METABOLITES OF 2,4-DICHLOROPHENOXYACETIC ACID FROM BEAN STEMS ' MICHAEL K. BACH 2 RESEARCH DEPARTMENT, UNION CARBIDE CHEMICALS COMTPANY, SOUTH CHARLESTON, WEST VIRGINIA 3 There is considerable contradiction in the litera- ture concerning the nature and number of radioactive metabolites which can be isolated from bean plants following exposture to C14-labeled 2,4-dichlorophen- oxyacetic acid (2,4-D). Thus the isolation of one principal product which was water-soluble and ether- insoluble has been reported (8, 9, 10), while on the other hand, Weintraub et al. (17) have suggested that there is an extensive metabolism of the side-chain of 2,4-D, yielding a multiplicity of products. The acid hydrolysis of the water-soluble material gives rise to a radioactive, ether-soluble product. which has been variously identified as 2,4-D (9) or as a sub- stance different from 2,4-D and possibly 6-hydroxy- 2,4-dichlorophenoxyacetic acid (6-OH-2.4-D) (8) suggesting that metabolism of the ring occurs. Wein- traul) (15) has presented evidence that, in micro- organisms, one of the chlorine atoms of the 2,4-D is lost. The report by Butts and Fang (6) of the pres- ence of 12 amino acids in the hydrolysis product of the ether-insoluble material suggests the formation of amino acid conjugates of the metabolites of 2,4-D as well. In connection with a study of the induction of callus growth in bean stem sections by 2.4-D (2, 3), several metabolites of this growth substance were found. The isolation and attempted clharacterization of these metabolites is presented in this paper. A preliminary report has been published elsewhere (4). ExPERIMTENTAL GROWTH, EXPOSURE, & EXTRACTION OF BEANS. The beans [Phaseolufs zulgaris (L.) var. 'Giant Stringless Pod Bean'] were purchased locally and grown in the greenhouse as described previously (3). Stems were harvested when approximately four weeks old, and all leaves, petioles, and developing flower stalks were removed. The stems were briefly washed, cut into 6 inch lengths, and vacuum infiltrated with a 2 % sucrose solution containing 10-4 M 2,4-D-1-C4 (2-22 mc/mmole). The excess infiltration solution was removed from the stems by draining and by evaporating with the aid of a blower. The surface of the dried stems was sterilized by immersion in a 1 Received Feb. 14, 1961. 2 Present address: Research Division, The Upjohn Co., Kalamazoo, Mich. 3 A Division of Union Carbide Corp. 10 % solution of Chlorox for 2 minutes, followved by two successive rinses in sterile distilled water. They were then incubated at room temperature for 3 days in the dark with their basal ends dipping into 100 ml modified V\hite's liquid medium (3) in sterile, wide- necked 500 ml Erlenmever flasks wvhich were plugged with cotton. At the end of the incubation period the stems were frozen in liquid nitrogen, dried by lyophilization, and stored at -20 C. After 200 g of dried stems (about four batches) had been accumulated they were passed through a granulating machine and the chips packed into a large soxhlet extractor. They were then moistened with ca. 50 ml water andl extracted for 24 hours with acetonitrile. At this time a second addition of water was made, and the extraction con- tinued for another 24 hours. This method of extrac- tion was used (in preference to alcohol) because it had been found to give almost quantitative extraction of radioactivity while minimizing the extraction of other cell constituents. ETHER-SOLUBLE FRACTION. The acetonitrile and most of the water were removed from the initial ex- tract by evaporation under re(luced pressure. The concentrate, after adjustment to pH 2 with HCl, was extracted five times with equal volumes of ether. A precipitate which formed in the water layer was re- moved by centrifugation, washed with acidified, ether- saturated water, and then discar(led. The ether layer was pooled, washed once with water, dried over an- hydrous Na2SO4, and concentrated under nitrogen. All the solutions were stored at -20 C. The ether extract was partitioned in a 100 tube automatic counter-current distribution (CCD) ap- paratus (E-C Apparatus Co., Swvarthmore, Pa.) be- tween 1 M sodium phosphate buffer, pH 5.1, and ether (8). The ether layers from CCD were collected and evaporated to dryness. Two milliliters of methanol were added to the dry samples and aliquots plated and counted. Thie solutions corresponding to a single peak of radioactivity were pooled and the radioactivity recovered from the correspondingly pooled aqueous layers by acidification and extraction with ether. The pooled CCD fractions were chromatographed on Whatman No. 1 filter paper in a descending man- ner using the system n-butanol-butanone-triethyla- mine-water (20: 20: 2: 10 v/v/v/v) (solvent A). Compounds were eluted from paper with 50 % aque- ous methanol. The following tests were carried out on these eluates. 558 Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.
