cases. This is interesting as it involves much

more elaborate mechanisms than hydroxylation in a vacant position.

The formation of these hydroxy acids requires a chlorine shift to

one of the meta positions and hydroxylation in the para position.

The object of this work is to determine whether hydroxylated deriv-

atives are formed by excised roots from 2,4-D or related herbicides.

Some workers have also found muconic acids and gamma-lactones

as succeeding stages in the metabolism of these herbicides. This

would follow from an oxidative degradation of the ring structure.

The lactones found are of interest from the public health stand-

point. The gamma-lactones isolated contain double bonds in the





2



even numbered positions and compounds of this type have been found

to be carcinogenic. If these compounds appear in any appreciable

amounts and are not very transitory in nature, the use of the herbi-

cides involved might require reevaluation by regulating organiza-

tions.















LITERATURE REVIEW

The literature review will be restricted to the metabolism of

chloro- and methylphenoxyaliphatic and naphthyl- and naphthyloxy-

aliphatic acids by higher plants and microorganisms. Animal metab-

olism is covered by Williams (1950, 1959). He states that phenoxy-

acetic acid and the ortho- and para-chloro-derivatives are unchanged

in animals. General classes of compounds which are considered to be

metabolites, such as phenols, catechols, etc., are discussed in his

publications.

Newman and Thomas (1949) discovered that the persistence of

2,4-dichlorophenoxyacetic acid (2,4-D) in soils was decreased by

pretreatment of the soil with 2,4-D and certain other compounds

having similar substituent groups such as 2,4-dichlorophenol. How-

ever, pretreatment with some compounds such as 2- or 3-chlorophenol

or phenoxyacetic acid had no effect on the persistence of 2,4-D in

the soil. Burger et al. (1962) measured the persistence of some

phenoxyaliphatic acids by a bioassay technique using alfalfa seed-

lings. Acetic, alpha-propionic, alpha-butyric and gamma-butyric

acid homologs were toxic when applied to soil planted to alfalfa,

but beta-propionic acid analogs were not toxic to the test species.

Prolonged persistence was exhibited when tho soil was treated with

3,4-dichloro- and 2,4,5-trichlorophenoxyaliphatic acids. The dura-

tion of phytotoxicity in soil receiving 4-chloro-, 2,4-dichloro- and









2-methyl-4-chlorophenoxyaliphatic acids was governed by the type and

linkage of the aliphatic side chain. Audus (1952) found that 2,4-D

enriched soil metabolized 2-methyl-4-chlorophenoxyacetic acid (MCPA)

immediately but did not affect the detoxication of 2,4,5-trichloro-

phenoxyacetic acid (2,4,5-T). MCPA enriched soil degraded 2,4-D

and 2,4,5-T.

Audus (1950) isolated Bacterium globiforme which was capable

of detoxifying 2,4-D. A Flavobacterium sp. was found by Burger et

al. (1962) which degraded phenoxybutyric acids. Only phenoxybutyric

acids having no meta-chlorine on the aromatic ring were metabolized

by the organism. These results show that the persistence of these

herbicides in soils is governed by the specific structural charac-

teristics of the molecule. A Corynebacterium sp., capable of de-

grading 2,4-D and MCPA, was isolated by Rogoff and Reid (1956). From

all indications, the ring was ruptured and complete destruction of

the molecule followed.

Audus (1952) suggested that the first step in the degradation

of 2,4-D, 2,4,5-T and MCPA might be the removal of the two carbon

side chain leaving a phenol. However, he found no accumulation of

phenols in soil treated with any of these herbicides. If a phenol

is an intermediate, it must be metabolized as rapidly as it is

formed. This is a possibility because it was found that 2,4-

dichlorophenol decomposed rapidly when added to a 2,4-D enriched

soil.

Leafe (1962) suggested two possibilities for the metabolism

of MCPA by Galium aparine. The first postulate listed for the





5



degradation of MCPA was the removal of the two carbon side chain

either as a two carbon fragment or its loss as carbon dioxide.

Secondly, he suggested that the metabolism or detoxication might

involve conjugation to another cellular component. Leafe stated

that the detoxication of the molecule by loss of both carbon atoms

of the side chain has been established as the reason Galium aparine

is resistant to MCPA.

Measurements were made by Canny and Markus (1960) of the rate

of evolution of labelled carbon dioxide from the shoots and roots

of Vicia faba which had been treated on one leaflet with 2,4-D

labelled in the carboxyl group. The carbon dioxide evolved from the

roots was more radioactive than that from the shoot. Labelled car-

bon was shown to be incorporated into many compounds in the root.

This is evidence that the degradation may take place mainly in the

roots. It was also concluded that although metabolism of the alkyl

side chain was rapid, this was not the main pathway for inactiva-

tion of the herbicide.

Bach (1961), working with stems of Phaseolus vulgaris, isolated

one half of the original radioactivity from carboxyl labelled 2,4-D

in an ether extract. The activity was found to be distributed among

ten components. Tests for functional groups showed that these com-

ponents retained the aromatic character of 2,4-D. Neither 2,4-D

nor 6-hydroxy-2,4-dichlorophenoxyacetic acid were isolated. Some

ten amino acids were also labelled in the treated stem tissue.

When red kidney beans were treated with labelled 2,4-D, Holley

(1952) found that in one week two thirds of the radioactivity was









recovered in a water soluble fraction. He stated that the water

soluble material must be a derivative of an unknown acid rather than

of 2,4-D. Holley suggested that the unknown acid might be 3-, 5-

or 6-hydroxy-2,4-dichlorophenoxyacetic acid.

Steenson and Walker (1956) isolated Flavobacterium peregrinum

and two Achromobacter sp. which were capable of decomposing 2,4-D,

MCPA or 4-chlorophenoxyacetic acid. They found that pretreatment

of the soil with one of the compounds accelerated the rate of de-

composition of the other two. In a liquid medium, F. peregrinum

metabolized 2,4-D and liberated 76 per cent of the chlorine in ionic

form. They suggested that 4-chloro-2-hydroxyphenoxyacetic acid and

4-chlorocatechol might be intermediates in the degradation of 4-

chlorophenoxyacetic acid.

A small, Gram-negative, motile organism was isolated from the

soil by Evans and Smith (1954) which utilized 4-chlorophenoxyacetic

acid and 2,4-D and produced 4-chlorophenol and 2,4-dichlorophenol,

respectively. A phenolic acid metabolite was found and it was

suggested to be 6-hydroxy-2,4-dichlorophenoxyacetic acid. It was

also observed that the chlorine atoms were retained until the aro-

matic character of the molecule was destroyed.

Using a replacement culture technique, Byrde and Woodcock

(1957) showed that phenoxyacetic and beta-phenoxypropionic acids

were hydroxylated mainly in the para position by Aspergillus niger.

The compounds were also hydroxylated in the ortho position but to

a lesser extent. Gamma-phenoxy-n-butyric acid also was metabolized









mainly to 4-hydroxyphenoxyacetic acid by hydroxylation and beta-

oxidation. Delta-phenoxy-n-valeric acid was degraded in a similar

manner to beta-(4-hydroxyphenoxy)propionic acid.

Wilcox et al. (1963) found that barley, oats and corn had the

ability to produce a ring hydroxylated intermediate from a phenoxy-

n-aliphatic acid with an even number of carbons in the side chain

when incubated in aerated water. Odd chain homologs yielded little

of a hydroxylated metabolite. In soybeans, alfalfa and peanuts, no

hydroxylated intermediates were detected. The phenolic product,

produced by the test plants when treated with various phenoxy-n-

aliphatic acids having an even number of carbons in the side chain,

was identical with 4-hydroxyphenoxyacetic acid. These data suggest

that ring hydroxylation by the resistant grass species coincides

with the suggested role of hydroxylation as a detoxication mech-

anism.

Working with Aspergillus niger, Byrde and Woodcock (1957) demon-

strated that phenoxyacetic and beta-phenoxypropionic acids were

hydroxylated in the para position mainly and to a lesser extent in

the ortho position. Faulkner and Woodcock (1961a, 1961b) alsc

found phenoxyacetic acid to be hydroxylated in the ortho and para

positions. Clifford and Woodcock (1964), however, found 2-hydroxy-

phenoxyacetic acid to be the main metabolite from phenoxyacetic

acid. Bocks et al. (1964) also found 2-hydroxyphenoxyacetic acid

to be the main metabolite from phenoxyacetic acid. All other isomers

were formed but in much smaller amounts. Bocks also found phenol to b(

a minor product. Faulkner and Woodcock (1961b) found that 2-chloro-









and 4-chlorophenoxyacetic acids were metabolized to 2-chloro-4-

hydroxy- and 4-chloro-2-hydroxyphenoxyacetic acids, respectively.

The original starting materials were monohydroxylated in all vacant

positions but the above were produced in much larger amounts. Also

found was 2-hydroxyphenoxyacetic acid. This was the first example

of fungal replacement of chlorine by a hydroxyl group. Bocks et al.

used methoxybenzene as a substrate and observed that 2-hydroxy-

methoxybenzene was produced. If the incubation was stopped after

three days, phenol was present in almost the same proportions as

2-hydroxymethoxybenzene. If allowed to proceed for a longer period,

the yield of phenol relative to 2-hydroxymethoxybenzene decreased.

Evans and Moss (1957), working with a Pseudomonas s2., extracted

2-hydroxy-4-chlorophenoxyacetic acid and 4-chlorocatechol from cul-

tures grown on a medium containing 4-chlorophenoxyacetic acid. This

was a strong indication that these were two of the possible inter-

mediates in the degradation of this herbicide.

Bell (1960), studying an Achromobacter sp., found an adaptive

system that is capable of oxidizing 2,4-D. The organism can also

oxidize many 2,4-D analogs. For the oxidation rate to be comparable

to 2,4-D, the organism appeared to require the following: a free

ortho position; a free carboxyl group on the side chain, prefer-

ably beta to the ethereal linkage; a chlorine atom in the para

position; and no more than two chlorine atoms on the ring whether

or not an ortho position is free. He suggested that the probable

degradation route was to 2,4-dichlorophenol but that action by a

decarboxylase to 2,4-dichloromethoxybenzene might be involved. He










also suggested that the metabolites from 2,4-dichlorophenol might

be 4-chlorocatechol or 3,5-dichlorocatechol.

The metabolism of 2,4-D and MCPA by a strain of Flavobacterium

peregrinum and an Achromobacter sp. was studied by Steenson and

Walker (1957). The evidence presented indicated that adapted

organisms metabolize 2,4-D through 2,4-dichlorophenol and 4-chloro-

catechol and that MCPA is degraded to 4-chloro-2-methylphenol. Bac-

teria, grown on a medium containing 2,4-D, did not oxidize any of

the five possible isomers but would oxidize 2,4-dibromo-, 4-bromo-

2-chloro-, 4-chloro- and to a small extent, 2-chlorophenoxyacetic

acids.

Thomas et al. (1963, 1964a) studied the hydroxylation of some

phenoxyacetic acids by stem tissue of Avena sativa. They found that

phenoxyacetic acid was converted to 4-hydroxyphenoxyacetic acid.

Phenoxyacetic acids with an unsubstituted para position were hydrox-

ylated in that position. The phenolic acids were accumulated as the

4-0-beta-D-glucosides. Phenoxyacetic acids with a chlorine atom in

the para position, however, were not hydroxylated to an appreciable

extent. These acids accumulated as neutral products which proved

to be phenoxyacetylglucoses. In the case of 2,4,6-trichlorophenoxy-

acetic acid, however, hydroxylation took place at the meta position

and the glucoside was isolated.

Thomas et al. (1964b) studied the metabolism of 2,4-D in stem

tissue of Phaseolus vulgaris. They found the predominant metabolite

to be 4-hydroxy-2,5-dichlorophenoxyacetic acid. This shows that the

hydroxylation and chlorine shift discovered first in Asoergillus










niger also occurs in higher plants. A minor metabolite was shown

to be 4-hydroxy-2,3-dichlorophenoxyacetic acid which also requires

the hydroxylation and chlorine shift. The products accumulated

within the tissue as beta-glucosides.

Evans and Moss (1957), using a Gram-negative, motile organism

of the Pseudomonas type, found that when it was incubated with 4-

chlorocatechol, beta-chloromuconic acid accumulated. The beta-

chloromuconic acid was further metabolized and the chlorine lib-

erated in ionic form. Later Fernley and Evans (1959) isolated

alpha-chloromuconic acid during the metabolism of 2,4-D which was

identical with that prepared by the oxidation of 2-chlorophenol.

They observed the shift of the 283 mP. peak of 2,4-D in the ultra-

violet range to another peak at 272 mpt.

Schemes for the metabolism of chlorophenoxyacetic acids by soil

bacteria were reported by Rogoff (1961), in which 4-chlorophenoxy-

acetic acid was metabolized through 2-hydroxy-4-chlorophenoxyacetic

acid and 4-chlorocatechol to beta-chloromuconic acid and 2,4-D was

degraded through 2,4-dichlorophenol and 3,5-dichlorocatechol to

alDha-chloromuconic acid. Evans and Smith (1951), Evans et al.

(1951), Dagley et al. (1960), Ribbons and Evans (1962) and Rogoff

all show schemes for further microbial oxidation of catechol type

derivatives. In all cases, the schemes show the catechol to cis-

cis-muconic acid step and terminate with beta-ketoadipic acid. The

steps between the cis-cis-muconic acid and beta-ketoadipic acid

appear to be somewhat uncertain. The uncertainty seems to be

whether the intermediates are lactonized or not when the double










bond migrates, which must occur if this scheme is to be correct.

The beta-ketoadipic acid is presumed to be split into acetic and

succinic acids which would pass into the tricarboxylic acid cycle.

Working with a Gram-negative soil bacterium, Gaunt and Evans

(1961) found that the ultraviolet peaks of MCPA at 228 and 279 mp.

are replaced by a peak at 277 mn. The properties of this compound

were consistent with the lactonic acid, alpha-methyl-gamma-carboxy-

methylene-A -butenolide. Peracetic acid oxidation of 4-chloro-2-

methylphenol produced a compound with the same properties. A

phenolic acid was also isolated and found to have properties con-

sistent with a hydroxy-2-methyl-4-chlorophenoxyacetic acid. It was

believed to be the 6-hydroxy-derivative. 4-Chloro-2-methylphenol

was also isolated from the extracts. The following metabolic path-

way for the degradation of MCPA was suggested: MCPA through 6-

hydroxy-4-chloro-2-methylphenoxyacetic acid; 5-chloro-3-methyl-

catechol; alpha-methyl-gamma-chloromuconic acid; alpha-methyl-amrma-

carboxymethylene-A -butenolide; alpha-methylmaleylacetate and to

smaller fragments prior to entering a terminal respiratory cycle.

