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Acifluorfen metabolism in soybean: Diphenylether bond cleavage and the formation of homoglutathione, cysteine, and glucose conjugates
PESTICIDE
BIOCHEMISTRY
AND
PHYSIOLOGY
20,
299-310 (1983)
Acifluorfen Metabolism in Soybean: Diphenylether Bond Cleavage and the Formation of Homoglutathione, Cysteine, and Glucose Conjugates1s2 D. S. FREAR, H. R. SWANSON, AND E. R. MANSAGER U.S. Department of Agriculture,
Agricultural Research Service, Metabolism Laboratory, Fargo, North Dakota 58105
and Radiation Research
Received February 15, 1983; accepted May 27, 1983 Metabolism of the substituted diphenylether herbicide, acitluorfen [sodium 5-(2-chloro-4-trifluoromethylphenoxy)-2nitrobenzoate], was studied in excised leaf tissues of soybean [Glycine mar (L.) Merr. ‘Evans’]. Studies with [chlorophenyl-‘4C]- and [nitrophenyl-t4C]acifluorfen showed that the diphenylether bond was rapidly cleaved. From 85 to 95% of the absorbed [t4C]acifluorfen was metabolized in less than 24 hr. Major polar metabolites were isolated and purified by solvent partitioning, adsorption, thin layer, and high-performance liquid chromatography. The major [chlorophenyPC]-labeled metabolite was identified as a malonyl-S-D-ghrcoside (I) of 2-chloro-4-trifluoromethylphenol. Major [nitrophenyPC]-labeled metabolites were identified as a homoglutathione conjugate [S-(3-carboxy+titrophenyl) y-glutamyl-cysteinyl+-alanine] (II), and a cysteine conjugate [S-(3-carboxy-4-nitrophenyl)cysteine] (III). INTRODUCTION
Acifluorfen [sodium 5-(2-chloro-4-trifluoromethylphenoxy) - 2 - nitrobenzoate] is a substituted diphenylether herbicide for selective preemergence and postemergence weed control in soybean (1, 2). Diphenylether herbicides require light absorption for their action (3). As potent inhibitors of photosynthesis, they block electron transport (4, 5), inhibit energy transfer (6), and affect plasma membrane systems (7). Although a recent paper (8) has indicated that differential metabolism may be a factor in the selective action of acifluorfen, detailed information on the nature of acifluorfen metabolites and metabolic pathways in susceptible and tolerant plants has not been reported. The objective of the present study was to establish the pathway of acifluorfen metab’ Presented in part at the North Central Weed Control Conference, Omaha, Neb., December, 1980. 2 Mention of .a trademark or proprietary product does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable.
olism in tolerant and identification MATERIALS
soybean by the isolation of major metabolites. AND METHODS
Chemicals. Sodium 5-(2-chloro-4-u-&oromethyl[U - 14C]phenoxy) - 2 - nitrobenzoate ([chlorophenyl-14C]acifluorfen, 1.27 $i/pmol), ethyl 5-(2-chloro-4-trifluoromethylphenoxy)-2-nitro[U-t4C]benzoate (1.95 pCi/pmol), and methyl-2-nitro-5-fluorobenzoate were provided by the Rohm and Haas Company. [Nitrophenyl-‘4C]acifluorfen was prepared by the reaction of ethyl 5-(Zchloro4 - trifluoromethylphenoxy) - 2 - nitro[U *4C]benzoate (40 FCi in 0.5 ml EtOH) with 1 ml of 1 N KOH at 80°C for 16 hr. The reaction mixture was acidifed (pH 2.0), extracted with Et,O, and the [nitrophenyl14C]acifluorfen (~95% yield) was purified by thin-layer chromatography (TLC) [Rf 0.5, benzene/HOAc (50:8)]. Plant materials and treatments. Soybeans (Glycine max Merril, var. Evans) were seeded in vermiculite, subirrigated with nutrient solution and grown in the greenhouse (9). Soybean leaf disks were
299 0048-3575183 $3.OU
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FREAR,
SWANSON.
used to compare the metabolism of [chlorophenyl-i4C]and [nitrophenyl-‘4C]acifluorfen. Seven disks (1 cm diameter) from fully expanded trifoliate leaves were placed in 50-ml beakers and treated with aqueous sodium salt solutions (0.5 ml) of either [chlorophenyl-14C]or [nitrophenyl-14C]acifluorfen (1.1 x lo6 dpm; 7.36 x lo3 dpm/pg). Treated disks were incubated at room temperature for 8 hr under cool white fluorescent lights (50-60 kEm-2 set-I), rinsed with distilled water, and extracted with 80% MeOH. Filtered MeOH extracts were concentrated in vucuo (30°C) and metabolites were separated by TLC [2-butanone:H20:HOAc (10/1/l) 2 x]. Excised leaves from 5- to 6-week-old plants were used for metabolite isolation and identification studies. Fully expanded trifoliate leaves were pulse-treated via cut petioles with aqueous sodium salt solutions (2 ml) of either [chlorophenyl-14C]or [nitrophenyl-*4C]acifluorfen (4.8 x 10e3 M; 0.14-0.23 @i/pmol). After 2-3 hr, the [14C]acifluorfen was absorbed and distilled water was added. Treated leaves were maintained at room temperature under cool white fluorescent lights (50-60 kErnp2 set -I) for 12 hr and either extracted directly (metabolites II and III) or transferred to the dark for an additional 12 hr and extracted (metabolite I). Under these conditions, 98-99% of the [14C]acifluorfen was absorbed. Six to eight trifoliate leaves, with a combined fresh weight of 30-40 g, were used for each [i4C]acifluorfen treatment. Chromatography. Silica gel HF plates (250 or 500 Frn, Analabs) were used for TLC of metabolites and derivatives. i4CLabeled metabolites were located with a Packard 7201 radiochromatogram scanner or by fluorescence quenching under uv light. Whatman LKSF silica gel plates (250 km) and ninhydrin were used for the TLC and detection of amino acids. A Waters Associates liquid chromatograph system with a 254-nm uv detector and a 1055 CA1 radioactivity monitor was used for high-performance liquid chroma-
AND
MANSAGER
tography (HPLC). Solvent flow rates of 2.0 ml/min were used with 10 km Cl8 RadialPak cartridges (Waters Associates). A Tracer-Microtek gas chromatograph with FID and a glass column (150 cm x 4 mm i.d.) packed with a 3% SP 2340 (Supelco) on 100/120 Supelcoport was used for gas chromatographic (GLC) analysis of dimethylmalonate. Injector and detector temperatures were maintained at 250°C. The oven temperature was 85°C and the nitrogen carrier gas flow rate was 60 cc/min. Under these conditions, the retention time for standard dimethylmalonate was 1.6 min. A glass column (1.8 m x 2 mm) packed with 0.65% EGA (ethylene glycol adipate) on 60180 acid washed Chromosorb W was used for the GLC analysis of amino acids as N-trifluoroacetyl n-butyl ester derivatives (IO, 11). An initial oven temperature (1OO’C) was held for 2 min. This was followed by a temperature program (3YYmin) to 185°C and a final I-min hold at 185°C. The nitrogen carrier gas flow rate was 40 cc/min. Under these conditions, derivatives of glycine, l3-alanine, and glutamic acid had retention times of 9.2, 11.4, and 28.7 min, respectively. Instrumentation. Mass spectra were obtained with a Varian MAT 112s spectrometer equipped with a combination EIKI source and a SS-200 data system. Samples were inserted with a solid sample probe. Ammonia and isobutane were used as ionizing gases for chemical ionization (CI) spectra. Proton FT-NMR spectra were obtained with a JEOL FX-90Q Fourier transform spectrometer equipped with a microprobe and a NV-VTS variable temperature controller. Tetramethylsilane was used as the internal reference and samples were analyzed in I.&mm-o.d. tubes. Quantitative t4C measurements were made with Insta-Gel (Packard Instrument Company) counting cocktail and a Packard 3375 liquid scintillation spectrometer. Synthesis and derivatization of reference glutathione and cysteine conjugates. S-(3-
ACIFLUORFEN
METABOLISM
IN
GSH + F FIG.