Transcript
METABOLITES OF 2,4-DICHLOROPHENOXYACETIC ACID FROM BEAN STEMS 'MICHAEL K. BACH 2
RESEARCH DEPARTMENT, UNION CARBIDE CHEMICALS COMTPANY, SOUTH CHARLESTON, WEST VIRGINIA 3
There is considerable contradiction in the litera-ture concerning the nature and number of radioactivemetabolites which can be isolated from bean plantsfollowing exposture to C14-labeled 2,4-dichlorophen-oxyacetic acid (2,4-D). Thus the isolation of one
principal product which was water-soluble and ether-insoluble has been reported (8, 9, 10), while on theother hand, Weintraub et al. (17) have suggestedthat there is an extensive metabolism of the side-chainof 2,4-D, yielding a multiplicity of products. Theacid hydrolysis of the water-soluble material givesrise to a radioactive, ether-soluble product. which hasbeen variously identified as 2,4-D (9) or as a sub-stance different from 2,4-D and possibly 6-hydroxy-2,4-dichlorophenoxyacetic acid (6-OH-2.4-D) (8)suggesting that metabolism of the ring occurs. Wein-traul) (15) has presented evidence that, in micro-organisms, one of the chlorine atoms of the 2,4-D islost. The report by Butts and Fang (6) of the pres-
ence of 12 amino acids in the hydrolysis product ofthe ether-insoluble material suggests the formationof amino acid conjugates of the metabolites of 2,4-Das well.
In connection with a study of the induction ofcallus growth in bean stem sections by 2.4-D (2, 3),several metabolites of this growth substance were
found. The isolation and attempted clharacterizationof these metabolites is presented in this paper. Apreliminary report has been published elsewhere (4).
ExPERIMTENTAL
GROWTH, EXPOSURE, & EXTRACTION OF BEANS.The beans [Phaseolufs zulgaris (L.) var. 'GiantStringless Pod Bean'] were purchased locally andgrown in the greenhouse as described previously (3).Stems were harvested when approximately four weeksold, and all leaves, petioles, and developing flowerstalks were removed. The stems were briefly washed,cut into 6 inch lengths, and vacuum infiltrated witha 2 % sucrose solution containing 10-4 M 2,4-D-1-C4(2-22 mc/mmole). The excess infiltration solutionwas removed from the stems by draining and byevaporating with the aid of a blower. The surfaceof the dried stems was sterilized by immersion in a
1 Received Feb. 14, 1961.2 Present address: Research Division, The Upjohn
Co., Kalamazoo, Mich.3 A Division of Union Carbide Corp.
10 % solution of Chlorox for 2 minutes, followved bytwo successive rinses in sterile distilled water. Theywere then incubated at room temperature for 3 daysin the dark with their basal ends dipping into 100 mlmodified V\hite's liquid medium (3) in sterile, wide-necked 500 ml Erlenmever flasks wvhich were pluggedwith cotton.
At the end of the incubation period the stems werefrozen in liquid nitrogen, dried by lyophilization, andstored at -20 C. After 200 g of dried stems (aboutfour batches) had been accumulated they were passedthrough a granulating machine and the chips packedinto a large soxhlet extractor. They were thenmoistened with ca. 50 ml water andl extracted for24 hours with acetonitrile. At this time a secondaddition of water was made, and the extraction con-tinued for another 24 hours. This method of extrac-tion was used (in preference to alcohol) because ithad been found to give almost quantitative extractionof radioactivity while minimizing the extraction ofother cell constituents.
ETHER-SOLUBLE FRACTION. The acetonitrile andmost of the water were removed from the initial ex-tract by evaporation under re(luced pressure. Theconcentrate, after adjustment to pH 2 with HCl, wasextracted five times with equal volumes of ether. Aprecipitate which formed in the water layer was re-moved by centrifugation, washed with acidified, ether-saturated water, and then discar(led. The ether layerwas pooled, washed once with water, dried over an-hydrous Na2SO4, and concentrated under nitrogen.All the solutions were stored at -20 C.
The ether extract was partitioned in a 100 tubeautomatic counter-current distribution (CCD) ap-paratus (E-C Apparatus Co., Swvarthmore, Pa.) be-tween 1 M sodium phosphate buffer, pH 5.1, and ether(8). The ether layers from CCD were collected andevaporated to dryness. Two milliliters of methanolwere added to the dry samples and aliquots plated andcounted. Thie solutions corresponding to a singlepeak of radioactivity were pooled and the radioactivityrecovered from the correspondingly pooled aqueouslayers by acidification and extraction with ether.
The pooled CCD fractions were chromatographedon Whatman No. 1 filter paper in a descending man-ner using the system n-butanol-butanone-triethyla-mine-water (20: 20: 2: 10 v/v/v/v) (solvent A).Compounds were eluted from paper with 50 % aque-ous methanol. The following tests were carried outon these eluates.