The lactones mentioned in the degradation schemes are of con-

siderable interest because butenolides of this general type with

double bonds in even numbered positions have been shown to be car-

cinogenic by Dickens and Jones (1961, 1963).

Byrde et al. (1956), using a replacement culture technique of

Aspergillus niger, found that omega-(2-naphthyloxy)-n-aliphatic acids

having an even number of carbons in the side chain were beta-oxidized

to (6-hydroxy-2-naphthyloxy)acetic acid. Beta-(2-naphthyloxy)propionic








acid was transformed to beta-(6-hydroxy-2-naphthyloxy)propionic acid.

The n-b.utyric homolog was hydroxylated in the six position before

beta-oxidation to (6-hydroxy-2-naphthyloxy)acetic acid. Hydroxyla-

tion of the acetic and propionic acids led to a decrease in toxicity

to the fungus. Byrde and Woodcock (1958), working with Sclerotinia

laxa, found that nuclear hydroxylation was virtually absent with this

organism. They did find that gamma-(2-naphthyloxy)-n-butyric and

delta-(2-naphthyloxy)-n-valeric acids underwent beta-oxidation to

the corresponding acetic and propionic acids, respectively. They

also found beta-naphthol was produced from beta-(2-naphthyloxy)-

propionic acid.

Fawcett et al. (1954) found that omega-phenoxy-n-aliphatic acids

with an odd number of carbons in the side chain yielded a substantial

amount of phenol when applied to flax plants. Compounds with an

even number of carbon atoms in the side chain were beta-oxidized to

the acetic derivative. The decanoic homolog, however, was found to

produce a large amount of phenol. This was best explained from the

fact that omega-oxidation has been shown in animals for some com-

pounds with fatty acids of nine or ten carbon atoms. It was assumed

that this was also occurring in the flax plant.

Omega-(4-chlorophenoxy)aliphatic and omega-(2,4-dichlorophenoxy)-

aliphatic acids showed the characteristic alternation of activity

in work by Wain and Wightman (1954), when used in the Went pea test,

the tomato leaf epinasty and the wheat cylinder test. Only the wheat

cylinder test showed activity with even numbered aliphatic acids of

the 2,4,5-trichlorophenoxy series. When solutions of the










2,4,5-trichlorophenoxyaliphatic acids with side chains containing an

even number of carbon atoms were pretreated with wheat cylinders,

however, the alternation of activity was shown by the Went pea test

and the tomato leaf epinasty test. This evidence showed that wheat

possessed the ability to beta-oxidize this series but that pea and

tomato did not. When omega-l-naphthylaliphatic acids were tested,

the Went pea test and wheat cylinder test showed the alternation of

activity but only the acetic homolog was active in the tomato leaf

epinasty test. It was found that the substitution of alkyl groups

in the alpha position did not hinder beta-oxidation, but substitu-

tion in the beta position did prevent beta-oxidation.

Webley et al. (1955) studied the breakdown of omega-phenyl-

aliphatic acids by Nocardia opaca. They found that acids with an

odd number of carbon atoms in the side chain were converted to ben-

zoic acid and that cinnamic acid was an intermediate. Ortho-

hydroxyphenylacetic acid was found to be a metabolite when acids

with an even number of carbon atoms in the side chain were used in

the growth medium. Benzoic acid was produced essentially quantita-

tively but ortho-hydroxyphenylacetic acid was not, indicating that

it must arise from some side reaction. They stated that the latter

accumulates because it is not metabolized further by this organism.

The evidence suggested that N. opaca was capable of beta-oxidation

of the fatty acid side chains of these phenylaliphatic acids.

Webley et al. (1958) expanded their study to examine the effect

of change of various groups in the molecule and how beta-oxidation

would be affected. They found the following five factors to affect









beta-oxidation: 1) an oxygen bridge between the fatty acid and the

ring; 2) ring substitution, particularly in the ortho position; 3)

the nature of the ring substituents; 4) the type of ring structure

to which the fatty acid is attached; and 5) the position of attach-

ment of the fatty acid. The side chain of phenylbutyric acid was

beta-oxidized more readily than phenoxybutyric acid. The position

of substituted chlorine atoms in the ring affected beta-oxidation

as follows: gamma-(3-chlorophenoxy)butyric acid was degraded most

rapidly; the 4-chloro-homolog was intermediate; and the 2-chloro-

compound was the least reactive. Monomethyl homologs reacted in the

same manner as the corresponding monochloro-compounds. The methyl

homologs, in general, were beta-oxidized more readily than the

corresponding chloro-compounds. The rate of conversion of the beta-
wa3
hydroxy-intermediate is reduced with the chlorinated materials.

2-Methyl-4-chloro- and 2,4-dichlorophenoxybutyric acids were tested

to evaluate the effect of disubstitution. It was found that disub-

stitution decreased the rate of beta-oxidation. The oxidation of

the disubstituted compounds proceeded to the beta-hydroxy-derivative

but the acetic homologs were not produced. The caproic homologs

were rapidly converted to the butyrics but again none of the acetic

homologs were obtained. Gamma-(2-naphthyloxy)butyric and beta-(2-

naphthyloxy)propionic acids were beta-oxidized to the corresponding

acetate and 2-naphthol, respectively. The l-naphthyloxy-derivatives

remained unchanged throughout the experiment. Gamma-(l-naphthyl)-

butyric acid was converted to the acetate while the corresponding

naphthyloxy-compound was unchanged. Beta-oxidation of the fatty









acid side chain was most rapid when attached to a phenyl ring and

slowest when attached to a 1-naphthyl group. The indolyl group was

intermediate.

Fawcett et al. (1959), using the wheat cylinder, pea curvature,

pea -ogment and tomato leaf epinasty tests, assayed the growth-

regulating activities of 18 homologous series of omega-phenoxy-

aliphatic acids with two to seven carbon atoms in the side chain and

with chlorine and methyl groups substituted in various ring posi-

tions. The degradation of some homologs was investigated and

identification of metabolites made by chromogenic methods and bio-

assay. They noted that there were four patterns of biological

activity as follows: 1) where beta-oxidation was operative, and

the acetic acid was active while the propionic acid was not, only

the even numbered homologs showed activity; 2) where beta-oxidation

was hindered at the butyric stage, and the acetic acid was active

but the propionic acid inactive, only the acetic acid showed activ-

ity; 3) where beta-oxidation was operative, and both the acetic

acid and propionic acid were active, all members showed activity;

and 4) where beta-oxidation was hindered at the butyric stage, and

the acetic acid was less active than the propionic acid, the acetic

acid plus all odd numbered homologs showed activity. The results

indicated that beta-oxidation is hindered at the butyric stage in

pea and tomato tissue. The hindrance at the butyric stage was shown

to be associated with an ortho chlorine or methyl group on the ring.

This effect was largely removed by a further chlorine atom in the

para position. Addition of a chlorine atom in tho .ota position





16


had no effect. The beta-oxidation sequence was not changed when a

methyl group was substituted for a chlorine atom.















MATERIALS AND METHODS

The excised roots were prepared in the following manner: a

single layer of seed was placed between double thicknesses of

cheesecloth supported on aluminum mesh over a dishpan of aerated

water. These were germinated in a growth chamber in the dark at

71 F for a period of 4 to 7 days depending on the species. At the

end of this period, except in the case of alfalfa, the roots pro-

truding below the aluminum mesh were excised with a razor blade

and used in subsequent incubations. The alfalfa roots did not grow

through the cheesecloth and, therefore, the top layer of cheese-

cloth was removed and the seed cut off with scissors, after which

the roots were used in the incubation studies. The species used

were as follows: Coker 67 field corn (Zea mays L.), Hadden wheat

(Triticum aestivum L.), Moregrain oats (Avena sativa L.), Manchuria

X Rabat barley (Hordeum vulgare L.) and Hairy Peruvian alfalfa

(Medicago sativa L.).

Erlenmeyer flasks (300 ml.) were used as incubation vessels.

Tetracycline and streptomycin at concentrations of 2 and 30 ppm,

respectively, were used to prevent buildup of microbial populations.

Sets were run using 0.4 M tris [2-amino-2-(hydroxymethyl)-1,3-

propanediol] buffer (pH 7.4) and 0.4 M phosphate -buffer (pH 5.2) at
-2
a final concentration of 2 X 10-2 M. The treatment flasks were made
1 X 10- M in the potassium salt of 2,4-D. All flasks were brought
1 X 10 -M in the potassium salt of 2,4-D. All flasks were brought









to a final volume of 100 ml. Roots (10 g. fresh weight) were then

added to the flasks. The flasks were covered with a tissue and

put on a shaker at approximately 60 cycles per minute. A total of

12 flasks were used for each species. Six blank flasks were used

as well as six containing 2,4-D.

The incubations using tris buffer were incubated for 10 hours.

At the end of this time, the roots were removed from the flasks and

frozen in cold methanol. The roots were then ground in an omni-

mixer, keeping them frozen by immersing the vessel in a bath at

-500C or colder. After grinding, the solution was centrifuged at

17,500 rpm (37,000 X g) for 30 minutes. The supernatant fraction

was concentrated to approximately 10 ml. by freeze drying. Each

blank and treatment was split in half. To one portion of each, 40

per cent sodium hydroxide was added so that the final solution was

10 per cent in sodium hydroxide. This solution was refluxed for 20

minutes in order to hydrolyze any esters present. The hydrolyzed

sample was brought to pH 3 with sulfuric acid and freeze dried.

Methanol was added to dissolve any hydroxylated derivatives that

might be present. The hydrolyzed and unhydrolyzed samples were then

reduced under a stream of nitrogen to a volume of approximately 1 ml.

and applied to 8 X 8 inch plates spread to a thickness of 1 mm. with

silica gel G (E. Merck Ag., Darmstadt) for thin layer chromatog-

raphy. These plates were run in chambers with an ether:ligroin

(b.p. 66-750C):formic acid (50:50:2) solvent system. After drying,

the silica gel G between the origin and the solvent front was

scraped from the plate. This carrier was then eluted with acetone.









The acetone extracts were reduced under a stream of nitrogen to

approximately 10 ml. and split again. One portion was set aside.

The others were reduced under a stream of nitrogen to approximately

.5 ml. after which the volume was adjusted to 1 ml. with methanol.

These samples were treated with an ethereal solution of diazo-n-

propane. More of the diazo-n-propane solution was added as necessary

to keep the color of the solutions yellow. After 30 minutes, two

drops of a solution of boron trifluoride (0.7 per cent) in methanol

were added to further catalyze the reaction and the solutions were

kept yellow for an additional 30 minutes. At the end of this time,

the samples were again concentrated under nitrogen and adjusted to

a volume of 1 ml. These solutions were used for analysis by gas

chromatography.

The incubation mixtures containing phosphate buffer were treated

similarly except that the extraction of the roots was carried out

by dropping the roots into boiling methanol and boiling an additional

20 minutes. Both halves of the split samples were also used in this

case as two diazo compounds were used. One portion was treated with

diazomethane while the other was treated with diazo-n-propane as in

the previous case. Otherwise, both sets were handled as described

previously.

Solutions of 2,4-, 6-hydroxy-2,4-, 5-hydroxy-2,4-, 4-hydroxy-

2,3- and 4-hydroxy-2,5-dichlorophenoxyacetic acids in methanol were

prepared and treated with diazomethane and diazo-n-propane as de-

scribed previously. The final concentration of these solutions was

2.5 mg./ml. These solutions were used as standards for gas chromatog-

raphy.










Mixtures of some synthesized compounds (10 mg.) and lanolin

(4 g.) were prepared for tomato epinasty tests. The following com-

pounds were tested: 5-nitro- and 5-amino-4-chloro-2-methylphenoxy-

acetic acids; ethyl 3-amino-2,4-dichlorophenoxyacetate; 4-hydroxy-

2,3-, 4-hydroxy-2,5-, 6-hydroxy-2,4- and 5-hydroxy-2,4-dichloro-

phenoxyacetic acids. 2,4-D was also included as a standard.

Approximately 300 mg. of the lanolin mixtures of these compounds

were spotted on the petiole of the youngest fully developed leaf

of two month old tomato plants in the greenhouse. Daily observa-

tions were made to observe the effects of the compounds on the

plants.

All melting points were run on a Kofler micro hot stage apparatus

and the temperatures corrected. The compounds were synthesized as

follows:

Diazomethane. Potassium hydroxide (40 ml. of 30 per cent

solution) and anhydrous ethyl ether (160 ml.) were placed in a large

test tube and immersed in an insulated container holding an anti-

freeze solution at -100C or lower. The contents of the tube were

allowed to cool for 20 minutes. N-methyl-N-nitro-N-nitrosoguanidine

(8 g.) was added to the tube. The bath temperature was allowed to

rise only to 00C. The reaction is complete when the ether assumes

a yellow color and the solid matter disappears and usually is com-

plete in about 30 minutes. The ethereal solution was poured off

into a polyethylene bottle containing potassium hydroxide pellets.

The solution should be stored in a freezer and used within 24 hours

for best results.









Diazo-n-propane. Potassium hydroxide (20 ml. of 60 per cent

solution) and anhydrous ethyl ether (160 ml.) were placed in a large

test tube and cooled as described above. N-propyl-N-nitrosourea

(8 g.), prepared previously by M. Wilcox, was added to the tube.

The reaction is complete when the ether assumes a deep yellow color

and the solid matter suspended in the potassium hydroxide loses its

yellow color, becoming white. The ethereal solution was poured off

as in the case of diazomethane. This solution should be stored in

a freezer and used within 24 hours for best results.

Monoperphthalic acid. An ethereal solution of monoperphthalic

acid was prepared as described by Payne (1962). The solution was

standardized by adding 30 ml. of 20 per cent potassium iodide solu-

tion to 2 ml. of the ethereal solution and, after 10 minutes, titrating

the liberated iodine with 0.06268 N sodium thiosulfate. This solution

was used in subsequent attempted preparations.

Isopropyl 4-chloro-2-methylphenoxyacetate. To 95 per cent

4-chloro-2-methylphenoxyacetic acid (212 g.) was added 2-propanol

(264 g.). Boron fluoride ethyl ether (178 g.) was added dropwise

and the mixture refluxed for 4 hours. The excess alcohol was dis-

tilled off and the solution neutralized with a 10 per cent solution

of sodium carbonate. The solution was then extracted with three

portions of ethyl ether and the ether removed under vacuum. The

remaining liquid was vacuum distilled (15 mm. Hg) and the portions

distilling over between 170 and 1740C collected and combined. The

weight of these portions was 193.6 g. (79.8 per cent yield).