1. Synthesis
301
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+ HF of reference
S-(3-carbomethoxy4nirropheny~glutathione
Carbomethoxy - 4 - nitrophenyl)glutathione and S - (3 - carbomethoxy - 4 - nitrophenyl)cysteine were synthesized by the reaction of either glutathione or cysteine with methyl 2 - nitro - 5 - fluorobenzoate (Fig. 1). Methyl 2-nitro-5fluorobenzoate (0.55 mmol) was dissolved in MeOH (2 ml) and mixed with an equal volume of an aqueous solution (pH 10) of either glutathione (0.5 mmol) or cysteine (0.5 mmol). After 4 hr at 30°C the reaction mixture was adjusted to pH 5.0 and the MeOH removed by a stream of Nz. The aqueous solution was acidified (pH < l.O), washed with Et,O, placed on a 1 x IO-cm XAD-2 column and eluted stepwise with 25 ml volumes of H20, HPO:CH$N (9/l), and H20:CHsCN (l/l). Reaction products in the HzO:CHsCN (l/l) eluate were taken to dryness in vucuo (30°C) and weighed. Reaction yields were 80-W% and the chemical purity of the glutathione or cysteine products was >95% by TLC analysis, [Rf 0.37 and 0.47, respectively; BuOH:HOAc:H20 (120/30/50)]. Glutathione and cysteine conjugates of 3carbomethoxy-4nitrobenzene (5 mg each) were methylated with 2 ml of methanolicHCl (3 N) at 80°C for 30 min, evaporated to dryness under a stream of Nz, and acetylated with 500 ~1 of acetic anhydride at 40°C for 16 hr. Derivatized products were concentrated under a stream of NZ, dissolved in MeOH (500 pl), and chromatographed by preparative TLC [CH2C12/ MeOH (9: l)]. Wo glutathione conjugate fractions (Z$ 0.46 and 0.53) and one cysteine conjugate fraction (Rf 0.80) were eluted with CHsCN. Further purification of these fractions was achieved by HPLC [isocratic CHsCN/H20 (35/65)]. The glutathione coniuaate derivative (RJ 0.46) and
conjugate.
the cysteine conjugate derivative (Rf 0.80) produced single sharp HPLC peaks at 2.20 and 3.46 min, respectively. The glutathione conjugate fraction at Rf0.53, however, produced two sharp HPLC peaks of almost equal intensity (2.38 and 2.92 min). Mass spectra (Wisobutane) of the isolated derivative fractions confirmed the structures of the reference glutathione and cysteine conjugates as well as the structures of two glutathione conjugate degradation products (dipeptides) formed during derivatization (Table 1). Characteristic derivative ions included protonated molecular ions (MH)+ with (M-31)+ or (M-59)+ ion fragments, strong peptide or cysteine derivative ion fragments (M-212)+ formed by cleavage of the cysteine thioether bond, and methyl 2nitro-5-thiobenzoate ion fragments at m/z 214 and 182. Extraction
and isoiation
of metaboiite
I.
Procedures developed for the extraction and isolation of metabolite I are summarized in Fig. 2. Initial partitioning procedures were similar to those of Dutton et al. (12). [Chlorophenyl-14C]acifluorfen-treated leaves (30-40 g fresh wt) were ground in an Omnimixer with 5 vol of 50% aqueous acetone. Extracts were vacuum filtered (10 pm, LC Millipore) and concentrated in vacua (30°C). The aqueous concentrate (~30 ml) was adjusted to pH 9.0 (1 N KOH), extracted twice with equal volumes of EtOAc, adjusted to pH 4.0 (1 N HCl), and extracted with equal volumes of CHC13(2 x ) and EtOAc(2 x ). The pH 4.0 EtOAc extract fraction was taken to dryness in vucuo (30°C). Only l-3% of the absorbed i4C was present in the insoluble residue and the aqueous phase after EtOAc extraction at pH 4.0. Small amounts of unreacted 114Clacifluorfen (3-4%) were
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Characterization
AND
MANSAGER
1
of Reference S-(3-Carbomethoxy-?-nitrophenyl.Elutathione
and Cysteine Conjugates
Conjugate derivatives HPLC retention time
Conjugates Glutathione
TLC RF
(Rf 0.37)
(Cysteinylglycine)
0.46 I
(mill) 2.20 2.38
0.53 (y-Glutamylcysteine) Cysteine
(Rr 0.47)’
n BuOH:HOAc:H20 (120/30/50). b CH#&:MeOH (9/l). c Isocratic CH$N:H20 (35/65),
2.92 0.80
3.46
Characteristic
U/MS
ions
(m/z)
557(MH) + ,525(M-31) + ,495, 344(M-212)+, 312, 255, 214 and 182. 414(MH)+, 354(M-59)+, 315(M-88)+, 214, 201(M-212)+ and 182. 5OO(MH)+, 287(M-212)+, 214, 186 and 182. 357(MH) + , 325(M-3 1) + , 295(M-59) 182 and 144(M-212)+.
+ , 214,
2 ml/min.
present in the pH 9.0 EtOAc and the pH 4.0 CHC13 fractions. Further purification was achieved by a sequence of preparative TLC and reverse-phase chromatography steps. Preparative TLC-separated fractions [RfO.20 and 0.60, respectively, with CHCIJ: MeOH:H20 (65/25/4)2 x or CHC13: MeOH:HzO:HOAc (65/25/4/4)2 x ] were eluted with MeOH, taken to dryness in vucuo (30°C) and dissolved in Hz0 prior to adsorption on preconditioned Cl8 Sep-Pak cartridges (Waters Associates) and elution with 40% aqueous MeOH. Metabolite recoveries were >90% for each chromatographic purification step. Derivatization of metabolite I. Metabolite I was dissolved in a small volume of MeOH:EtzO (l/4) and esterified with a slight excess of CH2N2 in MeOH:Et*O (1:9) at 4°C for 15 min (12). Excess CHzNz was removed with a stream of N2 and the reaction products were purified by TLC [CHC13:MeOH:H20 (65/25/4)]. The methylated product (ZQ 0.62) was eluted with CHsCN and chromatographed by HPLC [isocratic MeOH:HzO (3/2)]. A single sharp peak (R, 4.5 min) was eluted and taken to dryness in vucuo (30°C) prior to FT-NMR analysis, malonate determination, and acetylation for CI/MS analysis. Methylated me-
tabolite I was acetylated with acetic anhydride/pyridine (9: 1) at 35°C for 3 hr. Excess reagents were removed with a stream of N2 and several additions of CH&N. Malonate determinations. Methylated metabolite I (75-100 p+g)was dissolved in 0.1 ml MeOH and reacted at room temperature for 3 hr with an excess of CH2N2 dissolved in Et20 (12). At the end of the reaction, excess CHzN2 was removed with a gentle stream of Nz and aliquots of the reaction mixture were analyzed for dimethylmalonate (GLC). Extraction and isolation of metabolites ZZ and ZZZ. [Nitrophenyl-14C]acifluorfen treatment times prior to extraction were important in the isolation of metabolites II and III. A 24-hr treatment period resulted in >90% metabolism, but metabolite II was present only as a minor product (~10%). Shorter treatment times reduced overall metabolism, but increased the amounts of II in relation to III. As a compromise, a 12hr treatment period was selected for the isolation of both metabolites. Under these conditions, approximately 70% of the absorbed [nitrophenyl-14C]acifluorfen was metabolized, and the ratio of II to III was =1:2. The extraction and isolation of metabo-
ACIFLUORFEN
lj4 cg
METABOLISM
303
IN SOYBEAN
Le8f
Mdfhmfen-1roated
li88w
y-pzzyr
Soluble EfAc
lnroluble
‘-/G&w~~
(p;...,
-
Aqueou8 CHCI,/l-&O
(pH
4.0)
EtOAc
AQUOOW
-ITLC CHC~:M~OH:H~0~66/25/4~2x I ~2’8 Sop
Pek’R) H20:MeOH(3/2)
I TLC CHClyMeOH:H20:HOAc(6S/2S14/4)2x C’8
1 sep
P.kR’ H,O:MeOH(3/2)
I FIG.
2. Extraction and isolation of metabolite I.
lites II and III are summarized in Fig. 3. [Nitrophenyl-‘4C]acio~en-treated leaves (30-40 g fresh wt) were extracted with 80% aqueous methanol as described for metabolite I. Only l-3% of the absorbed r4C remained in the insoluble residue fraction. The aqueous concentrate (~30 ml) was adjusted to pH 3.5 with 1 N HCl extracted three times with equal volumes of Et20 to remove unreacted [14C]acifluorfen, adjusted to pH 2.0 with additional 1 N HCl, and placed on a 2.5 x 25cm column of XAD-2. Metabolites II and III were separated on the XAD column by stepwise elution with 200 ml of Hz0 and 200 ml of aqueous 20% CH$N. Column eluate frac-
tions were concentrated in vacua (30°C) and each metabolite was purified separately by a sequence of preparative TLC and reverse-phase chromatography steps. Metabolites were eluted from Cl* Sep-Pak cartridges with Hz0 and taken to dryness in wcuo (30°C). Preparative TLC-separated metabolites were eluted with 50% aqueous CHSCN and taken to dryness in vacua (30”). Metabolite recoveries from XAD and subsequent reverse-phase or thin-layer chromatographic purification steps were 30%. Derivatization
of metabolites
ZZ and ZZZ.