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BACH-METABOLITES OF 2,4-D FROM BEAN STEMS
Hydrogenations were carried out in 15 ml War-burg flasks. The samples were placed in the side-arm and 10 mg PtO suspended in 2 ml methanol inthe main chamber. After the catalyst was saturatedwith H2, the reaction was allowed to proceed for 24hours at 25 C. The catalyst was removed by centri-fugation and washed once with methanol. Themethanol solutions and washings were concentratedfor further use.
For HI cleavage of ether linkages the solutionswere placed in 50 ml pear-shaped flasks and evapor-ated to dryness. A small amount of monoiodoaceticacid was added as carrier to aid in isolation, followedby 0.2 ml 57 % HI from a freshly opened bottle.The mixture was refluxed in an oil bath for 4 hours,cooled, and then reacted with an excess of saturatedCuSO4 solution in 1% HCl. Both the '2 which wasformed and the organic acids were extracted intoether. The organic acids were recovered by extract-ing the ethereal solution with 5 % NaHCOx solution.The radioactive reaction products were finally re-covered with sufficiently low 12 contamination byacidifying the NaHCO3 solution and repeating theether-NaHCO3 extraction cycle. The solutions wereconcentrated to a small volume maintaining alkalinitythroughout this step. The concentrates were chro-matographed on paper in n-butanol saturated with1.5 N NH4OH in a descending manner.
Reactions with monochloroacetic acid, benzoylchloride, KMnO4, Br2, and alkali were carried outas described by McElvain (13) using a tenth thequantities of reagents. Reaction times for the lastthree reactions were 30 minutes, 10 minutes, and 2hours, respectively. The unreacted benzoyl chloridewas decomposed with solid NaHCO, and a few dropsof water, and the remaining KMnO4 and Br, withsolid NaHSO3. The solutions were acidified withHCI and the organic acids extracted into ether asdescribed above.
ETHER-INSOLUBLE EXTRACT. The aqueous resi-due from the acetonitrile and ether extractions wasimmediately neutralized with dilute KOH and con-centrated to a small volume in vacuo. The syrupyconcentrate was divided into two equal volumes andeach was put on a 17 x 90 mm column of Dowex-1x8(formate, 200-400 mesh). Elution with a graduallyincreasing concentration of formic acid was accom-plished as described by Busch et al. (5). The mix-ing flask contained 450 ml water and the secondflask contained 8.0 M formic acid. Twenty milliliterfractions were collected. The radioactive peaks werepooled as rapidly as possible and concentrated to dry-ness under reduced pressure.
A solution of the major radioactive componentfrom the Dowex-1 column was dried onto 1.25 g ofwashed silicic acid in a vacuum desiccator. Thismaterial was applied to the top of a 12.5 g, 100 mmcolumn of water-saturated silicic acid. The radio-activity was eluted with a water-saturated mixture of35 % n-butanol and 65 % chloroform (v/v).
Paper chromatography was carried out using sol-vent A. In addition the material was run withn-butanol-propionic acid-water (12:5.6: 8 v/v/v) asdescribed by Jaworski et al. (10) (solvent B) to con-firm the identity of the preparation with the previous-ly reported material from unhydrolyzed bean extract.
PROPERTIES OF PURIFIED METABOLITES. Distri-bution coefficients of the major Dowex-1 fractionwere determined for different solvent systems insingle tube experiments by shaking small aliquots withequal volumes of the pre-equilibrated solvents. Fol-lowing CCD experiments the aqueous layers wereacidified and extracted three times with butanone.For determining radioactivity this solvent was evap-orated to dryness, and the residue was dissolved in asmall volume of water, plated, and counted.
An acid hydrolysis of the major Dowex-l fractionwas carried out by reacting a solution of this materialin 1 N HCI for 3 hours on the steam bath. The prod-uct was extracted five times with ether, and the etherextract was dried and concentrated. The aqueousresidue from the extraction was evaporated to dry-ness several times with the intermittent addition ofwater to remove the HCl, and used for the chromat-ographic identification of the amino acids whichwere present. For this purpose the following sol-vents were used: solvent system B, benzyl alcohol-acetic acid-water (50: 10: 13 v/v/v) (11), phenol-phosphate buffer, pH 12 (11), and butanol-acetic acid-water (4: 1: 5 v/v/v, organic layer). The firsttwo solvents were used in a descending manner, whilethe last two solvents were used for two dimensionalseparations which were carried out in an ascendingmanner.