5-Nitro-4-chloro-2-methylphenoxyacetic acid. This compound

was prepared as described by Eckstein et al. (1964). The compound

melted at 151-1520C. Faulkner and Woodcock (1965) reported 152-

1530C.

5-Amino-4-chloro-2-methylphenoxyacetic acid. This compound

was also prepared as described by Eckstein et al. (1964). The com-

pound melted at 194-1970C. Eckstein et al. (1964) reported 197-1980C.

The ethyl ester, prepared by the previously described boron tri-

fluoride method, melted at 76-780C (Found: C, 54.51; H, 5.96; N,

5.95; Cl, 14.38. C H12NO requires C, 54.22; H, 5.79; N, 5.75;

Cl, 14.55 per cent). Faulkner and Woodcock (1965) reported 79-

800C.

6-Nitro-4-chloro-2-methylphenol. This compound was prepared

as described by Zincke (1918). He reported the melting point to be

1070C. Our preparation melted at 1060C (Found: C, 44.96; H, 3.47;

Cl, 18.32; N, 7.42. C7H6CNO3 requires C, 44.82; H, 3.22; Cl, 18.90;

N, 7.47 per cent).

Ethyl 6-nitro-4-chloro-2-methylphenoxyacetate. 6-Nitro-4-

chloro-2-methylphenol (30 g.) was added to a solution of sodium (3.7

g.) in 300 ml of absolute ethanol and heated to reflux. Ethyl bromo-

acetate (20 g.) was added dropwise through the condenser and the

mixture refluxed for 3.5 hours. Most of the ethanol was removed

under vacuum. The mixture was then diluted with water and the pre-

cipitate filtered off. The yield was 32 g. (75 per cent yield).

The compound melted at 700C. Faulkner and Woodcock (1965) also re-

ported a melting point of 700C.









6-Nitro-4-chloro-2-methylphenoxyacetamide. Ethyl 6-nitro-4-

chloro-2-methylphenoxyacetate (5 g.) was added to 20 ml. of absolute

ethanol and refluxed with 5 ml. of concentrated ammonium hydroxide.

The mixture was allowed to reflux for 8 hours and 5 ml. of concen-

trated ammonium hydroxide was added at 2 hour intervals. The yield

of product was 1.2 g. (27 per cent yield). The compound melted at

153-1560C.

5-Chloro-3-methylcatechol. Attempts were made to prepare this

compound through two intermediates described by Zincke (1918). The

structures given for the intermediates were dubious. In one trial,

however, a small amount of the desired product was obtained which

agreed with the melting point of 890C (Found: C, 53.31; H, 4.54;

Cl, 22.05. C H C102 requires C, 53.02; H, 4.45; Cl, 22.36 per cent)

reported by Zincke. The last step in the synthesis was very low

yielding, the product unstable and results inconsistent. Since the

preparation of 2-methyl-l,4-muconolactone from this compound failed,

further attempts to prepare it were terminated.

4-Chloro-2-methylmuconic acid. 4-Chloro-2-methylphenol (4.0

g.) was added to 175 ml. of ether containing monoperphthalic acid

(17 g.) and allowed to stand at room temperature for four days.

None of the desired product was obtained when the preparation was

treated as described by Testa (1952).

2-Methyl-l,4-muconolactone. 3-Methyl-5-chlorocatechol (1.08

g.) was dissolved in 50 ml. of ether and 88 ml. of ether containing

monoperphthalic acid (4.34 g.) added. This mixture was allowed to









stand at room temperature for five days. None of the desired product

was obtained when the preparation was treated as described by Testa

(1952).

6-Hydroxy-2,4-dichlorophenoxyacetic acid (6-OH-2,4-D). This

compound was prepared as described by Cavill and Ford (1954) using

3,5-dichlorocatechol previously prepared in this laboratory. The

compound after two recrystallizations from water melted at 1330C.

Cavill and Ford reported the melting point to be 1320C.

2-Chloro-l,4-muconolactone. 2-Chlorophenol (6.0 g.) was added

to 164 ml. of ether containing monoperphthalic acid (9.1 g.). On

the following day, 90 ml. of ether containing monoperphthalic acid

(5.0 g.) was added and the solution allowed to stand for four days

at room temperature. This preparation, when worked up, failed to

yield the desired product.

5-Hydroxy-2,4-dichlorophenoxyacetic acid hydrate (5-OH-2,4-D). -

This compound was prepared as described by Moszew and Wojciechowski

(1945). The product after two recrystallizations from water melted

at 1660C (Found: C, 37.62; H, 3.03; Cl, 27.42. C H C120-H20 requires

C, 37.67; H, 3.16; Cl, 27.80 per cent). Moszew and Wojciechowski

(1945) reported the melting point to be 175.50C.

3-Nitro-2,4-dichlorophenol. This compound was prepared as

described by Groves et al. (1929). After two recrystallizations

from 2,2,4-trimethylpentane, the compould melted at 720C. Groves

et al. found their preparation to melt at 70-720C.

Ethyl 3-nitro-2,4-dichlorophenoxyacetate. This compound

was prepared as described by Faulkner and Woodcock (1965). It was









found to melt from 80-82 C. Faulkner and Woodcock reported the

melting point to be 850C.

Ethyl 3-amino-2,4-dichlorophenoxyacetate. Ethyl 3-nitro-2,4-

dichlorophenoxyacetate (36 g.), 10 per cent palladium on charcoal

(2 g.) and 150 ml. of tetrahydrofuran were added to a bomb with a

total volume of 430 ml. The bomb was charged with nitrogen and re-

leased several times to remove air. The bomb was then charged with

hydrogen to a maximum pressure of 150 p.s.i. several times and the

pressure drops recorded. The final total pressure drop was 478

p.s.i. The theoretical pressure drop required was 473 p.s.i. The

bomb was fastened on a shaker and shaken during the course of the

reaction. When the temperature of the bomb rose noticeably, it was

cooled to room temperature in an ice bath. At the end of the reac-

tion, the material was poured out of the bomb and the charcoal fil-

tered off. The tetrahydrofuran and the water produced were taken

off under vacuum. The residue was heated with 2,2,4-trimethyl-

pentane and the solvent cooled. After several extractions, a yield

of 20.6 g. (64 per cent yield) was obtained. After recrystalliza-

tion from ligroine (b.p. 66-75C), the product melted from 56-580C.

Trials using Faulkner and Woodcock's (1965) procedure for this com-

pound were unsuccessful. They reported that their preparation melted

from 57-580C.

3-Hydroxy-2,4-dichlorophenoxyacetic acid. Attempts to diazotize

ethyl 3-amino-2,4-dichlorophenoxyacetate and obtain 3-hydroxy-2,4-

dichlorophenoxyacetic acid have been unsuccessful in this laboratory,

although Faulkner and Woodcock (1965) have prepared this compound.









2,3-Dichlorohydroquinone. This compound was prepared using

the procedure described by Conant and Fieser (1923). The product

melted at 1430C which agreed with the author's work. Attempts to

prepare this compound by the rearrangement of 2,5-dichlorobenzo-

quinone and subsequent saponification as described by Dimroth et al.

(1926) failed to yield a usable product.

Ethyl 4-hydroxy-2,3-dichlorophenoxyacetate. 2,3-Dichlorohydro-

quinone (10 g.) was dissolved in 100 ml. of absolute ethanol con-

taining sodium (1.28 g.) and refluxed for 2 hours during the dropwise

addition of ethyl bromoacetate (9.3 g.). The excess ethanol was then

removed under vacuum and the solution diluted with water. The prod-

uct, which precipitated out of the solution, was filtered off. The

yield was 12.7g. (86.5 per cent yield) and after recrystallization

from ligroine (b.p. 60-900C) melted at 790C.

4-Hydroxy-2,3-dichlorophenoxyacetic acid (4-OH-2,3-D). Attempts

to hydrolyze the above ester with acid or base have failed to yield

a significant amount of product with sufficient purity for use. Con-

densation of 2,3-dichlorohydroquinone with bromoacetic acid has also

failed to give the desired product. A small sample, however, was

supplied by Thomas et al. (1963) and melted at 167-1680C.

Ethyl 4-hydroxy-2,5-dichlorophenoxyacetate. This compound was

prepared in the same manner as the corresponding 2,3-dichloro- analog

starting with 2,5-dichlorohydroquinone. The product melted at 950C.

4-Hydroxy-2,5-dichlorophenoxyacetic acid (4-OH-2,5-D). Ethyl

4-hydroxy-2,5-dichlorophenoxyacetate (4 g.) was added to 50 ml. of

10 per cent aqueous sodium hydroxide and refluxed for 2 hours. The




27



solution was then acidified and cooled. White crystals precipitated

from the solution. After two recrystallizations from water, 0.6 g.

(16.6 per cent yield) of product melting at 166-1670C was obtained.

The infrared spectrum of this material was identical with that of a

sample supplied by Thomas et al. (1963). Their preparation melted

at 161-1620C. Attempts to prepare this compound by condensation of

2,5-dichlorohydroquinone with bromoacetic acid failed, as mentioned

previously in the case of the 2,3-dichloro- analog.
















EXPERIMENTAL RESULTS

Portions of the treated solutions were injected into the gas

chromatograph as described in table 1. Ten pl. injections were

used for the root extracts and 1 pl. for the standards. The reten-

tion times of the standards are given in table 2. 2,4-D was de-

tectable when as much as 0.1 pg. was present in the sample injected

into the gas chromatograph. A sample containing 0.25 Pg. of the

hydroxy-derivatives was necessary for reasonable detection on the

equipment used in this study. Since each alkylated extract repre-

sented 66 mg. of 2,4-D substrate and recovery was estimated at

75 per cent or better, the detection threshold of 0.25 pg. hydroxy-

dichlorophenoxyacetic acid represented excellent sensitivity. In

the samples treated with diazomethane, a peak corresponding to

2,4-D was present in all of the 2,4-D treated samples. This peak

was masked by the solvent peak due to the higher sensitivity and

large sample injection used when diazo-n-propane was the reactant.

All other peaks in the 2,4-D treated samples did not correspond to

any of the standards and were also present in the control extracts.

To determine the disappearance of 2,4-D from the incubation

media, three 1 ml. samples were taken from one of the flasks at

3 hour intervals during the incubation. These solutions were con-

centrated under nitrogen almost to dryness and adjusted to 1 ml.

with methanol. They were then treated with an ethereal solution of











TABLE 1

Operating Conditions for Gas Chromatography


Manufacturer


F and M


400


Hydrogen flame ionization


15 per cent SE-30 on Chromosorb W

(60/80 mesh)

3/16 inch

6 feet


Helium

60 ml./min.


diameter

length


Temperatures

flash heater

column

detector


Electrometer settings

knowns

diazomethane treatment

diazo-n-propane treatment


2350

1900C

240C


Range

10

10

10


Attenuation

4

8

1


Model


Detector


Column


Carrier

flow














TABLE 2

Retention Times of Standard Solutions in Methanol


Parent
compound


2,4-D


6-OH-2,4-D


5-OH-2,4-D


4-OH-2,3-D


4-OH-2,5-D


Methyl ester
methyl ether


min.


4.0


6.7


8.1


10.2


8.0


Methyl ester
propyl ether


min.





9.4


10.9


13.5


11.4


Propyl ester
propyl ether


min.


6.5


16.4


19.8


16.0


21.5








diazo-n-propane, reduced under a stream of nitrogen and readjusted

to 1 ml. with methanol. These solutions were run on the gas chro-

matograph. The size of the peaks representing 2,4-D were not re-

producible between replicates but there was a continuous decrease

in the average peak size as the length of the incubation time in-

creased. There was no peak representing 2,4-D after 12 hours of

incubation. This showed that 2,4-D was being removed from the solu-

tion.

Samples (2.5 mg.) of the synthesized hydroxylated derivatives

were added to a blank extract and carried through the purification

procedure. It is estimated from the relative size of the resulting

peaks on the gas chromatograph that more than 75 per cent of each

known was recovered in the final solutions. It was observed that

the retention times of the knowns added to the blank did not agree

with the standards when treated with diazo-n-propane. Since the

methanolic extracts had been agitated for several hours, it was

suspected that the acids had been converted to methyl esters. In

order to resolve this discrepancy, methanolic solutions of the

standards in tightly capped tubes were heated in a water bath at

75 C for 2 hours. The object of this procedure was to determine

whether the phenolic acids could be esterified under very mild

conditions. At the end of this time, they were treated with diazo-

n-propane and analyzed on the gas chromatograph. Partial methyl

esterification was indicated by the appearance of a second peak

other than the propyl ether ester from each of the standards, as

shown in table 3. The retention times of the second peaks agreed




















TABLE 3

Relative Peak Heights of Methyl and Propyl Esters

of the Propyl Ethers1


Parent compound Methyl ester Propyl ester



6-OH-2,4-D 11.0 14.5


5-OH-2,4-D 6.6 22.1


4-OH-2,3-D 3.3 23.0


4-OH-2,5-D 2.8 1-9.6


1
Anhydrous methanolic solutions of the samples,
were heated in a water bath at 750C for 2 hours and
with the appropriate ethereal diazoalkane.


in closed tubes,
then alkylated









with those subjected to the purification procedure and the diazo-

n-propane treatment. The diazomethane treatment gave retention

times which agreed with the standards.

4-Hydroxy-2,3-, 4-hydroxy-2,5-, 6-hydroxy-2,4- and 5-hydroxy-

2,4-dichlorophenoxyacetic acids produced no epinasty on the test

plants. 5-Nitro- and 5-amino-4-chloro-2-methylphenoxyacetates

showed positive epinasty on the test plants on the day following

treatment. Ethyl 6-nitro-4-chloro-2-methylphenoxyacetate produced

no epinasty during twelve days following treatment. Ethyl 3-amino-

2,4-dichlorophenoxyacetic acid showed positive epinasty within 2

hours of treatment, as did 2,4-D.















DISCUSSION

The greatest difficulty in this work was the lack of reference

compounds for use as standards. The next problem was the deter-

mination of an analytical procedure for detection of minute amounts

of these substances from biological materials. Preparations for

some of the compounds involved in this study were published by

others during the course of this work.

Using the procedure of Faulkner and Woodcock (1965), the

yield of 3-amino-2,4-dichlorophenoxyacetic acid was very low. In

an attempt to obtain a better yield, we used hydrogen in a bomb

with palladium on charcoal as a catalyst and obtained a yield of

64 per cent. Faulkner and Woodcock reported a yield of 56 per cent

but in this laboratory this yield was never approached using their

procedure.

Difficulty was also experienced in the procedures of diazotiza-

tion of aromatic amines to phenols reported by the above workers.