Metabolites II and III were methylated with 0.5 ml of methanolic HCl (3 N) at
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Soluble c
Insoluble
~Et,OIH,O
,.;,,I
Aqueous
I320
-
b
HzO:CHJCN
(4/t)
H20
c TLC
P-Butanone:H,O:HOAc
TLC
BuOH:HOAc:H,O
i TLC
2-Butanone:H,O:HOAc
(10/1/1)4x (120/30/50)2x (10/l
TLC /1)4x
TLC
mtabolite FIG.
I(
3. Extraction and isolation of metabolites II and III.
80” C for 30 and 60 min, respectively. A shortened reaction time for the methylation of metabolite II was selected to reduce the formation of peptide hydrolysis products and still achieve reasonable (ZO-30%) methylation of the nitrobenzoic acid moiety. Methylation reaction mixtures were evaporated to dryness under a stream of NZ, acetylated with 200 ~1 of acetic anhydride at 40°C for 12 hr, taken to dryness again, and dissolved in CHsCN. Methylated and acetylated reaction products were purified by preparative TLC [CH$&:MeOH (9/l)] and HPLC [isocratic CHsCN/H20 (35/65)] for CI/MS analysis and comparison with methylated and acetylated derivatives of reference glutathione and cysteine conjugates. Amino acid determination. Metabolite II
(Fig. 3) was purified further by HPLC [initial 1% HOAC:CHsCN @O/10)to (85/U) in 2 min and (80/20) in 15 min at 2 ml/min; R, 5.65 min] and hydrolyzed under N2 with 0.5 ml of constant boiling HCl at 105°C for 20 hr. Hydrolysates were analyzed directly for amino acids by TLC or converted to N-trifluoroacetyl n-butyl ester derivatives (11) and analyzed by GLC (10). TLC [2 butanone:HOAC:HzO (10/1/l) 4 X] Rf values for reference amino acids were: glycine (0.06); glutamic acid (0.12); @-alanine (0.17); and alanine (0.23). RESULTS
AND
DISCUSSION
Acif2uorfen Metabolism
Acifluorfen was metabolized rapidly by excised soybean leaf tissues. Within 24 hr
ACIFLUORFEN
METABOLISM
after treatment, 90-95% of the absorbed [14C]acifluorfen was metabolized to soluble products. Insoluble residues were negligible. Thin-layer chromatography [2-butanone:HzO:HOAc (10/l/l) 2 x] of methanol-soluble metabolites from leaf disks treated with either [chlorophenyl-14C]- or [nitrophenyI-r4C]acifluorfen clearly demonstrated that the diphenylether bond was rapidly cleaved. In [chlorophenyl-14Claci fluorfen-treated leaf disks, one major metabolite (I; Rf 0.72) was formed. In sharp contrast, two more polar [14C]-labeled metabolites (II and III; Rfls 0.13 and 0.17, respectively) were formed in leaf tissues treated with [nitrophenyl-‘4Clacifluorfen. Time course studies (4-72 hr) with either leaf disks or excised leaf tissues showed that metabolite II was rapidly converted to metabolite III. After 24 hr, III was the major (>90%) metabolite in leaf tissues that were treated with [nitrophenyl-14C]acifluorfen. Metabolite I, however, was not altered during similar time course studies and simply accumulated as the absorbed [chlorophenyl-14C]acifluorfen was metabolized. Characterization of Metabolite Z Increased mobility in an acidic TLC solvent system (Fig. 2) and adsorption on an anion-exchange column [l x 5-cm DEAESephacel (Pharmacia)] demonstrated that metabolite I was acidic. Partial hydrolysis during the isolation of metabolite I fractions resulted in the formation of a less polar product in both neutral and acidic TLC solvent systems. In contrast to metabolite I, this product was not adsorbed on DEAESephacel, but was hydrolyzed by p-glucosidase. These observations indicated that metabolite I was an acidic acylated glucoside. Indeed, when metabolite I was separated with an acidic TLC solvent system, eluted with MeOH and reacted directly with CHzNz at 25°C (12), it was hydrolyzed (>90%) to a neutral product. Mass spectra (CIMS) of the acetylated neutral product [acetic anhydride:pyridine (l:l); 16 hr at 4O”C] after chromatographic purification by
IN SOYBEAN
30s
TLC (R, 0.4; Et,O) and HPLC [isocratic CH3CN:Hz0 (3/2); 3.0 ml/min; R, 4.14 min] showed unequivocally that the metabolite I was hydrolyzed to the O-glucoside of 2chloro-4-trifluoromethylphenol. With ammonia as the ionizing gas, a base peak pseudomolecular ion (M + NH& + at m/z 544 was observed for the glucoside tetraacetate derivative. Additional evidence that the neutral hydrolysis product was a glucoside was obtained by analysis of acid hydrolysis products after reaction with 1N HCl at 100°C for 2 hr (13). Qualitative TLC and GLC analyses showed that glucose was the only carbohydrate present. Quantitative analyses showed that the molar ratio of glucose to the [14C]aglycone was 1:1. Identification of Metabolite Z Metabolite I was identified unequivocally as a malonyl-p-D-glucoside of 2-chloro-4trifluoromethylphenol, by CUMS analysis of the methylated and acetylated derivative, by FT-NMR analysis of the methylated derivative, and by qualitative GLC analysis of dimethylmalonate formed by the reaction of methylated metabolite I with CH2N2 at 25°C (12). A mass spectrum (CIMS) of the methylated and acetylated metabolite I derivative is shown in Fig. 4. With ammonia as the ionizing gas, the pseudomolecular ion (M+NH,)+ at m/z 602 was the base peak. The intact malonyl glucose moiety was identified by the ion fragment at m/z 389. Ion fragments at m/z 502 and 289 appear to be the result of a proton transfer together with the loss of a methyl malonate fragment (- OCCH&OOCHs) from the ions at m/z 602 and 389, respectively. Other peaks are consistent with ion fragment patterns associated with acetylated glucosides (14). FT-NMR spectra of methylated metabolite I in acetone-d6 showed singlet proton peaks at 3.46 ppm (2H) and 3.67 ppm (3H). These peaks were assigned as the methylene and methyl protons of the methyl malonate moiety, respectively. Assignments were based on a comparison with reference
306
FREAR,
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MANSAGER
100$02 H+NHJ
90:
OAc
30;
1
100
200
300
FIG.
of Metabolite
500
600
700
4. Mass spectrum of metabolite I derivative.
dimethylmalonate spectra that showed singlet proton peaks at 3.41 ppm (2H) and 3.68 ppm (6H), and by the fact that the exchangeable methylene proton peak at 3.46 ppm (2H) was lost when D20 was added to the methylated metabolite I sample. Methylated metabolite I spectra also showed a doublet proton peak at 5.23 ppm (1H) with a coupling constant of 7.2 Hz. This peak is characteristic for the anomeric proton of a g-D-ghtcoside. Identification
400
II
Metabolite II was identified as a tripeptide (homoglutathione) conjugate formed by the metabolic cleavage of the acitluorfen diphenylether bond. Characterization was based primarily on the qualitative analysis of amino acid hydrolysis products and mass spectroscopic analysis (CUisobutane) of methylated and acetylated derivatives. Qualitative amino acid analysis (TLC and
GLC) of acid hydrolysates from metabolite II showed the presence of glutamic acid and @alanine. Occasionally, traces of glytine were also detected. Acid hydrolysates of the reference glutathione conjugate contained only glutamic acid and glycine. The cysteine thioether bond was resistant to acid hydrolysis. Metabolite II was clearly different from previously reported fluorodifen glutathione conjugates in pea (Pisum sativum) and peanut (Arachis hypogea) (15, 16). The glytine moiety appeared to be replaced by palanine. Mass spectra (Wisobutane) of methylated and acetylated derivatives confirmed the identity of metabolite II as a homoglutathione conjugate, S-(3-carboxy-4-nitrophenyl) y-glutamyl-cysteinyl-p-alanine. A mass spectrum of the isolated metabolite II derivative is shown in Fig. 5. A base peak protonated molecular ion (MH)+ was ob-
ACIFLUORFEN
METABOLISM
IN
307
SOYBEAN
571
@bIbI)+
80
0 100
rhrl 150
200
250
FIG.