RESULTSETHER-SOLUBLE FRACTION. A summary of the
purification steps used on the ether-soluble portion ofthe bean extract is illustrated in figure 1. The Rfvalues and distribution coefficients (18) of the frac-tions as well as their relative concentrations areshown. The figures in the square boxes are thefraction numbers. Note that 60 % of the totalradioactivity of the ether extract is represented byone component (fraction 8). A second component(fraction 10) accounted for another 20 %, and theremaining eight components accounted for only 20 %of the total radioactivity. None of these compoundswas volatile as was suggested by Weintraub (17).A typical CCD pattern for the total ether extract
is shown in figure 2. The results were corrected forthe amount of radioactivity remaining in the aqueouslayers at equilibrium. Four components were found.Fraction 1 is plotted at one quarter of its actualheight. The disproportionate width of fraction 2suggested that this material was heterogeneous. Arerun of this peak through 300 transfers resulted inalmost complete separation into fractions 5 and 6.
Paper chromatography of the six CCD fractionsresulted in separation of the radioactive peaks from
559
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PLANT PHYSIOLOGY
[Ether Sol'n.|
54
100 transter CCD betweenether and phosphate pH 5.l
] 2E142
1.63
300transter, 100 tube CCDas above
10.8 33
IAq, Solln.
42
SEE DIAG.E
0.061.3
0.47
p a p e r C h r o m a t o g r a p h y s olv e n t A
A I SEAtA~~~D1% of Total 1.5 32.4 024 10.4 3.3 3 i8 1.6 0.8 0.2
Rf 0.96 078 0.75 0.40 0.42 0.53 085 0.52 037 0.27
FIG. 1. Purification scheme of ether-soluble metabo-lites of 2,4-D-1-C'4. The percentage figures indicatepercentage of total radioactivity in the extract which was
found in the fractions. Figures in sqluare boxes refer tofraction numbers.
the yellow color whiclh had accompanied them up tothat point. The small amounts of some of the isolatedfractions which were available prevented their furtherexamination. Others proved to be quite unstable.When fractions 14 and 15 were rechromatographedafter a few weeks' storage, their Rf values hadchangedI and had become in(listinguishable. It ispossible that some of the eight minor fractions were
degradation products of the two major components
or possibly representecl an original third major com-ponent.
An investigation of the chemical properties of theisolated fractions was preceded by a study of the
proposed reactions with model compounds. Benzoyl-ation and the reaction with chloroacetic acid went to
completion when 6-OH-2,4-D was used as a test com-
pound. As anticipated, malic and citric acids were
completely benzoylated but their aliphatic hydroxylgroups were not attacked by chloroacetic acid. Thephenoxy group of 2,4-D was very rapidly attackedby bromine. For this reason this test was used pri-marily as supporting evidence for the presence of
this group rather than serving as supporting evidencefor aliphatic unsaturation. In conjunction with
benzoylation, the reaction with KMnU4 was used to,indicate possible aliphatic unsaturation.
It was found that hydrogenation with PtO, ascontrasted to Pd on carbon, went to 80 % of com-pletion with 8-chloro-cis-crotonic acid but did notattack 2,4-D. This was evidenced by the ultravioletabsorption spectrum of the products, their Rf valuein solvent A and micro titrations using bromthymolblue as indicator. This indicator turns red in thepresence of HCl. Under the conditions used thedehydrochlorination of only 10 % of the added chloro-crotonic acid would have produced this color change.
The test for the ether linkage by cleavage withHI did not give conclusive results in all cases becauseof the large mechanical losses of radioactivity duringthe isolation of the reaction products. Therefore, theresults of the bromination test were also considered inarriving at a conclusion. It is noteworthy that inevery instance the products obtained from the beanfractions with HI differed chromatographically fromthose from authentic 2,4-D (table I).
The occurrence of several products in benzoylationand in the reaction with chloroacetic acid was un-expected. This may indicate that these materialspossessed several active groups or the presence ofmore than one component in each fraction. It shouldbe noted that fraction 8, the major metabolite of2,4-D, was chromatographically indistinguishablefronm 2,4-D in some 30 different solvents as well a,in CCD experiments. However, it clearly diffe-from 2,4-D by its reaction with benzoyl chloride,lack of reaction with bromine (table II).