In attempts to follow their procedures of diazotizing ethyl 3-

amino-2,4-dichlorophenoxyacetate and ethyl 5-amino-4-chloro-2-

methylphenoxyacetate to the corresponding hydroxy-acids, none of

the desired products were obtained.

6-Hydroxy-4-chloro-2-methylphenoxyacetic acid has never been

synthesized but Gaunt and Evans (1961), using a soil bacterium, iso-

lated a hydroxy-4-chloro-2-methylphenoxyacetic acid believed to be









the 6-hydroxy-analog. They also isolated a gamma-lactone which

presumably would be produced via the former compound. For this

reason, an unambiguous synthesis of this compound is of considerable

interest. We prepared 6-nitro-4-chloro-2-methylphenoxyacetamide as

it is possible that amide substitution would prevent the expected

cyclization to a lactam during the reduction of the corresponding

nitro compound to the amino analog. This amide may prove to be a

useful intermediate in the unambiguous synthesis of 6-hydroxy-4-

chloro-2-methylphenoxyacetic acid.

Difficulty was also encountered in the last step of the syn-

thesis of 5-chloro-3-methylcatechol as described by Zincke (1918).

Due to the dubious structures reported for the intermediates and

the low-yielding last step, the preparation of this compound was

abandoned. This compound would probably be an intermediate between

6-hydroxy-4-chloro-2-methylphenoxyacetic acid and the previously

mentioned gamma-lactone. Because of these difficulties any future

attempts to prepare 5-chloro-3-methyl-catechol should await an un-

ambiguous synthesis of 6-hydroxy-4-chloro-2-methylphenoxyacetic

acid such as by the above suggested method. Cleavage of 6-hydroxy-

4-chloro-2-methylphenoxyacetic acid prepared in this manner with

hydriodic acid or a similar reagent would be expected to give in

turn 5-chloro-3-methylcatechol unambiguously.

Under the conditions of this work, 2,4-D was not metabolized to

4-hydroxy-2,3-, 4-hydroxy-2,5-, 6-hydroxy-2,4- or 5-hydroxy-2,4-

dichlorophenoxyacetic acid in detectable amounts. Conversion at

any one time of 0.4 per cent of the original 2,4-D in the incubation








mixtures to any of the possible hydroxylated intermediates would

have been readily detectable. If any of these derivatives were

formed, they must have been further metabolized as rapidly as they

were produced. It is possible, however, that under some other con-

ditions these derivatives might be detected.

Preparation of more volatile derivatives is necessary for

analysis of phenolic acids by gas chromatography. The use of -solu-

tions of diazoalkanes in ether to alkylate the carboxyl and hydroxyl

groups of the possible metabolites proceeded smoothly during this

work. This method has the advantage that one reactant will quickly

convert both carboxylic and phenolic hydroxyls to more volatile

groups at room temperature. After the initial reaction with the

diazoalkane had subsided, a small amount of'boron trifluoride was

added to further catalyze the reaction and destroy the excess diazo-

alkane as the methyl ether.

By treating methanolic solutions of standards of the above mono-

hydroxydichlorophenoxyacetic acids with diazomethane and diazo-n-

propane, it is possible to prepare derivatives which allow separation

by means of gas chromatography. Use of only one of the reagents will

not produce separable derivatives of all four of the hydroxy-acids

under the conditions of this work. Treatment with diazomethane did

not separate derivatives of 5-hydroxy-2-4- and 4-hydroxy-2,5-di-

chlorophenoxyacetic acids. Diazo-n-propane treatment did not sepa-

rate derivatives of 6-hydroxy-2,4- and 4-hydroxy-2,3-dichloro-

phenoxyacetic acids.










Attempted preparations of gamma-lactones mentioned as metabo-

lites of 2,4-D and MCPA by Fernley and Evans (1959) and Gaunt and

Evans (1961), respectively, were unsuccessful. Synthesis of these

lactones was attempted by oxidation of the corresponding catechol

or phenols with monoperphthalic acid as described by Testa (1952).

The only compound which could be extracted and identified from

these solutions was phthalic acid.

Hydrolysis of ethyl 2,3-dichlorophenoxyacetate to the corre-

sponding acid also failed to yield a usable product. Hydrolysis by

both acid and base was attempted but the desired hydroxy-acid was

not produced. A high melting compound was extracted which may have

been a polymer. Rearrangement of 2,5-dichlorobenzoquinone to the

2,3-dichloro-analog desired as a possible precursor in the synthesis

of the previously mentioned compound by the method of Dimroth et al.

(1926) also proved unsuccessful. We suspect Dimroth et al. sepa-

rated the isomers in a mixture rather than actually causing a re-

arrangement of the molecule.

No detectable amounts of substrate (1 X 10-3 M 2,4-D) were

present after 12 hours incubation of excised roots of the plant

species studied. The root extracts, however, did give a peak

representing 2,4-D which showed that the roots had taken up the

herbicide. This indicated that the incubation had not been long

enough for all of the 2,4-D to have been converted to some product

further removed metabolically from the herbicide than those for

which analysis was being made.









One method which might make identification easier would be the

use of methanol as the extraction solvent with addition of boron

trifluoride, and subsequent heating to cause esterification of the

carboxylic groups. The resulting esters could be treated with

various diazoalkanes which would etherify the phenolic groups. This

method would enhance dissimilarities in the molecules and might pos-

sibly give better separation in gas chromatography.

Otherwise, it would seem advisable to use some extraction sol-

vent other than methanol which would not react with the acids being

analyzed. This would eliminate some of the identification problems

encountered in this work. Ethyl acetate would be a good solvent to

consider.

Another modification in the procedure which might improve the

technique would be the use of an electron capture detector on the

gas chromatograph. This would probably eliminate most of the peaks

noted except those of the compounds containing chlorine. Elimina-

tion of peaks for most of the natural products would increase the

worker's ability to identify the compounds being analyzed. Another

advantage would be the higher sensitivity to be expected with an

electron capture detector.

In the tomato epinasty tests, it is possible that the 5-nitro-

and 5-amino-4-chloro-2-methylphenoxyacetic acids were contaminated

with MCPA since they were prepared from the parent herbicide. How-

ever, the purification procedures should reduce this possibility

to a minimum. This is particularly true for the amino compound, as

its preparation from MCPA requires two steps with associated





39



purifications. There should have been no 2,4-D contamination in the

ethyl 3-amino-2,4-dichlorophenoxyacetate as it was prepared from

m-nitrophenol and had no common precursor with 2,4-D. Epinasty was

not observed for 6-hydroxy-2,4-, 5-hydroxy-2,4-, 4-hydroxy-2,3- and

4-hydroxy-2,5-dichlorophenoxyacetic acids. 6-Hydroxy-2,4-dichloro-

phenoxyacetic acid previously had been shown to be inactive by

Cavill and Ford (1954). This might be expected as hydroxylation is

known to be a prime method of detoxication by biological systems.















SUMMARY

The object of this work was to determine whether hydroxylated

metabolites or further derived compounds were formed during the

degradation of 2,4-dichlorophenoxyacetic acid by excised roots of

several species of higher plants. Excised roots were incubated in

a solution of 2,4-D for periods of 10 and 12 hours at two different

hydrogen ion concentrations. The roots were then extracted with

methanol. After prepurification by thin layer chromatography, the

solutions were treated with diazomethane and diazo-n-propane to

alkylate phenolic acids and analyzed by gas chromatography. Syn-

thetic 6-hydroxy-2,4-, 5-hydroxy-2,4-, 4-hydroxy-2,3- and 4-hydroxy-

2,5-dichlorophenoxyacetic acids were alkylated similarly and used

as standards in the analysis.

The extracts from the roots contained no detectable amounts of

the possible metabolites for which the analysis was being made. All

of the extracts from 2,4-D treatments did show a peak with a reten-

tion time identical with 2,4-D.

Tomato epinasty tests showed that none of the previously men-

tioned hydroxylated analogs tested were active. Ethyl 6-nitro-4-

chloro-2-methylphenoxyacetate also showed no activity in the test.

Ethyl 3-amino-2,4-dichlorophenoxyacetate and 2,4-D were very active

and began showing epinasty effects within 2 hours of treatment. 5-

Nitro- and 5-amino-4-chloro-2-methylphenoxyacetic acids also gave




41



positive results in the test. The former compound was almost as

active as 2,4-D. The latter was less active and the effects didn't

appear until the second day after treatment.














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Fawcett, C. H., R. M. Pascal, M. B. Pybus, H. F. Taylor, R. L. Wain,
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Gaunt, J. K., and W. C. Evans. 1961. Metabolism of 4-chloro-2-
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of Avena sativa. Nature 204:286.

Thomas, E. W., B. C. Loughman, and R. G. Powell. 1964b. Metabolic
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Phaseolus vulgaris. Nature 204:884.

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46



Williams, R. T. 1950. Biological oxidation of aromatic rings.
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BIOGRAPHY

Norman Glaze was born in Washington, D. C., on January 16,

1934. He attended public schools in Hyattsville, Maryland, and

was graduated from Northwestern High School in 1952. He obtained

the Bachelor of Science degree from the University of Maryland in

1957. Upon completion, he was inducted into the U. S. Army and

served for two years. After completion, he reentered the University

of Maryland and received the Master of Science degree in 1963. He

then transferred to the University of Florida for work on the Doctor

of Philosophy degree.

/'











This dissertation was prepared under the direction of the

chairman of the candidate's supervisory committee and has bee.

approved by all members of that committee. It was submitted to

the Dean of the College of Agriculture and to the Graduate

Council, and was approved as partial fulfillment of the require-

ments for the degree of Doctor of Philosophy.





April, 1966





ar-/ean, College of Agriculture





Dean, Graduate School



Supervisory Committee:




Chairman




Full Text

PAGE 1

ANALYSIS OF 2,4-D METABOLITES IN HIGHER PLANTS BY GAS CHROMATOGRAPHY By NORMAN CLINE GLAZE A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA April, 1966

PAGE 2

G £T 5" 2 a. AGKICUITURAL UNARY UNIVERSITY OF FLORIDA 3 1262 08552 3339

PAGE 3

'fr* ACKNOWLEDGEMENTS The author wishes to express his sincere appreciation to Dr. M. Wilcox, Dr. S. H. West, Dr. T. E. Humphreys, and Dr. H. C. Harris for their advice and guidance during the course of these studies. Special acknowledgement is made to Professor Zygmunt Eckstein, Katedra Technologii Organicznej II Politechniki, Warsaw, for supplying reprints and translations of necessary passages from his works, to Dr. G. R. Powell, Unit of Experimental Agronomy, University of Oxford, who supplied samples and physical properties of 4-hydroxy-2,3-dichloroand 4-hydroxy-2,5-dichlorophenoxyacetic acids and to Dr. R. H. Biggs for use of his gas chromato graph. This work was made possible by a grant from the American Cancer Society. ii

PAGE 4

TABLE OF CONTENTS Page ACKNOWLEDGEMENTS ii LIST OF TABLES iv INTRODUCTION 1 LITERATURE REVIEW 3 MATERIALS AND METHODS 17 EXPERIMENTAL RESULTS 28 DISCUSSION 34 SUMMARY 40 LITERATURE CITED 42 iii

PAGE 5

LIST OF TABLES Table Page 1 Operating Conditions for Gas Chromatography. . . . 2 Retention Times of Standard Solutions in Methanol 30 3 Relative Peak Heights of Methyl and Propyl Esters of the Propyl Ethers 32 iv

PAGE 6

INTRODUCTION The use of herbicides is increasing rapidly year by year. 2,4-Dichlorophenoxyacetic acid (2,4-D) is the most widely used of the herbicides offered on the market. Although 2,4-D was one of the first selective herbicides discovered and has been investigated by many workers over the years, there are still many questions concerning its metabolism. Hydroxylated derivatives have been found in some microorganisms and plants . This might be expected as hydroxylation is a prime method of detoxication in biological systems. 2,4-D has been found to be hydroxylated predominantly in the 5-position. However, 4hydroxy-2,3and 4-hydroxy-2,5-dichlorophenoxyacetic acids have been identified in some cases. This is interesting as it involves much more elaborate mechanisms than hydroxylation in a vacant position. The formation of these hydroxy acids requires a chlorine shift to one of the meta positions and hydroxylation in the para position. The object of this work is to determine whether hydroxylated derivatives are formed by excised roots from 2,4-D or related herbicides. Some workers have also found muconic acids and gamma -lac tones as succeeding stages in the metabolism of these herbicides. This would follow from an oxidative degradation of the ring structure. The lactones found are of interest from the public health standpoint. The gammalactones isolated contain double bonds in the

PAGE 7

even numbered positions and compounds of this type have been found to be carcinogenic. If these compounds appear in any appreciable amounts and are not very transitory in nature, the use of the herbicides involved might require reevaluation by regulating organizations.