5.
300
350
400
450
500
550
600
Mass spectrum of metabolite II derivative.
served at m/z 571 together with ion fragments at m/z 539 (M-31)+ and 468 (M102)+. Prominent tripeptide and protonated p-alanine ion fragments were found at m/z 358 (M-212)+, and 104, respectively. Other peptide ion fragments at m/z 326, 255, 186, 173 and 144 were present together with characteristic methyl 2-nitro-thiobenzoate ion fragments at m/z 214 and 182. Mass spectra of two dipeptide degradation product derivatives also supported the above identification of metabolite II. One dipeptide product was identified as the yglutamylcysteine conjugate. Chromatographic behavior (TLC and HPLC) and mass spectra (CIfisobutane) were identical to those obtained for the y-glutamylcysteine degradation product of the reference glutathione conjugate (‘lhble 1). The second dipeptide product was identified as the cysteinyl-P-alanine conjugate. Mass spectra (CYisobutane) showed a base peak protonated molecular ion (MH)+ at m/z 428 with ion fragments at m/z 397 (MN-31)+ and 368 (M-59). Prominent dipeptide and proton-
ated @alanine ions were observed at m/z 215 (M-212)+ and 104, respectively. Characteristic methyl 2-nitro-thiobenzoate ion fragments were also present at m/z 214 and 182. Occasionally, mass spectra of isolated metabohte II derivative fractions also contained weak ion fragments that were characteristic of a glutathione conjugate derivative and a cysteinylglycine derivative product. In these instances, small amounts (<5%) of a glutathione conjugate metabolite with very similar chromatographic properties (TLC and HPLC) may have been isolated together with metabolite II. Homoglutathione has been isolated from Phaseolus aureus (17) and characterized as the thiol tripeptide, u-L-glutamyl-L-cysteinyl-lkxlanine (18). It appears to be the major thiol tripeptide (>95%) present in white clover (Trifoiium ripens), soybean (Glycine max), and several bean (Phaseolus) species, but does not appear to be present in peanut (Aruchis hypogea), or pea (Pisum sativum) (17, 19). Indeed, the recent isolation and identification of an analogous
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FREAR,
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357
100
150
200
FIG. 6.
r
250
300
(MH)+
350
I 400
Mass spectrum of metabolite Ill derivative.
Cl1
I
,COOH
CH,OOCCH,COOH ml) HoQpcF3 OH
(I) 7. Proposed scheme for the metabolism of acifluorfen by soybean. Intermediate are shown in brackets. FIG.
metabolites
ACIFLUORFEN
METABOLISM
metabolite II fraction from [nitrophenyl“C]acifluorfen-treated peanut hypocotyl sections showed that it was a glutathione conjugate (unpublished data). Identification
of Metabolite
III
Metabolite III increased with time and appeared to result from the metabolism of II. A tentative identification of metabolite III as a catabolic cysteine conjugate [S-(3carboxy-4nitrophenyl)cysteinel was supported by the fact that the methylated and acetylated derivative of metabolite III cochromatographed (TLC and HPLC) with the derivatized reference cysteine conjugate (Table 1). This identification was confirmed by mass spectroscopic analysis (Fig. 6). Mass spectra (CI/isobutane) showed a base peak protonated molecular ion (MH) + at m/z 357. A prominent ion fragment at m/z 325 (M-31) was also evident together with other characteristic ion fragments at m/z 297 (M-59), 214, 182, and 144 (M-212). Identical mass spectra were obtained with the reference cysteine conjugate derivative (Table 1). CONCLUSION
A proposed scheme for the metabolism of acifluorfen in tolerant soybean is shown in Fig. 7. A rapid initial cleavage of the diphenylether bond results in the formation of a reactive phenolic intermediate (2chloro-4-trifluoromethylphenol) and S-(3carboxy4nitrophenyl) y-glutamyl-cysteinyl-@alanine (II). The phenolic cleavage product is rapidly conjugated as an O-glucoside intermediate and acylated to form a malonyl-P-D-glucoside (I). Further metabolism of II results in the rapid formation of S-(3-carboxy-4-nitrophenyl)cysteine (III). Intermediate dipeptide metabolites were not detected and further metabolism of III to a possible malonylcysteine endproduct (20) was not observed. ACKNOWLEDGMENTS The authors thank Dr. Fred S. Tanaka and Mrs. CaroleJean Lamoureux for their assistance in FT-
IN SOYBEAN
309
NMR and CYMS measurements and the Rohm and Haas Company for providing radioactive acifluorfen and necessary reference compounds. REFERENCES 1. Weed Science Society of America, “Herbicide Handbook,” 4th ed., pp. l-3, 1979. 2. R. Y. Yih, Today’s herbicide: BlazeeM-A new postemergence herbicide, Weeds Today 12 (11, 14 (1980). 3. S. Matsunaka, Diphenyl ethers. In “HerbicidesChemistry, Degradation and Mode of Action” (P. C. Keamey and D. D. Kaufman, Eds.), 2nd ed., pp. 709-739, Dekker, New York, 1976. 4. D. Moreland, W. Blackman, H. Todd, and F. Farmer, Effects of diphenyl ether herbicides on reactions of chloroplasts and mitochondria, Weed Sci. 18, 636 (1970). 5. M. W. Bugg, J. Whitmarsh, C. E. Rieck, and W. S. Cohen, Inhibition of photosynthetic electron transport by diphenyl ether herbicides, Plant Physiol. 65, 47 (1980). 6. G. Sandermann, R. Lambert, and P. Boger, Multifuctional mode of action of substituted nitrodiphenylethers in Scenedesmus cells, Z. Naturforsck. Sect. C Biosci. 36, 633 (1981). 7. T. Y. Leong and W. R. Briggs, Evidence from studies with acifluorfen for particitpation of a flavin-cytochrome complex in blue light photoreception for phototropism of oat coleoptiles, Plant Pkysiol. 70, 875 (1982). 8. R. L. Ritter and H. D. Coble, Penetration, translocation and metabolism of acifluorfen, in “Weed Science Society of America,” Abstr. No. 17, p. 8, 1980. 9. M. Blankendaal, R. H. Hodgson, D. G. Davis, R. A. Hoerauf, and R. H. Shimabukuro, “Growing Plants Without Soil for Experimental Use,” USDA, ARS, Miscellaneous Publication No. 1251, 1972. 10. D. Roach and C. W. Gehrke, The gas-liquid chromatography of amino acids, .I. Ckromatogr. 43, 303 (1969). 11. D. Roach and C. W. Gehrke, Direct esterification of the protein amino acids gas-liquid chromatography of N-TFA n-butyl esters, J. Ckromatogr. 44, 269 (1969). 12. A. J. Dutton, T. R. Roberts, and A. N. Wright, Characterization of acidic conjugates of flamprop in wheat, Chemosphere 5, 195 (1976). 13. D. S. Frear and H. R. Swanson, New metabolites of monuron in excised cotton leaves, Pkytochemistry 11, 1919 (1972). 14. J. Lonngren and S. Svensson, Mass spectrometry in structural analysis of natural carbohydrates. Advan. Carbokydr. Ckem. Biockem. 29, 41 (1979). 15. D. S. Frear and H. R. Swanson, Metabolism of substituted diphenylether herbicides in plants.
FREAR, SWANSON,
310
1. Enzymatic cleavage of fluorodifen Pestic.
Biochem.
Physiol.
in peas,
3, 473, (1973).
16. R. H. Shimabukuro, G. L. Lamoureux, H. R. Swanson, W. C. Walsh, L. E. Stafford, and D. S. Frear, Metabolism of substituted diphenylether herbicides in plants. II. Identification of a new fluorodifen metabohte, S-(2-nitro-4-trifluoromethyphenyl)-glutathione in Peanut, Pestic. Biochem. Physiol. 3, 483 (1973). 17. P. R. Carnegie, Isolation of a homologue of glutathione and other peptides from seedlings of Phnseolus
aureus,
Biochem.