ETHER-INSOLUBLE FRACTION. The purificatihsteps used on the ether-insoluble fraction are showinin figure 3. The elution pattern of this fractionifrom a colum of Dowex-l (formate) is shown infigure 4. The recovery of radlioactivity from thiscolumn was in excess of 90 %. The main peak ofactivity (fraction 18) represents almost all this radio-activity (95 %). Fraction 17 (loes not seem to be
FIG. 2. Countercurrent distribution of ether-solublemetabolites of 2,4-D between ether and 1 Mi phosphatebuffer, pH 5.1.
acetonitrile extraction ;acetonitrile re moved,
odjust pH to 5-extract with ether
I~~~~~~~~~~~~~~
IPrec ipitate% of Totet 4
DISCARDED
%of Total 34.1
DOlributlos Coeff.6.14
% of Total
r-
560
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BACH-METABOLITES OF 2,4-D FROM BEAN STEMS 561
TABLE IPRODUCTS OF REACTION OF ETHER-SOLUBLE RADIOACTIVE 2,4-D METABOLITES WITH ALKALI, HI, & H2
* See figure 1 for classification of fractions.** Rf corresponding to unreacted starting material, as confirmed by co-chromatography, is underlined.*** Reaction products were chromatographed in butanol saturated with 1.5 N NH40H.t Distribution coefficients between ether and 1 M phosphate buffer, pH 5.1, in a 25 transfer countercurrent dis-
tribution experiment.
bound to the resin at all. The very small amounts ofthis fraction and its gross contamination with sugarsmade it impossible to characterize the ether-insolublehydrolysis products which were obtained from it.The radioactive hydrolysis products were only slight-ly soluble in ether and continuous extraction for 24ours was required for their complete removal fromie acid aqueous phase. The Rf values in solvent A
and solubilities in ether of fractions 26 and 27 closelyresembled those of fractions 14 and 16. It seemsreasonable to conclude that fractions 26 and 27 wereidentical to fractions 14 and 16, respectively.
The main peak on the Dowex-l columns (fraction18) was partly resolved into two components. Underthe mild conditions of the chromatography, both thesecomponents were stable in formic acid. Chromatog-
TABLE IIPRODUCTS OF REACTION OF ETHER-SOLUBLE RADIOACTIVE 2,4-D METABOLITES
WITH VARIOUS FLTNCTIONAL GROUP SPECIFIC REAGENTS
Rf OF MAJOR PRODUCTS IN ORDER OF RELATIVE ABUNDANCE**REAGENTFRACTION* NONE C6H5COC1 + Cl-CH2-CO2H + KMnO4 Br WATER
acid hydrolysis of Individual fractions, ether oxtractlon
A. ETHER A. ETHE Q EHE
p a p e r c h r o m a t o g r a p h y n
solve nt A varIous solvents solvent A
At LeAc st2d2R 0.58 0.25 0.78 0.4 0
% f Total 03 I.T 0-8 40-32
FIG. 3. Purification of ether-insoluble metabolites of2,4-D.
raphy of fraction 18 on silicic acid removed a majorportion of the color which accompanied this materialthrough the previous purification steps but did noteffect any separation of the radioactive oomponents.Recovery of radioactivity from the silicic acid wasessentially quantitative. The separation of fraction18 on charcoal (4) was not complete but sufficient todemonstrate the heterogeneity of the original material.
FIG. 4. Elution pattern of ether-insoluble metabolitesof 2,4-D from a Dowex-1 (formate) column using a
gradually increasing concentration of formic acid as eluent.
Distribution coefficients for fraction 18 were de-termined in several solvent systems using butanone,2-methyl-4-pentanone, 2-heptanone, various butyl andamyl alcohols, and various ethers as the organicphases and phosphate buffers (pH 3-7) as the aqueousphases. Attempts to resolve fraction 18 by CCDusing some of these solvent pairs failed in all caseseven though useful dlistribution coefficients had beenfound. Similarly no resolution was achieved by theuse of high-voltage paper electrophoresis (pH range2.5-9.8).
Of some 25 different solvents for paper chromatog-raphy only solvent A effected a separation into sixpoorly resolved peaks which covered most of thepaper. Elution of the peaks and rechromatographyin the same solvent resulted in distinct peaks, eachhaving a characteristic Rf value. Figure 5 showsthe radioactivity tracing of a chromatogram to whichwere applied aliquots of fractions 20 to 24. Each ofthese fractions had an Rf value of 0.5 in solvent B,thus confirming that they were unchanged subfrac-tions of the original ether-insoluble extract. Acidhydrolysis of these fractions completely converted theradioactivity into an ether-soluble form (fig 3).Chromatography and CCD of the ether extracts
FIG. 5. Radioactivity tracing of a paper chromato-gram of fractions 20 to 24 developed in Solvent A.
showed the presence of only two components (frac-tions 28 & 29); their identity with fractions 8 and10, respectively, was confirmed by co-chromatog-raphy with authentic samples. In addition, the prod-ucts obtained from fractions 28 and 29 after reactionwith benzoyl chloride, chloroacetic acid, and KMnO4were identical to those obtained from fractions 8 and10, respectively.
Fractions 20 to 25 differed in the relative amountsof the two radioactive products which they contained.This distribution is shown in table III. While frac-tion 8 was more abundant in the original ether ex-tract, fraction 10 was predominant in the hydrolyzedether-insoluble fractions.