PAGE 8

LITERATURE REVIEW The literature review will be restricted to the metabolism of chloroand methylphenoxyaliphatic and naphthyland naphthyloxyaliphatic acids by higher plants and microorganisms. Animal metabolism is covered by Williams (1950, 1959)* He states that phenoxyacetic acid and the orthoand para -chloro-derivatives are unchanged in animals. General classes of compounds which are considered to be metabolites, such as phenols, catechols, etc., are discussed in his publications. Newman and Thomas (19^9) discovered that the persistence of 2,4-dichlorophenoxyacetic acid (2,4-D) in soils was decreased by pretreatment of the soil with 2,4-D and certain other compounds having similar substituent groups such as 2,4dichlorophenol. However, pretreatment with some compounds such as 2or 3-chlorophenol or phenoxyacetic acid had no effect on the persistence of 2,4-D in the soil. Burger et al. (1962) measured the persistence of some phenoxyaliphatic acids by a bioassay technique using alfalfa seedlings. Acetic, alpha -propionic, alphabutyric and gamma -butyric acid homologs were toxic when applied to soil planted to alfalfa, but beta-propionic acid analogs were not toxic to the test species. Prolonged persistence was exhibited when the soil was treated with 3,4-dichloroand 2,4,5-trichlorophenoxyaliphatic acids. The duration of phytotoxicity in soil receiving 4-chloro-, 2,4-dichloroand

PAGE 9

2-methyl-4-chlorophenoxyaliphatic acids was governed by the type and linkage of the aliphatic side chain. Audus (1952) found that 2,4-D enriched soil metabolized 2-methyl-4-chlorophenoxyacetic acid (MCPA) immediately but did not affect the detoxication of 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). MCPA enriched soil degraded 2,4-D and 2,4,5-T. Audus (1950) isolated Bacterium globiforme which was capable of detoxifying 2,kD. A Flavobacterium sp . was found by Burger e_t al . (1962) which degraded phenoxybutyric acids. Only phenoxybutyric acids having no meta -chlorine on the aromatic ring were metabolized by the organism. These results show that the persistence of these herbicides in soils is governed by the specific structural characteristics of the molecule. A Corynebacterium sp ., capable of degrading 2,4-D and MCPA, was isolated by Rogoff and Reid (1956). From all indications, the ring was ruptured and complete destruction of the molecule followed. Audus (1952) suggested that the first step in the degradation of 2,4-D, 2,4,5-T and MCPA might be the removal of the two carbon side chain leaving a phenol. However, he found no accumulation of phenols in soil treated with any of these herbicides. If a phenol is an intermediate, it must be metabolized as rapidly as it is formed. This is a possibility because it was found that 2,4dichlorophenol decomposed rapidly when added to a 2,4-D enriched soil. Leafe (1962) suggested two possibilities for the metabolism of MCPA by Galium aparine . The first postulate listed for the

PAGE 10

degradation of MCPA was the removal of the two carbon side chain either as a two carbon fragment or its loss as carbon dioxide. Secondly, he suggested that the metabolism or detoxication might involve conjugation to another cellular component. Leafe stated that the detoxication of the molecule by loss of both carbon atoms of the side chain has been established as the reason Galium aparine is resistant to MCPA. Measurements were made by Canny and Markus (i960) of the rate of evolution of labelled carbon dioxide from the shoots and roots of Vlcia faba which had been treated on one leaflet with 2,4-D labelled in the carboxyl group. The carbon dioxide evolved from the roots was more radioactive than that from the shoot. Labelled carbon was shown to be incorporated into many compounds in the root. This is evidence that the degradation may take place mainly in the roots. It was also concluded that although metabolism of the alkyl side chain was rapid, this was not the main pathway for inactivation of the herbicide. Bach (I96I), working with stems of Phap^olus vulgaris , isolated one half of the original radioactivity from carboxyl labelled 2,4D in an ether extract. The activity was found to be distributed among ten components. Tests for functional groups showed that these components retained the aromatic character of 2,4-D. Neither 2,4-D nor 6-hydroxy-2,4-dichlorophenoxyacetic acid were isolated. Some ten amino acids were also labelled in the treated stem tissue. When red kidney beans were treated with labelled 2,4-D, Holley (1952) found that in one week two thirds of the radioactivity was

PAGE 11

recovered in a water soluble fraction. He stated that the water soluble material must be a derivative of an unknown acid rather than of 2,4-D. Holley suggested that the unknown acid might be 3-> 5or 6-hydroxy-2,4— dichlorophenoxyacetic acid. Steenson and Walker (1956) isolated Flavobacterium peregrin urn and two Achromobacter sp . which were capable of decomposing 2,4-D, MCPA or 4-chlorophenoxyacetic acid. They found that pretreatment of the soil with one of the compounds accelerated the rate of decomposition of the other two. In a liquid medium, F. peregrinum metabolized 2 f kD and liberated 76 per cent of the chlorine in ionic form. They suggested that *4— chloro-2-hydroxyphenoxyacetic acid and 4-chlorocatechol might be intermediates in the degradation of hchlorophenoxyacetic acid. A small, Gram-negative, motile organism was isolated from the soil by Evans and Smith (195*0 which utilized kchlorophenoxyacetic acid and 2,k—B and produced 4-chlorophenol and 2,4-dichlorophenol, respectively. A phenolic acid metabolite was found and it was suggested to be 6-hydroxy-2, ^-dichlorophenoxyacetic acid. It was also observed that the chlorine atoms were retained until the aromatic character of the molecule was destroyed. Using a replacement culture technique, Byrde and Woodcock (1957) showed that phenoxyacetic and beta-phenoxypropionic acids were hydroxylated mainly in the para position by Aspergillus niger . The compounds were also hydroxylated in the ortho position but to a lesser extent. Gamma -phenoxy-n-butyric acid also was metabolized

PAGE 12

mainly to 4-hydroxyphenoxyacetic acid by hydroxylation and betaoxidation. Delta-phenoxy-n-valeric acid was degraded in a similar manner to beta-(4-hydroxyphenoxy)propionic acid. Wilcox et al. (I963) found that barley, oats and corn had the ability to produce a ring hydroxylated intermediate from a phenoxyn-aliphatic acid with an even number of carbons in the side chain when incubated in aerated water. Odd chain homologs yielded little of a hydroxylated metabolite. In soybeans, alfalfa and peanuts, no hydroxylated intermediates were detected. The phenolic product, produced by the test plants when treated with various phenoxy-naliphatic acids having an even number of carbons in the side chain, was identical with 4-hydroxyphenoxyacetic acid. These data suggest that ring hydroxylation by the resistant grass species coincides with the suggested role of hydroxylation as a detoxication mechanism. Working with Aspergillus niger, Byrde and Woodcock (1957) demonstrated that phenoxyacetic and beta-phenoxypropionic acids were hydroxylated in the para position mainly and to a lesser extent in the ortho position. Faulkner and Woodcock (l96la, 196lb) also found phenoxyacetic acid to be hydroxylated in the ortho and para positions. Clifford and Woodcock (1964), however, found 2-hydroxyphenoxyacetic acid to be the main metabolite from phenoxyacetic acid. Bocks e_t al. (1964) also found 2-hydroxyphenoxyacetic acid to be the main metabolite from phenoxyacetic acid. All other isomers were formed but in much smaller amounts. Bocks also found phenol to be a minor product. Faulkner and Woodcock (I96lb) found that 2-chloro-

PAGE 13

8 and 4-chlorophenoxyacetic acids were metabolized to 2-chloro-4hydroxyand 4-chloro-2-hydroxyphenoxyacetic acids, respectively. The original starting materials were monohydroxylated in all vacant positions but the above were produced in much larger amounts. Also found was 2-hydroxyphenoxyacetic acid. This was the first example of fungal replacement of chlorine by a hydroxyl group. Bocks et al . used methoxybenzene as a substrate and observed that 2-hydroxymethoxybenzene was produced. If the incubation was stopped after three days, phenol was present in almost the same proportions as 2-hydroxymethoxybenzene. If allowed to proceed for a longer period, the yield of phenol relative to 2-hydroxymethoxybenzene decreased. Evans and Moss (1957) t working with a Pseudomonas sp ., extracted 2-hydroxy-4-chlorophenoxyacetic acid and 4chlorocatechol from cultures grown on a medium containing 4-chlorophenoxyacetic acid. This was a strong indication that these were two of the possible intermediates in the degradation of this herbicide. Bell (i960), studying an Achromobacter sp ., found an adaptive system that is capable of oxidizing 2,4-D. The organism can also oxidize many 2,4-D analogs. For the oxidation rate to be comparable to 2,4-D, the organism appeared to require the following: a free ortho position; a free carboxyl group on the sj.de chain, preferably beta to the ethereal linkage; a chlorine atom in the para position; and no more than two chlorine atoms on the ring whether or not an ortho position is free. He suggested that the probable degradation route was to 2,4-dichlorophenol but that action by a decarboxylase to 2,4-dichloromethoxybenzene might be involved. He

PAGE 14

also suggested that the metabolites from 2,4-dichlorophenol might be 4chlorocatechol or 3j5-dichlorocatechol. The metabolism of 2,4-D and MCPA by a strain of Flavobacterium peregrinum and an Achromobacter sp . was studied by Steenson and Walker (1957) • The evidence presented indicated that adapted organisms metabolize 2,4-D through 2,4-dichlorophenol and 4-chlorocatechol and that MCPA is degraded to ^-chloro-2-methylphenol. Bacteria, grown on a medium containing 2,4-D, did not oxidize any of the five possible isomers but would oxidize 2,4dibromo-, 4-bromo2-chloro-, 4chloroand to a small extent, 2-chlorophenoxyacetic acids . Thomas et al. (1963? 1964a) studied the hydroxylation of some phenoxyacetic acids by stem tissue of Avena sativa . They found that phenoxyacetic acid was converted to 4-hydroxyphenoxyacetic acid. Phenoxyacetic acids with an unsubstituted para position were hydroxylated in that position. The phenolic acids were accumulated as the 4-0-beta-D-gluco sides. Phenoxyacetic acids with a chlorine atom in the para position, however, were not hydroxylated to an appreciable extent. These acids accumulated as neutral products which proved to be phenoxyacetylglucoses. In the case of 2,4,6-trichlorophenoxyacetic acid, however, hydroxylation took place at the meta position and the glucoside was isolated. Thomas et al. (1964b) studied the metabolism of 2,4— D in stem tissue of Phaseolus vulgaris . They found the predominant metabolite to be 4-hydroxy-2,5-dichlorophenoxyacetic acid. This shows that the hydroxylation and chlorine shift discovered first in Aspergillus

PAGE 15

10 niger also occurs in higher plants . A minor metabolite was shown to be 4-hydroxy-2,3-dichlorophenoxyacetic acid which also requires the hydroxylation and chlorine shift. The products accumulated within the tissue as beta glue o sides. Evans and Moss (1957) > using a Gram-negative, motile organism of the Pseudomonas type, found that when it was incubated with k— chlorocatechol, beta -chloromuconic acid accumulated. The beta chloromuconic acid was further metabolized and the chlorine liberated in ionic form. Later Fernley and Evans (1959) isolated aloha -chloromuconic acid during the metabolism of 2,^4— D which was identical with that prepared by the oxidation of 2-chlorophenol. They observed the shift of the 283 mp-* peak of 2,4-D in the ultraviolet range to another peak at 272 mo-. Schemes for the metabolism of chlorophenoxyacetic acids by soil bacteria were reported by Rogoff (1961), in which 4-chlorophenoxyacetic acid was metabolized through 2-hydroxy-4-chlorophenoxyacetic acid and 4-chlorocatechol to beta -chloromuconic acid and 2,4-D was degraded through 2,h~ dichlorophenol and 3»5-dichlorocatechol to albha -chloromuconic acid. Evans and Smith (l95l) 5 Evans et al. (1951), Dagley et al. (I9o0), Ribbons and Evans (1962) and Rogoff all show schemes for further microbial oxidation of catechol type derivatives. In all cases, the schemes show the catechol to ciscis -muconic acid step and terminate with beta-ketoadipic acid. The steps between the cis cis -muconic acid and beta -ketoadipic acid appear to be somewhat uncertain. The uncertainty seems to be whether the intermediates are lactonized or not when the double

PAGE 16

11 bond migrates, which must occur if this scheme is to be correct. The beta-ketoadipic acid is presumed to be split into acetic and succinic acids which would pass into the tricarboxylic acid cycle. Working with a Gram-negative soil bacterium, Gaunt and Evans (1961) found that the ultraviolet peaks of MCPA at 228 and 279 mu.. are replaced by a peak at 277 W-The properties of this compound were consistent with the lactonic acid, alphameth.ylgamma -carboxymethylene-A -butenolide. Peracetic acid oxidation of 4-chloro-2methylphenol produced a compound with the same properties. A phenolic acid was also isolated and found to have properties consistent with a hydroxy-2-methyl-^chlorophenoxyacetic acid. It was believed to be the 6-hydroxy-derivative. 4-Chloro-2-methylphenol was also isolated from the extracts. The following metabolic pathway for the degradation of MCPA was suggested: MCPA through 6hydroxy-4-chloro-2-methylphenoxyacetic acid; 5-chloro-3-methylcatechol; alphamethylgammachloromuconic acid; alphamethylparamacarboxymethylene-A -butenolide; alpha -me thy lmaleylacetate and to smaller fragments prior to entering a terminal respiratory cycle. The lactones mentioned in the degradation schemes are of considerable interest because butenolides of this general type with double bonds in even numbered positions have been shown to be carcinogenic by Dickens and Jones (I96I, I963). Byrde et al. (1956), using a replacement culture technique of Aspergillus niger , found that omega ( 2-naphth,yloxy ) -n-alipha tic acids having an even number of carbons in the side chain were beta -oxidized to (6-hydroxy-2-naphthyloxy)acetic acid. Beta(2-naphthyloxy) propionic

PAGE 17

12 acid was transformed to beta( 6-hydroxy-2-naphthyloxy )propionic acid. The n-b,utyric homolog was hydroxylated in the six position before betaoxidation to (6-hydroxy-2-naphthyloxy)acetic acid. Hydroxylation of the acetic and propionic acids led to a decrease in toxicity to the fungus. Byrde and Woodcock (1958), working with Sclerotinia laxa, found that nuclear hydroxylation was virtually absent with this organism. They did find that gamma( 2-naphthyloxy ) -n-butyric and delta(2-naphthyloxy)-nvaleric acids underwent beta -oxidation to the corresponding acetic and propionic acids, respectively. They also found beta-naphthol was produced from beta( 2-naphthyloxy ) propionic acid. Fawcett et al. (195^) found that omega_-phenoxy-n-aliphatic acids with an odd number of carbons in the side chain yielded a substantial amount of phenol when applied to flax plants. Compounds with an even number of carbon atoms in the side chain were beta -oxidized to the acetic derivative. The decanoic homolog, however, was found to produce a large amount of phenol. This was best explained from the fact that omega oxidation has been shown in animals for some compounds with fatty acids of nine or ten carbon atoms. It was assumed that this was also occurring in the flax plant. Omega(^—chlorophenoxy ) aliphatic and omega -(2,4-dichlorophenoxy)aliphatic acids showed the characteristic alternation of activity in work by Wain and Wightman (195^) » when used in the Went pea test, the tomato leaf epinasty and the wheat cylinder test. Only the wheat cylinder test showed activity with even numbered aliphatic acids of the 2,4,5-trichlorophenoxy series. When solutions of the

PAGE 18

13 2,4,5-trichlorophenoxyaliphatic acids with side chains containing an even number of carbon atoms were pretreated with wheat cylinders, however, the alternation of activity was shown by the Went pea test and the tomato leaf epinasty test. This evidence showed that wheat possessed the ability to beta -oxidize this series but that pea and tomato did not. When omega-1-naphthylaliphatic acids were tested, the Went pea test and wheat cylinder test showed the alternation of activity but only the acetic homolog was active in the tomato leaf epinasty test. It was found that the substitution of alkyl groups in the alpha position did not hinder beta-oxidation, but substitution in the beta position did prevent betaoxidation. Webley e_t al. (1955) studied the breakdown of omega-phenylaliphatic acids by Nocardia opaca . They found that acids with an odd number of carbon atoms in the side chain were converted to benzoic acid and that cinnamic acid was an intermediate. Orthohydroxyphenylacetic acid was found to be a metabolite when acids with an even number of carbon atoms in the side chain were used in the growth medium. Benzoic acid was produced essentially quantitatively but orthohydroxypheny lace tic acid was not, indicating that it must arise from some side reaction. They stated that the latter accumulates because it is not metabolized further by this organism. The evidence suggested that N. opaca was capable of beta -oxidation of the fatty acid side chains of these phenylaliphatic acids. Webley et al. (1958) expanded their study to examine the effect of change of various group;in the molecule and how beta -oxidation would be affected. They found the following five factors to affect