J. 89, 459 (1963).
AND MANSAGER 18. P. R. Carnegie, Structure and properties of a homologue of glutathione, Biochem. J. 89, 471 (1963). 19. C. A. Price, A new thiol in legumes, Nature (London) 180, 148 (1957). 20. G. L. Lamoureux and D. G. Rusness, Malonylcysteine conjugates as end-products of glutathione conjugate metabolism in plants, in “Pesticide Chemistry: Human Welfare and the Environment,” Vol. 3, (S. Matsunaka, D. H. Hudson, and S. D. Murphy, Eds.), pp. 2953(K), Pergamon Press, New York, 1983.
BIOCHEMISTRY
AND
PHYSIOLOGY
20,
299-310 (1983)
Acifluorfen Metabolism in Soybean: Diphenylether Bond Cleavage and the Formation of Homoglutathione, Cysteine, and Glucose Conjugates1s2 D. S. FREAR, H. R. SWANSON, AND E. R. MANSAGER U.S. Department of Agriculture,
Agricultural Research Service, Metabolism Laboratory, Fargo, North Dakota 58105
and Radiation Research
Received February 15, 1983; accepted May 27, 1983 Metabolism of the substituted diphenylether herbicide, acitluorfen [sodium 5-(2-chloro-4-trifluoromethylphenoxy)-2nitrobenzoate], was studied in excised leaf tissues of soybean [Glycine mar (L.) Merr. ‘Evans’]. Studies with [chlorophenyl-‘4C]- and [nitrophenyl-t4C]acifluorfen showed that the diphenylether bond was rapidly cleaved. From 85 to 95% of the absorbed [t4C]acifluorfen was metabolized in less than 24 hr. Major polar metabolites were isolated and purified by solvent partitioning, adsorption, thin layer, and high-performance liquid chromatography. The major [chlorophenyPC]-labeled metabolite was identified as a malonyl-S-D-ghrcoside (I) of 2-chloro-4-trifluoromethylphenol. Major [nitrophenyPC]-labeled metabolites were identified as a homoglutathione conjugate [S-(3-carboxy+titrophenyl) y-glutamyl-cysteinyl+-alanine] (II), and a cysteine conjugate [S-(3-carboxy-4-nitrophenyl)cysteine] (III). INTRODUCTION
Acifluorfen [sodium 5-(2-chloro-4-trifluoromethylphenoxy) - 2 - nitrobenzoate] is a substituted diphenylether herbicide for selective preemergence and postemergence weed control in soybean (1, 2). Diphenylether herbicides require light absorption for their action (3). As potent inhibitors of photosynthesis, they block electron transport (4, 5), inhibit energy transfer (6), and affect plasma membrane systems (7). Although a recent paper (8) has indicated that differential metabolism may be a factor in the selective action of acifluorfen, detailed information on the nature of acifluorfen metabolites and metabolic pathways in susceptible and tolerant plants has not been reported. The objective of the present study was to establish the pathway of acifluorfen metab’ Presented in part at the North Central Weed Control Conference, Omaha, Neb., December, 1980. 2 Mention of .a trademark or proprietary product does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable.
olism in tolerant and identification MATERIALS
soybean by the isolation of major metabolites. AND METHODS
Chemicals. Sodium 5-(2-chloro-4-u-&oromethyl[U - 14C]phenoxy) - 2 - nitrobenzoate ([chlorophenyl-14C]acifluorfen, 1.27 $i/pmol), ethyl 5-(2-chloro-4-trifluoromethylphenoxy)-2-nitro[U-t4C]benzoate (1.95 pCi/pmol), and methyl-2-nitro-5-fluorobenzoate were provided by the Rohm and Haas Company. [Nitrophenyl-‘4C]acifluorfen was prepared by the reaction of ethyl 5-(Zchloro4 - trifluoromethylphenoxy) - 2 - nitro[U *4C]benzoate (40 FCi in 0.5 ml EtOH) with 1 ml of 1 N KOH at 80°C for 16 hr. The reaction mixture was acidifed (pH 2.0), extracted with Et,O, and the [nitrophenyl14C]acifluorfen (~95% yield) was purified by thin-layer chromatography (TLC) [Rf 0.5, benzene/HOAc (50:8)]. Plant materials and treatments. Soybeans (Glycine max Merril, var. Evans) were seeded in vermiculite, subirrigated with nutrient solution and grown in the greenhouse (9). Soybean leaf disks were
299 0048-3575183 $3.OU
300
FREAR,
SWANSON.
used to compare the metabolism of [chlorophenyl-i4C]and [nitrophenyl-‘4C]acifluorfen. Seven disks (1 cm diameter) from fully expanded trifoliate leaves were placed in 50-ml beakers and treated with aqueous sodium salt solutions (0.5 ml) of either [chlorophenyl-14C]or [nitrophenyl-14C]acifluorfen (1.1 x lo6 dpm; 7.36 x lo3 dpm/pg). Treated disks were incubated at room temperature for 8 hr under cool white fluorescent lights (50-60 kEm-2 set-I), rinsed with distilled water, and extracted with 80% MeOH. Filtered MeOH extracts were concentrated in vucuo (30°C) and metabolites were separated by TLC [2-butanone:H20:HOAc (10/1/l) 2 x]. Excised leaves from 5- to 6-week-old plants were used for metabolite isolation and identification studies. Fully expanded trifoliate leaves were pulse-treated via cut petioles with aqueous sodium salt solutions (2 ml) of either [chlorophenyl-14C]or [nitrophenyl-*4C]acifluorfen (4.8 x 10e3 M; 0.14-0.23 @i/pmol). After 2-3 hr, the [14C]acifluorfen was absorbed and distilled water was added. Treated leaves were maintained at room temperature under cool white fluorescent lights (50-60 kErnp2 set -I) for 12 hr and either extracted directly (metabolites II and III) or transferred to the dark for an additional 12 hr and extracted (metabolite I). Under these conditions, 98-99% of the [14C]acifluorfen was absorbed. Six to eight trifoliate leaves, with a combined fresh weight of 30-40 g, were used for each [i4C]acifluorfen treatment. Chromatography. Silica gel HF plates (250 or 500 Frn, Analabs) were used for TLC of metabolites and derivatives. i4CLabeled metabolites were located with a Packard 7201 radiochromatogram scanner or by fluorescence quenching under uv light. Whatman LKSF silica gel plates (250 km) and ninhydrin were used for the TLC and detection of amino acids. A Waters Associates liquid chromatograph system with a 254-nm uv detector and a 1055 CA1 radioactivity monitor was used for high-performance liquid chroma-
AND
MANSAGER
tography (HPLC). Solvent flow rates of 2.0 ml/min were used with 10 km Cl8 RadialPak cartridges (Waters Associates). A Tracer-Microtek gas chromatograph with FID and a glass column (150 cm x 4 mm i.d.) packed with a 3% SP 2340 (Supelco) on 100/120 Supelcoport was used for gas chromatographic (GLC) analysis of dimethylmalonate. Injector and detector temperatures were maintained at 250°C. The oven temperature was 85°C and the nitrogen carrier gas flow rate was 60 cc/min. Under these conditions, the retention time for standard dimethylmalonate was 1.6 min. A glass column (1.8 m x 2 mm) packed with 0.65% EGA (ethylene glycol adipate) on 60180 acid washed Chromosorb W was used for the GLC analysis of amino acids as N-trifluoroacetyl n-butyl ester derivatives (IO, 11). An initial oven temperature (1OO’C) was held for 2 min. This was followed by a temperature program (3YYmin) to 185°C and a final I-min hold at 185°C. The nitrogen carrier gas flow rate was 40 cc/min. Under these conditions, derivatives of glycine, l3-alanine, and glutamic acid had retention times of 9.2, 11.4, and 28.7 min, respectively. Instrumentation. Mass spectra were obtained with a Varian MAT 112s spectrometer equipped with a combination EIKI source and a SS-200 data system. Samples were inserted with a solid sample probe. Ammonia and isobutane were used as ionizing gases for chemical ionization (CI) spectra. Proton FT-NMR spectra were obtained with a JEOL FX-90Q Fourier transform spectrometer equipped with a microprobe and a NV-VTS variable temperature controller. Tetramethylsilane was used as the internal reference and samples were analyzed in I.&mm-o.d. tubes. Quantitative t4C measurements were made with Insta-Gel (Packard Instrument Company) counting cocktail and a Packard 3375 liquid scintillation spectrometer. Synthesis and derivatization of reference glutathione and cysteine conjugates. S-(3-
ACIFLUORFEN
METABOLISM
IN
GSH + F FIG.