562
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BACH-METABOLITES OF 2,4-D FROM BEAN STEMS
Paper chromatography of aliquots of the aqueouslayers from the hydrolysis of fraction 20 to 25 andfrom the fractions obtained by chromatography oncharcoal (4) revealed the presence of at least 10amino acids in differing proportions. The followingcompounds were identified by co-chromatographywith known amino acids in four different solvents:aspartic acid, glutamic acid, glycine, alanine, serine,tyrosine, phenylalanine, and leucine or isoleucine.Identification of the other amino acids present wasprevented by the small amounts of material whichwere available.
DISCUSSIONOur previous results had clearly demonstrated the
aerobic nature of 2,4-D metabolism, the requirementfor sucrose, and the time parameters involved (2).Furthermore, we had demonstrated the very limitedtransport of radioactivity from the point of applica-tion of 2,4-D, and the difference in response to theherbicide between stem tissue and leaf petioles (3).It was felt for these reasons that vacuum infiltrationoffered the only workable method for uniformly ex-posing large amounts of tissue. In view of the largenumber of metabolites of 2,4-D which have been re-ported in the literature, there is concern whethersome of these might be due to the action of micro-organisms. Under the conditions used in the experi-ments described here, it did not prove possible tomaintain sterility. However, visible contaminationwas generally small, and isolated products were iden-tical to those obtained in small-scale experiments un-der completely aseptic conditions (2) in as far asthese were characterized. Thus, the metaboliteswhich were isolated are likely to have originated fromthe metabolism of 2,4-D by the plants.
The results presented here point to the presenceof two main classes of radioactive metabolites of thecarboxyl labeled 2,4-D: Compounds which travelwith Rf 0.5 in solvent B and those which move closeto the solvent front. The slower moving materials
TABLE IIIETHER-SOLUBLE HYDROLYSIS PRODUCTS OF WATER-
SOLUBLE FRACTIONS OF 2,4-D METABOLITESFROM BEANS*
% OF TOTAL RADIOACTIvITY
FRACTION** FRACTION 28 FRAcTION 29
20 0 10021 18 8222 17 8323 22 7824 13 8725 5 95
* Fractions were hydrolyzed with HC1 and extractedwith ether. Ether layers were concentrated and chro-matographed in solvent A. Only peaks corresponding tofractions 28 and 29, figure 3, were found.
** See figure 3.
were present in the ether-insoluble fraction while thefaster moving components constituted the ether-soluble fraction. Closer examination revealed thatboth these components were mixtures of a numberof compounds; two compounds (fractions 8 & 10)accounted for 80 % of the total radioactivity.
The demonstration of at least 10 amino acids inthe hydrolysis of the ether-insoluble fractions sup-ports the observations of Butts and Fang (6). Theassumption that these products were polypeptides isdifficult to reconcile with their demonstrated be-havior on ion-exchange chromatography, paper chro-matography, and extraction into organic solvents.Thus the detection of many amino acids along withtwo radioactive materials in each of the purified frac-tions (20-25) might be explained best if it is as-sumed that the purified, ether-insoluble, radioactiveproducts were still contaminated with relatively largeamounts of otherwise bound amino acids, such as,for example, amino acid amides of miscellaneous plantmetabolites, and that there is no direct correlationbetween the amino acids which have been identifiedand the amino acids which were presumably boundto the radioactive acids.
The ether-soluble radioactive metabolites of 2,4-Dprobably did not originate from the cleavage of thephenol ether linkage. The product of the reaction,glycolic acid, would be incorporated into the normaldicarboxylic acids of the plant; but none of theseacids possessed Rf values in solvent A, similar tothose of the radioactive compounds. Furthermore,the selective formation of amino acid amides of thesenormal plant metabolites is improbable. In view ofthe previously reported slow evolution of C1402 fromthe radioactive 2,4-D in the beans (2,16) it is alsounlikely that these acids were derived from the fixa-tion of C1402. It is concluded, therefore, that at
O-CH2-1
1
a Ib
O-CH2-cy2
B -'
ci
.1I*O-CH2-Co2HQ6 ci
H02C
O-CH2 -(C1ci
IO-CH2Ci
.~-ci
.OHOH
co2H
*
O-CH2-CO2HOH
C1 \*
co2H O-CH2-CO2H
CO2H
'O2H
bi'
O-CH2CO2H02H
ci2
*
O-CH2-C 2H
H
c
-CH2-co2H
co2HCo2R
ci
FIG. 6. Postulated pathways for the oxidation of thearomatic nucleus of 2,4-D-l-C14.
563
Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.
PLANT PHYSIOLOGY
least part of the carbon skeleton of the benzene ringremains attached to the radioactive carbon of thesemetabolites of 2,4-D.