PAGE 19

14 beta -oxidation : l) an oxygen bridge between the fatty acid and the ring; 2) ring substitution, particularly in the ortho position; 3) the nature of the ring substituents ; 4) the type of ring structure to which the fatty acid is attached; and 5) the position of attachment of the fatty acid. The side chain of phenylbutyric acid was betaoxidized more readily than phenoxybutyric acid. The position of substituted chlorine atoms in the ring affected beta -oxidation as follows: gamma(3-chlorophenoxy)butyric acid was degraded most rapidly; the 4-chloro-homolog was intermediate; and the 2-chlorocompound was the least reactive. Monomethyl homologs reacted in the same manner as the corresponding monochloro-compounds. The methyl homologs, in general, were betaoxidized more readily than the corresponding chloro-compounds. The rate of conversion of the betahydroxyintermediate is reduced with the chlorinated materials. 2-Methyl-4-chloroand 2,4dichlorophenoxybutyric acids were tested to evaluate the effect of disubstitution. It was found that disubstitution decreased the rate of beta -oxidation. The oxidation of the disubstituted compounds proceeded to the beta -hydroxy-derivative but the acetic homologs were not produced. The caproic homologs were rapidly converted to the butyrics but again none of the acetic homologs were obtained. Gamma ( 2-naphthyloxy )butyr ic and beta -(2naphthyloxy)propionic acids were betaoxidized to the corresponding acetate and 2-naphthol, respectively. The 1-naphthyloxy-derivatives remained unchanged throughout the experiment. Gamma ( 1-naphthyl )butyric acid was converted to the acetate while the corresponding naphthyloxycompound was unchanged. Beta -oxidation of the fatty

PAGE 20

15 acid side chain was most rapid when attached to a phenyl ring and slowest when attached to a 1-naphthyl group. The indolyl group was intermediate . Fawcett et al . (1959), using the wheat cylinder, pea curvature, pea . cjgment and tomato leaf epinasty tests, assayed the growthregulating activities of 18 homologous series of omega-phenoxyaliphatic acids with two to seven carbon atoms in the side chain and with chlorine and methyl groups substituted in various ring positions. The degradation of some homologs was investigated and identification of metabolites made by chromogenic methods and bioassay. They noted that there were four patterns of biological activity as follows: 1) where betaoxidation was operative, and the acetic acid was active while the propionic acid was not, only the even numbered homologs showed activity; 2) where beta -oxidation was hindered at the butyric stage, and the acetic acid was active but the propionic acid inactive, only the acetic acid showed activity; 3) where betaoxidation was operative, and both the acetic acid and propionic acid were active, all members showed activity; and 4) where beta -oxidation was hindered at the butyric stage, and the acetic acid was less active than the propionic acid, the acetic acid plus all odd numbered homologs showed activity. The results indicated that betaoxidation is hindered at the butyric stage in pea and tomato tissue. The hindrance at the butyric stage was shown to be associated with an ortho chlorine or methyl group on the ring. This effect was largely removed by a further chlorine atom in the para position. Addition of a chlorine atom in tho nota position

PAGE 21

16 had no effect. The beta-oxidation sequence was not changed when a methyl group was substituted for a chlorine atom.

PAGE 22

MATERIALS AND METHODS The excised roots were prepared in the following manner: a single layer of seed was placed between double thicknesses of cheesecloth supported on aluminum mesh over a dishpan of aerated water. These were germinated in a growth chamber in the dark at 71 F for a period of 4 to 7 days depending on the species. At the end of this period, except in the case of alfalfa, the roots protruding below the aluminum mesh were excised with a razor blade and used in subsequent incubations. The alfalfa roots did not grow through the cheesecloth and, therefore, the top layer of cheesecloth was removed and the seed cut off with scissors, after which the roots were used in the incubation studies. The species used were as follows: Coker 67 field corn ( Zea mays L.), Hadden wheat ( Triticum aestivum L.). Moregrain oats ( Avena sativa L.), Manchuria X Rabat barley ( Hordeum vulgare L.) and Hairy Peruvian alfalfa ( Medicago sativa L. ) . Erlenmeyer flasks (300 ml.) were used as incubation vessels. Tetracycline and streptomycin at concentrations of 2 and 30 ppm, respectively, were used to prevent buildup of microbial populations. Sets were run using 0.4 M tris [2-amino-2-(hydroxymethyl)-l,3propanediol] buffer (pH 7.4) and 0.4 M phosphate buffer (pH 5.2) at _2 a final concentration of 2 X 10 M. The treatment flasks were made 1 X 10 "* M in the potassium salt of 2,4D. All flasks were brought 17

PAGE 23

18 to a final volume of 100 ml. Roots (10 g. fresh weight) were then added to the flasks. The flasks were covered with a tissue and put on a shaker at approximately 60 cycles per minute. A total of 12 flasks were used for each species. Six blank flasks were used as well as six containing 2,4-D. The incubations using tris buffer were incubated for 10 hours. At the end of this time, the roots were removed from the flasks and frozen in cold methanol. The roots were then ground in an omnimixer, keeping them frozen by immersing the vessel in a bath at -50 C or colder. After grinding, the solution was centrifuged at 17,500 rpm (37,000 X g) for 30 minutes. The supernatant fraction was concentrated to approximately 10 ml. by freeze drying. Each blank and treatment was split in half. To one portion of each, 40 per cent sodium hydroxide was added so that the final solution was 10 per cent in sodium hydroxide. This solution was refluxed for 20 minutes in order to hydrolyze any esters present. The hydrolyzed sample was brought to pH 3 with sulfuric acid and freeze dried. Methanol was added to dissolve any hydroxylated derivatives that might be present. The hydrolyzed and unhydrolyzed samples were then reduced under a stream of nitrogen to a volume of approximately 1 ml. and applied to 8 X 8 inch plates spread to a thickness of 1 mm. with silica gel G (E. Merck Ag., Darmstadt) for thin layer chromatography. These plates were run in chambers with an ether :ligro in (b.p. 66-75 C):formic acid (50:50:2) solvent system. After drying, the silica gel G between the origin and the solvent front was scraped from the plate. This carrier was then eluted with acetone.

PAGE 24

19 The acetone extracts were reduced under a stream of nitrogen to approximately 10 ml. and split again. One portion was set aside. The others were reduced under a stream of nitrogen to approximately •5 ml. after which the volume was adjusted to 1 ml. with methanol. These samples were treated with an ethereal solution of diazo-npropane. More of the diazo-n-propane solution was added as necessary to keep the color of the solutions yellow. After 30 minutes, two drops of a solution of boron trifluoride (0.7 per cent) in methanol were added to further catalyze the reaction and the solutions were kept yellow for an additional 30 minutes. At the end of this time, the samples were again concentrated under nitrogen and adjusted to a volume of 1 ml. These solutions were used for analysis by gas chromatography. The incubation mixtures containing phosphate buffer were treated similarly except that the extraction of the roots was carried out by dropping the roots into boiling methanol and boiling an additional 20 minutes. Both halves of the split samples were also used in this case as two diazo compounds were used. One portion was treated with diazome thane while the other was treated with diazo-n-propane as in the previous case. Otherwise, both sets were handled as described previously. Solutions of 2,^-, 6-hydroxy-2,4-, 5-hydroxy-2,4-, 4-hydroxy2,3and *J—hydroxy-2,5-dichlorophenoxyacetic acids in methanol were prepared and treated with diazomethane and diazo-n-propane as described previously. The final concentration of these solutions was 2.5 mg./ml. These solutions were used as standards for gas chromatography.

PAGE 25

20 Mixtures of some synthesized compounds (10 mg.) and lanolin (k g.) were prepared for tomato epinasty tests. The following compounds were tested: 5-nitroand 5-amino-4-chloro-2-methylphenoxyacetic acids; ethyl 3-amino-2,4-dichlorophenoxyacetate; i|--hydroxy2,3-, Jj-hydroxy-2,5-, 6-hydroxy-2,4and 5-hydroxy-2,4-dichlorophenoxyacetic acids. 2,k-D was also included as a standard. Approximately 300 mg. of the lanolin mixtures of these compounds were spotted on the petiole of the youngest fully developed leaf of two month old tomato plants in the greenhouse. Daily observations were made to observe the effects of the compounds on the plants. All melting points were run on a Kofler micro hot stage apparatus and the temperatures corrected. The compounds were synthesized as follows : Diazomethane. Potassium hydroxide (^0 ml. of 30 per cent solution) and anhydrous ethyl ether (160 ml.) were placed in a large test tube and immersed in an insulated container holding an antifreeze solution at -10°C or lower. The contents of the tube were allowed to cool for 20 minutes. N-methyl-N-nitro-N-nitrosoguanidine (8 g.) was added to the tube. The bath temperature was allowed to rise only to 0°C. The reaction is complete when the ether assumes a yellow color and the solid matter disappears and usually is complete in about 30 minutes. The ethereal solution was poured off into a polyethylene bottle containing potassium hydroxide pellets. The solution should be stored in a freezer and used within 2k hours for best results.

PAGE 26

21 Diazo-n-propane. Potassium hydroxide (20 ml. of 60 per cent solution) and anhydrous ethyl ether (160 ml.) were placed in a large test tube and cooled as described above. N-propyl-N-nitrosourea (8 g.), prepared previously by M. Wilcox, was added to the tube. The reaction is complete when the ether assumes a deep yellow color and the solid matter suspended in the potassium hydroxide loses its yellow color, becoming white. The ethereal solution was poured off as in the case of diazomethane. This solution should be stored in a freezer and used within 2k hours for best results. Monoperphthalic acid. An ethereal solution of monoperphthalic acid was prepared as described by Payne (1962). The solution was standardized by adding 30 ml. of 20 per cent potassium iodide solution to 2 ml. of the ethereal solution and, after 10 minutes, titrating the liberated iodine with 0.06268 N sodium thiosulfate. This solution was used in subsequent attempted preparations. Isopropyl kchloro-2-methylphenoxyacetate. To 95 V ev cent 4-chloro-2-methylphenoxyacetic acid (212 g.) was added 2-propanol (26^ g.). Boron fluoride ethyl ether (178 g.) was added dropwise and the mixture refluxed for 4 hours. The excess alcohol was distilled off and the solution neutralized with a 10 per cent solution of sodium carbonate. The solution was then extracted with three portions of ethyl ether and the ether removed under vacuum. The remaining liquid was vacuum distilled (15 mm. Hg) and the portions distilling over between 170 and 17^ C collected and combined. The weight of these portions was 193*6 g. (79*8 per cent yield).

PAGE 27

22 5-Nitro-4-chloro-2-methylphenoxyacetic acid. This compound was prepared as described by Eckstein et al. (196*0 « The compound melted at ±'j±-152°C. Faulkner and Woodcock (1965) reported 152153°C 5-Amino-4-chloro-2-methylphenoxyacetic acid. This compound was also prepared as described by Eckstein e_t al. (1964). The compound melted at 194-197°C Eckstein et al. (1964) reported 197-198°C. The ethyl ester, prepared by the previously described boron trifluoride method, melted at 76-78°C (Found: C, 54.51; H, 5.96; N, 5.95; CI, 14.38. C n H 12 N0 requires C, 54.22; H, 5*79; N, 5-75; CI, 14.55 per cent). Faulkner and Woodcock (1965) reported 7980°C. 6-Nitro-4-chloro-2-methylphenol. This compound was prepared as described by Zincke (1918). He reported the melting point to be 107°C. Our preparation melted at 106°C (Found: C, 44.96; H, 3*^7; CI, 18.32; N, 7.42. CJ^CINO requires C, 44.82; H, 3.22; CI, 18. 90; N, 7.47 per cent). Ethyl 6-nitro-4-chloro-2-methylphenoxyacetate. 6-Nitro-4chloro-2-methylphenol (30 g.) was added to a solution of sodium (3. 7 g.) in 300 nil of absolute ethanol and heated to reflux. Ethyl bromoacetate (20 g.) was added dropwise through the condenser and the mixture refluxed for 3*5 hours. Most of the ethanol was removed under vacuum. The mixture was then diluted with water and the precipitate filtered off. The yield was 32 g. (75 per cent yield). The compound melted at 70 C. Faulkner and Woodcock (1965) also reported a melting point of 70 C.