1. Synthesis
301
SOYBEAN
+ HF of reference
S-(3-carbomethoxy4nirropheny~glutathione
Carbomethoxy - 4 - nitrophenyl)glutathione and S - (3 - carbomethoxy - 4 - nitrophenyl)cysteine were synthesized by the reaction of either glutathione or cysteine with methyl 2 - nitro - 5 - fluorobenzoate (Fig. 1). Methyl 2-nitro-5fluorobenzoate (0.55 mmol) was dissolved in MeOH (2 ml) and mixed with an equal volume of an aqueous solution (pH 10) of either glutathione (0.5 mmol) or cysteine (0.5 mmol). After 4 hr at 30°C the reaction mixture was adjusted to pH 5.0 and the MeOH removed by a stream of Nz. The aqueous solution was acidified (pH < l.O), washed with Et,O, placed on a 1 x IO-cm XAD-2 column and eluted stepwise with 25 ml volumes of H20, HPO:CH$N (9/l), and H20:CHsCN (l/l). Reaction products in the HzO:CHsCN (l/l) eluate were taken to dryness in vucuo (30°C) and weighed. Reaction yields were 80-W% and the chemical purity of the glutathione or cysteine products was >95% by TLC analysis, [Rf 0.37 and 0.47, respectively; BuOH:HOAc:H20 (120/30/50)]. Glutathione and cysteine conjugates of 3carbomethoxy-4nitrobenzene (5 mg each) were methylated with 2 ml of methanolicHCl (3 N) at 80°C for 30 min, evaporated to dryness under a stream of Nz, and acetylated with 500 ~1 of acetic anhydride at 40°C for 16 hr. Derivatized products were concentrated under a stream of NZ, dissolved in MeOH (500 pl), and chromatographed by preparative TLC [CH2C12/ MeOH (9: l)]. Wo glutathione conjugate fractions (Z$ 0.46 and 0.53) and one cysteine conjugate fraction (Rf 0.80) were eluted with CHsCN. Further purification of these fractions was achieved by HPLC [isocratic CHsCN/H20 (35/65)]. The glutathione coniuaate derivative (RJ 0.46) and
conjugate.
the cysteine conjugate derivative (Rf 0.80) produced single sharp HPLC peaks at 2.20 and 3.46 min, respectively. The glutathione conjugate fraction at Rf0.53, however, produced two sharp HPLC peaks of almost equal intensity (2.38 and 2.92 min). Mass spectra (Wisobutane) of the isolated derivative fractions confirmed the structures of the reference glutathione and cysteine conjugates as well as the structures of two glutathione conjugate degradation products (dipeptides) formed during derivatization (Table 1). Characteristic derivative ions included protonated molecular ions (MH)+ with (M-31)+ or (M-59)+ ion fragments, strong peptide or cysteine derivative ion fragments (M-212)+ formed by cleavage of the cysteine thioether bond, and methyl 2nitro-5-thiobenzoate ion fragments at m/z 214 and 182. Extraction
and isoiation
of metaboiite
I.
Procedures developed for the extraction and isolation of metabolite I are summarized in Fig. 2. Initial partitioning procedures were similar to those of Dutton et al. (12). [Chlorophenyl-14C]acifluorfen-treated leaves (30-40 g fresh wt) were ground in an Omnimixer with 5 vol of 50% aqueous acetone. Extracts were vacuum filtered (10 pm, LC Millipore) and concentrated in vacua (30°C). The aqueous concentrate (~30 ml) was adjusted to pH 9.0 (1 N KOH), extracted twice with equal volumes of EtOAc, adjusted to pH 4.0 (1 N HCl), and extracted with equal volumes of CHC13(2 x ) and EtOAc(2 x ). The pH 4.0 EtOAc extract fraction was taken to dryness in vucuo (30°C). Only l-3% of the absorbed i4C was present in the insoluble residue and the aqueous phase after EtOAc extraction at pH 4.0. Small amounts of unreacted 114Clacifluorfen (3-4%) were
302
FREAR,
SWANSON, TABLE
Characterization
AND
MANSAGER
1
of Reference S-(3-Carbomethoxy-?-nitrophenyl.Elutathione
and Cysteine Conjugates
Conjugate derivatives HPLC retention time
Conjugates Glutathione
TLC RF
(Rf 0.37)
(Cysteinylglycine)
0.46 I
(mill) 2.20 2.38
0.53 (y-Glutamylcysteine) Cysteine
(Rr 0.47)’
n BuOH:HOAc:H20 (120/30/50). b CH#&:MeOH (9/l). c Isocratic CH$N:H20 (35/65),
2.92 0.80
3.46
Characteristic
U/MS
ions
(m/z)
557(MH) + ,525(M-31) + ,495, 344(M-212)+, 312, 255, 214 and 182. 414(MH)+, 354(M-59)+, 315(M-88)+, 214, 201(M-212)+ and 182. 5OO(MH)+, 287(M-212)+, 214, 186 and 182. 357(MH) + , 325(M-3 1) + , 295(M-59) 182 and 144(M-212)+.
+ , 214,
2 ml/min.
present in the pH 9.0 EtOAc and the pH 4.0 CHC13 fractions. Further purification was achieved by a sequence of preparative TLC and reverse-phase chromatography steps. Preparative TLC-separated fractions [RfO.20 and 0.60, respectively, with CHCIJ: MeOH:H20 (65/25/4)2 x or CHC13: MeOH:HzO:HOAc (65/25/4/4)2 x ] were eluted with MeOH, taken to dryness in vucuo (30°C) and dissolved in Hz0 prior to adsorption on preconditioned Cl8 Sep-Pak cartridges (Waters Associates) and elution with 40% aqueous MeOH. Metabolite recoveries were >90% for each chromatographic purification step. Derivatization of metabolite I. Metabolite I was dissolved in a small volume of MeOH:EtzO (l/4) and esterified with a slight excess of CH2N2 in MeOH:Et*O (1:9) at 4°C for 15 min (12). Excess CHzNz was removed with a stream of N2 and the reaction products were purified by TLC [CHC13:MeOH:H20 (65/25/4)]. The methylated product (ZQ 0.62) was eluted with CHsCN and chromatographed by HPLC [isocratic MeOH:HzO (3/2)]. A single sharp peak (R, 4.5 min) was eluted and taken to dryness in vucuo (30°C) prior to FT-NMR analysis, malonate determination, and acetylation for CI/MS analysis. Methylated me-
tabolite I was acetylated with acetic anhydride/pyridine (9: 1) at 35°C for 3 hr. Excess reagents were removed with a stream of N2 and several additions of CH&N. Malonate determinations. Methylated metabolite I (75-100 p+g)was dissolved in 0.1 ml MeOH and reacted at room temperature for 3 hr with an excess of CH2N2 dissolved in Et20 (12). At the end of the reaction, excess CHzN2 was removed with a gentle stream of Nz and aliquots of the reaction mixture were analyzed for dimethylmalonate (GLC). Extraction and isolation of metabolites ZZ and ZZZ. [Nitrophenyl-14C]acifluorfen treatment times prior to extraction were important in the isolation of metabolites II and III. A 24-hr treatment period resulted in >90% metabolism, but metabolite II was present only as a minor product (~10%). Shorter treatment times reduced overall metabolism, but increased the amounts of II in relation to III. As a compromise, a 12hr treatment period was selected for the isolation of both metabolites. Under these conditions, approximately 70% of the absorbed [nitrophenyl-14C]acifluorfen was metabolized, and the ratio of II to III was =1:2. The extraction and isolation of metabo-
ACIFLUORFEN
lj4 cg
METABOLISM
303
IN SOYBEAN
Le8f
Mdfhmfen-1roated
li88w
y-pzzyr
Soluble EfAc
lnroluble
‘-/G&w~~
(p;...,
-
Aqueou8 CHCI,/l-&O
(pH
4.0)
EtOAc
AQUOOW
-ITLC CHC~:M~OH:H~0~66/25/4~2x I ~2’8 Sop
Pek’R) H20:MeOH(3/2)
I TLC CHClyMeOH:H20:HOAc(6S/2S14/4)2x C’8
1 sep
P.kR’ H,O:MeOH(3/2)
I FIG.