The partial degradation of the benzene ring with-out cleavage of the phenol ether linkage, might pro-ceed via one of the pathways in figure 6, by analogy tothe proposed microbial pathways (7). Thus oxida-tion via (a) and (c) results in esters of glycolic acidwhich yield radioactive glycolate upon saponification.Glycolate had a distribution coefficient of 49 in theCCD system. However, no radioactivity was foundat this location after saponification (table I). More-over, substituted chloromuconic acids result from thedegradation of 2,4-D by any other oxidative path-ways, although none of them are very plausible. Byanalogy to B-chloro-cis-crotonic acid these compoundsshould be hydrogenated under the conditions em-ployed and the products should differ significantlyin their solubility properties from the original com-pounds. No evidence for significant change uponhydrogenation was obtained in any of the fractions.It is evident that these mechanisms, by themselves,cannot explain the properties of the isolated metabo-lites.
Further examination of table II shows that severalof the isolated fractions (fractions 8, 9, 11) formedmore products on reaction with benzoyl chloride thanon reaction with chloroacetic acid, suggesting thepresence of non-phenolic susceptible groups in addi-tion to at least some phenolic hydroxyl groups inthese fractions. Because of their solubility char-acteristics and the treatments used during isolationit is more likely that these compounds contained ali-phatic hydroxyl groups rather than sulfhydryl oramino groups. The hemiacetal of glyoxylic acidwith dichlorophenol is the only possible compoundwhich has a hydroxyl group on the carbon side chainof 2,4-D and retains the carboxyl group. However,this linkage would split with the mildest acid to giveglyoxylic acid. In order to accommodate a side chainhydroxyl group, it is postulated that the side chainof 2,4-D is lengthened in the course of 2,4-D metabo-lism. Further support for this interpretation comesfrom the HI cleavage results. While the variousmetabolites were attacked by III none of them yieldeda material having chromatographic properties similarto the cleavage products obtained from 2,4-D. Alengthened side chain could account for this finding.(Note in passing that since iodoacetic acid is de-composed by HI, the isolated cleavage product of2,4-D was not iodoacetic acid. The structure of theactual product is unknown, but it is clearly not aceticacid, glycolic acid, glyoxylic acid, glycine, or diglycol-ic acid.)
It has been reported (19) that 2,4-D, as well asother herbicides and auxins, can be converted totheir coenzyme A thioesters. The isolation of indole-3-acetylaspartate as a metabolite of indoleacetic acid(1) and the isolation of amino acid amide metabolitesof 3-amino-1, 2, 4-triazole (12) also suggest thatcarboxyl-activation is a common pathway in the
metabolism, if not a step in the mode of action, of thesecompounds. If it is assumed that 2,4-dichlorophen-oxyacetyl coenzyme A can react with acetyl coenzymeA, CO2, and ATP, in a manner similar to that in-volved in fatty acid biosynthesis (14), a lengtheningof the side chain in the metabolism of 2,4-D is ex-plained. Thus, depending on the point of termina-tion of this chain-lengthening reaction, it is possibleto obtain mono- or dicarboxylic acids with potentialsites for hydroxyl or ketone groups on alternatecarbons along the chain. Unfortunately, the amountof metabolites obtained from the present experi-ments did not permit confirmation of this hypothesis.The use of high specific activity ring-labeled 2,4-Dwould enable an unequivocal test for a retention ofthe phenol ether linkage of 2,4-D during these met-abolic conversions. Finally, the implications of theseresults on herbicide metabolism, in general, as wellas on herbicide structure-activity studies. remain tobe explored. If lengthening of the side chain of2,4-D is verified, this new pathway to detoxicationshould receive more attention.
SUMMARYThe radioactive metabolites of 2,4-dichlorophen-
oxyacetic acid-1-C14 have been isolated from beanstems. One half of the original radioactivitv wasfound in ten components of an ether extract. Testsfor functional groups indicate that these componentsretained the aromatic nucleus of 2,4-D. Some ofthem possessed phenol and alcohol groups but therewas no evidence for aliphatic unsaturation. Neither2,4-D nor 6-hydroxy-2,4-D was isolated.
The ether-insoluble fraction contained at leastsix major components. Acid hydrolysis of each ofthese yielded two radioactive, ether-soluble, com-pounds which were identical to the two major com-ponents of the original ether extract. In addition,some ten amino acids were isolated and seven ofthem identified. The implications of these resultsare discussed.