PAGE 28

23 6-Nitro-4-chloro-2-methylphenoxyacetamide. Ethyl 6-nitro-4chloro-2-methylphenoxyacetate (5 g.) was added to 20 ml. of absolute ethanol and refluxed with 5 ml. of concentrated ammonium hydroxide. The mixture was allowed to reflux for 8 hours and 5 ml. of concentrated ammonium hydroxide was added at 2 hour intervals. The yield of product was 1.2 g. (27 per cent yield). The compound melted at 153-156°C. 5-Chloro-3-methylcatechol. Attempts were made to prepare this compound through two intermediates described by Zincke (1918). The structures given for the intermediates were dubious. In one trial, however, a small amount of the desired product was obtained which agreed with the melting point of 89°C (Found: C, 53.31; H, 4.5^; CI, 22.05. C 7 H 7 C10 2 squires C, 53.02; H, k.k5\ CI, 22.36 per cent) reported by Zincke. The last step in the synthesis was very low yielding, the product unstable and results inconsistent. Since the preparation of 2-methyl-l,4-muconolactone from this compound failed, further attempts to prepare it were terminated. hChloro-2-methylmuconic acid. 4Chloro-2-methylphenol (4.0 g.) was added to 175 ml. of ether containing monoperphthalic acid (17 g«) and allowed to stand at room temperature for four days. None of the desired product was obtained when the preparation was treated as described by Testa (1952). 2-Methyl-l,4-muconolactone. 3-Methyl-5-chlorocatechol (1.08 g.) was dissolved in 50 ml. of ether and 88 ml. of ether containing monoperphthalic acid (4.3^ g.) added. This mixture was allowed to

PAGE 29

24 stand at room temperature for five days. None of the desired product was obtained when the preparation was treated as described by Testa (1952). 6-Hydroxy-2,4-dichlorophenoxyacetic acid (6-0H-2,4-D). This compound was prepared as described by Cavill and Ford (195*0 using 3,5-dichlorocatechol previously prepared in this laboratory. The compound after two recrystallizations from water melted at 133 C. Cavill and Ford reported the melting point to be 132 C. 2-Chloro-l,4-muconolactone. 2-Chlorophenol (6.0 g.) was added to 164 ml. of ether containing monoperphthalic acid (9*1 g«). On the following day, 90 ml. of ether containing monoperphthalic acid (5»0 g.) was added and the solution allowed to stand for four days at room temperature. This preparation, when worked up, failed to yield the desired product. 5-Hydroxy-2,4-dichlorophenoxyacetic acid hydrate (5-0H-2,4D). This compound was prepared as described by Moszew and Wojciechowski (1945). The product after two recrystallizations from water melted at 166°C (Found: C, 37.62; H, 3. 03; CI, 27.42. CghVCl O'h^O requires C, 37.67; H, 3*16; CI, 27.80 per cent). Moszew and Wojciechowski (1945) reported the melting point to be 175 «5 C. 3-Nitro-2,4-dichlorophenol. This compound was prepared as described by Groves e_t al. (1929). After two recrystallizations from 2,2,4-trimethylpentane, the compould melted at 72 C. Groves et al . found their preparation to melt at 70-72 C. Ethyl 3-nitro-2,4-dichlorophenoxyacetate. This compound was prepared as described by Faulkner and Woodcock (1965). It was

PAGE 30

25 found to melt from 80-82°C. Faulkner and Woodcock reported the melting point to be 85°C. Ethyl 3-amino-2,4-dichlorophenoxyacetate. Ethyl 3-nitro-2,4dichlorophenoxyacetate (36 g.), 10 per cent palladium on charcoal (2 g.) and 150 ml. of tetrahydrofuran were added to a bomb with a total volume of ^30 ml. The bomb was charged with nitrogen and released several times to remove air. The bomb was then charged with hydrogen to a maximum pressure of 150 p.s.i. several times and the pressure drops recorded. The final total pressure drop was ^78 p.s.i. The theoretical pressure drop required was ^73 p.s.i. The bomb was fastened on a shaker and shaken during the course of the reaction. When the temperature of the bomb rose noticeably, it was cooled to room temperature in an ice bath. At the end of the reaction, the material was poured out of the bomb and the charcoal filtered off. The tetrahydrofuran and the water produced were taken off under vacuum. The residue was heated with 2,2,4-trimethylpentane and the solvent cooled. After several extractions, a yield of 20.6 g. (6^ per cent yield) was obtained. After recrystallization from ligroine (b.p. 66-75°C), the product melted from 56-58°C. Trials using Faulkner and Woodcock's (1965) procedure for this compound were unsuccessful. They reported that their preparation melted from 57-58°C 3-Hydroxy-2,4-dichlorophenoxyacetic acid. Attempts to diazotize ethyl 3-amino-2,4-dichlorophenoxyacetate and obtain 3-hydroxy-2,A— dichlorophenoxyacetic acid have been unsuccessful in this laboratory, although Faulkner and Woodcock (1965) have prepared this compound.

PAGE 31

26 2,3-Dichlorohydroquinone. This compound was prepared using the procedure described by Conant and Fieser (1923). The product melted at 1^3°C which agreed with the author's work. Attempts to prepare this compound by the rearrangement of 2,5-dichlorobenzoquinone and subsequent saponification as described by Dimroth et al. (1926) failed to yield a usable product. Ethyl ^-hydroxy-2,3-dichlorophenoxyacetate. 2,3-Dichlorohydroquinone (10 g.) was dissolved in 100 ml. of absolute ethanol containing sodium (1.28 g.) and refluxed for 2 hours during the dropwise addition of ethyl bromoacetate (9. 3 g«)« The excess ethanol was then removed under vacuum and the solution diluted with water. The product, which precipitated out of the solution, was filtered off. The yield was 12.7 g. (86.5 per cent yield) and after recrystallization from ligroine (b.p. 60-90°C) melted at 79°C 4-Hydroxy-2,3-dichlorophenoxyacetic acid (4-0H-2,3-D)« Attempts to hydrolyze the above ester with acid or base have failed to yield a significant amount of product with sufficient purity for use. Condensation of 2,3-dichlorohydroquinone with bromoacetic acid has also failed to give the desired product. A small sample, however, was supplied by Thomas et al. (1963) and melted at l67-l68°C. Ethyl ^-hydroxy-2,5-dichlorophenoxyacetate. This compound was prepared in the same manner as the corresponding 2,3-dichloroanalog starting with 2,5-dichlorohydroquinone. The product melted at 95 C. kHydroxy-2,5-dichlorophenoxyacetic acid (40H-2,5-D). Ethyl 4-hydroxy-2,5-dichlorophenoxyacetate (^ g.) was added to 50 ml. of 10 per cent aqueous sodium hydroxide and refluxed for 2 hours. The

PAGE 32

27 solution was then acidified and cooled. White crystals precipitated from the solution. After two recrystallizations from water, 0.6 g. (16.6 per cent yield) of product melting at I66-I67 C was obtained. The infrared spectrum of this material was identical with that of a sample supplied by Thomas et al. (1963). Their preparation melted at 161-162 C. Attempts to prepare this compound by condensation of 2,5-dichlorohydroquinone with bromoacetic acid failed, as mentioned previously in the case of the 2,3-dichloroanalog.

PAGE 33

EXPERIMENTAL RESULTS Portions of the treated solutions were injected into the gas chromatograph as described in table 1. Ten u.1. injections were used for the root extracts and 1 ul. for the standards. The retention times of the standards are given in table 2. 2,^-D was detectable when as much as 0.1 u.g. was present in the sample injected into the gas chromatograph. A sample containing 0.25 l^g* of the hydroxy-derivatives was necessary for reasonable detection on the equipment used in this study. Since each alkylated extract represented 66 mg. of 2,^-D substrate and recovery was estimated at 75 per cent or better, the detection threshold of 0.25 M.g. hydroxydichlorophenoxyacetic acid represented excellent sensitivity. In the samples treated with diazome thane, a peak corresponding to 2,4-D was present in all of the 2,4-D treated samples. This peak was masked by the solvent peak due to the higher sensitivity and large sample injection used when diazo-n-propane was the reactant. All other peaks in the 2,4-D treated samples did not correspond to any of the standards and were also present in the control extracts. To determine the disappearance of 2,4-D from the incubation media, three 1 ml. samples were taken from one of the flasks at 3 hour intervals during the incubation. These solutions were concentrated under nitrogen almost to dryness and adjusted to 1 ml. with methanol. They were then treated with an ethereal solution of 28

PAGE 34

29 TABLE 1 Operating Conditions for Gas Chromatography Manufacturer Model F and M ij-00 Detector Column diameter length Carrier flow Hydrogen flame ionization 15 per cent SE-30 on Chromosorb W (60/80 mesh) 3/16 inch 6 feet Helium 60 ml./min. Temperatures flash heater column detector 235°C 190°C 240°C Electrometer settings knowns diazomethane treatment diazo-n-propane treatment Range 10 10 10 Attenuation 8 1

PAGE 35

30 TABLE 2 Retention Times of Standard Solutions in Methanol Parent compound Methyl ester methyl ether Methyl ester propyl ether Propyl ester propyl ether man. 2,4-D

PAGE 36

31 diazo-n-propane, reduced under a stream of nitrogen and readjusted to 1 ml. with methanol. These solutions were run on the gas chromatograph. The size of the peaks representing 2,4— D were not reproducible between replicates but there was a continuous decrease in the average peak size as the length of the incubation time increased. There was no peak representing 2,4— D after 12 hours of incubation. This showed that 2,4-D was being removed from the solution. Samples (2.5 mg.) of the synthesized hydroxylated derivatives were added to a blank extract and carried through the purification procedure. It is estimated from the relative size of the resulting peaks on the gas chromatograph that more than 75 per cent of each known was recovered in the final solutions. It was observed that the retention times of the knowns added to the blank did not agree with the standards when treated with diazo-n_-propane. Since the methanolic extracts had been agitated for several hours, it was suspected that the acids had been converted to methyl esters. In order to resolve this discrepancy, methanolic solutions of the standards in tightly capped tubes were heated in a water bath at 75 C for 2 hours. The object of this procedure was to determine whether the phenolic acids could be esterified under very mild conditions. At the end of this time, they were treated with diazon-propane and analyzed on the gas chromatograph. Partial methyl esterification was indicated by the appearance of a second peak other than the propyl ether ester from each of the standards, as shown in table 3« The retention times of the second peaks agreed

PAGE 37

32 1 TABLE 3 Relative Peak Heights of Methyl and Propyl Esters of the Propyl Ethers Parent compound Methyl ester Propyl ester 6-0H-2,4-D 11.0 1^.5 5-0H-2,4-D 6.6 22.1 M)H-2,3-D 3.3 23*0 4-0H-2,5-D 2.8 19.6 1 Anhydrous methanolic solutions of the samples, in closed tubes, were heated in a water bath at 75°C for 2 hours and then alkylated with the appropriate ethereal diazoalkane.

PAGE 38

33 with those subjected to the purification procedure and the diazon-propane treatment. The diazomethane treatment gave retention times which agreed with the standards. 4-Hydroxy-2,3-, ^--hydroxy-2,5-, 6-hydroxy-2,4and 5-hydroxy2,4-dichlorophenoxyacetic acids produced no epinasty on the test plants. 5-Nitroand 5-amino-^-chloro-2-methylphenoxyacetates showed positive epinasty on the test plants on the day following treatment. Ethyl 6-nitro-4-chloro-2-methylphenoxyacetate produced no epinasty during twelve days following treatment. Ethyl 3-amino2,kdichlorophenoxyacetic acid showed positive epinasty within 2 hours of treatment, as did 2,4-D.

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DISCUSSION The greatest difficulty in this work was the lack of reference compounds for use as standards. The next problem was the determination of an analytical procedure for detection of minute amounts of these substances from biological materials. Preparations for some of the compounds involved in this study were published by others during the course of this work. Using the procedure of Faulkner and Woodcock (1965), the yield of 3-amino-2,4-dichlorophenoxyacetic acid was very low. In an attempt to obtain a better yield, we used hydrogen in a bomb with palladium on charcoal as a catalyst and obtained a yield of 64 per cent. Faulkner and Woodcock reported a yield of 56 per cent but in this laboratory this yield was never approached using their procedure. Difficulty was also experienced in the procedures of diazotization of aromatic amines to phenols reported by the above workers. In attempts to follow their procedures of diazotizing ethyl 3amino-2,4-dichlorophenoxyacetate and ethyl 5-amino-4chloro-2methylphenoxyacetate to the corresponding hydroxy-acids , none of the desired products were obtained. 6-Hydroxy-4-chloro-2-methylphenoxyacetic acid has never been synthesized but Gaunt and Evans (I96I), using a soil bacterium, isolated a hydroxy-4-chloro-2-methylphenoxyacetic acid believed to be 34

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35 the 6-hydroxy-analog. They also isolated a gammalactone which presumably would be produced via the former compound. For this reason, an unambiguous synthesis of this compound is of considerable interest. We prepared 6-nitro-4-chloro-2-methylphenoxyacetamide as it is possible that amide substitution would prevent the expected cyclization to a lactam during the reduction of the corresponding nitro compound to the amino analog. This amide may prove to be a useful intermediate in the unambiguous synthesis of 6-hydroxy-4chloro-2-methylphenoxyacetic acid . Difficulty was also encountered in the last step of the synthesis of 5-chloro-3-methylcatechol as described by Zincke (1918). Due to the dubious structures reported for the intermediates and the low-yielding last step, the preparation of this compound was abandoned. This compound would probably be an intermediate between 6-hydroxy-4-chloro-2-methylphenoxyacetic acid and the previously mentioned gammalac tone . Because of these difficulties any future attempts to prepare 5-chloro-3-methyl-catechol should await an unambiguous synthesis of 6-hydroxy-^-chloro-2-methylphenoxyacetic acid such as by the above suggested method. Cleavage of 6-hydroxy4-chloro-2-methylphenoxyacetic acid prepared in this manner with hydriodic acid or a similar reagent would be expected to give in turn 5-chloro-3-methylcatechol unambiguously. Under the conditions of this work, 2,4-D was not metabolized to 4-hydroxy-2,3-, 4hydroxy-2,5-, 6-hydroxy-2,4or 5-hydroxy-2,4dichlorophenoxyacetic acid in detectable amounts. Conversion at any one time of 0.4 per cent of the original 2,4-D in the incubation

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36 mixtures to any of the possible hydroxylated intermediates would have been readily detectable. If any of these derivatives were formed, they must have been further metabolized as rapidly as they were produced. It is possible, however, that under some other conditions these derivatives might be detected. Preparation of more volatile derivatives is necessary for analysis of phenolic acids by gas chromatography. The use of solutions of diazoalkanes in ether to alkylate the carboxyl and hydroxyl groups of the possible metabolites proceeded smoothly during this work. This method has the advantage that one reactant will quickly convert both carboxylic and phenolic hydroxyls to more volatile groups at room temperature. After the initial reaction with the diazoalkane had subsided, a small amount of 'boron trifluoride was added to further catalyze the reaction and destroy the excess diazoalkane as the methyl ether. By treating methanolic solutions of standards of the above monohydroxydichlorophenoxyacetic acids with diazomethane and diazo-npropane, it is possible to prepare derivatives which allow separation by means of gas chromatography. Use of only one of the reagents will not produce separable derivatives of all four of the hydroxy-acids under the conditions of this work. Treatment with diazomethane did not separate derivatives of 5-hydroxy-2-^— and 4-hydroxy-2,5-dichlorophenoxyacetic acids. Diazo-n_-propane treatment did not separate derivatives of 6-hydroxy-2,4and 4-hydroxy-2,3-dichlorophenoxyacetic acids .

PAGE 42

37 Attempted preparations of gamma -lactones mentioned as metabolites of 2,4-D and MCPA by Fernley and Evans (1959) and Gaunt and Evans (196l), respectively, were unsuccessful. Synthesis of these lactones was attempted by oxidation of the corresponding catechol or phenols with monoperphthalic acid as described by Testa (1952). The only compound which could be extracted and identified from these solutions was phthalic acid. Hydrolysis of ethyl 2,3-dichlorophenoxyacetate to the corresponding acid also failed to yield a usable product. Hydrolysis by both acid and base was attempted but the desired hydroxy-acid was not produced. A high melting compound was extracted which may have been a polymer. Rearrangement of 2,5-dichlorobenzoquinone to the 2,3-dichloro-analog desired as a possible precursor in the synthesis of the previously mentioned compound by the method of Dimroth et al. (1926) also proved unsuccessful. We suspect Dimroth et al. separated the isomers in a mixture rather than actually causing a rearrangement of the molecule. No detectable amounts of substrate (l X 10"^ M 2,4-D) were present after 12 hours incubation of excised roots of the plant species studied. The root extracts, however, did give a peak representing 2,4-D which showed that the roots had taken up the herbicide. This indicated that the incubation had not been long enough for all of the 2,4-D to have been converted to some product further removed metabolically from the herbicide than those for which analysis was being made.