2. Extraction and isolation of metabolite I.
lites II and III are summarized in Fig. 3. [Nitrophenyl-‘4C]acio~en-treated leaves (30-40 g fresh wt) were extracted with 80% aqueous methanol as described for metabolite I. Only l-3% of the absorbed r4C remained in the insoluble residue fraction. The aqueous concentrate (~30 ml) was adjusted to pH 3.5 with 1 N HCl extracted three times with equal volumes of Et20 to remove unreacted [14C]acifluorfen, adjusted to pH 2.0 with additional 1 N HCl, and placed on a 2.5 x 25cm column of XAD-2. Metabolites II and III were separated on the XAD column by stepwise elution with 200 ml of Hz0 and 200 ml of aqueous 20% CH$N. Column eluate frac-
tions were concentrated in vacua (30°C) and each metabolite was purified separately by a sequence of preparative TLC and reverse-phase chromatography steps. Metabolites were eluted from Cl* Sep-Pak cartridges with Hz0 and taken to dryness in wcuo (30°C). Preparative TLC-separated metabolites were eluted with 50% aqueous CHSCN and taken to dryness in vacua (30”). Metabolite recoveries from XAD and subsequent reverse-phase or thin-layer chromatographic purification steps were 30%. Derivatization
of metabolites
ZZ and ZZZ.
Metabolites II and III were methylated with 0.5 ml of methanolic HCl (3 N) at
304
FREAR,
SWANSON,
AND
MANSAGER
Soluble c
Insoluble
~Et,OIH,O
,.;,,I
Aqueous
I320
-
b
HzO:CHJCN
(4/t)
H20
c TLC
P-Butanone:H,O:HOAc
TLC
BuOH:HOAc:H,O
i TLC
2-Butanone:H,O:HOAc
(10/1/1)4x (120/30/50)2x (10/l
TLC /1)4x
TLC
mtabolite FIG.
I(
3. Extraction and isolation of metabolites II and III.
80” C for 30 and 60 min, respectively. A shortened reaction time for the methylation of metabolite II was selected to reduce the formation of peptide hydrolysis products and still achieve reasonable (ZO-30%) methylation of the nitrobenzoic acid moiety. Methylation reaction mixtures were evaporated to dryness under a stream of NZ, acetylated with 200 ~1 of acetic anhydride at 40°C for 12 hr, taken to dryness again, and dissolved in CHsCN. Methylated and acetylated reaction products were purified by preparative TLC [CH$&:MeOH (9/l)] and HPLC [isocratic CHsCN/H20 (35/65)] for CI/MS analysis and comparison with methylated and acetylated derivatives of reference glutathione and cysteine conjugates. Amino acid determination. Metabolite II
(Fig. 3) was purified further by HPLC [initial 1% HOAC:CHsCN @O/10)to (85/U) in 2 min and (80/20) in 15 min at 2 ml/min; R, 5.65 min] and hydrolyzed under N2 with 0.5 ml of constant boiling HCl at 105°C for 20 hr. Hydrolysates were analyzed directly for amino acids by TLC or converted to N-trifluoroacetyl n-butyl ester derivatives (11) and analyzed by GLC (10). TLC [2 butanone:HOAC:HzO (10/1/l) 4 X] Rf values for reference amino acids were: glycine (0.06); glutamic acid (0.12); @-alanine (0.17); and alanine (0.23). RESULTS
AND
DISCUSSION
Acif2uorfen Metabolism
Acifluorfen was metabolized rapidly by excised soybean leaf tissues. Within 24 hr
ACIFLUORFEN
METABOLISM
after treatment, 90-95% of the absorbed [14C]acifluorfen was metabolized to soluble products. Insoluble residues were negligible. Thin-layer chromatography [2-butanone:HzO:HOAc (10/l/l) 2 x] of methanol-soluble metabolites from leaf disks treated with either [chlorophenyl-14C]- or [nitrophenyI-r4C]acifluorfen clearly demonstrated that the diphenylether bond was rapidly cleaved. In [chlorophenyl-14Claci fluorfen-treated leaf disks, one major metabolite (I; Rf 0.72) was formed. In sharp contrast, two more polar [14C]-labeled metabolites (II and III; Rfls 0.13 and 0.17, respectively) were formed in leaf tissues treated with [nitrophenyl-‘4Clacifluorfen. Time course studies (4-72 hr) with either leaf disks or excised leaf tissues showed that metabolite II was rapidly converted to metabolite III. After 24 hr, III was the major (>90%) metabolite in leaf tissues that were treated with [nitrophenyl-14C]acifluorfen. Metabolite I, however, was not altered during similar time course studies and simply accumulated as the absorbed [chlorophenyl-14C]acifluorfen was metabolized. Characterization of Metabolite Z Increased mobility in an acidic TLC solvent system (Fig. 2) and adsorption on an anion-exchange column [l x 5-cm DEAESephacel (Pharmacia)] demonstrated that metabolite I was acidic. Partial hydrolysis during the isolation of metabolite I fractions resulted in the formation of a less polar product in both neutral and acidic TLC solvent systems. In contrast to metabolite I, this product was not adsorbed on DEAESephacel, but was hydrolyzed by p-glucosidase. These observations indicated that metabolite I was an acidic acylated glucoside. Indeed, when metabolite I was separated with an acidic TLC solvent system, eluted with MeOH and reacted directly with CHzNz at 25°C (12), it was hydrolyzed (>90%) to a neutral product. Mass spectra (CIMS) of the acetylated neutral product [acetic anhydride:pyridine (l:l); 16 hr at 4O”C] after chromatographic purification by
IN SOYBEAN
30s
TLC (R, 0.4; Et,O) and HPLC [isocratic CH3CN:Hz0 (3/2); 3.0 ml/min; R, 4.14 min] showed unequivocally that the metabolite I was hydrolyzed to the O-glucoside of 2chloro-4-trifluoromethylphenol. With ammonia as the ionizing gas, a base peak pseudomolecular ion (M + NH& + at m/z 544 was observed for the glucoside tetraacetate derivative. Additional evidence that the neutral hydrolysis product was a glucoside was obtained by analysis of acid hydrolysis products after reaction with 1N HCl at 100°C for 2 hr (13). Qualitative TLC and GLC analyses showed that glucose was the only carbohydrate present. Quantitative analyses showed that the molar ratio of glucose to the [14C]aglycone was 1:1. Identification of Metabolite Z Metabolite I was identified unequivocally as a malonyl-p-D-glucoside of 2-chloro-4trifluoromethylphenol, by CUMS analysis of the methylated and acetylated derivative, by FT-NMR analysis of the methylated derivative, and by qualitative GLC analysis of dimethylmalonate formed by the reaction of methylated metabolite I with CH2N2 at 25°C (12). A mass spectrum (CIMS) of the methylated and acetylated metabolite I derivative is shown in Fig. 4. With ammonia as the ionizing gas, the pseudomolecular ion (M+NH,)+ at m/z 602 was the base peak. The intact malonyl glucose moiety was identified by the ion fragment at m/z 389. Ion fragments at m/z 502 and 289 appear to be the result of a proton transfer together with the loss of a methyl malonate fragment (- OCCH&OOCHs) from the ions at m/z 602 and 389, respectively. Other peaks are consistent with ion fragment patterns associated with acetylated glucosides (14). FT-NMR spectra of methylated metabolite I in acetone-d6 showed singlet proton peaks at 3.46 ppm (2H) and 3.67 ppm (3H). These peaks were assigned as the methylene and methyl protons of the methyl malonate moiety, respectively. Assignments were based on a comparison with reference
306
FREAR,
SWANSON,
AND
MANSAGER
100$02 H+NHJ
90:
OAc
30;
1
100
200
300
FIG.
of Metabolite
500
600
700
4. Mass spectrum of metabolite I derivative.
dimethylmalonate spectra that showed singlet proton peaks at 3.41 ppm (2H) and 3.68 ppm (6H), and by the fact that the exchangeable methylene proton peak at 3.46 ppm (2H) was lost when D20 was added to the methylated metabolite I sample. Methylated metabolite I spectra also showed a doublet proton peak at 5.23 ppm (1H) with a coupling constant of 7.2 Hz. This peak is characteristic for the anomeric proton of a g-D-ghtcoside. Identification
400
II
Metabolite II was identified as a tripeptide (homoglutathione) conjugate formed by the metabolic cleavage of the acitluorfen diphenylether bond. Characterization was based primarily on the qualitative analysis of amino acid hydrolysis products and mass spectroscopic analysis (CUisobutane) of methylated and acetylated derivatives. Qualitative amino acid analysis (TLC and
GLC) of acid hydrolysates from metabolite II showed the presence of glutamic acid and @alanine. Occasionally, traces of glytine were also detected. Acid hydrolysates of the reference glutathione conjugate contained only glutamic acid and glycine. The cysteine thioether bond was resistant to acid hydrolysis. Metabolite II was clearly different from previously reported fluorodifen glutathione conjugates in pea (Pisum sativum) and peanut (Arachis hypogea) (15, 16). The glytine moiety appeared to be replaced by palanine. Mass spectra (Wisobutane) of methylated and acetylated derivatives confirmed the identity of metabolite II as a homoglutathione conjugate, S-(3-carboxy-4-nitrophenyl) y-glutamyl-cysteinyl-p-alanine. A mass spectrum of the isolated metabolite II derivative is shown in Fig. 5. A base peak protonated molecular ion (MH)+ was ob-
ACIFLUORFEN
METABOLISM
IN
307
SOYBEAN
571
@bIbI)+
80
0 100
rhrl 150
200
250
FIG.