ACKNOWLEDGMENTSWe are grateful to Dr. D. L. Ford, Chemicals
Division, Union Carbide of Australia, Ltd., Rhodes,N.S.W., for a supply of 6-OH-2,4-D, and to Mr.Walter J. Skraba of these Laboratories for the prepa-ration of high specific activity C14-labeled 2,4-D.The able and devoted assistance of Mrs. J. T. Hilland Miss M. J. Persohn, and the critical help of Dr.I. W. F. Davidson in the preparation of the manu-script are also acknowledged.
LITERATURE CITFD1. ANDREAE, W. A. & N. E. GOOD. 1957. Studies on
3-indoleacetic acid metabolism. IV. Conjugationwith aspartic acid & ammonia as processes in themetabolism of carboxylic acids. Plant Physiol.32: 566-572.
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BACH-METABOLITES OF 2,4-D FROM BEAN STEMS
2. BACH, M. K. & J. FELLIG. 1961. Correlation be-tween inactivation of 2,4-dichlorophenoxyacetic acid& cessation of callus growth in bean stem sections.Plant Physiol. 36: 89-91.
3. BACH, M. K. & J. FELLIG. 1961. The uptake &fate of C14-labeled 2,4-dichlorophenoxyacetic acidin bean stem sections. In: Plant Growth Regu-lation, R. M. Klein, chief editor, Iowa State Univ.Press, Ames. Pp. 273-287.
4. BACH, M. K. & J. FELLIG. 1961. Metabolism ofcarboxyl-C14-labeled 2,4-dichlorophenoxyacetic acidby bean stems: Heterogeneity of the ethanol-soluble, ether-insoluble products. Nature 189:763.
5. EUSCH, H., R. B. HURLBERT, & V. R. POTTER. 1952.Anion exchange chromatography of acids of thecitric acid cycle. J. Biol. Chem. 196: 717-727.
6. BUTTS, J. S. & S. C. FANG. 1956. Tracer studieson the mechanism of action of hormone herbicides.In: A Conference on Radioisotopes in Agriculture,E. Lansing, Mich. Pp. 209-214. U. S. Govern-ment Printing Office, Washington, D. C.
7. FERNLEY, H. N. & W. C. EVANS. 1959. Metabolismof 2,4-dichlorophenoxyacetic acid by soil Pseudo-monas: Isolation of a-chloromuconic acid as anintermediate. Biochem. J. 73: 22 Proc.
8. HOLLEY, R. W., F. P. BOYLE, & D. B. HAND. 1950.Studies on the fate of radioactive 2,4-dichloro-phenoxyacetic acid in bean plants. Arch. Biochem.Biophys. 27: 143-151.
9. JAWORSKI, E. G. & J. S. BuTTs. 1952. Studies inplant metabolism. II. The metabolism of C'14-labeled 2,4-D in bean plants. Arch. Biochem. Bio-phys. 38: 207-218.
10. JAWORSKI, E. G., S. C. FANG, & V. H. FREED. 1955.Studies in plant metabolism. V. The metabolismof radioactive 2,4-D in etiolated bean plants. PlantPhysiol. 30: 272-275.
11. LiNSKENs, H. F. 1955. Papierchromatographie inder Botanik. Springer-Verlag, Berlin.
12. MASSINI, P. 1959. Synthesis of 3-amino-1,2,4-triazolylalanine from 3-aminotriazole in plants.Biochim. Biophys. Acta 36: 548-549.
13. McELvAIN, S. M. 1953. The Characterization ofOrganic Compounds. The McMillan Co., NewYork.
14. WAKIL, S. J. & J. GANGULY. 1959. On the mech-anism of fatty acid synthesis. J. Am. Chem. Soc.81: 2597-2598.
15. WEINTRAUB, R. L. 1953. Metabolism of 2,4-D bymicroorganisms & higher plants. Proc. N. CentralWeed Control Conf. P. 6.
16. WEINTRAUB, R. L., J. W. BROWN, M. FIELDS, &J. ROHAN. 1952. Metabolism of 2,4-dichloro-phenoxyacetic acid. I. C'402 production by beanplants treated with labeled 2,4-dichlorophenoxy-acetic acids. Plant, Physiol. 27: 292-301.
17. WEINTRAUB, R. L., J. N. YEATMAN, J. A. LOCKHART,J. H. REINHART, & M. FIELDs. 1952. Metabolismof 2,4-dichlorophenoxyacetic acid. II. Metabolismof the side chain by bean plants. Arch. Biochem.Biophys. 40: 277-285.
18. WEISIGER, J. R. 1954. Countercurrent distribu-tion. In: Organic Analysis, J. Mitchell, Jr., I. M.Kolthoff, E. S. Proskauer, & A. Weissberger, eds.2: 277-326. Interscience Publishers. N. Y.
19. ZENK, M. H. 1960. Enzymatische Aktivierung vonAuxinen und ihre Konjugierung mit Glycin. Z.Naturforsch. 15b: 436-441.
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