PAGE 43

38 One method which might make identification easier would be the use of methanol as the extraction solvent with addition of boron trifluoride, and subsequent heating to cause esterification of the carboxylic groups. The resulting esters could be treated with various diazoalkanes which would etherify the phenolic groups. This method would enhance dissimilarities in the molecules and might possibly give better separation in gas chromatography. Otherwise, it would seem advisable to use some extraction solvent other than methanol which would not react with the acids being analyzed. This would eliminate some of the identification problems encountered in this work. Ethyl acetate would be a good solvent to consider. Another modification in the procedure which might improve the technique would be the use of an electron capture detector on the gas chromatograph. This would probably eliminate most of the peaks noted except those of the compounds containing chlorine. Elimination of peaks for most of the natural products would increase the worker's ability to identify the compounds being analyzed. Another advantage would be the higher sensitivity to be expected with an electron capture detector. In the tomato epinasty tests, it is possible that the 5-nitroand 5-amino-4-chloro-2-methylphenoxyacetic acids were contaminated with MCPA since they were prepared from the parent herbicide. However, the purification procedures should reduce this possibility to a minimum. This is particularly true for the amino compound, as its preparation from MCPA requires two steps with associated

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39 purifications. There should have been no 2,4-D contamination in the ethyl 3-amino-2,4-dichlorophenoxyacetate as it was prepared from m-nitrophenol and had no common precursor with 2,4-D. Epinasty was not observed for 6-hydroxy-2,4-, 5-hydroxy-2,4-, 4-hydroxy-2,3and 4-hydroxy-2,5-dichlorophenoxyacetic acids . 6-Hydroxy-2, i J— dichlorophenoxyacetic acid previously had been shown to be inactive by Cavill and Ford (1954). This might be expected as hydroxylation is known to be a prime method of detoxication by biological systems.

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SUMMARY The object of this work -was to determine whether hydroxylated metabolites or farther derived compounds were formed during the degradation of 2,4-dichlorophenoxyacetic acid by excised roots of several species of higher plants. Excised roots were incubated in a solution of 2,4-D for periods of 10 and 12 hours at two different hydrogen ion concentrations. The roots were then extracted with methanol. After prepurification by thin layer chromatography, the solutions were treated with diazomethane and diazo-n-propane to alkylate phenolic acids and analyzed by gas chromatography. Synthetic 6-hydroxy-2,4-, 5-hydroxy-2,4-, A-hydroxy-2,3and 4-hydroxy2,5-dichlorophenoxyacetic acids were alkylated similarly and used as standards in the analysis. The extracts from the roots contained no detectable amounts of the possible metabolites for which the analysis was being made. All of the extracts from 2,4-D treatments did show a peak with a retention time identical with 2,4-D. Tomato epinasty tests showed that none of the previously mentioned hydroxylated analogs tested were active. Ethyl 6-nitro-4chloro-2-methylphenoxyacetate also showed no activity in the test. Ethyl 3-amino-2,4-dichlorophenoxyacetate and 2,4-D were very active and began showing epinasty effects within 2 hours of treatment. 5Nitroand 5-&mino-4-chloro-2-methylphenoxyacetic acids also gave 40

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41 positive results in the test. The former compound was almost as active as 2,4-D. The latter was less active and the effects didn't appear until the second day after treatment.

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LITERATURE CITED Audus, L. J. 1950. Biological detoxication of 2,4-dichlorophenoxyacetic acid in soils. Isolation of an effective organism. Nature 166:356. Audus, L. J. 1952. The decomposition of 2,4-dichlorophenoxyacetic acid and 2-methyl-4chlorophenoxyacetic acid in the soil. J. Sci. Food Agr. 3:268. Bach, M. K. 196l. Metabolites of 2,4-dichlorophenoxyacetic acid from bean stems. Plant Physiol. 36:558. Bell, G. R. i960. Studies on a soil Achromobacter which degrades 2,4-dichlorophenoxyacetic acid. Can. J. Microbiol. 6:325. Bocks, S. M., J. R. L. Smith, and R. 0. C. Norman. 1964. Hydroxylation of phenoxyacetic acid and anisole by Aspergillus niger (van Tiegh). Nature 201:398. Burger, K., I. C. MacRae, and M. Alexander. I962. Decomposition of phenoxyalkylcarboxylic acids. Soil Sci. Soc. Am. Proc. 26:243. Byrde, R. J. •. , J. F. Harris, and D. Woodcock. 1956. Fungal detoxication. The metabolism of (ju-(2-naphthyloxy)-n-alkylcarboxylic acids by Aspergillus niger . Biochem. J. 64:154. Byrde, R. J. W. , and D. Woodcock. 1957* Fungal detoxication. 2. The metabolism of some phenoxy-n_-alkylcarboxylic acids by Aspergillus niger . Biochem. J. 65:682. Byrde, R. J. W. , and D. Woodcock. 1958. Fungal detoxication. J>. The metabolism of cju-(2-naphthyloxy)-n-alkylcarboxylic acids by Sclerotinia laxa . Biochem. J. 69:19* Canny, M. J., and K. Markus . i960. The breakdown of 2,4-dichlorophenoxyacetic acid in shoots and roots. Australian J. Biol. Sci. 4:486. Cavill, G. W. K., and D. L. Ford. 1954. The chemistry of plantgrowth regulators. Part I. 2:4-Dichloro-6-hydroxyphenoxyacetic acid and related compounds. J. Chem. Soc.:565« Clifford, D. R., and D. Woodcock. 1964. Metabolism of phenoxyacetic acid by Aspergillus niger van Tiegh. Nature 203: 763. 42

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43 Conant, J. B., and L. F. Fieser. 1923* Reduction potentials of quinone. I. The effect of the solvent on the potentials of certain benzoquinones . J. Am. Chem. Soc. 45:2194. Dagley, S., •. C. Evans, and D. W. Ribbons, i960. New pathways in the oxidative metabolism of aromatic compounds by microorganisms. Nature 188:560. Dickens, F., and H. E. H. Jones. 196l. Carcinogenic activity of a series of reactive lactones and related substances. Brit. J. Cancer 15:85. Dickens, F., and H. E. H. Jones. 19&3Further studies on the carcinogenic and growthinhibitory activity of lactones and related substances. Brit. J. Cancer 17:100. Dimroth, 0., H. Eber, and K. Wehr. 1926. III. Uber das benzochinondichlorid. Ann. 446:132. Eckstein, Z., E. Dyszer, and T. Niedzwialowska. 1964. nitrowaniu estrow kwasow 2-metylooraz 2-etylo-4-chlorofenoksy-octowego. I. Poszukiwanie metody syntezy analogow alkilowych 2,4,5-T. Roczniki Chemii 38:51. Evans, W. C, and B. S. •. Smith. 1951* The oxidation of aromatic compounds by soil bacteria. Biochem. J. 49 :x. Evans, •. C, B. S. W. Smith, R. P. Linstead, and J. A. Elvidge. 1951* Chemistry of the oxidative metabolism of certain aromatic compounds by micro-organisms. Nature 168:772. Evans, •. C, and B. S. W. Smith. 1954. The photochemical inactivation and microbial metabolism of the chlorophenoxyacetic acid herbicides. Biochem. J. 57 :xxx. Evans, •. C. 1956. Biochemistry of the oxidative metabolism of aromatic compounds by micro-organisms. Ann. Rept. Progr. Chem. (Chem. Soc. London) 53:279. Evans, •. C, and P. Moss. 1957. The metabolism of the herbicide, p_-chlorophenoxyacetic acid by a soil micro-organism — the formation of a P-chloromuconic acid on ring fission. Biochem. J. 65:8P. Faulkner, J. K., and D. •oodcock. 196la. Metabolism of chlorophenoxyacetic acids by Aspergillus niger . Chem. Ind. (London): 1366. Faulkner, J. K., and D. •oodcock. 19olb. Fungal detoxication. Part V. Metabolism of 0and p_-chlorophenoxyacetic acids by Aspergillus niger. J. Chem. Soc.:5397«

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44 Faulkner, J. K., and D. Woodcock. 1964. Metabolism of 2,4-dichlorophenoxyacetic acid ("2,4-D") by Aspergillus niger van Tiegh. Nature 203:865. Faulkner, J. K., and D. Woodcock. 1965. Fungal detoxication. Part VII. Metabolism of 2,4-dichlorophenoxyacetic and 4-chloro-2methylphenoxyacetic acids by Aspergillus niger . J. Chem. Soc: 1187. Fawcett, C. H., J. M. A. Ingram, and R. L. Wain. 1954. The Boxidation of phenoxyalkylcarboxylic acids in the flax plant in relation to their growth-regulating activity. Proc. Roy. Soc. (London), Ser. B 142:60. Fawcett, C. H., R. M. Pascal, M. B. Pybus, H. F. Taylor, R. L. Wain, and F. Wightman. 1959. Plant growth-regulating activity in homologous series of tu-phenoxyalkanecarboxylic acids and the influence of ring substitution on their breakdown by B-oxidation within plant tissues. Proc. Roy. Soc. (London), Ser. B 150:95. Fernley, H. N., and W. C. Evans. 1959. Metabolism of 2,4-dichlorophenoxyacetic acid by soil Pseudomonas : Isolation of a-chloromuconic acid as an intermediate. Biochem. J. 73:22P. Gaunt, J. K., and W. C. Evans. 1961. Metabolism of 4-chloro-2methylphenoxyacetic acid by a soil micro-organism. Biochem. J. 79:25P. Groves, L. G., E. E. Turner, and G. I. Sharp. 1929. LXXVT. The scission of diaryl ethers and related compounds by means of piperidine. Part II. The nitration of 2:4:4'-trichlorodiphenyl ether, and of 2:4-dichlorophenyl p_-toluenesulphonate and benzoate. J. Chem. Soc.:512. Holley, R. W. 1952. Studies on the fate of radioactive 2,4-dichlorophenoxyacetic acid in bean plants. II. A water soluble transformation product of 2,4-D. Arch. Biochem. Biophys. 35:171. Leafe, E. L. I962. Metabolism and selectivity of plant-growth regulator herbicides. Nature 193:485. Moszew, J., and J. Wo jcie chows ki. 1945* Studia nad synteza regulatorow wzrostu roslin, pochodnych fenoli duruvodorotlenowych. Roczniki Chemii 28:445. Newman, A. S., and J. R. Thomas. 1949. Decomposition of 2,4dichlorophenoxyacetic acid in soil and liquid media. Soil Sci. Soc. Am. Proc. 14:160. Payne, G. B. I962. Monoperphthalic acid. Org. Syn. 42:77.

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45 Ribbons, D. •. , and W. C. Evans. 19 62. Oxidative metabolism of protocatechuic acid by certain soil Pseadomonads : A new ringfission mechanism. Biochem. J. 83:482. Rogoff, M. H., and J. J. Reid. 1956. Bacterial decomposition of 2,4-dichlorophenoxyacetic acid. J. Bacteriol. 71:303* Rogoff, M. H. 1961. Oxidation of aromatic compounds by bacteria. Advan. Appl. Microbiol. 3:193. Steenson, T. I., and N. Walker. 1956. Observations on the bacterial oxidation of chlorophenoxyacetic acids. Plant Soil 8:17. Steenson, T. I., and N. Walker. 1957* The pathway of breakdown of 2,4dichloroand 4-chloro-2-methylphenoxyacetic acid by bacteria. J. Gen. Microbiol. 16:146. Testa, E. 1952. Oxydationen durch Wasserstoffsuperoxyd und Persauren die zun spaltung von C-C bindungen fuhren. JurisVerlag, Zurich. 49 p. Thomas, E. W. , 3. C. Loughman, and R. G. Powell. 1963* Hydroxylation of phenoxyacetic acid by stem tissue of Avena sativa . Nature 199:73* Thomas, E. W. , B. C. Loughman, and R. G. Powell. 1964a. Metabolic fate of some chlorinated phenoxyacetic acids in the stem tissue of 1 vena sativa . Nature 204:286. Thomas, E. W., B. C. Loughman, and R. G. Powell. 1964b. Metabolic fate of 2,4-dichlorophenoxyacetic acid in the stem tissue of Phassolus vulgaris . Nature 204:884. Wain, R. L., and F. Wightman. 1954. The growth-regulating activity of certain uu-substituted alkylcarboxylic acids in relation to their B-oxidation within the plant. Proc. Roy. Soc. (London), Ser. B 142:525. Webley, D. M., R. B. Duff, and V. C. Farmer. 1955* Beta -oxidation of fatty acids by Nocardia opaca . J. Gen. Microbiol. 13:361. Webley, D. M., R. B. Duff, and V. C. Farmer. 1958. The influence of chemical structure on B-oxidation by soil Nocardias . J. Gen. Microbiol. 18:733. Wilcox, M., D. E. Moreland, and G. C. Klingman. 1963. Aryl hydroxylation of phenoxyaliphatic acids by excised roots. Physiol. Plantar um 16:565*

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h6 Williams, R. T. 1950* Biological oxidation of aromatic rings. Biochem. Soc. Symp. (Cambridge, Engl.) Ho. 5« 96 p. Williams, R. T. 1959* Detoxication mechanisms. The metabolism and detoxication of drugs, toxic substances and other organic compounds. Chapman and Hall, London795 P« Zincke, T. 1918. Uber die einwirkung von salpetersaure auf halogenderivate von o-alkylphenolen. Ann. kl7: 191 •

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BIOGRAPHY Norman Glaze was born in Washington, D. C, on January 16, 193^« He attended public schools in Hyattsville, Maryland, and was graduated from Northwestern High School in 1952. He obtained the Bachelor of Science degree from the University of Maryland in 1957* Upon completion, he was inducted into the U. S. Army and served for two years. After completion, he reentered the University of Maryland and received the Master of Science degree in 1963* He then transferred to the University of Florida for work on the Doctor of Philosophy degree. 47

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This dissertation was prepared under the direction of the chairman of the candidate's supervisory committee and has bet approved by all members of that committee. It was submitted to the Daan of the College of Agriculture and to the Graduate Council, and was approved as partial fulfillment of the requirements for the degree of Doctor of Philosophy. April, 1966 ^^/TTean, College of Agriculture Supervisory Committee : hair man / Cha Dean, Graduate School

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