5.
300
350
400
450
500
550
600
Mass spectrum of metabolite II derivative.
served at m/z 571 together with ion fragments at m/z 539 (M-31)+ and 468 (M102)+. Prominent tripeptide and protonated p-alanine ion fragments were found at m/z 358 (M-212)+, and 104, respectively. Other peptide ion fragments at m/z 326, 255, 186, 173 and 144 were present together with characteristic methyl 2-nitro-thiobenzoate ion fragments at m/z 214 and 182. Mass spectra of two dipeptide degradation product derivatives also supported the above identification of metabolite II. One dipeptide product was identified as the yglutamylcysteine conjugate. Chromatographic behavior (TLC and HPLC) and mass spectra (CIfisobutane) were identical to those obtained for the y-glutamylcysteine degradation product of the reference glutathione conjugate (‘lhble 1). The second dipeptide product was identified as the cysteinyl-P-alanine conjugate. Mass spectra (CYisobutane) showed a base peak protonated molecular ion (MH)+ at m/z 428 with ion fragments at m/z 397 (MN-31)+ and 368 (M-59). Prominent dipeptide and proton-
ated @alanine ions were observed at m/z 215 (M-212)+ and 104, respectively. Characteristic methyl 2-nitro-thiobenzoate ion fragments were also present at m/z 214 and 182. Occasionally, mass spectra of isolated metabohte II derivative fractions also contained weak ion fragments that were characteristic of a glutathione conjugate derivative and a cysteinylglycine derivative product. In these instances, small amounts (<5%) of a glutathione conjugate metabolite with very similar chromatographic properties (TLC and HPLC) may have been isolated together with metabolite II. Homoglutathione has been isolated from Phaseolus aureus (17) and characterized as the thiol tripeptide, u-L-glutamyl-L-cysteinyl-lkxlanine (18). It appears to be the major thiol tripeptide (>95%) present in white clover (Trifoiium ripens), soybean (Glycine max), and several bean (Phaseolus) species, but does not appear to be present in peanut (Aruchis hypogea), or pea (Pisum sativum) (17, 19). Indeed, the recent isolation and identification of an analogous
308
FREAR,
SWANSON,
AND
MANSAGER
357
100
150
200
FIG. 6.
r
250
300
(MH)+
350
I 400
Mass spectrum of metabolite Ill derivative.
Cl1
I
,COOH
CH,OOCCH,COOH ml) HoQpcF3 OH
(I) 7. Proposed scheme for the metabolism of acifluorfen by soybean. Intermediate are shown in brackets. FIG.
metabolites
ACIFLUORFEN
METABOLISM
metabolite II fraction from [nitrophenyl“C]acifluorfen-treated peanut hypocotyl sections showed that it was a glutathione conjugate (unpublished data). Identification
of Metabolite
III
Metabolite III increased with time and appeared to result from the metabolism of II. A tentative identification of metabolite III as a catabolic cysteine conjugate [S-(3carboxy-4nitrophenyl)cysteinel was supported by the fact that the methylated and acetylated derivative of metabolite III cochromatographed (TLC and HPLC) with the derivatized reference cysteine conjugate (Table 1). This identification was confirmed by mass spectroscopic analysis (Fig. 6). Mass spectra (CI/isobutane) showed a base peak protonated molecular ion (MH) + at m/z 357. A prominent ion fragment at m/z 325 (M-31) was also evident together with other characteristic ion fragments at m/z 297 (M-59), 214, 182, and 144 (M-212). Identical mass spectra were obtained with the reference cysteine conjugate derivative (Table 1). CONCLUSION
A proposed scheme for the metabolism of acifluorfen in tolerant soybean is shown in Fig. 7. A rapid initial cleavage of the diphenylether bond results in the formation of a reactive phenolic intermediate (2chloro-4-trifluoromethylphenol) and S-(3carboxy4nitrophenyl) y-glutamyl-cysteinyl-@alanine (II). The phenolic cleavage product is rapidly conjugated as an O-glucoside intermediate and acylated to form a malonyl-P-D-glucoside (I). Further metabolism of II results in the rapid formation of S-(3-carboxy-4-nitrophenyl)cysteine (III). Intermediate dipeptide metabolites were not detected and further metabolism of III to a possible malonylcysteine endproduct (20) was not observed. ACKNOWLEDGMENTS The authors thank Dr. Fred S. Tanaka and Mrs. CaroleJean Lamoureux for their assistance in FT-
IN SOYBEAN
309
NMR and CYMS measurements and the Rohm and Haas Company for providing radioactive acifluorfen and necessary reference compounds. REFERENCES 1. Weed Science Society of America, “Herbicide Handbook,” 4th ed., pp. l-3, 1979. 2. R. Y. Yih, Today’s herbicide: BlazeeM-A new postemergence herbicide, Weeds Today 12 (11, 14 (1980). 3. S. Matsunaka, Diphenyl ethers. In “HerbicidesChemistry, Degradation and Mode of Action” (P. C. Keamey and D. D. Kaufman, Eds.), 2nd ed., pp. 709-739, Dekker, New York, 1976. 4. D. Moreland, W. Blackman, H. Todd, and F. Farmer, Effects of diphenyl ether herbicides on reactions of chloroplasts and mitochondria, Weed Sci. 18, 636 (1970). 5. M. W. Bugg, J. Whitmarsh, C. E. Rieck, and W. S. Cohen, Inhibition of photosynthetic electron transport by diphenyl ether herbicides, Plant Physiol. 65, 47 (1980). 6. G. Sandermann, R. Lambert, and P. Boger, Multifuctional mode of action of substituted nitrodiphenylethers in Scenedesmus cells, Z. Naturforsck. Sect. C Biosci. 36, 633 (1981). 7. T. Y. Leong and W. R. Briggs, Evidence from studies with acifluorfen for particitpation of a flavin-cytochrome complex in blue light photoreception for phototropism of oat coleoptiles, Plant Pkysiol. 70, 875 (1982). 8. R. L. Ritter and H. D. Coble, Penetration, translocation and metabolism of acifluorfen, in “Weed Science Society of America,” Abstr. No. 17, p. 8, 1980. 9. M. Blankendaal, R. H. Hodgson, D. G. Davis, R. A. Hoerauf, and R. H. Shimabukuro, “Growing Plants Without Soil for Experimental Use,” USDA, ARS, Miscellaneous Publication No. 1251, 1972. 10. D. Roach and C. W. Gehrke, The gas-liquid chromatography of amino acids, .I. Ckromatogr. 43, 303 (1969). 11. D. Roach and C. W. Gehrke, Direct esterification of the protein amino acids gas-liquid chromatography of N-TFA n-butyl esters, J. Ckromatogr. 44, 269 (1969). 12. A. J. Dutton, T. R. Roberts, and A. N. Wright, Characterization of acidic conjugates of flamprop in wheat, Chemosphere 5, 195 (1976). 13. D. S. Frear and H. R. Swanson, New metabolites of monuron in excised cotton leaves, Pkytochemistry 11, 1919 (1972). 14. J. Lonngren and S. Svensson, Mass spectrometry in structural analysis of natural carbohydrates. Advan. Carbokydr. Ckem. Biockem. 29, 41 (1979). 15. D. S. Frear and H. R. Swanson, Metabolism of substituted diphenylether herbicides in plants.
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1. Enzymatic cleavage of fluorodifen Pestic.
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AND MANSAGER 18. P. R. Carnegie, Structure and properties of a homologue of glutathione, Biochem. J. 89, 471 (1963). 19. C. A. Price, A new thiol in legumes, Nature (London) 180, 148 (1957). 20. G. L. Lamoureux and D. G. Rusness, Malonylcysteine conjugates as end-products of glutathione conjugate metabolism in plants, in “Pesticide Chemistry: Human Welfare and the Environment,” Vol. 3, (S. Matsunaka, D. H. Hudson, and S. D. Murphy, Eds.), pp. 2953(K), Pergamon Press, New York, 1983.